Current and new frontiers in hereditary cancer surveillance: Opportunities for liquid biopsy.

At least 5% of cancer diagnoses are attributed to a causal pathogenic or likely pathogenic germline genetic variant (hereditary cancer syndrome-HCS). These individuals are burdened with lifelong surveillance monitoring organs for a wide spectrum of cancers. This is associated with substantial uncertainty and anxiety in the time between screening tests and while the individuals are awaiting results. Cell-free DNA (cfDNA) sequencing has recently shown potential as a non-invasive strategy for monitoring cancer. There is an opportunity for high-yield cancer early detection in HCS. To assess clinical validity of cfDNA in individuals with HCS, representatives from eight genetics centers from across Canada founded the CHARM (cfDNA in Hereditary and High-Risk Malignancies) Consortium in 2017. In this perspective, we discuss operationalization of this consortium and early data emerging from the most common and well-characterized HCSs: hereditary breast and ovarian cancer, Lynch syndrome, Li-Fraumeni syndrome, and Neurofibromatosis type 1. We identify opportunities for the incorporation of cfDNA sequencing into surveillance protocols; these opportunities are backed by examples of earlier cancer detection efficacy in HCSs from the CHARM Consortium. We seek to establish a paradigm shift in early cancer surveillance in individuals with HCSs, away from highly centralized, regimented medical screening visits and toward more accessible, frequent, and proactive care for these high-risk individuals.

Psoralen mapping reveals a bacterial genome supercoiling landscape dominated by transcription.

DNA supercoiling is a key regulator of all DNA metabolic processes including replication, transcription, and recombination, yet a reliable genomic assay for supercoiling is lacking. Here, we present a robust and flexible method (Psora-seq) to measure whole-genome supercoiling at high resolution. Using this tool in Escherichia coli, we observe a supercoiling landscape that is well correlated to transcription. Supercoiling twin-domains generated by RNA polymerase complexes span 25 kb in each direction - an order of magnitude farther than previous measurements in any organism. Thus, ribosomal and many other highly expressed genes strongly affect the topology of about 40 neighboring genes each, creating highly integrated gene circuits. Genomic patterns of supercoiling revealed by Psora-seq could be aptly predicted from modeling based on gene expression levels alone, indicating that transcription is the major determinant of chromosome supercoiling. Large-scale supercoiling patterns were highly symmetrical between left and right chromosome arms (replichores), indicating that DNA replication also strongly influences supercoiling. Skew in the axis of symmetry from the natural ori-ter axis supports previous indications that the rightward replication fork is delayed several minutes after initiation. Implications of supercoiling on DNA replication and chromosome domain structure are discussed.

Occurrence of bisphosphonate-associated osteonecrosis of the jaws in individuals with rheumatoid arthritis - a systematic review.

BACKGROUND: To access the occurrence of bisphosphonate-associated osteonecrosis of the jaw (BAONJ) in individuals with rheumatoid arthritis (RA). MATERIAL AND METHODS: Observational studies that evaluated the occurrence of BAONJ in individuals with RA (BAONJ-RA) were considered for inclusion. Electronic searches were performed up to December 2022 in six databases and in the grey literature. The study selection, data extraction, and quality assessment of the included studies according to the Joanna Briggs Institute Critical Appraisal Checklists was performed. The certainty of evidence was evaluated using the GRADE approach. RESULTS: Five studies were included three cohort and two cross-sectional. The sample size of subjects with RA ranged from 16 to 3201. Together, the studies presented 36 cases of BAONJ-RA. Prevalence of BAONJ-RA ranged from 0.094% to 56.25%. The incidence ranged from 0.4% to 2.21. Women between the 6th and 8th decade of life were the most affected. Alendronate (n=5) and zoledronic acid (n=9), orally and intravenously, respectively, were the most used bisphosphonates. The duration of bisphosphonates use ranged from 2.7 to 8 years. The certainty of evidence was very low. CONCLUSIONS: The occurrence of BAONJ-RA is low. However, the certainty of the evidence was very low for this outcome.

Tricyclic antidepressants target FKBP51 SUMOylation to restore glucocorticoid receptor activity.

