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At least 19 recordsLinked to original sources

Fluorometric and myothermic studies of cardiac energetics.

Energy production during isometric and isotonic contractions of cardiac muscle has been studied using the isolated papillary muscle of the rabbit at room temperature. Muscles were mounted on thermopiles to record heat production associated with small numbers of contractions and then were mounted in an organ bath for fluorometric recording of the level of reduction of mitochondrial NAD. After each twitch there was a decrease in the level of fluorescence followed by an exponential return to the resting level, or further decreases after subsequent twitches. The fluorescence-time integral (integral delta Fl-dt), or area enclosed by a fluorometric transient during and after a series of twitches, was found to vary in magnitude in a manner similar to the magnitude of the total heat production when the mechanical conditions of contraction were altered by varying the load. The influence of the metabolic state of the muscle was also studied by using either glucose or sodium pyruvate as the exogenous substrates in the bathing medium. When pyruvate was substituted for glucose, maximum external work, maximum isometric force, and maximum mechanical efficiency were all increased. The kinetics of oxidative recovery metabolism, either measured fluorometrically or derived from the heat production, was accelerated when pyruvate replaced glucose.

Animals

Simplifying multiplex genome engineering in Saccharomyces cerevisiae with intron-mediated Random Assembly and INtegration (RAIN).

Engineering of multistep enzymatic pathways often involves extensive optimization of heterologous gene expression levels and requires cloning of promoter and open reading frames (ORFs) to generate expression cassettes. We present work on a nascent method for multiplex genome engineering in Saccharomyces cerevisiae that negates the requirement for cloning of expression cassettes. Our system, Random Assembly and INtegration (RAIN), uses intron-mediated homologous recombination (HR) for random in vivo assembly of exogenous promoter and ORF libraries, which are combined and cotransformed in a one-pot method. The libraries include consensus homology arms which target long terminal repeat regions of the Ty1 retrotransposon, providing over a hundred possible integration loci. In this way, our developmental system aims to negate the need for in vitro combinatorial cloning of promoters and ORFs to generate expression cassettes, simplifying in vitro DNA preparation before multiplex genome engineering. This paper presents findings from a series of experiments to demonstrate a proof of concept for the RAIN system. These include: the first reported use of intron-mediated assembly of promoters and ORFs for expression of a functional gene product; up to three markerless genomic integrations; and up to five integrations with antibiotic selection. We also present a number of innovations to improve integration efficiency during multiplex engineering in S. cerevisiae including: SGS1 gene knockout; disruption of heteroduplex rejection; modified Cas9 expression architecture; and overexpression of HR genes RAD52, MRE11, and RAD59. To demonstrate how our system can be used for single transformation phenotype engineering of multiple strains, we also transformed a library of methylotrophy associated genes to generate four new strains that were able to grow on a solid minimal medium with methanol as the sole additional carbon source. Our findings contribute to the ongoing efforts to improve multiplex genome engineering tools in S. cerevisiae, and provide the foundations for further development of a novel toolbox for generating useful genetic diversity for metabolic pathway engineering.

Saccharomyces cerevisiae

Insertion of bacteriophage lambda into the deo operon of Escherichia coli K-12 and isolation of plaque-forming lambdadeo+ transducing bacteriophages.

