Many noncoding RNAs are known to play a role in the cell directly linked to their structure. Structure prediction based on the sole sequence is, however, a challenging task. On the other hand, thanks to the low cost of sequencing technologies, a very large number of homologous sequences are becoming available for many RNA families. In the protein community, the idea of exploiting the covariance of mutations within a family to predict the protein structure using the direct-coupling-analysis (DCA) method has emerged in the last decade. The application of DCA to RNA systems has been limited so far. We here perform an assessment of the DCA method on 17 riboswitch families, comparing it with the commonly used mutual information analysis and with state-of-the-art R-scape covariance method. We also compare different flavors of DCA, including mean-field, pseudolikelihood, and a proposed stochastic procedure (Boltzmann learning) for solving exactly the DCA inverse problem. Boltzmann learning outperforms the other methods in predicting contacts observed in high-resolution crystal structures.

Respiratory support of the critically ill neonate has steadily shifted from invasive to noninvasive forms of support. There have recently been a number of important advances in our understanding of the changes to neonatal resuscitation practices as they pertain to clinically important outcomes, mechanisms of gas exchange for high-flow nasal cannula, and best use of noninvasive ventilation and predicting response. Although the proportion of infants requiring intubation and mechanical ventilation has decreased, the most severely ill often still require intubation and ventilation. Recently, volume-targeted ventilation, high-frequency ventilation, and different methods of assessing weaning and extubation have been investigated. This review summarizes a number of important advances that have been made in the management of prematurity and neonatal respiratory distress syndrome.

Formate oxidation to carbon dioxide is a key reaction in one-carbon compound metabolism, and its reverse reaction represents the first step in carbon assimilation in the acetogenic and methanogenic branches of many anaerobic organisms. The molybdenum-containing dehydrogenase FdsABG is a soluble NAD+-dependent formate dehydrogenase and a member of the NADH dehydrogenase superfamily. Here, we present the first structure of the FdsBG subcomplex of the cytosolic FdsABG formate dehydrogenase from the hydrogen-oxidizing bacterium Cupriavidus necator H16 both with and without bound NADH. The structures revealed that the two iron-sulfur clusters, Fe4S4 in FdsB and Fe2S2 in FdsG, are closer to the FMN than they are in other NADH dehydrogenases. Rapid kinetic studies and EPR measurements of rapid freeze-quenched samples of the NADH reduction of FdsBG identified a neutral flavin semiquinone, FMNH•, not previously observed to participate in NADH-mediated reduction of the FdsABG holoenzyme. We found that this semiquinone forms through the transfer of one electron from the fully reduced FMNH−, initially formed via NADH-mediated reduction, to the Fe2S2 cluster. This Fe2S2 cluster is not part of the on-path chain of iron-sulfur clusters connecting the FMN of FdsB with the active-site molybdenum center of FdsA. According to the NADH-bound structure, the nicotinamide ring stacks onto the re-face of the FMN. However, NADH binding significantly reduced the electron density for the isoalloxazine ring of FMN and induced a conformational change in residues of the FMN-binding pocket that display peptide-bond flipping upon NAD+ binding in proper NADH dehydrogenases.

Allostery exploits the conformational dynamics of enzymes by triggering a shift in population ensembles toward functionally distinct conformational or dynamic states. Allostery extensively regulates the activities of key enzymes within biosynthetic pathways to meet metabolic demand for their end products. Here, we have examined a critical enzyme, 3-deoxy-d-arabino-heptulosonate 7-phosphate synthase (DAH7PS), at the gateway to aromatic amino acid biosynthesis in Mycobacterium tuberculosis, which shows extremely complex dynamic allostery: three distinct aromatic amino acids jointly communicate occupancy to the active site via subtle changes in dynamics, enabling exquisite fine-tuning of delivery of these essential metabolites. Furthermore, this allosteric mechanism is co-opted by pathway branchpoint enzyme chorismate mutase upon complex formation. In this study, using statistical coupling analysis, site-directed mutagenesis, isothermal calorimetry, small-angle X-ray scattering, and X-ray crystallography analyses, we have pinpointed a critical node within the complex dynamic communication network responsible for this sophisticated allosteric machinery. Through a facile Gly to Pro substitution, we have altered backbone dynamics, completely severing the allosteric signal yet remarkably, generating a nonallosteric enzyme that retains full catalytic activity. We also identified a second residue of prime importance to the inter-enzyme communication with chorismate mutase. Our results reveal that highly complex dynamic allostery is surprisingly vulnerable and provide further insights into the intimate link between catalysis and allostery.

