Feeding behavior modifies the circadian variation in RR and QT intervals by distinct mechanisms in mice.

Rhythmic feeding behavior is critical for regulating phase and amplitude in the ≈24-h variation of heart rate (RR intervals), ventricular repolarization (QT intervals), and core body temperature in mice. We hypothesized changes in cardiac electrophysiology associated with feeding behavior were secondary to changes in core body temperature. Telemetry was used to record electrocardiograms and core body temperature in mice during ad libitum-fed conditions and after inverting normal feeding behavior by restricting food access to the light cycle. Light cycle-restricted feeding modified the phase and amplitude of 24-h rhythms in RR and QT intervals, and core body temperature to realign with the new feeding time. Changes in core body temperature alone could not account for changes in phase and amplitude in the ≈24-h variation of the RR intervals. Heart rate variability analysis and inhibiting ß-adrenergic and muscarinic receptors suggested that changes in the phase and amplitude of 24-h rhythms in RR intervals were secondary to changes in autonomic signaling. In contrast, changes in QT intervals closely mirrored changes in core body temperature. Studies at thermoneutrality confirmed that the daily variation in QT interval, but not RR interval, primarily reflected daily changes in core body temperature (even in ad libitum-fed conditions). Correcting the QT interval for differences in core body temperature helped unmask QT interval prolongation after starting light cycle-restricted feeding and in a mouse model of long QT syndrome. We conclude feeding behavior alters autonomic signaling and core body temperature to regulate phase and amplitude in RR and QT intervals, respectively.NEW & NOTEWORTHY We used time-restricted feeding and thermoneutrality to demonstrate that different mechanisms regulate the 24-h rhythms in heart rate and ventricular repolarization. The daily rhythm in heart rate reflects changes in autonomic input, whereas daily rhythms in ventricular repolarization reflect changes in core body temperature. This novel finding has major implications for understanding 24-h rhythms in mouse cardiac electrophysiology, arrhythmia susceptibility in transgenic mouse models, and interpretability of cardiac electrophysiological data acquired in thermoneutrality.

Single-cell analysis delineates a trajectory toward the human early otic lineage.

Efficient pluripotent stem cell guidance protocols for the production of human posterior cranial placodes such as the otic placode that gives rise to the inner ear do not exist. Here we use a systematic approach including defined monolayer culture, signaling modulation, and single-cell gene expression analysis to delineate a developmental trajectory for human otic lineage specification in vitro. We found that modulation of bone morphogenetic protein (BMP) and WNT signaling combined with FGF and retinoic acid treatments over the course of 18 days generates cell populations that develop chronological expression of marker genes of non-neural ectoderm, preplacodal ectoderm, and early otic lineage. Gene expression along this differentiation path is distinct from other lineages such as endoderm, mesendoderm, and neural ectoderm. Single-cell analysis exposed the heterogeneity of differentiating cells and allowed discrimination of non-neural ectoderm and otic lineage cells from off-target populations. Pseudotemporal ordering of human embryonic stem cell and induced pluripotent stem cell-derived single-cell gene expression profiles revealed an initially synchronous guidance toward non-neural ectoderm, followed by comparatively asynchronous occurrences of preplacodal and otic marker genes. Positive correlation of marker gene expression between both cell lines and resemblance to mouse embryonic day 10.5 otocyst cells implied reasonable robustness of the guidance protocol. Single-cell trajectory analysis further revealed that otic progenitor cell types are induced in monolayer cultures, but further development appears impeded, likely because of lack of a lineage-stabilizing microenvironment. Our results provide a framework for future exploration of stabilizing microenvironments for efficient differentiation of stem cell-generated human otic cell types.

Nicotine-induced molecular alterations are modulated by GABAB receptor activity.

