Massively parallel analysis of single-molecule dynamics on next-generation sequencing chips.

Single-molecule techniques are ideally poised to characterize complex dynamics but are typically limited to investigating a small number of different samples. However, a large sequence or chemical space often needs to be explored to derive a comprehensive understanding of complex biological processes. Here we describe multiplexed single-molecule characterization at the library scale (MUSCLE), a method that combines single-molecule fluorescence microscopy with next-generation sequencing to enable highly multiplexed observations of complex dynamics.

Molecular mechanism of plasmid elimination by the DdmDE defense system.

Seventh-pandemic strains contain two pathogenicity islands that encode the DNA defense modules DdmABC and DdmDE. In this study, we used cryogenic electron microscopy to determine the mechanistic basis for plasmid defense by DdmDE. The helicase-nuclease DdmD adopts an autoinhibited dimeric architecture.

An alpha-helical lid guides the target DNA toward catalysis in CRISPR-Cas12a.

CRISPR-Cas12a is a powerful RNA-guided genome-editing system that generates double-strand DNA breaks using its single RuvC nuclease domain by a sequential mechanism in which initial cleavage of the non-target strand is followed by target strand cleavage. How the spatially distant DNA target strand traverses toward the RuvC catalytic core is presently not understood. Here, continuous tens of microsecond-long molecular dynamics and free-energy simulations reveal that an α-helical lid, located within the RuvC domain, plays a pivotal role in the traversal of the DNA target strand by anchoring the crRNA:target strand duplex and guiding the target strand toward the RuvC core, as also corroborated by DNA cleavage experiments.

Continuous directed evolution of a compact CjCas9 variant with broad PAM compatibility.

CRISPR-Cas9 genome engineering is a powerful technology for correcting genetic diseases. However, the targeting range of Cas9 proteins is limited by their requirement for a protospacer adjacent motif (PAM), and in vivo delivery is challenging due to their large size. Here, we use phage-assisted continuous directed evolution to broaden the PAM compatibility of Campylobacter jejuni Cas9 (CjCas9), the smallest Cas9 ortholog characterized to date.

Structural basis for the assembly of the type V CRISPR-associated transposon complex.

CRISPR-Cas systems have been co-opted by Tn7-like transposable elements to direct RNA-guided transposition. Type V-K CRISPR-associated transposons rely on the concerted activities of the pseudonuclease Cas12k, the AAA+ ATPase TnsC, the Zn-finger protein TniQ, and the transposase TnsB. Here we present a cryo-electron microscopic structure of a target DNA-bound Cas12k-transposon recruitment complex comprised of RNA-guided Cas12k, TniQ, a polymeric TnsC filament and, unexpectedly, the ribosomal protein S15.

Mutations in DNA polymerase δ subunit 1 co-segregate with CMD2-type resistance to Cassava Mosaic Geminiviruses.

Cassava mosaic disease (CMD) suppresses cassava yields across the tropics. The dominant CMD2 locus confers resistance to cassava mosaic geminiviruses. It has been reported that CMD2-type landraces lose resistance after regeneration through de novo morphogenesis.

The haplotype-resolved chromosome pairs of a heterozygous diploid African cassava cultivar reveal novel pan-genome and allele-specific transcriptome features.

Background: Cassava (Manihot esculenta) is an important clonally propagated food crop in tropical and subtropical regions worldwide. Genetic gain by molecular breeding has been limited, partially because cassava is a highly heterozygous crop with a repetitive and difficult-to-assemble genome.

Findings: Here we demonstrate that Pacific Biosciences high-fidelity (HiFi) sequencing reads, in combination with the assembler hifiasm, produced genome assemblies at near complete haplotype resolution with higher continuity and accuracy compared to conventional long sequencing reads.

Target site selection and remodelling by type V CRISPR-transposon systems.