Laterites preserved on both sides of the Western Ghats Escarpment of Peninsular India have formed by long-term lateritic weathering essentially after India–Seychelles continental break-up following Deccan Traps emplacement (c. 63 myr ago). Supergene manganese ores of the Western Ghats were formed on Late Archean manganese protores. Among Mn oxides composing the ores, cryptomelane (K-rich Mn oxide) was characterized and dated by ⁴⁰Ar/³⁹Ar geochronology. Measured ages complement those previously obtained in other South Indian manganese ores from the hinterland plateau and further document three major weathering periods, c. 53–44, c. 39–22 and c. 14–10 Ma, the last being documented for the first time in India. These periods coincide with global palaeoclimatic proxies and date the lateritic weathering of three successive palaeolandscapes of the Western Ghats that evolved under slow denudation (c. 8 m Ma^–1) over the last 44 myr and were mostly incised during the Neogene (<22 Ma). This indicates that the Western Ghats are a relict of a South Indian plateau preserved at the headwaters of very long east-flowing river systems and above the Western Ghats Escarpment. Topography and denudation history of this landscape do not require Neogene tilt of the Peninsula as recently proposed.

Supplementary material: Full details of field and sample description, methods and analytical data including electron probe microanalyses of cryptomelane, and isotopic analyses and degassing spectra of irradiated cryptomelane grains are available at https://doi.org/10.6084/m9.figshare.c.4726661

Objective

To study the immunomodulatory effect of dimethyl fumarate (DF) on granulocyte macrophage colony-stimulating factor (GM-CSF) production in CD4⁺ T cells in experimental autoimmune encephalomyelitis (EAE) and human peripheral blood mononuclear cells (PBMCs).

Mutations in X-linked methyl-CpG-binding protein 2 (MECP2) cause Rett syndrome (RTT). To identify functional pathways that could inform therapeutic entry points, we carried out a genetic screen for secondary mutations that improved phenotypes in Mecp2/Y mice after mutagenesis with N-ethyl-N-nitrosourea (ENU). Here, we report the isolation of 106 founder animals that show suppression of Mecp2-null traits from screening 3177 Mecp2/Y genomes. Whole-exome sequencing, genetic crosses, and association analysis identified 22 candidate genes. Additional lesions in these candidate genes or pathway components associate variant alleles with phenotypic improvement in 30 lines. A network analysis shows that 63% of the genes cluster into the functional categories of transcriptional repression, chromatin modification, or DNA repair, delineating a pathway relationship with MECP2. Many mutations lie in genes that modulate synaptic signaling or lipid homeostasis. Mutations in genes that function in the DNA damage response (DDR) also improve phenotypes in Mecp2/Y mice. Association analysis was successful in resolving combinatorial effects of multiple loci. One line, which carries a suppressor mutation in a gene required for cholesterol synthesis, Sqle, carries a second mutation in retinoblastoma binding protein 8, endonuclease (Rbbp8, also known as CtIP), which regulates a DDR choice in double-stranded break (DSB) repair. Cells from Mecp2/Y mice have increased DSBs, so this finding suggests that the balance between homology-directed repair and nonhomologous end joining is important for neuronal cells. In this and other lines, two suppressor mutations confer greater improvement than one alone, suggesting that combination therapies could be effective in RTT.

Capicua (CIC) is a transcriptional repressor that counteracts activation of genes in response to receptor tyrosine kinase (RTK)/Ras/ERK signaling. Following activation of RTK, ERK enters the nucleus and serine-phosphorylates CIC, releasing it from its targets to permit gene expression. We recently showed that ERK triggers ubiquitin-mediated degradation of CIC in glioblastoma (GBM). In this study, we examined whether another important downstream effector of RTK/EGFR, the non-RTK c-Src, affects CIC repressor function in GBM. We found that c-Src binds and tyrosine-phosphorylates CIC on residue 1455 to promote nuclear export of CIC. On the other hand, CIC-mutant allele (CIC-Y1455F), that escapes c-Src–mediated tyrosine phosphorylation, remains localized to the nucleus and retains strong repressor function against CIC targets, the oncogenic transcription factors ETV1 and ETV5. Furthermore, we show that the orally available Src family kinase inhibitor, dasatinib, which prevents EGF-mediated tyrosine phosphorylation of CIC and attenuates elevated ETV1 and ETV5 levels, reduces viability of GBM cells and glioma stem cells (GSC), but not of their control cells with undetectable c-Src activity. In fact, GBM cells and GSC expressing the tyrosine-defective CIC mutant (Y1455F) lose sensitivity to dasatinib, further endorsing the effect of dasatinib on Src-mediated tyrosine phosphorylation of CIC. These findings elucidate important mechanisms of CIC regulation and provide the rationale to target c-Src alongside ERK pathway inhibitors as a way to fully restore CIC tumor suppressor function in neoplasms such as GBM.