It has been demonstrated that GABAB receptors modulate nicotine (NIC) reward effect; nevertheless, the mechanism implicated is not well known. In this regard, we evaluated the involvement of GABAB receptors on the behavioral, neurochemical, biochemical and molecular alterations associated with the rewarding effects induced by NIC in mice, from a pharmacological and genetic approach. NIC-induced rewarding properties (0.5 mg/kg, subcutaneously, sc) were evaluated by conditioned place preference (CPP) paradigm. CPP has three phases: preconditioning, conditioning and postconditioning. GABAB receptor antagonist 2-hydroxysaclofen (0.25, 0.5 and 1 mg/kg; intraperitoneally, ip) or the GABAB receptor agonist baclofen (3 mg/kg; ip) was injected before NIC during the conditioning phase. GABAB1 knockout (GABAB1 KO) mice received NIC during the conditioning phase. Vehicle and wild-type controls were employed. Neurochemical (dopamine, serotonin and their metabolites), biochemical (nicotinic receptor α4ß2, α4ß2nAChRs) and molecular (c-Fos) alterations induced by NIC were analyzed after the postconditioning phase by high-performance liquid chromatography (HPLC), receptor-ligand binding assays and immunohistochemistry, respectively, in nucleus accumbens (Acb), prefrontal cortex (PFC) and ventral tegmental area (VTA). NIC induced rewarding effects in the CPP paradigm and increased dopamine levels in Acb and PFC, α4ß2nAChRs density in VTA and c-Fos expression in Acb shell (AcbSh), VTA and PFC. We showed that behavioral, neurochemical, biochemical and molecular alterations induced by NIC were prevented by baclofen. However, in 2-hydroxysaclofen pretreated and GABAB1 KO mice, these alterations were potentiated, suggesting that GABAB receptor activity is necessary to control alterations induced by NIC-induced rewarding effects. Therefore, the present findings provided important contributions to the mechanisms implicated in NIC-induced rewarding effects.

Practical splicing of poly-methyl-methacrylate plastic optical fibers.

This Engineering and Laboratory Note describes a simple, fast, cost-effective, and practical splicing technique for poly-methyl-methacrylate (PMMA) plastic optical fibers. We believe this technique can be useful for home networks, laboratory research, and educational uses. It employs a widely available, low-cost transparent adhesive combined with a suitable alignment sleeve. The fiber tips did not need to be cleaned or polished, and the polyethylene jacket did not need to be removed. The alignment between the cleaved fibers was performed by means of the jacket. Low insertion losses were measured for the main PMMA-based fiber types.

Noninvasive proteome analysis of psoriatic stratum corneum reflects pathophysiological pathways and is useful for drug profiling.

BACKGROUND: Protein expression is disturbed in the psoriatic stratum corneum (SC). Noninvasive methods for the description of pathophysiological changes and drug profiling in psoriasis are desirable. OBJECTIVES: Undertake large-scale noninvasive protein expression studies in psoriatic SC to identify biomarkers of pathophysiological processes and use them for drug profiling. METHODS: Psoriatic SC was harvested through repetitive tape-stripping. Nonlesional and lesional SC, as well as vehicle-treated and drug-treated lesional SC samples were collected. Protein extracts from nonlesional and lesional skin biopsies were used for comparison. Calcipotriol-betamethasone (CB) was used as a reference medication. Proteins extracted from pooled tape strips were quantified using mass spectrometry (MS), Western blotting, enzyme-linked immunosorbent assay and Luminex technologies. RESULTS: MS-based methods identified 140 proteins differentially expressed in psoriatic SC. Epidermis development, glycolysis, regulation of apoptosis, cytoskeleton organization and peptide cross-linking were modulated, all reflecting perturbed epidermal differentiation. Using antibody-based techniques, increased levels of sICAM1, of CXCL1- and CXCL8-attracting neutrophils, of CXCL10- and CCL4-attracting T helper (Th) 1 cells, and of CCL2- and CCL4-attracting monocytes and dendritic cells were observed. Quantification of the Th1 and Th17 markers tumour necrosis factor, interleukin (IL) 12B, IL17A and IL17F in lesional SC was successful, while the Th2 cytokines IL4, IL5 and IL13, not involved in the disease process, were not detected. The pruritic cytokine IL31 was detected in lesional SC. CXCL1, CXCL8, CXCL10 and sICAM were used to investigate disease remission, ranking three topical treatments according to their known clinical efficacy. CONCLUSIONS: Protein biomarker quantification in psoriatic SC detects key pathophysiological mechanisms and enables noninvasive drug profiling in translational medicine settings.

Combined abdomino-sacral laparoscopically assisted approach for retrorectal mass resection in a patient with Currarino's Syndrome - video vignette.

A correct preoperative strategy is crucial when surgery is needed for retrorectal tumours (RRT).[1] Surgical approaches may be purely anterior-abdominal, posterior-sacrococcygeal or combined depending on the tumour's size and location.[2] We present the case of an 18-year-old female with Currarino Syndrome who underwent surgery by a combined abdominal laparoscopic-posterior Kraske approach for the resection of a large RRT. This article is protected by copyright. All rights reserved.

Profile of Steroid Receptors and Increased Aromatase Immunoexpression in Canine Inflammatory Mammary Cancer as a Potential Therapeutic Target.