Canonical CRISPR-Cas systems provide adaptive immunity against mobile genetic elements. However, type I-F, I-B and V-K systems have been adopted by Tn7-like transposons to direct RNA-guided transposon insertion. Type V-K CRISPR-associated transposons rely on the pseudonuclease Cas12k, the transposase TnsB, the AAA+ ATPase TnsC and the zinc-finger protein TniQ, but the molecular mechanism of RNA-directed DNA transposition has remained elusive.

Role of P-170 glycoprotein in colchicine brain uptake.

To study the role of P-glycoprotein (P-gp) in the delivery of colchicine from blood to brain, the pharmacokinetics of colchicine in plasma and brain was studied in the rat by an in vivo method and by the in situ brain perfusion technique. Colchicine was administered intravenously at three doses (1, 2.5, and 5 mg/kg) with or without an inhibitor of P-gp, verapamil (0.

Specific modulation of two neuronal digitalis receptors by anaesthesia.

Three isoenzymes of digitalis receptors (alpha 1, alpha 2, alpha 3) in the brain and only one in the kidney (alpha 1) can be distinguished by their ouabain affinities and their responsiveness to sodium. Since we have reported modulations for these digitalis receptors by their fatty acid membrane environment, anaesthesics could bind on and modulate either directly these receptors or indirectly by disturbing membrane lipids. The aim of this study was to evaluate this anaesthetic action on apparent ouabain affinities and sodium dependence of cerebral and renal Na+, K(+)-ATPase isoenzymes activities.

Developmental effects of intrauterine growth retardation on cerebral amino acid transport.

Early restriction of nutrients during the perinatal period of life can modify the development of the mammalian fetus and have marked repercussions on the ontogeny of the CNS. The brain is vulnerable to undernutrition, with delayed morphologic and biochemical maturation leading to impaired functions. The aim of the present investigation was to assess whether modified brain neurotransmitter and amino acid concentrations found in an animal model of intrauterine growth retardation were related to modified blood-brain amino acid transport properties.

Effect of fish oil diet on fatty acid composition of phospholipids of brain membranes and on kinetic properties of Na+,K(+)-ATPase isoenzymes of weaned and adult rats.

The influence of dietary (n-3) fatty acids (such as eicosapentaenoic and docosahexaenoic acids) as found in fish oil on Na+ sensitivity and ouabain affinity of Na+,K(+)-ATPase isoenzymes (alpha 1, alpha 2, alpha 3) was studied in whole brain membranes from weaned and adult rats fed diets for two generations. The long chain (n-3) fatty acids supplied by fish oil decreased the fatty acids of the (n-6) series compared with the standard diet, resulting in a decrease in the (n-6)/(n-3) molar ratio in both 21- and 60-day-old rats. On the basis of ouabain titration, three inhibitory processes with markedly different affinities were associated with isoenzymes, i.

Cerebral amino acid changes in an animal model of intrauterine growth retardation.

As part of a series of experiments to ascertain the effects of prenatal malnutrition on brain development, we measured brain amino acids in an animal model of intrauterine growth retardation (IUGR) obtained by restriction of blood supply to the fetus in utero during the last 5 days of gestation. In the present study, amino acids were measured during development by HPLC as their O-phthaldialdehyde derivatives in cerebral cortex, cerebellum and hippocampus. In rats with IUGR, significant increase of alanine (by 20% to 50%) and taurine (by 20% to 80%) were observed prior to weaning in the cerebellum and the cerebral cortex respectively.

Effect of dietary alpha-linolenic acid on functional characteristic of Na+/K(+)-ATPase isoenzymes in whole brain membranes of weaned rats.

The influence of dietary fatty acids on Na+ sensitivity and ouabain affinity of Na+/K(+)-ATPase isoenzymes of whole brain membranes were studied in weaned rats fed for two generations with diets either devoid of alpha-linolenic acid (sunflower oil diet) or rich in alpha-linolenic acid (soya oil diet). The (n--3) deficiency induced by the sunflower oil diet led to an increase in the (n--6)/(n--3) molar ratio in whole brain membranes. Na+/K(+)-ATPase isoenzymes were discriminated on the basis of their differential affinities for ouabain.