FKBP51 is an important inhibitor of the glucocorticoid receptor (GR) signaling. High FKBP51 levels are associated to stress-related disorders, which are linked to GR resistance. SUMO conjugation to FKBP51 is necessary for FKBP51's inhibitory action on GR. The GR/FKBP51 pathway is target of antidepressant action. Thus we investigated if these drugs could inhibit FKBP51 SUMOylation and therefore restore GR activity. Screening cells using Ni2+ affinity and in vitro SUMOylation assays revealed that tricyclic antidepressants- particularly clomipramine- inhibited FKBP51 SUMOylation. Our data show that clomipramine binds to FKBP51 inhibiting its interaction with PIAS4 and therefore hindering its SUMOylation. The inhibition of FKBP51 SUMOylation decreased its binding to Hsp90 and GR facilitating FKBP52 recruitment, and enhancing GR activity. Reduction of PIAS4 expression in rat primary astrocytes impaired FKBP51 interaction with GR, while clomipramine could no longer exert its inhibitory action. This mechanism was verified in vivo in mice treated with clomipramine. These results describe the action of antidepressants as repressors of FKBP51 SUMOylation as a molecular switch for restoring GR sensitivity, thereby providing new potential routes of antidepressant intervention.

Glucocorticoids rapidly activate cAMP production via Gαs to initiate non-genomic signaling that contributes to one-third of their canonical genomic effects.

Glucocorticoids are widely used for the suppression of inflammation, but evidence is growing that they can have rapid, non-genomic actions that have been unappreciated. Diverse cell signaling effects have been reported for glucocorticoids, leading us to hypothesize that glucocorticoids alone can swiftly increase the 3',5'-cyclic adenosine monophosphate (cAMP) production. We found that prednisone, fluticasone, budesonide, and progesterone each increased cAMP levels within 3 minutes without phosphodiesterase inhibitors by measuring real-time cAMP dynamics using the cAMP difference detector in situ assay in a variety of immortalized cell lines and primary human airway smooth muscle (HASM) cells. A membrane- impermeable glucocorticoid showed similarly rapid stimulation of cAMP, implying that responses are initiated at the cell surface. siRNA knockdown of Gαs virtually eliminated glucocorticoid-stimulated cAMP responses, suggesting that these drugs activate the cAMP production via a G protein-coupled receptor. Estradiol had small effects on cAMP levels but G protein estrogen receptor antagonists had little effect on responses to any of the glucocorticoids tested. The genomic and non-genomic actions of budesonide were analyzed by RNA-Seq analysis of 24 hours treated HASM, with and without knockdown of Gαs . A 140-gene budesonide signature was identified, of which 48 genes represent a non-genomic signature that requires Gαs signaling. Collectively, this non-genomic cAMP signaling modality contributes to one-third of the gene expression changes induced by glucocorticoid treatment and shifts the view of how this important class of drugs exerts its effects.

EAN consensus statement for management of patients with neurological diseases during the COVID-19 pandemic.

BACKGROUND AND PURPOSE: The recent SARS-CoV-2 pandemic has posed multiple challenges to the practice of clinical neurology including recognition of emerging neurological complications and management of coexistent neurological diseases. In a fast-evolving pandemic, evidence-based studies are lacking in many areas. This paper presents European Academy of Neurology (EAN) expert consensus statements to guide neurologists caring for patients with COVID-19. METHODS: A refined Delphi methodology was applied. In round 1, statements were provided by EAN scientific panels (SPs). In round 2, these statements were circulated to SP members not involved in writing them, asking for agreement/disagreement. Items with agreement >70% were retained for round 3, in which SP co-chairs rated importance on a five-point Likert scale. Results were graded by importance and reported as consensus statements. RESULTS: In round one, 70 statements were provided by 23 SPs. In round two, 259/1061 SP member responses were received. Fifty-nine statements obtained >70% agreement and were retained. In round three, responses were received from 55 co-chairs of 29 SPs. Whilst general recommendations related to prevention of COVID-19 transmission had high levels of agreement and importance, opinion was more varied concerning statements related to therapy. CONCLUSION: This is the first structured consensus statement on good clinical practice in patients with neurological disease during the COVID-19 pandemic that provides immediate guidance for neurologists. In this fast-evolving pandemic, a rapid response using refined Delphi methodology is possible, but guidance may be subject to change as further evidence emerges.