A procedure has been devised to isolate plaque-forming lambda cI857S7 transducing bacteriophage which carry the internal promoter, P3, of the deo operon of Escherichia coli and the deoB and deoD genes, while lacking the deoP and cytP promoters of the same operon, in order to study, specifically, regulation at the P3 site. This has been accomplished by selecting for the insertion of bacteriophage lambda into the deoA gene in a strain deleted for the normal lambda attachment site (delta att lambda) and isolating from this lysogen lambda spi- and lambda EDTAr phage. Among these, lambda pdeoB+D+ phage were identified by their transducing abilities. From in vivo enzyme induction experiments performed on a delta deo strain lysogenized with such phage, they were shown to carry the P3 promoter while lacking the deoP and cytP promoters. A lambdapdeo B+D+ phage phage was used to lysogenize a deo+ delta att lambda strain, integration of lambda occurring within the region of homology, and, from a heat-induced lysate of this strain, a plaque-forming lambda+ phage carrying the complete deo operon was obtained. Phage lambda was also inserted into the deoB and deoD genes and into the tdk gene. By isolating lambdaspi- and lambdaEDTAr phage from the deo::(lambda) mutants and determining which bacterial genes they carried and whether they retained the int gene of lambda, it was found that lambda had inserted into deoD with the same orientation as lambda inserted into attlambda, whereas lambda inserted into deoA and deoB had the opposite orientation. Deletions extending from the site of lambda insertion into the bacterial chromosome were isolated by selecting for heat-resistant revertants. These confirmed the order of markers to be deo-serB-trpR-thr and also placed a locus, msp, determining sensitivity or resistance of male strains to male-specific phages, between trpR and thr. For some reason unknown, but which may be related to the orientation of the lambda prophages, short deletions rendering the bacterium Ser- Thr+ were of much lower frequency from the deoD::(lambda) lysogen than from the other two lysogens. From an examination of the residual deoD enzyme levels in deoB::(lambda) mutants, it was deduced that there may be two promoter sites within the deoB::(lambda) mutants, it was deduced that there may be two promoter sites within the deoB gene, transcription from one of these being sufficient to account for the noncoordinate nature of the induction of deoB and deoD gene products.

Chromosome Mapping

[Premature and usual periods of "rapid" sleep during attacks of diurnal narcolepsy].

The authors studied some psychophysiological characteristics of precocious and usual periods of rapid sleep in 17 patients with polysymptomatic narcolepsy during 92 attacks of day sleep. Precocious rapid sleep duffers from usual one in an inclusion into the dream content the experimental situations and their emotional saturation. The patients underestimated the duration of sleep and were unsatisfied by its quality. These differences indicate to a functional interconnection of rapid sleep with delta-sleep in the process of brain integration of information and assist to understand the pathogenesis of some traits of narcolepsy.

Humans

Chemical energetics in repeated contractions of frog sartorius muscles at 0 degrees C.

1. Recovery oxygen consumption (DeltaO(2)) was measured in aerobic, unpoisoned frog sartorius muscles at 0 degrees C following a series of repeated tetani.2. For a series of tetani having nearly identical tension-time integrals, DeltaO(2) was dependent on the interval between tetani and reached a maximal value for intervals equal to or greater than 200 sec.3. For a series of tetani at short (5 sec) intervals, DeltaO(2) was not distinguishable from DeltaO(2) following a single long tetanus having a tension-time integral similar to that of the sum of the series.4. A model is proposed to account for these observations in terms of the initial chemical reactions (Delta approximately P) including a saturable non-steady state reaction which utilizes about 1 mumole Delta approximately P/g and which is superimposed on an energy utilization proportional to the tension-time integral. Measurements of DeltaO(2) and Delta approximately P as functions of tetanic duration and interval between tetani are consistent with such a model.5. These experiments show that, in addition to parameters usually considered, the previous contractile history of a muscle plays a significant role in determining the chemical energy cost of an isometric contraction.

Animals

[So-called coronary angiographic paradoxes].

The results of examination of 25 patients (18 with ischemic heart disease and 7 with cardialgia of other etiology) are discussed. The arteriolo-capillary bed of the coronary arteries was studied by intraarterial injection of albumin aggregates labelled with radioactive isotopes. A correlation was revealed between the degree of microcirculatory disorders and the results of electrocardiography. With increase in the degree of microcirculatory disorders, cicatricial changes in the left ventricular myocardium were found more frequently and a history of myocardial infarction (in 11 persons) and the development of left ventricular aneurysm (in 2 patients) were noted. Certain indices of integral function (EF, VcF, MNSER) and regional contractility (% delta r, L) of the left ventricle were determined in 14 patients. Among these parameters characterizing left ventricular function (EF, VCF, MNSER, % delta r, L), VCF, MNSER, and % delta r, L are most sensitive. No correlation was revealed between the state of the myocardial microcirculation bed and EF, VCF, and MNSER, but a definite correlation was found between it and % delta r, L (in the form of regional hypo- and hyperkinesia and paradoxical pulsation). Normal coronary arteriograms do not exclude the presence of ischemic heart disease and other primary diseases of the myocardium. Patients of this category should be examined more scrupulously to detect disorders of blood supply to the myocardium and function of the left ventricle.

