Plant DNA topoisomerase I is recognized and inhibited by human Scl-70 sera autoantibodies

Type I topoisomerases (EC 5.99.1.2) are those enzymes capable of relaxing negatively supercoiled DNA without the need for ATP. The central role played by these enzymes in cell function suggests that the structure of type I topoisomerases may be highly conserved in eukaryotic cells. However, the extent of the conservation among eukaryotes is unknown. Human DNA topoisomerase I is an autoimmune antigen (Scl-70) of scleroderma patients. We have found that the autoimmune antibodies in human Scl-70 sera recognize protein from various plants, and these proteins display DNA relaxation function. In addition, Scl-70 antibodies were able to inhibit enzymatic activity of plant topoisomerase I. Therefore, the immunological cross-reactivity of the plant topoisomerase with human antibodies demonstrates that, despite divergence of eukaryotic organisms, these plant and animal enzymes retain structurally similar enzymatic features.     

Repeated H2O2 exposure drives cell cycle progression in an in vitro model of ulcerative colitis

The production of hydrogen peroxide (H₂O₂) drives tumourigenesis in ulcerative colitis (UC). Recently, we showed that H₂O₂ activates DNA damage checkpoints in human colonic epithelial cells (HCEC) through c‐Jun N‐terminal Kinases (JNK) that induces p21^WAF1. Moreover, caspases circumvented the G1/S and intra‐S checkpoints, and cells accumulated in G2/M. The latter observation raised the question of whether repeated H₂O₂ exposures alter JNK activation, thereby promoting a direct passage of cells from G2/M arrest to driven cell cycle progression. Here, we report that increased proliferation of repeatedly H₂O₂‐exposed HCEC cells (C‐cell cultures) was associated with (i) increased phospho‐p46 JNK, (ii) decreased total JNK and phospho‐p54 JNK and (iii) p21^WAF1 down‐regulation. Altered JNK activation and p21^WAF1 down‐regulation were accompanied by defects in maintaining G2/M and mitotic spindle checkpoints through adaptation, as well as by apoptosis resistance following H₂O₂ exposure. This may cause increased proliferation of C‐cell cultures, a defining initiating feature in the inflammation‐carcinoma pathway in UC. We further suggest that dysregulated JNK activation is attributed to a non‐apoptotic function of caspases, causing checkpoint adaptation in C‐cell cultures. Additionally, loss of cell‐contact inhibition and the overcoming of senescence, hallmarks of cancer, contributed to increased proliferation. Furthermore, there was evidence that p54 JNK inactivation is responsible for loss of cell‐contact inhibition. We present a cellular model of UC and suggest a sinusoidal pattern of proliferation, which is triggered by H₂O₂‐induced reactive oxygen species generation, involving an interplay between JNK activation/inactivation, p21^WAF1, c‐Fos, c‐Jun/phospho‐c‐Jun, ATF2/phospho‐ATF2, β‐catenin/TCF4‐signalling, c‐Myc, CDK6 and Cyclin D2, leading to driven cell cycle progression.     

Potential antifouling strategies for marine finfish aquaculture: the effects of physical and chemical treatments on the settlement and survival of the hydroid Ectopleura larynx

The hydroid Ectopleura larynx is a common fouling organism on aquaculture nets. To contribute to the development of novel cleaning methods, laboratory and field studies determined the effects of heat (30, 40, 50 and 60°C for immersion times of 1 and 3 s) and acetic acid (0.2 and 2.0% for immersion times of 1, 3 and 10 s, 1 and 5 min) on the settlement of actinulae and the survival of juvenile and adult E. larynx. Laboratory studies showed that, regardless of immersion time, a temperature of 50°C was effective in preventing the settlement of actinulae and the survival of juveniles, while ≤12% of adult hydroids could survive. A temperature of 60°C killed all adult hydroids. For an acetic acid concentration of 0.2%, an immersion time of 1 min substantially reduced the settlement of actinulae and the survival of juvenile and adult hydroids, and none of the juvenile and adult hydroids survived after 5 min. For an acetic acid concentration of 2.0%, all immersion times were effective and reduced the mean settlement of actinulae and the survival of juvenile and adult hydroids to ≤10%. Field studies with fouled net panels exposed to selected heat or acetic acid treatments showed small reductions in mean wet weight and net aperture occlusion of the net panels 2 and 5 days after treatment. Visual inspections of the net panels showed that hydranths of the hydroids were shed, but the dead stolons of the hydroids remained on the treated net panels. Novel cleaning methods and devices may utilise these results to effectively kill E. larynx on aquaculture nets, while further studies are needed to determine the necessity of removing the dead hydroids before further biofouling accumulates on thenets.     