Effects of photobiomodulation and swimming on gene expression in rats with the tibialis anterior muscle injury.

The aim of the present study was to evaluate the effects of photobiomodulation (low-level laser therapy (LLLT)) and aquatic exercise on the expression of genes related to muscle regeneration in rats. Wistar rats were divided into five groups: control group (n = 15), non-treated injury group (n = 15), injury+LLLT group (n = 15), injury+aquatic exercise group (n = 15), and injury+LLLT+aquatic exercise group (n = 15). Cryoinjury was performed on the belly of the tibialis anterior (TA) muscle. LLLT was performed daily with an AlGaAs laser (830 nm; beam spot of 0.0324 cm2, output power of 100 mW, energy density of 180 J/cm2, and 58-s exposure time). Animals were euthanized at 7, 14, and 21 days. The TA muscles were removed for gene expression analysis of TGF-ß, Myogenin, and MyoD. The results were statistically analyzed at a significance level of 5%. The cryoinjury increased the expression of genes related to muscle regeneration-MyoD, Myogenin, and TGF-ß-compared to the control group (p < 0.05); the photobiomodulation increased the expression of these genes at day 7 (p < 0.05), decreasing until day 21; and the aquatic exercise increases the expression of the three genes over time. When the two treatments were combined, the expression of the analyzed genes also increased over time. In summary, the results of our study suggest that photobiomodulation (LLLT), when applied alone in cryoinjury, is able to increase the gene expression of MyoD, Myogenin, and TGF-ß at the acute phase, while when combined with aquatic exercises, there is an increase in expression of these genes specially at the long-term treatment.

Budesonide enhances agonist-induced bronchodilation in human small airways by increasing cAMP production in airway smooth muscle.

The nongenomic mechanisms by which glucocorticoids modulate ß2 agonist-induced-bronchodilation remain elusive. Our studies aimed to elucidate mechanisms mediating the beneficial effects of glucocorticoids on agonist-induced bronchodilation. Utilizing human precision-cut lung slices (hPCLS), we measured bronchodilation to formoterol, prostaglandin E2 (PGE2), cholera toxin (CTX), or forskolin in the presence and absence of budesonide. Using cultured human airway smooth muscle (HASM), intracellular cAMP was measured in live cells following exposure to formoterol, PGE2, or forskolin in the presence or absence of budesonide. We showed that simultaneous budesonide administration amplified formoterol-induced bronchodilation and attenuated agonist-induced phosphorylation of myosin light chain, a necessary signaling event mediating force generation. In parallel studies, cAMP levels were augmented by simultaneous exposure of HASM cells to formoterol and budesonide. Budesonide, fluticasone, and prednisone alone rapidly increased cAMP levels, but steroids alone had little effect on bronchodilation in hPCLS. Bronchodilation induced by PGE2, CTX, or forskolin was also augmented by simultaneous exposure to budesonide in hPCLS. Furthermore, HASM cells expressed membrane-bound glucocorticoid receptors that failed to translocate with glucocorticoid stimulation and that potentially mediated the rapid effects of steroids on ß2 agonist-induced bronchodilation. Knockdown of glucocorticoid receptor-α had little effect on budesonide-induced and steroid-dependent augmentation of formoterol-induced cAMP generation in HASM. Collectively, these studies suggest that glucocorticoids amplify cAMP-dependent bronchodilation by directly increasing cAMP levels. These studies identify a molecular mechanism by which the combination of glucocorticoids and ß2 agonists may augment bronchodilation in diseases such as asthma or chronic obstructive pulmonary disease.

Does SARS-Cov-2 invade the brain? Translational lessons from animal models.

