Replication forks of Escherichia coli are not the preferred sites for lysogenic integration of bacteriophage Mu.

The question of whether bacteriophage Mu prefers replication forks for lysogenic integration into Escherichia coli chromosomes was tested by using two different systems. In the first, inactivation of genes was scored in synchronized cultures infected by Mu at various times. No increase in the mutation frequency of a gene was found after infection at the time of its replication. In the second, the composition of colonies formed by bacteria lysogenized by Mu was determined; the newly formed lysogens should give rise to mixed colonies (containing lysogenized as well as nonlysogenized bacteria), uniform colonies, or both, depending on the mode of integration. Both types of colonies were found, and the fraction of uniform colonies was proportional to the relative length of the unreplicated segment of an average chromosome in the culture. The results in both systems clearly preclude the possibility that a lysogenizing Mu integrates with high preference at the chromosome replication forks.     

The effect of a moving foveal target on the subjective sensation of motion

In this paper, we report on two experiments concerning the effect of the visual field of fovea on the subjective estimation of angular velocity. Experiment 1 investigates the effect of a slow moving target on the perception of self motion. The result of this experiment can be summarized as follows: a slow moving target seen in the visual field of fovea by a stationary person generates in this person a sensation of self rotation in the same direction as the motion of the target. This phenomenon will be called foveal induced ego motion. Experiment 2 investigates the latency for the detection of a self angular acceleration when the person focusses his fovea on a slowly moving target. From the results of this experiment we conclude that the latency for detection of a small self angular acceleration is shorter if the person sees a small foveal target moving with respect to the person in the direction of self rotation than if that small foveal target is moving (with respect to the person) in the opposite direction. The results of these experiments help us in refining existing models of visual-vestibular interaction, by providing a model which accounts for the phenomenon of oculogyral illusion.This research was conducted while serving as a Visiting Professor at the Man Vehicle Laboratory, Department of Aeronautics and Astronautics, Massachusetts Institute of Technology, Cambridge, MA 02139, USA     

Characterization of PfiT/PfiA toxin–antitoxin system of Pseudomonas aeruginosa that affects cell elongation and prophage induction

Toxin–antitoxin (TA) systems are small genetic modules usually consisting of two elements—a toxin and an antitoxin. The abundance of TA systems among various bacterial strains may indicate an important evolutionary role. Pseudomonas aeruginosa, which can be found in a variety of niches in nature, is an opportunistic pathogen for various hosts. While P. aeruginosa strains are very versatile and diverse, only a few TA systems were characterized in this species. Here, we describe a newly characterized TA system in P. aeruginosa that is encoded within the filamentous Pf4 prophage. This system, named PfiT/PfiA, is a homologue of the ParE/YefM TA system. It is a type II TA system, in which the antitoxin is a protein that binds the toxic protein and eliminates the toxic effect. PfiT/PfiA carries several typical type II characteristics. Specifically, it constitutes two small genes expressed in a single operon, PfiT inhibits growth and PfiA eliminates this effect, PfiA binds PfiT, and PfiT expression results in elongated cells. Finally, we assigned a novel function to this TA system, where an imbalance between PfiT and PfiA, favouring the toxin, resulted in cell elongation and an increase in virion production.     

Identification of chironomid species as natural reservoirs of toxigenic Vibrio cholerae strains with pandemic potential

Vibrio cholerae causes the fatal cholera diarrhea. Chironomids (Diptera; Chironomidae) are abundant in freshwater aquatic habitats and estuaries and are natural reservoirs of V. cholerae. Until now, only the non-O1/O139 serogroups of V. cholerae were identified in chironomids. Here, we explored whether chironomids are natural reservoirs of V. cholerae O1/O139 serogroups, which are associated with cholera endemics and pandemics. All four life stages of chironomids were sampled from two rivers, and a laboratory culture in Pune, India, and from a pond in Israel. In total, we analyzed 223 chironomid samples. The presence of V. cholerae O1/O139 serogroups was verified using molecular tools. Nine chironomid species were identified; of them, Chironomus circumdatus was the most abundant. The presence of V. cholerae serogroup O1 and the cholera toxin genes were detected in samples from all chironomid species. However, serogroup O139 was detected in only two chironomid species. Besides PCR to detect specific genes, a metagenomic analysis that was performed in three selected C. ramosus larvae, identified a list of virulence genes associated with V. cholerae. The findings provide evidence that chironomids are natural reservoirs of toxigenic V. cholerae O1/O139. Chironomid populations and V. cholerae show biannual peak patterns. A similar pattern is found for cholera epidemics in the Bengal Delta region. Thus, we hypothesize that monitoring chironomids in endemic areas of the disease may provide a novel tool for predicting and preventing cholera epidemics. Moreover, serogroup O139 was detected only in two chironomid species that have a restricted distribution in the Indian subcontinent, possibly explaining why the distribution of the O139 serogroup is limited.     

