Functional RelBE-Family Toxin-Antitoxin Pairs Affect Biofilm Maturation and Intestine Colonization in Vibrio cholerae

Toxin–antitoxin (TA) systems are small genetic elements that typically encode a stable toxin and its labile antitoxin. These cognate pairs are abundant in prokaryotes and have been shown to regulate various cellular functions. Vibrio cholerae, a human pathogen that is the causative agent of cholera, harbors at least thirteen TA loci. While functional HigBA, ParDE have been shown to stabilize plasmids and Phd/Doc to mediate cell death in V. cholerae, the function of seven RelBE-family TA systems is not understood. In this study we investigated the function of the RelBE TA systems in V. cholerae physiology and found that six of the seven relBE loci encoded functional toxins in E. coli. Deletion analyses of each relBE locus indicate that RelBE systems are involved in biofilm formation and reactive oxygen species (ROS) resistance. Interestingly, all seven relBE loci are induced under the standard virulence induction conditions and two of the relBE mutants displayed a colonization defect, which was not due to an effect on virulence gene expression. Although further studies are needed to characterize the mechanism of action, our study reveals that RelBE systems are important for V. cholerae physiology.     

Methylation of KvDMR1 involved in regulating the imprinting of CDKN1C gene in cattle

The CDKN1C gene encodes a cyclin‐dependent kinase inhibitor and is one of the key genes involved in the development of Beckwith–Wiedemann syndrome and cancer. In this study, using a direct sequencing approach based on a single nucleotide polymorphism (SNP) at genomic DNA and cDNA levels, we show that CDKN1C exhibits monoallelic expression in all seven studied organs (heart, liver, spleen, lung, kidney, muscle and subcutaneous fat) in cattle. To investigate how methylation regulates imprinting of CDKN1C in cattle, allele‐specific methylation patterns in two putative differential methylation regions (DMRs), the CDKN1C DMR and KvDMR1, were analyzed in three tissues (liver, spleen and lung) using bisulfite sequencing PCR. Our results show that in the CDKN1C DMR both parental alleles were unmethylated in all three analyzed tissues. In contrast, KvDMR1 was differentially methylated between the two parental alleles in the same tissues. Statistical analysis showed that there is a significant difference in the methylation level between the two parental alleles (P < 0.01), confirming that this region is the DMR of KvDMR1 and that it may be correlated with CDKN1C imprinting.     

Plasminogen Deficiency Causes Reduced Corticospinal Axonal Plasticity and Functional Recovery after Stroke in Mice

Tissue plasminogen activator (tPA) has been implicated in neurite outgrowth and neurological recovery post stroke. tPA converts the zymogen plasminogen (Plg) into plasmin. In this study, using plasminogen knockout (Plg^-/-) mice and their Plg-native littermates (Plg^+/+), we investigated the role of Plg in axonal remodeling and neurological recovery after stroke. Plg^+/+ and Plg^-/- mice (n = 10/group) were subjected to permanent intraluminal monofilament middle cerebral artery occlusion (MCAo). A foot-fault test and a single pellet reaching test were performed prior to and on day 3 after stroke, and weekly thereafter to monitor functional deficit and recovery. Biotinylated dextran amine (BDA) was injected into the left motor cortex to anterogradely label the corticospinal tract (CST). Animals were euthanized 4 weeks after stroke. Neurite outgrowth was also measured in primary cultured cortical neurons harvested from Plg^+/+ and Plg^-/- embryos. In Plg^+/+ mice, the motor functional deficiency after stroke progressively recovered with time. In contrast, recovery in Plg^-/- mice was significantly impaired compared to Plg^+/+ mice (p<0.01). BDA-positive axonal density of the CST originating from the contralesional cortex in the denervated side of the cervical gray matter was significantly reduced in Plg^-/- mice compared with Plg^+/+ mice (p<0.05). The behavioral outcome was highly correlated with the midline-crossing CST axonal density (R²>0.82, p<0.01). Plg^-/- neurons exhibited significantly reduced neurite outgrowth. Our data suggest that plasminogen-dependent proteolysis has a beneficial effect during neurological recovery after stroke, at least in part, by promoting axonal remodeling in the denervated spinal cord.     

A New Glabrous Gene (csgl3) Identified in Trichome Development in Cucumber (Cucumis sativus L.)