Previous studies indicated that circular RNAs (circRNA) played vital roles in the development of non–small cell lung cancer (NSCLC). Although hsa_circ_0014130 might be a potential NSCLC biomarker, its function in NSCLC remains unknown. Thus, this study aimed to investigate the role of hsa_circ_0014130 in the progression of NSCLC. The levels of hsa_circ_0014130 in NSCLC tissues and adjacent normal tissues were determined by qRT-PCR. In addition, the expressions of Bcl-2 and cleaved caspase-3 in A549 cells were detected with Western blot analysis. Meanwhile, the dual luciferase reporter system assay was used to determine the interaction of hsa_circ_0014130 and miR-136-5p or Bcl-2 and miR-136-5p in NSCLC, respectively. The level of hsa_circ_0014130 was significantly upregulated in NSCLC tissues. Downregulation of hsa_circ_0014130 markedly inhibited the proliferation and invasion of A549 cells via inducing apoptosis. In addition, downregulation of hsa_circ_0014130 inhibited the tumorigenesis of subcutaneous A549 xenograft in mice in vivo. Meanwhile, mechanistic analysis indicated that downregulation of hsa_circ_0014130 decreased the expression of miR-136-5p–targeted gene Bcl-2 via acting as a competitive "sponge" of miR-136-5p. In this study, we found that hsa_circ_0014130 was upregulated in NSCLC tissues. In addition, hsa_circ_0014130 functions as a tumor promoter in NSCLC to promote tumor growth through upregulating Bcl-2 partially via "sponging" miR-136-5p.

Lisa Müller, Katrin Rietscher, Rene Keil, Marvin Neuholz, and Mechthild Hatzfeld

Desmosome remodeling is crucial for epidermal regeneration, differentiation and wound healing. It is mediated by adapting the composition, and by post-translational modifications, of constituent proteins. We have previously demonstrated in mouse suprabasal keratinocytes that plakophilin (PKP) 1 mediates strong adhesion, which is negatively regulated by insulin-like growth factor 1 (IGF1) signaling. The importance of PKP3 for epidermal adhesion is incompletely understood. Here, we identify a major role of epidermal growth factor (EGF), but not IGF1, signaling in PKP3 recruitment to the plasma membrane to facilitate desmosome assembly. We find that ribosomal S6 kinases (RSKs) associate with and phosphorylate PKP3, which promotes PKP3 association with desmosomes downstream of the EGF receptor. Knockdown of RSKs as well as mutation of an RSK phosphorylation site in PKP3 interfered with desmosome formation, maturation and adhesion. Our findings implicate a coordinate action of distinct growth factors in the control of adhesive properties of desmosomes through modulation of PKPs in a context-dependent manner.

The ₂ receptor is a potential in vivo target for measuring proliferative status in cancer. The feasibility of using N-(4-(6,7-dimethoxy-3,4-dihydroisoquinolin-2(1H)-yl)butyl)-2-(2-¹⁸F-fluoroethoxy)-5-methylbenzamide (¹⁸F-ISO-1) to image solid tumors in lymphoma, breast cancer, and head and neck cancer has been previously established. Here, we report the results of the first dedicated clinical trial of ¹⁸F-ISO-1 in women with primary breast cancer. Our study objective was to determine whether ¹⁸F-ISO-1 PET could provide an in vivo measure of tumor proliferative status, and we hypothesized that uptake would correlate with a tissue-based assay of proliferation, namely Ki-67 expression. Methods: Twenty-eight women with 29 primary invasive breast cancers were prospectively enrolled in a clinical trial (NCT 02284919) between March 2015 and January 2017. Each received an injection of 278–527 MBq of ¹⁸F-ISO-1 and then underwent PET/CT imaging of the breasts 50–55 min later. In vivo uptake of ¹⁸F-ISO-1 was quantitated by SUV_max and distribution volume ratios and was compared with ex vivo immunohistochemistry for Ki-67. Wilcoxon rank-sum tests assessed uptake differences across Ki-67 thresholds, and Spearman correlation tested associations between uptake and Ki-67. Results: Tumor SUV_max (median, 2.0 g/mL; range, 1.3–3.3 g/mL), partial-volume–corrected SUV_max, and SUV ratios were tested against Ki-67. Tumors stratified into the high–Ki-67 (≥20%) group had SUV_max greater than the low–Ki-67 (<20%) group (P = 0.02). SUV_max exhibited a positive correlation with Ki-67 across all breast cancer subtypes ( = 0.46, P = 0.01, n = 29). Partial-volume–corrected SUV_max was positively correlated with Ki-67 for invasive ductal carcinoma ( = 0.51, P = 0.02, n = 21). Tumor–to–normal-tissue ratios and tumor distribution volume ratio did not correlate with Ki-67 (P > 0.05). Conclusion: ¹⁸F-ISO-1 uptake in breast cancer modestly correlates with an in vitro assay of proliferation.

Metabolism and development must be closely coupled to meet the changing physiological needs of each stage in the life cycle. The molecular mechanisms that link these pathways, however, remain poorly understood. Here we show that the Drosophila estrogen-related receptor (dERR) directs a transcriptional switch in mid-pupae that promotes glucose oxidation and lipogenesis in young adults. dERR mutant adults are viable but display reduced locomotor activity, susceptibility to starvation, elevated glucose, and an almost complete lack of stored triglycerides. Molecular profiling by RNA-seq, ChIP-seq, and metabolomics revealed that glycolytic and pentose phosphate pathway genes are induced by dERR, and their reduced expression in mutants is accompanied by elevated glycolytic intermediates, reduced TCA cycle intermediates, and reduced levels of long chain fatty acids. Unexpectedly, we found that the central pathways of energy metabolism, including glycolysis, the tricarboxylic acid cycle, and electron transport chain, are coordinately induced at the transcriptional level in mid-pupae and maintained into adulthood, and this response is partially dependent on dERR, leading to the metabolic defects observed in mutants. Our data support the model that dERR contributes to a transcriptional switch during pupal development that establishes the metabolic state of the adult fly.