ALFY-Controlled DVL3 Autophagy Regulates Wnt Signaling,Determining Human Brain Size

Primary microcephaly is a congenital neurodevelopmental disorder of reduced head circumference and brain volume, with fewer neurons in the cortex of the developing brain due to premature transition between symmetrical and asymmetrical cellular division of the neuronal stem cell layer during neurogenesis. We now show through linkage analysis and whole exome sequencing, that a dominant mutation in ALFY, encoding an autophagy scaffold protein, causes human primary microcephaly. We demonstrate the dominant effect of the mutation in drosophila: transgenic flies harboring the human mutant allele display small brain volume, recapitulating the disease phenotype. Moreover, eye-specific expression of human mutant ALFY causes rough eye phenotype. In molecular terms, we demonstrate that normally ALFY attenuates the canonical Wnt signaling pathway via autophagy-dependent removal specifically of aggregates of DVL3 and not of Dvl1 or Dvl2. Thus, autophagic attenuation of Wnt signaling through removal of Dvl3 aggregates by ALFY acts in determining human brain size.     

Methanogens rapidly transition from methane production to iron reduction

Methanogenesis, the microbial methane (CH₄) production, is traditionally thought to anchor the mineralization of organic matter as the ultimate respiratory process in deep sediments, despite the presence of oxidized mineral phases, such as iron oxides. This process is carried out by archaea that have also been shown to be capable of reducing iron in high levels of electron donors such as hydrogen. The current pure culture study demonstrates that methanogenic archaea (Methanosarcina barkeri) rapidly switch from methanogenesis to iron‐oxide reduction close to natural conditions, with nitrogen atmosphere, even when faced with substrate limitations. Intensive, biotic iron reduction was observed following the addition of poorly crystalline ferrihydrite and complex organic matter and was accompanied by inhibition of methane production. The reaction rate of this process was of the first order and was dependent only on the initial iron concentrations. Ferrous iron production did not accelerate significantly with the addition of 9,10‐anthraquinone‐2,6‐disulfonate (AQDS) but increased by 11–28% with the addition of phenazine‐1‐carboxylate (PCA), suggesting the possible role of methanophenazines in the electron transport. The coupling between ferrous iron and methane production has important global implications. The rapid transition from methanogenesis to reduction of iron–oxides close to the natural conditions in sediments may help to explain the globally‐distributed phenomena of increasing ferrous concentrations below the traditional iron reduction zone in the deep ‘methanogenic’ sediment horizon, with implications for metabolic networking in these subsurface ecosystems and in past geological settings.     

Ca transients from Ca channel activity in rat cardiac myocytes reveal dynamics of dyad cleft and troponin C Ca binding