The current coronavirus disease (COVID-19) outbreak, caused by the novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has raised the possibility of potential neurotropic properties of this virus. Indeed, neurological sequelae of SARS-CoV-2 infection have already been reported and highlight the relevance of considering the neurological impact of coronavirus (CoV) from a translational perspective. Animal models of SARS and Middle East respiratory syndrome, caused by structurally similar CoVs during the 2002 and 2012 epidemics, have provided valuable data on nervous system involvement by CoVs and the potential for central nervous system spread of SARS-CoV-2. One key finding that may unify these pathogens is that all require angiotensin-converting enzyme 2 as a cell entry receptor. The CoV spike glycoprotein, by which SARS-CoV-2 binds to cell membranes, binds angiotensin-converting enzyme 2 with a higher affinity compared with SARS-CoV. The expression of this receptor in neurons and endothelial cells hints that SARS-CoV-2 may have higher neuroinvasive potential compared with previous CoVs. However, it remains to be determined how such invasiveness might contribute to respiratory failure or cause direct neurological damage. Both direct and indirect mechanisms may be of relevance. Clinical heterogeneity potentially driven by differential host immune-mediated responses will require extensive investigation. Development of disease models to anticipate emerging neurological complications and to explore mechanisms of direct or immune-mediated pathogenicity in the short and medium term is therefore of great importance. In this brief review, we describe the current knowledge from models of previous CoV infections and discuss their potential relevance to COVID-19.

The international European Academy of Neurology survey on neurological symptoms in patients with COVID-19 infection.

BACKGROUND AND PURPOSE: Although the main clinical features of COVID-19 infection are pulmonary, several associated neurological signs, symptoms and diseases are emerging. The incidence and characteristics of neurological complications are unclear. For this reason, the European Academy of Neurology (EAN) core COVID-19 Task Force initiated a survey on neurological symptoms observed in patients with COVID-19 infection. METHODS: A 17-question online survey was made available on the EAN website and distributed to EAN members and other worldwide physicians starting on 9 April 2020. RESULTS: By 27 April 2020, proper data were collected from 2343 responders (out of 4199), of whom 82.0% were neurologists, mostly from Europe. Most responders (74.7%) consulted patients with COVID-19 mainly in emergency rooms and in COVID-19 units. The majority (67.0%) had evaluated fewer than 10 patients with neurological manifestations of COVID-19 (neuro COVID-19). The most frequently reported neurological findings were headache (61.9%), myalgia (50.4%), anosmia (49.2%), ageusia (39.8%), impaired consciousness (29.3%) and psychomotor agitation (26.7%). Encephalopathy and acute cerebrovascular disorders were reported at 21.0%. Neurological manifestations were generally interpreted as being possibly related to COVID-19; they were most commonly recognized in patients with multiple general symptoms and occurred at any time during infection. CONCLUSION: Neurologists are currently and actively involved in the management of neurological issues related to the COVID-19 pandemic. This survey justifies setting up a prospective registry to better capture the prevalence of patients with neuro COVID-19, neurological disease characteristics and the contribution of neurological manifestations to outcome.

Does tobacco smoking predispose to apical periodontitis and endodontic treatment need? A systematic review and meta-analysis.