Autophagy captures intracellular components and delivers them to lysosomes for degradation and recycling. Conditional autophagy deficiency in adult mice causes liver damage, shortens life span to 3 mo due to neurodegeneration, and is lethal upon fasting. As autophagy deficiency causes p53 induction and cell death in neurons, we sought to test whether p53 mediates the lethal consequences of autophagy deficiency. Here, we conditionally deleted Trp53 (p53 hereafter) and/or the essential autophagy gene Atg7 throughout adult mice. Compared with Atg7^/ mice, the life span of Atg7^/p53^/ mice was extended due to delayed neurodegeneration and resistance to death upon fasting. Atg7 also suppressed apoptosis induced by p53 activator Nutlin-3, suggesting that autophagy inhibited p53 activation. To test whether increased oxidative stress in Atg7^/ mice was responsible for p53 activation, Atg7 was deleted in the presence or absence of the master regulator of antioxidant defense nuclear factor erythroid 2-related factor 2 (Nrf2). Nrf2^–/–Atg7^/ mice died rapidly due to small intestine damage, which was not rescued by p53 codeletion. Thus, Atg7 limits p53 activation and p53-mediated neurodegeneration. In turn, NRF2 mitigates lethal intestine degeneration upon autophagy loss. These findings illustrate the tissue-specific roles for autophagy and functional dependencies on the p53 and NRF2 stress response mechanisms.

It is important to identify, assess, and address current barriers to implementation of post-approval changes that are intended to ensure continued (uninterrupted) operations and drive innovation and continual improvement in a maximally efficient, agile, and flexible pharmaceutical manufacturing sector. Leveraging the International Conference for Harmonisation Quality Guideline Q10 provides regulatory relief when it comes to addressing changes related to excipients, specifically excipient supplier's name and address changes, which will ensure a sustainable, reliable global supply and the availability of high quality product to patients through the entire commercial lifecycle of a product without extensive regulatory oversight.

Background

People with pulmonary fibrosis often experience a protracted time to diagnosis, high symptom burden and limited disease information. This review aimed to identify the supportive care needs reported by people with pulmonary fibrosis and their caregivers.

An aberrant bi-apical Follicucullus specimen (Albaillellaria, Radiolaria) has been discovered from an upper Guadalupian (Middle Permian) chert block of the Kamiaso Unit of the Mino terrane, central Japan. If this specimen was formed with double malformation, it would be the oldest record of this phenomenon in radiolarians and the first record of its kind in Albaillellaria.

An exceptionally well-preserved specimen of the articulated rhodophyte Permocalculus, compared with P. tenellus sensu Elliott, 1955, is described from fine-grained Upper Permian limestones of the Khuff Formation of Saudi Arabia. Longitudinal medullary and sheaf-like cortical filaments extend through the uniserial series of elongate-globular, concave- and convex-terminating, interlocking segments for which they are interpreted to have functioned in articulation. The filaments tend to splay and branch laterally into the cortex where they terminate at the pores. At the terminal aperture, the filaments extend as bifurcating and possibly trifurcating branches and may serve as the origin of a new segment. Numerous elongate-globular chambers, up to five in each row and intimately involved with the filaments, are developed in the outer medulla and are considered to represent reproductive sporangia. The specimen is considered to have occupied predominantly low-energy, normal to slightly elevated salinity, shallow conditions within the subtidal regime of a lagoon.

ABSTRACTThe Standard Days Method (SDM), a modern fertility awareness-based family planning method, has been introduced in 30 countries since its development in 2001. It is still unclear to what extent the SDM was mainstreamed within the family planning method mix, particularly in low- and middle-income country (LMIC) settings, where the SDM had been introduced by donors and implementing partners. This review of implementation science publications on the SDM in LMICs first looked at community pilot studies of the SDM to determine the acceptability of the method; correct use and efficacy rates; demographics of users; and changes to contraceptive prevalence rates and family planning behaviors, especially among men and couples. Then, we examined the status of the SDM in the 16 countries that had attempted to scale up the method within national family planning protocols, training, and service delivery. At the community level, evidence demonstrated a high level of acceptability of the method; efficacy rates comparable to the initial clinical trials; diversity in demographic characteristics of users, including first-time or recently discontinued users of family planning; increased male engagement in family planning; and improved couple's communication. Nationally, few countries had scaled up the SDM due to uneven stakeholder engagement, lackluster political will, and competing resource priorities. Results of this review could help policy makers determine the added value of the SDM in the contraceptive method mix and identify potential barriers to its implementation moving forward.

OBJECTIVE

There is a controversy over the association between obesity and end-stage renal disease (ESRD) in people with or without type 2 diabetes; therefore, we examined the effect of BMI on the risk of ESRD according to glycemic status in the Korean population.