Heterogeneous Na+ sensitivity of Na+,K(+)-ATPase isoenzymes in whole brain membranes.

The Na+ sensitivity of whole brain membrane Na+,K(+)-ATPase isoenzymes was studied using the differential inhibitory effect of ouabain (alpha 1, low affinity for ouabain; alpha 2, high affinity; and alpha 3, very high affinity). At 100 mM Na+, we found that the proportion of isoforms with low, high, and very high ouabain affinity was 21, 38, and 41%, respectively. Using two ouabain concentrations (10(-5) and 10(-7) M), we were able to discriminate Na+ sensitivity of Na+,K(+)-ATPase isoenzymes using nonlinear regression.

Temperature-dependent immunoreactive assay to screen for digoxin-like immunoreactive factor(s).

Endogenous circulating digoxin-like immunoreactive factors (DLIF) are known to cross-react with antibodies to digoxin and to inhibit Na+/K(+)-transporting ATPase (Na+K+ATPase; EC 3.6.1.

Existence of a digitalis-like compound in the human fetus.

Digoxin-like inhibitors of Na+,K(+)-ATPase have been implicated in several pathophysiological problems in the perinatal period. Aqueous endogenous digoxin-like immunoreactive substance (DLIS) was extracted from 9 different organs of a 24-week-old human fetus whose mother died after paraquat poisoning. The results indicate that this endogenous DLIS has a wide distribution in fetal tissues.

Development of the cholinergic system in control and intra-uterine growth retarded rat brain.

The activity of choline acetyltransferase (ChAT), acetylcholinesterase (AChE), and muscarinic receptors was studied in control rats and in rats growth-retarded in utero because of reduction of the blood supply 5 days before birth. The different markers of the cholinergic system were estimated at P (postnatal day) 6, 9, 12, 15, 22 and 60 in cerebellum, hypothalamus, septum, striatum and CA1, CA3 and fascia dentata of the hippocampus. In control rats, there was a transient increase in ChAT activity in the septum during the second week of postnatal development.

Effect of organic and inorganic mercuric salts on Na+K+ATPase in different cerebral fractions in control and intrauterine growth-retarded rats: alterations induced by serotonin.

An intrauterine growth-retarded (IUGR) model based on restriction of blood supply to the rat fetus at the 17th day of pregnancy was studied. We investigated in vitro the effects of thimerosal and mercuric chloride on Na+K+ATPase activity in total brain homogenate, synaptosomes and myelin at weaning. In addition, we evaluated the reversal effect of serotonin on mercury-inhibited Na+K+ATPase activity.

Na(+) K(+) ATPase activity in synaptosomes and myelin of developing control and intra-uterine growth retarded rats: effects of lead and serotonin.

An intrauterine growth retarded (IUGR) model based on restriction of blood supply to fetuses at 17 days of pregnancy in rats was studied. We investigated in vitro the effects of lead on Na(+)K(+) ATPase activity in synaptosomes and myelin of IUGR and control rats from 6 to 60 days after birth. In both groups an age-dependent effects existed in synaptosomes for the lowest doses of lead.

Effect of lead on Na+,K+ATPase activity in the developing brain of intra-uterine growth-retarded rats.

Lead (Pb) intoxication in developing mammals, including humans, produces serious brain damage. In addition, it is known that nutritional status influences the susceptibility to Pb toxicity. We developed an in utero undernutrition model based on restriction of blood supply to fetuses on d 17 of pregnancy (IUGR rats).

Effect of intrauterine growth retardation on developmental changes in DNA and [14C]thymidine metabolism in different regions of rat brain: histological and biochemical correlations.

Intrauterine growth retardation was induced in the rat by clamping the uterine artery on day 17 of gestation. The effect of hypotrophy on DNA synthesis was studied in two different cerebral structures: hippocampus and cerebellum. Accumulation of DNA in these structures was biochemically measured in parallel to the incorporation of methyl-[14C]thymidine into nucleic acid at different ages and correlated with autoradiography.