Monitoring of gene knockouts: genome-wide profiling of conditionally essential genes

We have developed a new microarray-based genetic technique, named MGK (Monitoring of Gene Knockouts), for genome-wide identification of conditionally essential genes. MGK identified bacterial genes that are critical for fitness in the absence of aromatic amino acids, and was further applied to identify genes whose inactivation causes bacterial cell death upon exposure to the bacteriostatic antibiotic chloramphenicol. Our findings suggest that MGK can serve as a robust tool in functional genomics studies.     

Efficient gene inactivation in Bacillus anthracis

A procedure for high-efficiency gene inactivation in Bacillus anthracis has been developed. It is based on a highly temperature-sensitive plasmid vector carrying kanamycin resistance cassette surrounded by DNA fragments flanking the desired insertion site. The approach was tested by constructing glutamate racemase E1 (racE1), glutamate racemase E2 (racE2) and comEC knock-out mutants of B. anthracis strain DeltaANR. Allelic replacements were observed at high frequencies, ranging from approximately 0.5% for racE2 up to 50% for racE1 and comEC. The system can be used for genetic validation of potential drug targets.     

Study of the cytotoxicity of protein X amyloid fibrils

Amyloid oligomers, protofibrils, and fibrils of various amyloidogenic proteins are known to induce cell death. Tetracycline prevents the formation of fibrils of Aβ peptide and other amyloidogenic proteins and decomposes mature fibrils. It was previously shown that sarcomeric cytoskeletal proteins of the titin family (protein X, protein C, and protein H) in vitro form amyloid fibrils and tetracycline decomposes them. In this work, the concentration and time dependence of the survival of polymorphonuclear leukocytes in the presence of protein X amyloid fibrils is demonstrated. It is also shown that the survival rate increases as fibrils are decomposed by tetracycline. The antibiotic itself is found to be nontoxic. The results obtained show that this approach can be used to evaluate the efficiency of drugs that prevent or rectify amyloidoses.     

Characteristic properties of fluctuating particle fluxes near the last closed flux surface in the course of lower hybrid heating and during a transition into the improved confinement mode in the FT-2 tokamak

Results are presented from experimental studies of the time behavior of the transport processes in the edge plasma of the FT-2 tokamak during auxiliary lower hybrid heating when an internal transport barrier and then an external transport barrier form in the plasma. An analysis of the data on turbulent particle transport in the edge plasma shows that the radial electric field generated inside the plasma column during auxiliary heating plays an important role in both the formation of a transport barrier and the suppression of anomalous transport at the plasma periphery in the postheating phase of the discharge. The mechanism for the formation of a negative radial electric field E _r near the last closed flux surface after the end of the lower hybrid heating pulse is considered. Fluctuation spectra of the particle density and poloidal electric field are presented that characterize the process of suppression of microturbulence at the plasma periphery. The experimental data were obtained with the use of movable multielectrode Langmuir probes.     

Effect of the liposomal form of taxifolin complexes with metals of variable valence on skin regeneration after chemical burn

The effect of the liposomal form formed by taxifolin and metals of variable valency was investigated. It was shown that the application of preparations based on the free flavonoid and its complexes with Fe(II/III) and Cu(II) ions after chemical burns results in a more effective skin regeneration and the repair of hair follicles and sebaceous glands. A tendency for a more effective wound healing after the applications of taxifolin-Cu(II) and taxifolin-Fe(III) liposomal complexes versus control was observed. It was assumed that the mechanism of action of these preparations is based on the oxidative polymerization and conjugation of the flavonoid, which results in the utilization of toxic metabolites and lipid peroxidation products.     

Effect of the radial electric field on lower hybrid plasma heating in the FT-2 tokamak

Conditions for efficient ion heating in the interaction of lower hybrid waves with plasma are experimentally determined. Experiments show that efficient lower hybrid heating stimulates a transition to the improved confinement mode. The formation of internal and external transport barriers is associated with strong central ion heating, which results in a change of the radial electric field E _r and an increase in the shear of the poloidal plasma velocity. The improved confinement mode in the central region of the discharge is attained under the combined action of lower hybrid heating and an additional rapid increase in the plasma current. A new mechanism for the generation of an additional field E _r is proposed to explain the formation of a transport barrier.     

Activation of hypothalamic RIP‐Cre neurons promotes beiging of WAT via sympathetic nervous system

Activation of brown adipose tissue (BAT) and beige fat by cold increases energy expenditure. Although their activation is known to be differentially regulated in part by hypothalamus, the underlying neural pathways and populations remain poorly characterized. Here, we show that activation of rat‐insulin‐promoter‐Cre (RIP‐Cre) neurons in ventromedial hypothalamus (VMH) preferentially promotes recruitment of beige fat via a selective control of sympathetic nervous system (SNS) outflow to subcutaneous white adipose tissue (sWAT), but has no effect on BAT. Genetic ablation of APPL2 in RIP‐Cre neurons diminishes beiging in sWAT without affecting BAT, leading to cold intolerance and obesity in mice. Such defects are reversed by activation of RIP‐Cre neurons, inactivation of VMH AMPK, or treatment with a β3‐adrenergic receptor agonist. Hypothalamic APPL2 enhances neuronal activation in VMH RIP‐Cre neurons and raphe pallidus, thereby eliciting SNS outflow to sWAT and subsequent beiging. These data suggest that beige fat can be selectively activated by VMH RIP‐Cre neurons, in which the APPL2–AMPK signaling axis is crucial for this defending mechanism to cold and obesity.     

Transition into the improved core confinement mode as a possible mechanism for additional electron heating observed in the lower hybrid current drive experiments at the FT-2 tokamak

In experiments on lower hybrid current drive (LHCD) carried out at the FT-2 tokamak, a substantial increase in the central electron temperature T _e (r = 0 cm) from 550 to 700 eV was observed. A complex simulation procedure is used to explain a fairly high LHCD efficiency and the observed additional heating, which can be attributed to a transition into the improved core confinement (ICC) mode. For numerical simulations, data obtained in experiments with deuterium plasma at 〈n _e 〉 = 1.6 × 10¹⁹ m^–3 were used. Simulations by the GRILL3D, FRTC, and ASTRA codes have shown that the increase in the density and central temperature is apparently caused by a significant suppression of heat transport in the electron component. The mechanism for transition into the improved confinement mode at r < 3 cm can be associated with the broadening of the plasma current channel due to the lower hybrid drive of the current carried by superthermal and runaway electrons. In this case, the magnetic shear s = (r/q)(dq/dr) in the axial region of the plasma column almost vanishes during the RF pulse. In this study, the effect of lower hybrid waves on the plasma parameters, resulting in a transition into the ICC mode, is considered. New experimental and calculated data are presented that evidence in favor of such a transition. Special attention is paid to the existence of a threshold for the transition into the ICC mode in deuterium plasma.