Coronary Angiography

Indices of myocardial oxygen consumption for isotonic contraction.

To investigate the model-independent mechanical determinants of energy expenditure, a respirometer was constructed to study isolated feline papillary muscles. Mechanical parameters recorded were the distance of shortening (deltaL), peak velocity of shortening (Vp), mean velocity of shortening (V), tension-time index (TTI), afterload (P), and the integral of the contraction portion of the phase plane trajectory of velocity and length (integral of VdL). Oxygen consumption (Vo2) during 15-min isotonic contraction periods was monitored with a polarographic electrode. Vp, V, and delta L were inversely related to Vo2 in a curvilinear manner. P and TTI were directly related to Vo2 in a curvilinear fashion. Integral of VdL was inversely related to Vo2 in a linear manner. In several experiments the contractile state of the muscles was augmented by addition of norepinephrine (7 X 10(-8) M). The relationship between integral of VdL and Vo2 was shifted above and parallel to that for the control muscles. These experiments indicate that the index integral of VdL is linearly related to the oxygen consumed in isotonic contraction of isolated mammalian ventricular myocardium at a given level of contractile state.

Animals

[Change in the human electroencephalogram during brief mental loads].

Frequency-amplitude characteristics of EEG beta-, alpha-, theta- and delta-rhythms were studied in 33 male subjects with different maximal velocity of effective processing of visual information. Dosed mental load enhanced the total activity of EEG frequency components of parieto-occipital parts of the brain. The most substantial changes were revealed in the delta-rhythm range. A statistically significant correlative connection was found between integrative amplitude values of EEG slow rhythms and success in performing mental tasks. The higher the maximal velocity of effective processing of visual information, the greater the magnitudes of theta- and beta-activity. No statistically significant connection has been revealed between the maximal velocity of effective processing of visual information and the frequency-amplitude EEG characteristics before and after mental loads.

Adult

Changes in frequency and amplitude of delta activity during sleep.

Modified period analysis was applied to all-night sleep recordings from 14 young adult males. The modifications involved addition of measures of integrated amplitude and of time in frequency band to the zero crossings and zero counts of the first derivative. The analysis was directed toward changes in the characteristics of delta activity (0--3 c/sec) across the night. Delta shifted toward lower frequencies and decreased in amplitude as sleep progressed. These trends were apparent in mean values for successive periods of slow-wave sleep (SSWPs). For epochs of record classified as stage 4 EEG, these trends were seen both within and across SSWPs. The physiological significance of these changes is unknown. We suggest that they may reflect the kinetics of the metabolic processes underlying sleep. Whatever their theoretical significance, the present results provide new, normative features of the sleep EEG which are not evident in sleep stage classification. The trends we observed appear sufficiently reliable to merit empirical investigation as a function of age, clinical state, or the administration of drugs in addition to further studies aimed at determining their biological significance.

Adult

Postganglionic sympathetic activity with correlation to heart rhythm and central cortical rhythms.

1. Renal sympathetic nerve activity, ECG and parieto-occipital EEG were recorded in dogs anaesthetized with chloralose. The carotid sinus nerves were cut. Autocovariance functions and power spectra for these variables were computed. 2. During cooling of the vagus nerves, the integrated renal sympathetic activity exhibited rhythms which were correlated to the delta-theta rhythm of the EEG. This rhythm was also present with the vagus nerves functionally intact, but a cardiac rhythm was dominant. 3. Blood pressure-dependent neurones in the lower brain stem reticular formation have both cardiac and central cortical rhythms. This provides a hint that these neurones might be involved in the sympathetic tone generating network.

Animals

New insights into diagnostic values and mechanisms of ferroptosis associated with immune infiltration in diabetic kidney disease.