Canine inflammatory mammary cancer (IMC) has been proposed as a model for the study of human inflammatory breast cancer (IBC). The aims of this study were to compare the immunohistochemical expression of aromatase (Arom) and several hormone receptors [estrogen receptor α (ERα), estrogen receptor ß (ERß), progesterone receptor (PR) and androgen receptor (AR)], in 21 IMC cases vs 19 non-IMC; and to study the possible effect of letrozole on canine IMC and human inflammatory breast cancer (IBC) in vitro using IPC-366 and SUM-149 cell lines. Significant elevations of the means of Arom Total Score (TS), ERß TS and PR TS were found in the IMC group (p = 0.025, p = 0.038 and p = 0.037, respectively). Secondary IMC tumours expressed higher levels of Arom than primary IMC (p = 0.029). Non-IMC PR- tumours contained higher levels of Arom than non-IMC PR+ tumours (p = 0.007). After the addition of letrozole, the number of IMC and IBC cells dropped drastically. The overexpression of Arom found and the results obtained in vitro further support canine IMC as a model for the study of IBC and future approaches to the treatment of dogs with mammary cancer, and especially IMC, using Arom inhibitors.

Compressive imaging in scattering media.

One challenge that has long held the attention of scientists is that of clearly seeing objects hidden by turbid media, as smoke, fog or biological tissue, which has major implications in fields such as remote sensing or early diagnosis of diseases. Here, we combine structured incoherent illumination and bucket detection for imaging an absorbing object completely embedded in a scattering medium. A sequence of low-intensity microstructured light patterns is launched onto the object, whose image is accurately reconstructed through the light fluctuations measured by a single-pixel detector. Our technique is noninvasive, does not require coherent sources, raster scanning nor time-gated detection and benefits from the compressive sensing strategy. As a proof of concept, we experimentally retrieve the image of a transilluminated target both sandwiched between two holographic diffusers and embedded in a 6mm-thick sample of chicken breast.

Ortho and para hydrogen dimers on G/SiC(0001): combined STM and DFT study.

The hydrogen (H) dimer structures formed upon room-temperature H adsorption on single layer graphene (SLG) grown on SiC(0001) are addressed using a combined theoretical-experimental approach. Our study includes density functional theory (DFT) calculations for the full (6√3 × 6√3)R30° unit cell of the SLG/SiC(0001) substrate and atomically resolved scanning tunneling microscopy images determining simultaneously the graphene lattice and the internal structure of the H adsorbates. We show that H atoms normally group in chemisorbed coupled structures of different sizes and orientations. We make an atomic scale determination of the most stable experimental geometries, the small dimers and ellipsoid-shaped features, and we assign them to hydrogen adsorbed in para dimers and ortho dimers configuration, respectively, through comparison with the theory.

Baclofen prevented the changes in c-Fos and brain-derived neutrophic factor expressions during mecamylamine-precipitated nicotine withdrawal in mice.

Previous studies from our laboratory showed that baclofen (BAC, GABAB receptor agonist) prevented the behavioral and neurochemical alterations of nicotine (NIC) withdrawal syndrome. To further investigate the mechanisms underlying these effects, we analyzed the c-Fos and brain-derived neutrophic factor (BDNF) expression during NIC withdrawal and its prevention with BAC. Swiss-Webster mice received NIC (2.5 mg/kg, sc) four times daily, for 7 days. On the 8th day, NIC-treated mice received the nicotinic antagonist mecamylamine (MEC; 2 mg/kg, i.p.) 1 h after the last dose of NIC. A second group of NIC-treated mice received BAC (2 mg/kg, i.p.) prior to MEC administration. Thirty minutes after MEC, mice were sacrificed and the immunohistochemistry assays (c-Fos and BDNF) were performed at different anatomical levels. c-Fos expression decreased in the dentate gyrus of the hippocampus (DG) and the bed nucleus of the stria terminalis (BST), and increased in the habenular (Hb), accumbens shell (AcbSh) nuclei during NIC withdrawal. BAC re-established the modified c-Fos expression only in the DG, BST and AcbSh during NIC withdrawal. Conversely, BDNF expression decreased in the CA1 and CA3 area of the hippocampus, the Hb, and caudate putamen (CPu) during NIC withdrawal. Finally, BAC restored the decreased BDNF expression during NIC withdrawal in the CA1, CA3, Hb, and CPu. The results suggest a relationship between BAC's preventive effect of the expression of NIC withdrawal signs, and its ability to restore the changes in c-Fos and BDNF expression, observed in specific brain areas of NIC-withdrawn mice.