BACKGROUND: Controversial findings exist in the literature regarding the association between tobacco smoking and development of apical periodontitis or need for root canal treatment, with some studies reporting an increase in the prevalence of these outcomes in smokers, whilst others reporting no association. AIM: To evaluate if there is scientific evidence to support an association between tobacco smoking and a greater prevalence of apical periodontitis and/or root canal treatments. DATA SOURCES: A systematic search was performed using MeSH terms and free terms in the PubMed, Scopus, Virtual Health Library (VHL), Cochrane library and Open Gray databases. STUDY ELIGIBILITY CRITERIA, PARTICIPANTS AND INTERVENTIONS: Observational studies that evaluated the association between smoking and the development or healing of periapical lesion and/or the prevalence of root canal treatment in humans were included. STUDY APPRAISAL AND SYNTHESIS METHODS: A quality assessment of included studies was performed, and the relevant information and findings were evaluated. A meta-analysis using nine included studies was performed through RevMan software, and the certainty of evidence was evaluated through GRADE. RESULTS: Fifteen studies were included, of which 10 were classified as low risk of bias, 4 were considered as moderate risk of bias and 1 as high risk of bias. Ten studies reported that smoking was associated with a greater prevalence of periapical periodontitis and/or root canal treatment. Five studies reported no association. Nine studies were included in the pooled meta-analysis, comprising two subgroups: apical periodontitis and root canal treatment. From these nine studies, eight studies were included in the apical periodontitis subgroup meta-analysis and demonstrated that this condition was significantly more prevalent in smokers when compared to the nonsmokers (odds ratio = 2.78[CI: 1.60, 4.85], P < 0.001; I2  = 79%; no adjusted odds ratio). Three studies were included in the root canal treatment subgroup meta-analysis and demonstrated an increased prevalence in smokers when compared to the nonsmokers (odds ratio = 2.73 [CI: 1.06, 2.83], P < 0.001; I2  = 72%; no adjusted odds ratio). The pooled meta-analysis demonstrated that smokers had twice the chance of having apical periodontitis and/or root canal treatment when compared to nonsmokers (odds ratio of 2.42 [CI: 1.59, 3.68], P < 0.01; I2  = 85%). The GRADE analysis demonstrated moderate certainty of evidence. LIMITATIONS: Several confounding factors were identified such as misinformation regarding the period of time subjects had smoked, as well as the frequency of smoking and the number of cigarettes consumed. No adjustment in odds ratio data for confounding was performed. CONCLUSIONS AND IMPLICATIONS OF KEY FINDINGS: This systematic review and meta-analysis indicate that tobacco smokers have an increased prevalence of periapical periodontitis and root canal treatments with moderate certainty of evidence.

MTT versus other cell viability assays to evaluate the biocompatibility of root canal filling materials: a systematic review.

OBJECTIVES: This systematic review aimed to compare the cytotoxicity of root canal filling materials (RCFMs) assessed using tetrazolium salt-based tests (TSBT), including the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay, with those obtained using other cell viability assays. METHODS: A search was performed on PubMed, Scopus, Web of Science and OpenGrey up to March 2019, followed by a manual search. According to the Participants, Exposure, Comparator and Outcomes (PECO) criteria, in vitro studies that evaluated the cytotoxic effect of RCFMss on animal and/or human cells through TSBT and at least one other viability assay were compared. The methodological quality of selected papers was assessed using ToxRTool® and SciRAP® . Data were analysed using Wilcoxon's signed-rank test for paired samples and linear weighting kappa. RESULTS: A total of 230 non-duplicated records were identified. After applying the eligibility criteria, 55 studies were selected for methodological evaluation, seven were selected by manual searching, 22 were excluded for methodological reasons, and 40 were included. A total of 410 comparisons were performed between TSBT and distinct cell viability tests (DCVT). MTT had moderate concordance with DCVT using human cells (n = 138 samples) (P = 0.507; k = 0.4225) and animal cells (n = 122 samples) (P = 0.124; k = 0.5775). XTT had good concordance using human (n = 110 samples) (P = 0.507; k = 0.6336) and animal cells (n = 12 samples) (P = 0.564; k = 0.6604). MTT, XTT, WST and MTS assays showed moderate concordance with DCVT (n = 410 samples) (P = 0.375; k = 0.5138) and complete agreement in 226 samples. DISCUSSION: The included studies had methodological heterogeneity that was minimized by the systematic review methodology. CONCLUSIONS: MTT and XTT do not cause over- or underestimation of cell viability during cytotoxicity screening of root canal filling materials, implying that these assays can be considered reliable for this purpose. Nonetheless, the development of protocols for the cytotoxic screening of these materials on 3D tissue-like cultures aiming to improve their predictability in the clinical scenario is suggested.

Transforming Growth Factor-ß1 Decreases ß2-Agonist-induced Relaxation in Human Airway Smooth Muscle.