OBJECTIVE

Daily foot self-inspection may permit earlier detection and treatment of a foot lesion, reducing the risk of infection and lower-limb amputation (LLA). Though race and ethnicity are strongly associated with LLA risk, with higher risk seen in African Americans (AA), American Indians/Alaska Natives (AI/AN), and Native Hawaiians/Pacific Islanders (NH/PI), associations between foot self-inspection and racial and ethnic groups are inconsistent. We aimed to assess differences in foot self-inspection among people with diabetes by race/ethnicity.

B-cell receptor (BCR) signaling pathway components represent promising treatment targets in multiple B-cell malignancies including diffuse large B-cell lymphoma (DLBCL). In in vitro and in vivo model systems, a subset of DLBCLs depend upon BCR survival signals and respond to proximal BCR/phosphoinositide 3 kinase (PI3K) blockade. However, single-agent BCR pathway inhibitors have had more limited activity in patients with DLBCL, underscoring the need for indicators of sensitivity to BCR blockade and insights into potential resistance mechanisms. Here, we report highly significant transcriptional upregulation of C-X-C chemokine receptor 4 (CXCR4) in BCR-dependent DLBCL cell lines and primary tumors following chemical spleen tyrosine kinase (SYK) inhibition, molecular SYK depletion or chemical PI3K blockade. SYK or PI3K inhibition also selectively upregulated cell surface CXCR4 protein expression in BCR-dependent DLBCLs. CXCR4 expression was directly modulated by fork-head box O1 via the PI3K/protein kinase B/forkhead box O1 signaling axis. Following chemical SYK inhibition, all BCR-dependent DLBCLs exhibited significantly increased stromal cell-derived factor-1α (SDF-1α) induced chemotaxis, consistent with the role of CXCR4 signaling in B-cell migration. Select PI3K isoform inhibitors also augmented SDF-1α induced chemotaxis. These data define CXCR4 upregulation as an indicator of sensitivity to BCR/PI3K blockade and identify CXCR4 signaling as a potential resistance mechanism in BCR-dependent DLBCLs.

Autophagy is a genetically regulated process of adaptation to metabolic stress and was recently shown to be involved in the treatment response of chronic myeloid leukemia (CML). However, in vivo data are limited and the molecular mechanism of autophagy regulators in the process of leukemogenesis is not completely understood. Here we show that Beclin-1 knockdown, but not Atg5 deletion in a murine CML model leads to a reduced leukemic burden and results in a significantly prolonged median survival of targeted mice. Further analyses of murine cell lines and primary patient material indicate that active BCR-ABL directly interacts with BECLIN-1 and phosphorylates its tyrosine residues 233 and 352, resulting in autophagy suppression. By using phosphorylation-deficient and phosphorylation-mimic mutants, we identify BCR-ABL induced BECLIN-1 phosphorylation as a crucial mechanism for BECLIN-1 complex formation: interaction analyses exhibit diminished binding of the positive autophagy regulators UVRAG, VPS15, ATG14 and VPS34 and enhanced binding of the negative regulator Rubicon to BCR-ABL-phosphorylated BECLIN-1. Taken together, our findings show interaction of BCR-ABL and BECLIN-1 thereby highlighting the importance of BECLIN-1-mediated autophagy in BCR-ABL⁺ cells.

Interactions between platelets, leukocytes and the vessel wall provide alternative pathological routes of thrombo-inflammatory leukocyte recruitment. We found that when platelets were activated by a range of agonists in whole blood, they shed platelet-derived extracellular vesicles which rapidly and preferentially bound to blood monocytes compared to other leukocytes. Platelet-derived extracellular vesicle binding to monocytes was initiated by P-selectin-dependent adhesion and was stabilised by binding of phosphatidylserine. These interactions resulted in the progressive transfer of the platelet adhesion receptor GPIbα to monocytes. GPIbα^+-monocytes tethered and rolled on immobilised von Willebrand Factor or were recruited and activated on endothelial cells treated with TGF-β1 to induce the expression of von Willebrand Factor. In both models monocyte adhesion was ablated by a function-blocking antibody against GPIbα. Monocytes could also bind platelet-derived extracellular vesicle in mouse blood in vitro and in vivo. Intratracheal instillations of diesel nanoparticles, to model chronic pulmonary inflammation, induced accumulation of GPIbα on circulating monocytes. In intravital experiments, GPIbα⁺-monocytes adhered to the microcirculation of the TGF-β1-stimulated cremaster muscle, while in the ApoE^–/– model of atherosclerosis, GPIbα⁺-monocytes adhered to the carotid arteries. In trauma patients, monocytes bore platelet markers within 1 hour of injury, the levels of which correlated with severity of trauma and resulted in monocyte clearance from the circulation. Thus, we have defined a novel thrombo-inflammatory pathway in which platelet-derived extracellular vesicles transfer a platelet adhesion receptor to monocytes, allowing their recruitment in large and small blood vessels, and which is likely to be pathogenic.