Baclofen prevents morphine rewarding effects and associated biochemical alterations in male and female mice.

Previous studies from our laboratory have shown sex differences in the behavioral, molecular, and neurochemical manifestations of morphine withdrawal and they were related to an increased sensitivity to morphine effects in males. In addition, we observed an interaction between the GABAergic and opioid systems that could also be sex-dependent. Baclofen, a GABAB receptor agonist, prevented the somatic expression and the molecular and neurochemical changes induced by morphine withdrawal syndrome in mice. On the contrary, little is known about baclofen effects in the rewarding properties of morphine in male and female mice. The present study aimed to explore the effect of baclofen (1, 2 and 3 mg/kg, i.p.) pretreatment in the rewarding effects induced by morphine (7 mg/kg, s.c.) and its effect on c-Fos and brain-derived neurotrophic factor (BDNF) expression induced by the rewarding properties of morphine in prepubertal male and female mice. Baclofen (2 mg/kg) pretreatment prevented the rewarding effects of morphine only in male mice, while baclofen (3 mg/kg) reduced these effects in both sexes. Moreover, the rewarding effects of morphine were associated with a decrease of BDNF and c-Fos expression cingulate cortex, nucleus accumbens shell, cornu ammonis 1 (CA1), and cornu ammonis 3 (CA3) areas of the hippocampus only in male mice. In addition, baclofen pretreatment prevented these changes in BDNF, but not in c-Fos expression. In conclusion, our results show that GABAB receptors have a regulatory role in the rewarding effects of morphine that could be of interest for a potential future therapeutic application in opioid use disorders.

n-Alkanes formed by methyl-methylene addition as a source of meteoritic aliphatics.

Aliphatics prevail in asteroids, comets, meteorites and other bodies in our solar system. They are also found in the interstellar and circumstellar media both in gas-phase and in dust grains. Among aliphatics, linear alkanes (n-CnH2n+2) are known to survive in carbonaceous chondrites in hundreds to thousands of parts per billion, encompassing sequences from CH4 to n-C31H64. Despite being systematically detected, the mechanism responsible for their formation in meteorites has yet to be identified. Based on advanced laboratory astrochemistry simulations, we propose a gas-phase synthesis mechanism for n-alkanes starting from carbon and hydrogen under conditions of temperature and pressure that mimic those found in carbon-rich circumstellar envelopes. We characterize the analogs generated in a customized sputter gas aggregation source using a combination of atomically precise scanning tunneling microscopy, non-contact atomic force microscopy and ex-situ gas chromatography-mass spectrometry. Within the formed carbon nanostructures, we identify the presence of n-alkanes with sizes ranging from n-C8H18 to n-C32H66. Ab-initio calculations of formation free energies, kinetic barriers, and kinetic chemical network modelling lead us to propose a gas-phase growth mechanism for the formation of large n-alkanes based on methyl-methylene addition (MMA). In this process, methylene serves as both a reagent and a catalyst for carbon chain growth. Our study provides evidence of an aliphatic gas-phase synthesis mechanism around evolved stars and provides a potential explanation for its presence in interstellar dust and meteorites.

Parallel laser micromachining based on diffractive optical elements with dispersion compensated femtosecond pulses.

We experimentally demonstrate multi-beam high spatial resolution laser micromachining with femtosecond pulses. The effects of chromatic aberrations as well as pulse stretching on the material processed due to diffraction were significantly mitigated by using a suited dispersion compensated module (DCM). This permits to increase the area of processing in a factor 3 in comparison with a conventional setup. Specifically, 52 blind holes have been drilled simultaneously onto a stainless steel sample with a 30 fs laser pulse in a parallel processing configuration.

Prognostic value of histological grading in noninflammatory canine mammary carcinomas in a prospective study with two-year follow-up: relationship with clinical and histological characteristics.