Helper T effector cytokines implicated in asthma modulate the contractility of human airway smooth muscle (HASM) cells. We have reported recently that a profibrotic cytokine, transforming growth factor (TGF)-ß1, induces HASM cell shortening and airway hyperresponsiveness. Here, we assessed whether TGF-ß1 affects the ability of HASM cells to relax in response to ß2-agonists, a mainstay treatment for airway hyperresponsiveness in asthma. Overnight TGF-ß1 treatment significantly impaired isoproterenol (ISO)-induced relaxation of carbachol-stimulated, isolated HASM cells. This single-cell mechanical hyporesponsiveness to ISO was corroborated by sustained increases in myosin light chain phosphorylation. In TGF-ß1-treated HASM cells, ISO evoked markedly lower levels of intracellular cAMP. These attenuated cAMP levels were, in turn, restored with pharmacological and siRNA inhibition of phosphodiesterase 4 and Smad3, respectively. Most strikingly, TGF-ß1 selectively induced phosphodiesterase 4D gene expression in HASM cells in a Smad2/3-dependent manner. Together, these data suggest that TGF-ß1 decreases HASM cell ß2-agonist relaxation responses by modulating intracellular cAMP levels via a Smad2/3-dependent mechanism. Our findings further define the mechanisms underlying ß2-agonist hyporesponsiveness in asthma, and suggest TGF-ß1 as a potential therapeutic target to decrease asthma exacerbations in severe and treatment-resistant asthma.

Intraprocedural direct left atrial and wedge pressure correlation during transcatheter mitral valve repair: Results from a single center registry.

OBJECTIVES: This study investigates the correlation of occlusive wedge pressure (WP) with direct left atrial (LA) pressure in patients with severe mitral regurgitation (MR) undergoing transcatheter mitral valve repair (TMVr) with MitraClip. BACKGROUND: There is interest in acquiring objective hemodynamic parameters for intraprocedural guidance in patients undergoing MitraClip. METHODS: The study included 94 patients with severe MR at prohibitive surgical risk who underwent MitraClip at the University of California Davis Medical Center between 2014 and 2016. RESULTS: An average of 1.8 ± 0.7 clips were used to achieve MR grade of 2+ or less in 99% of patients. Correlation analysis of all (n = 236) pre-clip, inter-clip, and final-clip WP and LA pressures yielded a Pearson's R (r) of 0.85 and 0.79 for mean WP vs mean LA and WP V vs LA V, respectively. Median LA V to mean LA ratio (LAV:mLA) was 1.75 (IQR 1.5-1.9). 79% (n = 74) of patients had LAV:mLA ratio ≥ 1.5 with associated WP V vs LA V correlation (r) of 0.83. In patients with LAV:mLA ratio < 1.5, the correlation (r) was 0.69. Baseline characteristics were not significantly different between patients with LAV:mLA ratio ≥ 1.5 and patients with LAV:mLA ratio < 1.5. Post-procedure, median LA V: mean LA ratio decreased from 1.75 to 1.4, P = 0.0001. CONCLUSIONS: Correlation between WP and direct LA pressure in patients with severe MR undergoing Mitraclip is modest. Caution is advised when using WP to approximate LA pressure intraprocedurally, especially in patients with baseline low LAV:mLA ratios.

Natural vertical infection by dengue virus serotype 4, Zika virus and Mayaro virus in Aedes (Stegomyia) aegypti and Aedes (Stegomyia) albopictus.

Vertical transmission to progeny ensures the maintenance of arboviruses in their natural vectors. This mechanism is largely reported for dengue virus (DENV) and yellow fever virus (YFV). Few studies have addressed this mechanism for Zika virus (ZIKV), Mayaro virus (MAYV) and other arboviruses. The present study investigated the natural infection rate by arboviruses in 4490 Aedes (Stegomyia) aegypti and 296 Aedes (Stegomyia) albopictus (Diptera: Culicidae) reared from eggs collected with ovitraps in Cuiabá, Mato Grosso State, from February to July, 2017. After viral RNA extraction and reverse transcriptase-polymerase chain reaction protocols for 10 flaviviruses and five alphaviruses, nucleotide sequencing and three passages in C6/36 cells, eight pools of Ae. aegypti positive for DENV-4 genotype II, seven for ZIKV Asian genotype and two for MAYV genotype L were found. In addition, two Ae. albopictus pools were positive for DENV-4 genotype II and two were positive for ZIKV Asian genotype. Infection was confirmed by viral isolation in all positive pools for DENV-4 and for MAYV and in eight of nine for ZIKV. This mechanism may contribute to the spread of arboviruses during epidemics and also to their maintenance in natural vectors during interepidemic periods.