The properties of the dyad cleft can in principle significantly impact excitation-contraction coupling, but these properties are not easily amenable to experimental investigation. We simultaneously measured the time course of the rise in integrated Ca current (I_Ca) and the rise in concentration of fura 2 with Ca bound ([Ca-fura 2]) with high time resolution in rat myocytes for conditions under which Ca entry is only via L-type Ca channels and sarcoplasmic reticulum (SR) Ca release is blocked, and compared these measurements with predictions from a finite-element model of cellular Ca diffusion. We found that 1) the time course of the rise of [Ca-fura 2] follows the time course of integrated I_Ca plus a brief delay (1.36 ± 0.43 ms, n = 6 cells); 2) from the model, high-affinity Ca binding sites in the dyad cleft at the level previously envisioned would result in a much greater delay (=" BORDER="0">3 ms) and are therefore unlikely to be present at that level; 3) including ATP in the model promoted Ca efflux from the dyad cleft by a factor of 1.57 when low-affinity cleft Ca binding sites were present; 4) the data could only be fit to the model if myofibrillar troponin C (TnC) Ca binding were low affinity (4.56 µM), like that of soluble troponin C, instead of the high-affinity value usually used (0.38 µM). In a "good model," the rate constants for Ca binding and dissociation were 0.375 times the values for soluble TnC; and 5) consequently, intracellular Ca buffering at the rise of the Ca transient is inferred to be low. excitation-contraction coupling; adenosine triphosphate; fura 2; modeling; fuzzy space     

DCX, a new mediator of the JNK pathway

Mutations in the X-linked gene DCX result in lissencephaly in males, and abnormal neuronal positioning in females, suggesting a role for this gene product during neuronal migration. In spite of several known protein interactions, the involvement of DCX in a signaling pathway is still elusive. Here we demonstrate that DCX is a substrate of JNK and interacts with both c-Jun N-terminal kinase (JNK) and JNK interacting protein (JIP). The localization of this signaling module in the developing brain suggests its functionality in migrating neurons. The localization of DCX at neurite tips is determined by its interaction with JIP and by the interaction of the latter with kinesin. DCX is phosphorylated by JNK in growth cones. DCX mutated in sites phosphorylated by JNK affected neurite outgrowth, and the velocity and relative pause time of migrating neurons. We hypothesize that during neuronal migration, there is a need to regulate molecular motors that are working in the cell in opposite directions: kinesin (a plus-end directed molecular motor) versus dynein (a minus-end directed molecular motor).     

Small angle X-ray scattering of resistant starch type III

Starch fraction, which is resistant to enzymatic digestion, is produced during retrogradation. This fraction, termed resistant starch type III (RSIII), has health benefits such as pre-biotic effects, improving lipid and cholesterol metabolism, and reducing the risk of colon cancer. The work presented in this paper is aimed at investigating the colloidal structure of native high amylose corn starch (HACS) and the RSIII polymorphs produced from it using small angle X-ray scattering. Experimental scattering curves were fitted with appropriate theoretical models such as the "modified lamellar model". Our results show that retrogradation at low temperature leads to formation of polymorph B with crystallinity much lower than that of the granular form, but the lamellas are arranged in long-range periodicity. Conversely, retrogradation at high temperature leads to formation of polymorphs A and V with no defined periodicity. The degree of crystallinity is very low, and the system is better described as a dilute particulate system.     

Antifibrotic action of Cu/Zn SOD is mediated by TGF-beta1 repression and phenotypic reversion of myofibroblasts

Skin fibrosis is characterized by the proliferation and accumulation of activated fibroblasts called myofibroblasts. They exhibit specific cytoskeletal differentiation, overexpress the fibrogenic cytokine TGF-beta1, synthesize excess extracellular matrix compounds and exhibit a depleted antioxidant metabolism. Recently, SOD was successfully used as an antifibrotic agent in vivo, thus challenging the postulate of established fibrosis irreversibility. We postulated that myofibroblasts could be a direct target for this therapeutic effect. To test this hypothesis, we used three-dimensional co-culture models of skin, in which specific phenotypes of normal fibroblasts versus myofibroblasts are retained. These 3-D models were treated with liposomal and carrier-free Cu/Zn SOD, and examined for their effects on cell number, cell death, and phenotypic differentiation. The results show that SOD did not induce myofibroblast cell death, whereas it significantly reduced TGF-beta1 expression, thus demonstrating that SOD might be proposed as a potent antagonist of this major fibrogenic growth factor. We also found that SOD significantly lowered the levels of the myofibroblast marker alpha-sm actin, of beta-actin, and of the extracellular matrix components alpha1(I) collagen and tenascin-C. In conclusion, our results suggest that SOD antifibrotic action occurred in vitro through the reversion of myofibroblasts into normal fibroblasts.