In iron-depleted women without anemia, oral iron supplements induce an increase in serum hepcidin (SHep) that persists for 24 hours, decreasing iron absorption from supplements given later on the same or next day. Consequently, iron absorption from supplements is highest if iron is given on alternate days. Whether this dosing schedule is also beneficial in women with iron-deficiency anemia (IDA) given high-dose iron supplements is uncertain. The primary objective of this study was to assess whether, in women with IDA, alternate-day administration of 100 and 200 mg iron increases iron absorption compared to consecutive-day iron administration. Secondary objectives were to correlate iron absorption with SHep and iron status parameters. We performed a cross-over iron absorption study in women with IDA (n=19; median hemoglobin 11.5 mg/dL; mean serum ferritin 10 mg/L) who received either 100 or 200 mg iron as ferrous sulfate given at 8 AM on days 2, 3 and 5 labeled with stable iron isotopes 57Fe, 58Fe and 54Fe; after a 16-day incorporation period, the other labeled dose was given at 8 AM on days 23, 24 and 26 (days 2, 3 and 5 of the second period). Iron absorption on days 2 and 3 (consecutive) and day 5 (alternate) was assessed by measuring erythrocyte isotope incorporation. For both doses, SHep was higher on day 3 than on day 2 (P<0.001) or day 5 (P<0.01) with no significant difference between days 2 and 5. Similarly, for both doses, fractional iron absorption (FIA) on days 2 and 5 was 40-50% higher than on day 3 (P<0.001), while absorption on day 2 did not differ significantly from day 5. There was no significant difference in the incidence of gastrointestinal side effects comparing the two iron doses (P=0.105). Alternate day dosing of oral iron supplements in anemic women may be preferable because it sharply increases FIA. If needed, to provide the same total amount of iron with alternate day dosing, twice the daily target dose should be given on alternate days, as total iron absorption from a single dose of 200 mg given on alternate days was approximately twice that from 100 mg given on consecutive days (P<0.001). In IDA, even if hepatic hepcidin expression is strongly suppressed by iron deficiency and erythropoietic drive, the intake of oral iron supplements leads to an acute hepcidin increase for 24 hours. The study was funded by ETH Zürich, Switzerland. This study has been registered at www.clinicaltrials.gov as #NCT03623997.

Human bone marrow stromal cells (BMSC) are key elements of the hematopoietic environment and they play a central role in bone and bone marrow physiology. However, how key stromal cell functions are regulated is largely unknown. We analyzed the role of the immediate early response transcription factor EGR1 as key stromal cell regulator and found that EGR1 was highly expressed in prospectively-isolated primary BMSC, down-regulated upon culture, and low in non-colony-forming CD45^neg stromal cells. Furthermore, EGR1 expression was lower in proliferative regenerating adult and fetal primary cells compared to adult steady-state BMSC. Overexpression of EGR1 in stromal cells induced potent hematopoietic stroma support as indicated by an increased production of transplantable CD34⁺CD90⁺ hematopoietic stem cells in expansion co-cultures. The improvement in bone marrow stroma support function was mediated by increased expression of hematopoietic supporting genes, such as VCAM1 and CCL28. Furthermore, EGR1 overexpression markedly decreased stromal cell proliferation whereas EGR1 knockdown caused the opposite effects. These findings thus show that EGR1 is a key stromal transcription factor with a dual role in regulating proliferation and hematopoietic stroma support function that is controlling a genetic program to co-ordinate the specific functions of BMSC in their different biological contexts.

An aging global population combined with sedentary lifestyles and unhealthy diets has contributed to an increasing incidence of obesity and type 2 diabetes. These metabolic disorders are associated with perturbations to nitric oxide (NO) signaling and impaired glucose metabolism. Dietary inorganic nitrate, found in high concentration in green leafy vegetables, can be converted to NO in vivo and demonstrates antidiabetic and antiobesity properties in rodents. Alongside tissues including skeletal muscle and liver, white adipose tissue is also an important physiological site of glucose disposal. However, the distinct molecular mechanisms governing the effect of nitrate on adipose tissue glucose metabolism and the contribution of this tissue to the glucose-tolerant phenotype remain to be determined. Using a metabolomic and stable-isotope labeling approach, combined with transcriptional analysis, we found that nitrate increases glucose uptake and oxidative catabolism in primary adipocytes and white adipose tissue of nitrate-treated rats. Mechanistically, we determined that nitrate induces these phenotypic changes in primary adipocytes through the xanthine oxidoreductase–catalyzed reduction of nitrate to NO and independently of peroxisome proliferator–activated receptor-α. The nitrate-mediated enhancement of glucose uptake and catabolism in white adipose tissue may be a key contributor to the antidiabetic effects of this anion.