Spines or trichomes on the fruit of cucumbers enhance their commercial value in China. In addition, glabrous mutants exhibit resistance to aphids and therefore their use by growers can reduce pesticide residues. Previous studies have reported two glabrous mutant plants containing the genes, csgl1 and csgl2. In the present study, a new glabrous mutant, NCG157, was identified showing a gene interaction effect with csgl1 and csgl2. This mutant showed the glabrous character on stems, leaves, tendrils, receptacles and ovaries, and there were no spines or tumors on the fruit surface. Inheritance analysis showed that a single recessive gene, named csgl3, determined the glabrous trait. An F₂ population derived from the cross of two inbred lines 9930 (a fresh market type from Northern China that exhibits trichomes) and NCG157 (an American processing type with glabrous surfaces) was used for genetic mapping of the csgl3 gene. By combining bulked segregant analysis (BAS) with molecular markers, 18 markers, including two simple sequence repeats (SSR), nine insertion deletions (InDel) and seven derived cleaved amplified polymorphism sequences (dCAPs), were identified to link to the csgl3 gene. All of the linked markers were used as anchor loci to locate the csgl3 gene on cucumber chromosome 6. The csgl3 gene was mapped between the dCAPs markers dCAPs-21 and dCAPs-19, at genetic distances of 0.05 cM and 0.15 cM, respectively. The physical distance of this region was 19.6 kb. Three markers, InDel-19, dCAPs-2 and dCAPs-11, co-segregated with csgl3. There were two candidate genes in the region, Csa6M514860 and Csa6M514870. Quantitative real-time PCR showed that the expression of Csa6M514870 was higher in the tissues of 9930 than that of NCG157, and this was consistent with their phenotypic characters. Csa6M514870 is therefore postulated to be the candidate gene for the development of trichomes in cucumber. This study will facilitate marker-assisted selection (MAS) of the smooth plant trait in cucumber breeding and provide for future cloning of csgl3.     

Potentiation of harringtonine cytotoxicity by calcium antagonist diltiazem and biscoclaurine alkaloid cepharanthine in adriamycin-resistant P388 murine leukemia and K562 human leukemia cells

Harringtonine showed cross resistance in adriamycin-resistant murine leukemia P388 (P388/ADM) and human leukemia K562 (K562/ADM) cells. The relative resistance of the P388/ADM and K562/ADM cells to harringtonine was about 7 and 40, respectively. Calcium influx blockers, diltiazem and the biscoclaurine alkaloid cepharanthine enhanced the cytotoxicity of harringtonine in P388/ADM and K562/ADM cells. The extent of enhancement was different for the two drugs, and up to a 9- to 10-fold increase in harringtonine cytotoxicity occurred in P388/ADM cells, and 14- to 22-fold enhancement in K562/ADM cells with diltiazem or cepharanthine. Harringtonine resistance of P388/ADM was circumvented completely, and the resistance of K562/ADM was circumvented partially, by diltiazem or cepharanthine. The mechanism of enhanced cytotoxicity by diltiazem and cepharanthine is probably inhibition of active efflux of harringtonine in P388/ADM and K562/ADM cells.     

Bioenergetics of growth of a cyprinid, Phoxinus phoxinus: the effect of ration, temperature and body size on food consumption, faecal production and nitrogenous excretion

Food consumption, faecal production and nitrogen excretion by minnows, Phoxinus phoxinus , weighing 1–5.5 g were studied at five rations ranging from starvation to ad libitum and four temperatures ranging from 5 to 15°C.
The maximum rate of food consumption (C_max) was related to body weight ( W ) and temperature ( T ) by the relationship: C _max= aW^b1T^b2 . There were significant daily variations in C_max, which tended to decline over time. Absorption efficiency increased with increasing ration size and decreasing temperature. Body weight had no significant effect on the faecal production. The equation F = a C^b1e^b2 T described the relationship between faecal production ( F ), food consumption ( C ) and temperature. Ammonia-N predominated over urea-N in the excreta of most experimental fish. The proportion of urea-N in the total nitrogen excreted was generally higher at lower rations than at higher rations. Rates of nitrogen excretion increased with increased ration size and were, to a lesser extent, influenced by temperature. Body weight had no significant effect on the nitrogen excretion by feeding minnows. The equation N = a+b_lT+b₂C described the effects of food consumption and temperature on nitrogen excretion ( N ) other than urea-N excretion. The relationship between urea-N excretion ( N_u ), food consumption and temperature was described by the equation N_u= ae^b1T ((C+1) ^b 2.
On the average, 11 % of food energy was lost in faecal production and nitrogen excretion by minnows feeding on whiteworms, Enchytraeus spp.