The pathogenesis of diabetic kidney disease (DKD) is complex and closely related to ferroptosis and immune dysregulation, but the relevance is unclear. The present study investigates the potential mechanisms of ferroptosis-related genes (FRGs) in DKD and their relationship with the immune-inflammatory response. It searches for new diagnostic biomarkers to help diagnose and treat DKD. Four Gene Expression Omnibus (GEO) datasets, GSE30528, GSE30529 and GSE30122 as the test set, and GSE96804 for validation, were analyzed. FRGs were obtained from GeneCards, and 47 ferroptosis-related differentially expressed genes (FRDEGs) were identified by intersecting with DKD-related differentially expressed genes. Functional enrichment analyses, including Gene Ontology, Kyoto Encyclopedia of Genes and Genomes, Gene Set Enrichment Analysis and Gene Set Variation Analysis, revealed that these FRDEGs are primarily associated with ferroptosis, hypoxia response and immune inflammation. Subsequently, the weighted gene co-expression network analysis (WGCNA) was employed to expand the ferroptosis-related gene network, and intersection of the 47 FRDEGs with key WGCNA module genes yielded 10 key genes. Based on the 10 key genes, the least absolute shrinkage and selection operator and support vector machine algorithms identified three hub genes [chemokine ligand 5 (CCL5), forkhead box C1 (FOXC1) and lactotransferrin (LTF)] for DKD diagnosis. Receiver operating characteristic curves confirmed their diagnostic value, with FOXC1 and LTF validated in the independent dataset. Immune infiltration analysis via CIBERSORT revealed eight immune cell types with significantly different infiltration levels between the DKD and control group in the integrated GEO datasets. Notably, both LTF and CCL5 showed a significant positive correlation with gamma delta T cells (γδT). Quantitative PCR results confirmed differential expression of the three hub genes in the DKD group, with elevated expression observed in DKD mice following intervention with rosiglitazone and hyperoside.

bioinformatics analysis

Indirect estimation of physiological distribution functions.

Multicompartment models, such as sums of exponential decays and sums of effects of different ventilation-perfusion ratios, are cast in the form of integrals. Difficulties in obtaining the density function in such an integral from measured values of the integral are attributed to amplification of error in the inversion solution and to the limited number of measurement points. The present approach to control the effect of the error is regularization with the use of a non-negativity constraint on the density function. The answers are sums of the influence or kernel functions of the integral wherever the sum is positive, and zero elsewhere. Such non-negative answers not only ensure that true density functions are obtained but also permit the answer to fall abruptly to zero. For example, a delta function can be much more closely approximated with the non-negativity constraint than without. A rule is developed to choose the value of smoothing parameter so as to minimize an approximate upper bound on the integral of the squared error of the answer. This typically tends to result in some oversmoothing. Functions tested without error and with 2% relative error are as follows: one of the kernel functions (best results); rectangular boxes and delta functions (fair results); and wide boxes (poor results).

Models, Biological

Delta component variations in human sleep electroencephalogram.