Analysis of exposure margins in developmental toxicity studies for detection of human teratogens.

The draft Step 2 ICH S5(R3) guideline includes an exposure-based endpoint as an option for selecting the high-dose in reproductive and developmental toxicity studies. To help determine an appropriate exposure margin for embryofetal developmental toxicity testing, a retrospective analysis was undertaken to determine what threshold would have been sufficient to detect hazards to embryofetal development in rats and rabbits for 18 known and 4 presumed human teratogens. The analysis showed that using a high dose that provided at least a 6-fold exposure margin in the developmental toxicity studies would have been sufficient to detect the teratogenic hazard with relevance for humans for all these therapeutics. With regards to human risk assessment practices for developmental toxicity, the analysis showed that, after excluding lenalidomide and pomalidomide data in rats, all available AUC margins at the NOAEL for the induction of malformations or embryofetal lethality were <4-fold of the exposure at the MRHD for all 22 therapeutics. These data support the proposed general approach of increased level of concern for human risk when exposure margins of the NOAEL to the MRHD are <10-fold, reduced concern when the exposure margins are 10- to 25-fold, and minimal concern when the exposure margin is > 25-fold.

PDE8 Is Expressed in Human Airway Smooth Muscle and Selectively Regulates cAMP Signaling by ß2-Adrenergic Receptors and Adenylyl Cyclase 6.

Two cAMP signaling compartments centered on adenylyl cyclase (AC) exist in human airway smooth muscle (HASM) cells, one containing ß2-adrenergic receptor AC6 and another containing E prostanoid receptor AC2. We hypothesized that different PDE isozymes selectively regulate cAMP signaling in each compartment. According to RNA-sequencing data, 18 of 24 PDE genes were expressed in primary HASM cells derived from age- and sex-matched donors with and without asthma. PDE8A was the third most abundant of the cAMP-degrading PDE genes, after PDE4A and PDE1A. Knockdown of PDE8A using shRNA evoked twofold greater cAMP responses to 1 µM forskolin in the presence of 3-isobutyl-1-methylxanthine. Overexpression of AC2 did not alter this response, but overexpression of AC6 increased cAMP responses an additional 80%. We examined cAMP dynamics in live HASM cells using a fluorescence sensor. PF-04957325, a PDE8-selective inhibitor, increased basal cAMP concentrations by itself, indicating a significant basal level of cAMP synthesis. In the presence of an AC inhibitor to reduce basal signaling, PF-04957325 accelerated cAMP production and increased the inhibition of cell proliferation induced by isoproterenol, but it had no effect on cAMP concentrations or cell proliferation regulated by prostaglandin E2. Lipid raft fractionation of HASM cells revealed PDE8A immunoreactivity in buoyant fractions containing caveolin-1 and AC5/6 immunoreactivity. Thus, PDE8 is expressed in lipid rafts of HASM cells, where it specifically regulates ß2-adrenergic receptor AC6 signaling without effects on signaling by the E prostanoid receptors 2/4-AC2 complex. In airway diseases such as asthma and chronic obstructive pulmonary disease, PDE8 may represent a novel therapeutic target to modulate HASM responsiveness and airway remodeling.

Selection of candidate reference genes and validation for real-time PCR studies in rice plants exposed to low temperatures.