In this prospective study, a canine-adapted histological grading method was compared with histopathological and clinical characteristics and was evaluated as a prognostic indicator in canine mammary carcinomas (CMCs). Recruited dogs with at least 1 malignant mammary tumor (n = 65) were clinically evaluated, surgically treated, and followed up (minimum follow-up 28 months, maximum 38 months). Histopathological diagnoses were performed according to Goldschmidt et al (2011). Tumors were graded as grade I (29/65), grade II (19/65), and grade III (17/65). The tumor size, clinical stage, histological diagnosis, presence/absence of myoepithelial proliferation, and regional lymph node metastases at diagnosis were significantly associated with histological grade. The histological grade, age, clinical stage, tumor subtype group, and lymph node metastases at time of diagnosis were significantly associated with the development of recurrences and/or metastases, cancer-associated death, and survival times (disease-free survival and overall survival) in univariate analyses. A subdivision of clinical stage I (T1N0M0) into stages IA and IB was proposed in terms of prognosis. The clinical stage, histological grade, and spay status were selected as independent prognostic variables (multivariate analyses) with disease-free survival as the dependent variable. When overall survival was evaluated as a dependent variable, clinical stage and histological grade were selected as the independent covariates. This grading system is a useful prognostic tool, facilitates histological interpretation, and offers uniform criteria for veterinary pathologists. Comparative studies on CMCs performed in different countries should take into account possible changes in the prognoses due to different proportions of spayed females among the selected dog population.

Diffusion of hydrogen in Pd assisted by inelastic ballistic hot electrons.

Sykes et al. [Proc. Natl. Acad. Sci. U.S.A. 102, 17907 (2005)] have reported how electrons injected from a scanning tunneling microscope modify the diffusion rates of H buried beneath Pd(111). A key point in that experiment is the symmetry between positive and negative voltages for H extraction, which is difficult to explain in view of the large asymmetry in Pd between the electron and hole densities of states. Combining concepts from the theory of ballistic electron microscopy and electron-phonon scattering we show that H diffusion is driven by the s-band electrons only, which explains the observed symmetry.

GABA(B) receptors involvement in the effects induced by nicotine on anxiety-related behaviour in mice.

The aim of the present study was to evaluate the possible involvement of GABA(B) receptors in the anxiolytic- and anxiogenic-like responses induced by nicotine in mice. Animals were exposed to nicotine only once. The acute administration of low (0.05mg/kg, sc) or high (0.8mg/kg, sc) doses of nicotine produced opposite effects in the elevated plus maze test; respectively, anxiolytic- and anxiogenic-like responses. The effect of pretreatment with either the GABA(B) receptor antagonist 2-OH-saclofen (0.25, 0.5 and 1mg/kg; ip) or the GABA(B) receptor agonist baclofen (0.5, 1 and 2mg/kg; ip), was evaluated on the anxiolytic- and anxiogenic-like responses induced by nicotine. 2-OH-saclofen completely abolished both nicotine-induced effects (p<0.001) at the highest dose tested, suggesting an involvement of GABA(B) receptors in these behavioural responses. On the other hand, baclofen failed to modify the anxiety-related effects of nicotine. These results suggest that the GABA(B) receptors are involved in the regulation of nicotine-induced anxiety-related behavioural responses in mice, and provide new findings to support a potential pharmaco therapeutic use of GABAergic drugs in the treatment of tobacco addiction.

Poor zinc status is associated with increased risk of insulin resistance in Spanish children.

Zn plays a key role in the synthesis and action of insulin. The aim of the present work was to determine whether a poorer Zn status was associated with insulin resistance in a group of 357 Spanish schoolchildren. Zn intake was determined by using a 3 d food record (i.e. Sunday to Tuesday). The body weight, height and waist and hip circumferences of all subjects were recorded and fasting plasma glucose, insulin and Zn concentrations were determined. Insulin resistance was determined using the homoeostasis model assessment (HOMA) marker. Children (11·5 %) with Zn deficiency (serum Zn concentration < 10·7 µmol/l) had higher HOMA values than those with a more satisfactory Zn status (1·73 (sd 0·93)) compared with 1·38 (sd 0·90; P < 0·05). An inverse correlation was found between the HOMA value and the serum Zn concentration (r - 0·149, P < 0·05). The risk of having a greater insulin resistance value (HOMA greater than the 75th percentile) increased with age (OR 1·438; 95 % CI 1·021, 2·027) and BMI (OR 1·448; 95 % CI 1·294, 1·619) and decreased as Zn serum levels increased (OR 0·908; 95 % CI 0·835, 0·987; P < 0·001). Moreover, an inverse relationship was observed between HOMA values and Zn dietary density (r - 0·122), and the Zn intakes of male children with a HOMA value of >3·16 made a significantly smaller contribution to the coverage of those recommended (59·7 (sd 14·7) %) than observed in children with lower HOMA values (73·6 (sd 18·2) %; P < 0·05). Taking into account that Zn intake was below than that recommended in 89·4 % of the children, it would appear that increasing the intake of Zn could improve the health and nutritional status of these children, and thus contribute to diminish problems of insulin resistance.