All night sleep was recorded polygraphically on 14 healthy adults, once for each person, and EEG was analyzed with bandpass filters. The integrated values during successive 10 second epochs were recorded consecutively at 1 mm intervals, separately for each frequency band. The variations of integrated values for long time span were clearly observed. The findings on 1-2 Hz component are reported exclusively in this report. (1) Four variation patterns were classified on the variations of integrated values of 1-2 Hz band component; long undulation, short undulation, irregular undulation and slight fluctuation. Short undulation was superimposed on long undulation. Long undulation had a gradually increasing slope, a plateau and steeply decreasing slope. Irregular undulation showed generally irregular fluctuations, without any definite variation patterns. During slight fluctuation the variations were small. Long undulation and irregular undulation corresponded to slow-wave sleep and slight fluctuation corresponded to REM sleep. The mean duration of each long undulation and irregular undulation was 31.2 and 11.9 minutes, respectively. In long undulations, 62.8% of the time was occupied by Stage 2 and the lesser ratio by Stages 3 and 4. In irregular undulation 89.1% of the time corresponded to Stage 2. (2) One sleep cycle was composed of the three sleep states which were accompanied with variation patterns, long undulation, irregular undulation and slight fluctuation. The sequence of the appearance of the former two patterns in one sleep cycle was classified into four types. About half of the sleep cycles in all records exhibited Type 1; the pattern started with long undulation, after one or several long undulations followed by irregular undulation and proceeded to slight fluctuation. The average number of long indulation in one sleep cycle was 1.58. (3) On all records, the highest long undulations appeared in the first cycle of all night sleep, in both frontal and central areas. In the occipital area, some of the highest crests appeared in the second cycle. When the highest of long undulations appeared, the height tended to decrease prior to wakening. The crest line of long undulation in frontal and central areas crossed in the latter period of all night sleep, in five controls. The mean voltage of long undulation in frontal, central and occipital areas was 52.4, 42.5 and 23.5 microvolts, respectively. The underlying physiological mechanism of delta waves in human sleep EEG and possible brain structures essential for long undulation and irregular undulation were discussed from both the clinical and experimental aspects.

Adult

Determination of the number of superhelical turns in simian virus 40 DNA by gel electrophoresis.

Closed-circular, superhelical DNA from simian virus 40 (SV40 DNA I) was treated with an excess of DNA-relaxing enzyme in the presence of increasing amounts of ethidium bromide (EtdBr). After removal of the ethidium, each sample consisted of a group of close-circular DNA molecules differing in their number of superhelical turns (tau) around a mean value of tau in a Gaussian-like distribution. The DNA samples were analyzed by electrophoresis in agarose gels under conditions where the electrophoretic mobility was a function of the number of superhelical turns. Since the distributions around tau of DNA molecules of different samples overlappped, the difference in the mean number of superhelical turns from sample to sample, delta tau, could be determined and used to measure the mean number (tau) for native SV40 DNA I. By this criterion, SV40 DNA I contains a Gaussian-like distribution of molecules differing by integral numbers around a mean value of tau = -24 +/- 2 at 37 degree C [in 0.2 M NaC1, 10 mM Tris-HC1 (pH 7.9), and 0.2 mM EDTA]. The heterogeneity in tau is probably a consequence of thermal fluctuations in the DNA helix at the time when the last phospholiester bond is closed in vivo. When correlated to the buoyant shift of completely relaxed SV40 DNA in a CsC1-propidium diiodide gradient, the number of delta tau = 24 +/- 2 of superhelical versus relaxed DNA implies an unwinding of the DNA helix by 26-28 degree upon intercalation of one molecule of EtdBr.

DNA, Circular

A dual-dimensional CRISPR toolkit enables one-step high-efficiency multiplex genome editing in Komagataella phaffii.