Surface microtopographies of tropical sea stars: lack of an efficient physical defence mechanism against fouling

Abstract

The role of surface topography as a defence against fouling in tropical sea stars was investigated. The sea stars Linckia laevigata, Fromia indica, Cryptasterina pentagona and Archaster typicus are not fouled and have paxillae (modified ossicles with a median vertical pillar) on their aboral surfaces, which varied in diameter, height and distance depending on species and position on the aboral surface, providing unique and complex surface microtopographies for each species. The surfaces of the sea stars L. laevigata, F. indica and A. typicus were moderately wettable, with their mean seawater contact angles, calculated from captive bubble measurements, being 60.1°, 70.3° and 57.3°, respectively. The seawater contact angle of C. pentagona could not be measured. To evaluate the effectiveness of the surface microtopographies in deterring the settlement of fouling organisms, field experiments with resin replicas of the four sea star species were conducted at three sites around Townsville, Australia, for 8 weeks during the dry and wet seasons. The fouling community and total fouling cover did not differ significantly between replicas of L. laevigata, F. indica, C. pentagona, A. typicus and control surfaces at any site during the dry season. Significant differences between fouling communities on the replicas of the sea stars and control surfaces were detected at two sites during the wet season. However, these differences were transitory, and the total fouling cover did not differ significantly between replicas of sea stars and control surfaces at two of the three sites. In contrast to recent literature on the effects of biofouling control by natural surfaces in the marine environment, the surface microtopographies of tropical sea stars alone were not effective in deterring the settlement and growth of fouling organisms.     

A Lunar Clock Changes Shielding Pigment Transparency in Larval Ocelli of Clunio marinus

Living in the tidal zones of the sea requires synchronization with the dominant environmental influences of tidal, solar, and lunar periodicity. Endogenous clocks anticipate those geoclimatic changes and control the respective rhythms of vital functions. But the underlying mechanisms are only partly understood. While the circadian clocks in animals are investigated employing neurobiological, molecular, and genetic approaches, clocks with a lunar periodicity have been studied with reference to development and behavior only. Sites of their pacemakers, zeitgeber receptors, and coupled endocrine components are unknown. Here, a lunar‐rhythmic change of shielding pigment transparency in the larval ocelli of the intertidal midge Clunio marinus is demonstrated for the first time as a possible access to the neurobiology of lunar timing mechanisms. We studied third instar larvae (Vigo strain) throughout the lunar cycle by light‐ and electron-microscopy as well as by x‐ray fluorescence analysis for the identification of the pigment. Moonlight detection is a prerequisite for photic synchronization of the lunar clock. The larval ocelli of Clunio putatively may function as moonlight receptors and are also controlled by the circalunar clock itself, hence being primary candidates for tracing input and output pathways of the lunar pacemaker. Additionally, the demonstration of a reversible optical change of shielding pigment transparency in Clunio is a novel finding, not reported so far in any other animal species, and reveals a mechanism to enhance photosensitivity under the condition of very dim light. It represents a remarkable change of a sense organ from an imaging device to a radiometer. Its restriction to the developmental stage susceptible to lunar timing elucidates a unique sensory strategy evolved at the level of sensory input. It also raises basic questions about the biochemistry of optically active pigments, like melanin, and their intracellular control.     

Effect of increasing GnRH and PGF2α dose during Double-Ovsynch on ovulatory response,luteal regression,and fertility of lactating dairy cows