[Particulate matter PM10, NO2 and exacerbations of chronic respiratory diseases]. / Partículas en suspensión PM10, NO2 y agudizaciones de enfermedad respiratoria crónica.

OBJECTIVES: The new World Health Organization guidelines recommend studies with simultaneous exposure to multiple air pollutants. The main objective has been to analyze the strength of the association between different concentrations of PM10 and NO2 and the exacerbation of chronic respiratory diseases (ECRD), specifically asthma and chronic obstructive pulmonary disease. MATERIAL AND METHODS: Retrospective cross-sectional study. The population analyzed were adults treated in an urgent and primary health care center on certain lag+1 days in 2019. Three indices have been developed (1: high levels of PM10 and NO2; 2: high level of PM10 and low level of NO2, and 3: low levels of PM10 and NO2) and a logistic regression model for each of them, with ECRD as the outcome variable, and the progressive addition of adjustment variables (sex, age, tobacco, Charlson index, season, precipitation, wind and temperature). RESULTS: Four hundred and sixty-one people were analyzed, 17 with ECRD. Models 1 and 2 presented very similar values in the adjusted OR (4.28 [95% CI 1.05-17]), R2 (0.88) and the area under the ROC curve (>0.72). In both of them the significance was maintained after including the adjustment variables, while model 3 only allowed the addition of precipitation. The inclusion of the Charlson index and the tobacco consumption in the 3 models implied the loss of statistical significance of the PM10/NO2 combination regarding ECRD. CONCLUSIONS: High levels of PM10 are related to ECRD and have a greater impact than NO2, with tobacco use and comorbidities being the main precipitants of ECRD.

Cell adhesion molecules E-cadherin and CADM1 are differently expressed in canine inflammatory mammary cancer.

Human inflammatory breast cancer (IBC) and canine inflammatory mammary cancer (IMC) are the most aggressive and lethal types of mammary tumors with specific characteristics such as exacerbated angiogenesis, lymphangiogenesis and lymphangiotropism. E-cadherin expression is another specific feature of IBC not previously studied in canine IMC. In this study, the expression of E-cadherin and CADM1 (Cell Adhesion molecule 1) and their possible role as key molecules involved in the pathogenesis of IMC were immunohistochemically analyzed in 19 canine IMC and 15 grade III non-IMC cases. E-cadherin and CADM1 expression was higher in IMC cases (p = 0.002, p = 0.008, respectively). In the IMC group, E-cadherin cytoplasmic immunolabeling was more frequent (p = 0.035) and it was associated to the expression of the angiogenic and lymphangiogenic factors COX-2 (p = 0.009), VEGF-A (p = 0.031) and VEGF-D (p = 0.008). The differential mRNA expression between IMC and non-IMC was studied by microarray analysis in 6 cases. E-cadherin gene (CDH1) was not up-regulated in IMC cases at a transcriptional level; interestingly CADM1 was 7-fold upregulated. The differential expression of E-cadherin protein in IMC suggests a possible role of E-cadherin in the characteristic exacerbated angiogenesis and lymphangiogenesis and further support IMC as a natural model for the study of human IBC. Future studies in IBC and IMC including a broad panel of adhesion molecules are necessary to elucidate their role in the metastatic process and angiogenesis.

The Pisum sativum psp54 gene requires ABI3 and a chromatin remodeller to switch from a poised to a transcriptionally active state.

Aspects of transcriptional regulation in plants, such as the order in which transcriptional factors and the preinitiation complex are assembled, are obscure because studies carried out under conditions in which native chromatin structure is preserved are still few in comparison with those carried out under other conditions. In vivo chromatin immunoprecipitation (ChIP) experiments were used here to study the regulation of Pisum sativum psp54, which codes for the precursor of a chromatin-associated protein in dry seeds. Antibodies against PsSNF5, a component of the SWI/SNF remodelling complex, and against the transcriptional factor Pisum sativum abscisic acid insensitive 3 (PsABI3) were raised and used for ChIP experiments, which showed that both factors are bound to the psp54 promoter only when the gene is actively expressed during seed maturation and germination. However, RNA polymerase II appeared to be bound to the inactive promoter, which was poised for transcription, before the assembly of factors. Micrococcal nuclease protection assays showed that chromatin conformation at the proximal psp54 promoter changes in shifting from the active to inactive state. The changes in the promoter chromatin of psp54 are discussed. Stalled polymerase is described for the first time at the promoter of a non-heat-shock plant gene.