Nayeli G. Reyes-Nava, Hung-Chun Yu, Curtis R. Coughlin II, Tamim H. Shaikh, and Anita M. Quintana

We used whole-exome sequencing (WES) to determine the genetic etiology of a patient with a multi-system disorder characterized by a seizure phenotype. WES identified a heterozygous de novo missense mutation in the GABRA1 gene (c.875C>T). GABRA1 encodes the alpha subunit of the gamma-aminobutyric acid receptor A (GABA_AR). The GABA_AR is a ligand gated ion channel that mediates the fast inhibitory signals of the nervous system, and mutations in the subunits that compose the GABA_AR have been previously associated with human disease. To understand the mechanisms by which GABRA1 regulates brain development, we developed a zebrafish model of gabra1 deficiency. gabra1 expression is restricted to the nervous system and behavioral analysis of morpholino injected larvae suggests that the knockdown of gabra1 results in hypoactivity and defects in the expression of other subunits of the GABA_AR. Expression of the human GABRA1 protein in morphants partially restored the hypomotility phenotype. In contrast, the expression of the c.875C>T variant did not restore these behavioral deficits. Collectively, these results represent a functional approach to understand the mechanisms by which loss-of-function alleles cause disease.

Chalongrat Noree and Naraporn Sirinonthanawech

Previously, we have developed an extramitochondrial assembly system, where mitochondrial targeting signal (MTS) can be removed from a given mitochondrial enzyme, which could be used to characterize the regulatory factors involved in enzyme assembly/disassembly in vivo. Here, we demonstrate that addition of exogenous acetaldehyde can quickly induce the supramolecular assembly of MTS-deleted aldehyde dehydrogenase Ald4p in yeast cytoplasm. Also, by using PCR-based modification of the yeast genome, cytoplasmically targeted Ald4p cannot polymerize into long filaments when key functional amino acid residues are substituted, as shown by N192D, S269A, E290K and C324A mutations. This study has confirmed that extramitochondrial assembly could be a powerful external system for studying mitochondrial enzyme assembly, and its regulatory factors outside the mitochondria. In addition, we propose that mitochondrial enzyme assembly/disassembly is coupled to the regulation of a given mitochondrial enzyme activity.

Yu Liu, Shuhua Qi, Fridtjof Thomas, Brittany L. Correia, Angela P. Taylor, Roy V. Sillitoe, and Detlef H. Heck

Respiration is controlled by central pattern generating circuits in the brain stem, whose activity can be modulated by inputs from other brain areas to adapt respiration to autonomic and behavioral demands. The cerebellum is known to be part of the neuronal circuitry activated during respiratory challenges, such as hunger for air, but has not been found to be involved in the control of spontaneous, unobstructed breathing (eupnea). Here we applied a measure of intrinsic rhythmicity, the CV2, which evaluates the similarity of subsequent intervals and is thus sensitive to changes in rhythmicity at the temporal resolution of individual respiratory intervals. The variability of intrinsic respiratory rhythmicity was reduced in a mouse model of cerebellar ataxia compared to their healthy littermates. Irrespective of that difference, the average respiratory rate and the average coefficient of variation (CV) were comparable between healthy and ataxic mice. We argue that these findings are consistent with a proposed role of the cerebellum in modulating the duration of individual respiratory intervals, which could serve the purpose of coordinating respiration with other rhythmic orofacial movements, such as fluid licking and swallowing.

The American Board of Family Medicine routinely surveys its Diplomates in each national graduating cohort 3 years out of training. These data were used to characterize early career family physicians whose services include management of pregnancy and prescribing buprenorphine. A total of 261 (5.1%) respondents both provide maternity care and prescribe buprenorphine. Family physicians who care for pregnant women and also prescribe buprenorphine represented 50.4% of all buprenorphine prescribers. The family physicians in this group were trained in a small number of residency programs, with only 15 programs producing at least 25% of graduates who do this work.

The Stenotrophomonas maltophilia complex (Smc) comprises opportunistic environmental Gram-negative bacilli responsible for a variety of infections in both humans and animals. Beyond its large genetic diversity, its genetic organization in genogroups was recently confirmed through the whole-genome sequencing of human and environmental strains. As they are poorly represented in these analyses, we sequenced the whole genomes of 93 animal strains to determine their genetic background and characteristics. Combining these data with 81 newly sequenced human strains and the genomes available from RefSeq, we performed a genomic analysis that included 375 nonduplicated genomes with various origins (animal, 104; human, 226; environment, 30; unknown, 15). Phylogenetic analysis and clustering based on genome-wide average nucleotide identity confirmed and specified the genetic organization of Smc in at least 20 genogroups. Two new genogroups were identified, and two previously described groups were further divided into two subgroups each. Comparing the strains isolated from different host types and their genogroup affiliation, we observed a clear disequilibrium in certain groups. Surprisingly, some antimicrobial resistance genes, integrons, and/or clusters of attC sites lacking integron-integrase (CALIN) sequences targeting antimicrobial compounds extensively used in animals were mainly identified in animal strains. We also identified genes commonly found in animal strains coding for efflux systems. The result of a large whole-genome analysis performed by us supports the hypothesis of the putative contribution of animals as a reservoir of Stenotrophomonas maltophilia complex strains and/or resistance genes for strains in humans.