Rice is a cereal that presents a great ability to adapt to different soil and climate conditions. However, as it is a tropical crop with C3 metabolism, it performs better in warm temperatures with high solar radiation. Tolerance to stress caused by low temperatures is a highly complex process that involves various metabolic pathways and cellular compartments, resulting in general or specific effects on plant growth and development. In order to observe the true effect of a particular stress on genetic expression, reference genes need to be chosen for real-time PCRs, the expression levels of which should remain stable independent of the situation imposed. In this paper, the expression stability was evaluated of the actin 11 (ACT11), ubiquitin-conjugating enzyme 2 (UBC-E2), glyceraldehyde 3-phosphate dehydrogenase (GAPDH), beta tubilin (ß-Tubulin), eukaryotic initiation factor 4α (eIF-4-α), eukaryotic initiation factor 1α (eIF-1-α), ubiquitin 10 (UBQ10), ubiquitin 5 (UBQ5), aquaporin (TIP41), and cyclophilin genes, in two rice genotypes cultivated in low temperature (13°C) conditions in vegetative stage (V4). The analysis material (leaves) was collected after 0, 6, 24, 48, and 72 h of exposure to the stress. In this study, the geNorm, BestKeeper, ΔCt, NormFinder, and RefFinder methods were used to evaluate the expression stability of the candidate reference genes. The results revealed that the most indicated genes for all the analysis methods were UBQ10 and UBQ5 for BRS Bojuru and BRS Pampa, respectively. On the other hand, the eIF-1-α gene presents the least expression stability and is not indicated for studies of rice plants subjected to low temperatures. The validation with the antioxidant system genes SODCc1-Cu/Zn, CATC, APX2, and GR2 confirmed the importance of using previously tested normalizing genes for adequate real-time PCR results.

Estimates of genetic parameters and genotypic values prediction in maize landrace populations by REML/BLUP procedure.

The REML/BLUP statistics are analyses that can be used as selective criteria in the routine of maize breeding programs. The present study aims to determine the genetic potential in crosses of landrace populations applying the REML/BLUP methodology, and to identify populations for the synthesis of new populations and intrapopulation selection for family farming systems, as well as genetic constitutions for use in maize breeding programs. Nine top cross hybrids obtained in the 2012/2013 harvest were evaluated along with their testator, the landraces used as parents, and four commercial hybrids, in a randomized block design, with information taken from the average of each plot. The evaluated traits were: leaf angle, number of ramifications of the tassel, spike insertion height, plant height, spike diameter, number of grains per spike, mass of grains per spike, spike mass, spike length, prolificity, mass of one hundred grains, and grain yield per plot. The data were analyzed using the Selegen-REML/BLUP software. The top cross hybrids Cateto Branco x Planalto, Amarelão x Planalto and the population Cateto Branco are ranked among the ten best crosses, simultaneously, for the traits: leaf angle, number of ramifications of the tassel, spike insertion height, and plant height (Cateto Branco x Planalto), and leaf angle, spike insertion height, and plant height (Amarelão x Planalto and Cateto Branco). The top cross hybrids Criolão x Planalto, Branco 8 Carreiras x Planalto, Caiano Rajado x Planalto, Amarelão x Planalto, Branco Roxo Índio x Planalto stand out for their high genotypic value of the individual BLUP mean components among the ten best genotypes for grain yield, and by combining three or more traits of interest together, being, for effects of selection, the most indicated.

Diallel and prediction (REML/BLUP) for yield components in intervarietal maize hybrids.

Genetic improvement is essential to achieve increments in maize (Zea mays L.) grain yield components. It may be obtained through crosses, which enable to exploit the effects of intervarietal heterosis, allelic complementarity, as well as gene actions and effects. This study estimated the components of variance and genetic parameters (REML/BLUP) of an intervarietal diallel to select and predict the best genotypes for maize yield components. The experimental design was randomized blocks containing 60 intervarietal maize hybrids arranged in three repetitions. They were obtained through intervarietal crosses and evaluated in a diallel scheme, where 14 open-pollinated varieties were designated as parentals. Thus, 10 crosses were performed for each hybrid combination to obtain the number of seeds necessary for the competition test. The measured traits were: grain volume relative index, the mass of one hundred grains, and grain yield. The male parents and the additive genetic fraction were determinants for grain volume relative index. Mass of one hundred grains and grain yield were defined by the specific combining ability, and female parents revealed low narrow sense heritability. The female parent Taquarão and male parent Argentino Amarelo presented the best general combining abilities for the measured traits. The specific combining abilities were expressed for crosses AL 25 x Dente de Ouro Roxo, AL 25 x BRS Pampeano, and Taquarão x Argentino Branco. Genetic estimates and predictions were consistent and applicable to breeding programs and could be applied in future quantitative genetic studies of maize.