Against the backdrop of green biomanufacturing, engineering methanol-utilizing Komagataella phaffii (K. phaffii) represents an effective strategy to expand the one carbon (C1) product profile and speed up the industrialization of C1-based bioeconomy. To address the technical challenges of low efficiency and cumbersome experimental procedures for multiplex gene editing and precise large-fragment integration during the reconstruction of complex metabolic pathways in K. phaffii, this study established a CRISPR toolkit - Efficient Multi-Gene Editing System 3.0 (EMGES 3.0) - which enabled one-step large-fragment integration coupled with multiplex gene knockout. EMGES 3.0 was constructed through the synergistic optimization of a repair-engineered chassis and an episomal CRISPR vector. For chassis engineering, five DNA repair modules: Δlig4 (DNA Ligase IV, non-homologous end joining end ligation), ppMRE11(The endogenous MRE11 gene from Pichia pastoris) overexpression (The Meiotic Recombination 11, DNA double-strand break end resection), Δrad9 (Radiation-Sensitive 9, DNA damage checkpoint regulation), Δmph1 (Mutator Phenotype Helicase 1, improvement of homologous recombinant strand extension), and PapRecT-PaSSB co-expression (stabilization of recombination intermediates) were integrated to generate the highly recombinogenic strain Y09. For vector engineering, cenARS was replaced by panARS and the endogenous promoter PGAP was employed to drive the double hammerhead ribozyme-single guide RNA-hepatitis delta virus ribozyme (double HH-sgRNA-HDV: dHgH)-mediated sgRNA expression, yielding the optimized vector Nov_pGAP_panARS_pLAT1_Cas9. These two features on K. phaffii together enhanced the EMGES 3.0 to a higher standard of transformation rate and editing efficiency. According to our results, EMGES 3.0 achieved dual-functional gene knockout efficiencies between 76.6% and 100%. For insertion of medium-long fragments (>4.5 kb), the efficiency achieved 93.3%. In addition, the one-step integration of ultra-long fragments (>16 kb) achieved 14.8%, which was reported for the first time. Furthermore, the efficiency of simultaneous long-fragment integration at three neutral loci reached 38.4% (>15 kb). We applied the system for one-step production of free fatty acids (FFAs, yield: 5.82 ∼ 7.30 mg/L/OD600) and resveratrol (yield: 1.14 ∼ 1.28 mg/L) using methanol as the sole carbon source. EMGES 3.0 provides a robust technical foundation for complex compounds biosynthesis and high-yield industrial strains, while also advancing K. phaffii as an industrial synthetic biology chassis for efficient C1 utilization.

CRISPR-Cas Systems

NADH fluorescence and [K+]o changes during hippocampal electrical stimulation.

1. Short (2 s) trains of stimuli were applied to the dorsal hippocampal surface of cats, producing an increase in [K+]o and a decrease in NADH fluorescence (the latter being indicative of an increase in tissue oxygen utilization). 2. The [K+]o rose rapidly during stimulation (delta[K+]o values from 1 to 6 mM) with larger stimulus currents producing larger changes. The time course of the poststimulus decline of [K+]o was an exponential decay function, with T 1/2 values varying from 1.3 to 6.9s, and independent of the magnitude of the delta[K+]o. Consistent undershoots of [K+]o occurred following stimuli causing less than 1 mM change in [K+]o. 3. The maximum depression of fluorescence and the time integral of the fluorescence changes following each stimulus train were both highly correlated with the total increase of [K+]o occurring during the stimulus train. 4. Application of several stimulus trains in close succession resulted in more rapid potassium reuptake following the later trains and an unusually large undershoot after the last train. Concomitantly, there was a progressive decrease in the fluorescence level. 5. When afterdischarges were induced by prolonged (less than 2 s) stimulation, larger and more sustained increases in [K+]o and decreases of fluorescence were observed, and there was some indication that afterdischarges were followed by accelerated reuptake of extracellular potassium.

Animals

[Effect of coagulation of the reticular formation in the region of the NRP and NRT nuclei on the spread of brain biopotential synchronization in rabbits].

The influence of coagulation of NRP and NRT nuclei on the level of spatial synchronization of bioelectrical activity in the visual and motor cortical areas was studied in chronic experiments on alert rabbits. It was shown that elimination of these structures from activity diminishes the correlation of processes in the studied zones. The degree of the coagulation effect depends on the volume of destruction and on the degree of synchroneity of the background neocortical biopotentials. Destruction of the nuclei is attended with dominance of delta-frequencies in the power spectra and a drop of coherence function in the theta range. It is assumed that the NRP and NRT nuclei are functional links of the integral system which controls the level of spatial synchronization of bioelectrical processes in the rabbit cerebral cortex.

Animals

Nesidioblastosis and congenital neuroblastoma: a histologic and immunocytochemical study of a new complex neurocristopathy.

Congenital neuroblastoma and nesidioblastosis occurred simultaneously in a neonate with severe hypoglycemia. This combination may represent a rare type of complex neurocristopathy. Immunocytochemical staining revealed a diffuse proliferation of alpha, beta, and delta cells as clusters and individual cells. The total islet volume was less than control values and beta cell counts were not increased. This case tends to support the hypothesis that morphologic integrity of the normal islet architecture is important in the control of function and not merely excessive numbers of one cell type.

Antibodies