Ovsynch-type synchronization of ovulation protocols have suboptimal synchronization rates due to reduced ovulation to the first GnRH treatment and inadequate luteolysis to the prostaglandin F_2α (PGF_2α) treatment before timed artificial insemination (TAI). Our objective was to determine whether increasing the dose of the first GnRH or the PGF_2α treatment during the Breeding-Ovsynch portion of Double-Ovsynch could improve the rates of ovulation and luteolysis and therefore increase pregnancies per artificial insemination (P/AI). In experiment 1, cows were randomly assigned to a two-by-two factorial design to receive either a low (L) or high (H) doses of GnRH (Gonadorelin; 100 vs. 200 μg) and a PGF_2α analogue (cloprostenol; 500 vs. 750 μg) resulting in the following treatments: LL (n = 263), HL (n = 277), LH (n = 270), and HH (n = 274). Transrectal ultrasonography and serum progesterone (P4) were used to assess ovulation to GnRH1, GnRH2, and luteal regression after PGF_2α during Breeding-Ovsynch in a subgroup of cows (n = 651 at each evaluation). Pregnancy status was assessed 29, 39, and 74 days after TAI. In experiment 2, cows were randomly assigned to LL (n = 220) or HH (n = 226) treatment as described for experiment 1. For experiment 1, ovulation to GnRH1 was greater (P = 0.01) for cows receiving H versus L GnRH (66.6% [217/326] vs. 57.5% [187/325]) treatment, but only for cows with elevated P4 at GnRH1. Cows that ovulated to GnRH1 had increased (P < 0.001) fertility compared with cows that did not ovulate (52.2% vs. 38.5%); however, no effect of higher dose of GnRH on fertility was detected. The greater PGF_2α dose increased luteal regression primarily in multiparous cows (P = 0.03) and tended to increase fertility (P = 0.05) only at the pregnancy diagnosis 39 days after TAI. Overall, P/AI was 47.0% at 29 days and 39.7% at 74 days after TAI; P/AI did not differ (P = 0.10) among treatments at 74 days (LL, 34.6%; HL, 40.8%; LH, 42.2%; HH, 40.9%) and was greater (P < 0.001) for primiparous cows than for multiparous cows (46.1% vs. 33.8%). For experiment 2, P/AI did not differ (P = 0.21) between H versus L treatments (44.2% [100/226] vs. 40.5% [89/220]). Thus, despite an increase in ovulatory response to GnRH1 and luteal regression to PGF_2α, there were only marginal effects of increasing dose of GnRH or PGF_2α on fertility to TAI after Double-Ovsynch.     

Feeding the Probiotic Enterococcus faecium Strain NCIMB 10415 to Piglets Specifically Reduces the Number of Escherichia coli Pathotypes That Adhere to the Gut Mucosa

Feed supplementation with the probiotic Enterococcus faecium for piglets has been found to reduce pathogenic gut microorganisms. Since Escherichia coli is among the most important pathogens in pig production, we performed comprehensive analyses to gain further insight into the influence of E. faecium NCIMB 10415 on porcine intestinal E. coli. A total of 1,436 E. coli strains were isolated from three intestinal habitats (mucosa, digesta, and feces) of probiotic-supplemented and nonsupplemented (control) piglets. E. coli bacteria were characterized via pulsed-field gel electrophoresis (PFGE) for clonal analysis. The high diversity of E. coli was reflected by 168 clones. Multilocus sequence typing (MLST) was used to determine the phylogenetic backgrounds, revealing 79 sequence types (STs). Pathotypes of E. coli were further defined using multiplex PCR for virulence-associated genes. While these analyses discerned only a few significant differences in the E. coli population between the feeding groups, analyses distinguishing clones that were uniquely isolated in either the probiotic group only, the control group only, or both groups (shared group) revealed clear effects at the habitat level. Interestingly, extraintestinal pathogenic E. coli (ExPEC)-typical clones adhering to the mucosa were significantly reduced in the probiotic group. Our data show a minor influence of E. faecium on the overall population of E. coli in healthy piglets. In contrast, this probiotic has a profound effect on mucosa-adherent E. coli. This finding further substantiates a specific effect of E. faecium strain NCIMB 10415 in piglets against pathogenic E. coli in the intestine. In addition, these data question the relevance of data based on sampling fecal E. coli only.     

In vitro resistance mechanisms of Neisseria meningitidis against neutrophil extracellular traps

Neisseria meningitidis (Nm) is a leading cause of septicemia in childhood. Nm septicemia is unique with respect to very quick disease progression, high in vivo bacterial replication rate and its considerable mortality. Nm circumvents major mechanisms of innate immunity such as complement system and phagocytosis. Neutrophil extracellular traps (NETs) are formed from neutrophils during systemic infection and are suggested to contain invading microorganisms. Here, we investigated the interaction of Nm with NETs. Both, meningococci and spontaneously released outer membrane vesicles (SOMVs) were potent NET inducers. NETs were unable to kill NET bound meningococci, but slowed down their proliferation rate. Using Nm as model organism we identified three novel mechanisms how bacteria can evade NET‐mediated killing: (i) modification of lipid A of meningococcal LPS with phosphoethanolamine protected Nm from NET‐bound cathepsin G; (ii) expression of the high‐affinity zinc uptake receptor ZnuD allowed Nm to escape NET‐mediated nutritional immunity; (iii) binding of SOMVs to NETs saved Nm from NET binding and the consequent bacteriostatic effect. Escape from NETs may contribute to the most rapid progression of meningococcal disease. The induction of NET formation by Nm in vivo might aggravate thrombosis in vessels ultimately directing to disseminated intravascular coagulation (DIC).