IMPORTANCE Given its naturally large antimicrobial resistance profile, the Stenotrophomonas maltophilia complex (Smc) is a set of emerging pathogens of immunosuppressed and cystic fibrosis patients. As it is group of environmental microorganisms, this adaptation to humans is an opportunity to understand the genetic and metabolic selective mechanisms involved in this process. The previously reported genomic organization was incomplete, as data from animal strains were underrepresented. We added the missing piece of the puzzle with whole-genome sequencing of 93 strains of animal origin. Beyond describing the phylogenetic organization, we confirmed the genetic diversity of the Smc, which could not be estimated through routine phenotype- or matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF)-based laboratory tests. Animals strains seem to play a key role in the diversity of Smc and could act as a reservoir for mobile resistance genes. Some genogroups seem to be associated with particular hosts; the genetic support of this association and the role of the determinants/corresponding genes need to be explored.

Burkholderia sp. strain SG-MS1 and Pseudomonas sp. strain SG-MS2 have previously been found to mineralize (+)-pinoresinol through a common catabolic pathway. Here, we used comparative genomics, proteomics, protein semipurification, and heterologous expression to identify a flavoprotein from the vanillyl alcohol oxidase/p-cresol methyl hydroxylase (VAO/PCMH) enzyme family in SG-MS2 that carries out the initial hydroxylation of (+)-pinoresinol at the benzylic carbon. The cognate gene is translationally coupled with a downstream cytochrome gene, and the cytochrome is required for activity. The flavoprotein has a unique combination of cofactor binding and cytochrome requirements for the VAO/PCMH family. The heterologously expressed enzyme has a K_m of 1.17 μM for (+)-pinoresinol. The enzyme is overexpressed in strain SG-MS2 upon exposure to (+)-pinoresinol, along with 45 other proteins, 22 of which were found to be encoded by genes in an approximately 35.1-kb cluster also containing the flavoprotein and cytochrome genes. Homologs of 18 of these 22 genes, plus the flavoprotein and cytochrome genes, were also found in a 38.7-kb cluster in SG-MS1. The amino acid identities of four of the other proteins within the SG-MS2 cluster suggest they catalyze conversion of hydroxylated pinoresinol to protocatechuate and 2-methoxyhydroquinone. Nine other proteins upregulated in SG-MS2 on exposure to (+)-pinoresinol appear to be homologs of proteins known to comprise the protocatechuate and 2-methoxyhydroquinone catabolic pathways, but only three of the cognate genes lie within the cluster containing the flavoprotein and cytochrome genes.

IMPORTANCE (+)-Pinoresinol is an important plant defense compound, a major food lignan for humans and some other animals, and the model compound used to study degradation of the β-β' linkages in lignin. We report a gene cluster, in one strain each of Pseudomonas and Burkholderia, that is involved in the oxidative catabolism of (+)-pinoresinol. The flavoprotein component of the α-hydroxylase which heads the pathway belongs to the 4-phenol oxidizing (4PO) subgroup of the vanillyl alcohol oxidase/p-cresol methyl hydroxylase (VAO/PCMH) enzyme family but constitutes a novel combination of cofactor and electron acceptor properties for the family. It is translationally coupled with a cytochrome gene whose product is also required for activity. The work casts new light on the biology of (+)-pinoresinol and its transformation to other bioactive molecules. Potential applications of the findings include new options for deconstructing lignin into useful chemicals and the generation of new phytoestrogenic enterolactones from lignans.

This study showed that treatment with a therapeutic monoclonal immunoglobulin-G1 antibody against phosphorylcholine on oxidized phospholipids preserves coronary flow reserve and attenuates atherosclerotic inflammation as determined by the uptake of ¹⁸F-fluorodeoxyglucose in atherosclerotic mice. The noninvasive imaging techniques represent translational tools to assess the efficacy of phosphorylcholine-targeted therapy on coronary artery function and atherosclerosis in clinical studies.

Since lung cancer (LC) is still the leading cause of cancer deaths worldwide [1], early detection through screening represents an important opportunity to improve LC survival and is a priority area for cancer care. The National Lung Screening Trial (NLST) aimed to compare low-dose helical computed tomography (LDCT) with chest radiography in LC screening of current or former heavy smokers. The trial found a relative reduction in mortality from LC of 20% in those who had undergone LDCT screening. LC screening has regained prominence in the thoracic oncology literature with the completion of NELSON and other European trials, which support the role of LC screening in achieving early diagnosis and reducing mortality. A growing number of implementation pilots are providing an impetus towards organised, national programmes for LC screening, which are in need of long-term follow-up data such as those presented in this study.

A 78-year-old male presented at the emergency room complaining of dry cough, fever up to 38.5 °C and malaise for 1 month. He had visited a general practitioner and received amoxicillin 500 mg three times a day for 7 days for a presumed chest infection, without improvement. He had a history of diabetes and arterial blood hypertension, for which he was receiving metformin 1000 mg twice a day and amlodipine 10 mg a day for 7 years. He reported no alcohol abuse and was an ex-smoker of 20 pack-years (quit 30 years ago). He had no recent hospitalisations or any medical interventions.