Naturally Occurring Rock Type Influences the Settlement of Fucus spiralis L. zygotes

The settlement of spores and larvae on hard substrates has been shown to be influenced by many factors, but few studies have evaluated how underlying bedrock may influence recruitment. The characteristics of coastal rock types such as color, heat capacity, mineral size, and free energy have all been implicated in settlement success. We examined the influence of naturally occurring rock types on the initial attachment of zygotes of the brown alga Fucus spiralis Linnaeus 1753. We also assessed the dislodgment of zygotes on four bedrock types after initial attachment in laboratory experiments using wave tanks. Settling plates were prepared from limestone, basalt, schist, and granite, found in the region of Orrs Island, Maine, USA. The plate surfaces tested were either naturally rough or smooth-cut surfaces. We measured the density of attached zygotes after 1.5 h of settlement and subsequently after a wave treatment, in both winter and summer. The pattern of initial attachment was the same on natural and smooth surfaces regardless of season: highest on limestone (range 7.0–13.4 zygotes/cm2), intermediate on schist (1.8–8.5 zygotes/cm2) and basalt (3.5–14.0 zygotes/cm2), and lowest on granite (0.8–7.8 zygotes/cm2). Patterns of survivorship following the wave treatment were similar to those of initial settlement with the mean survivorship varying from 60.1% (SE = 3.8) (limestone, smooth substrate) to 31.8% (SE = 0.59) (granite, natural substrate), and with the highest mean survival on limestone, basalt, and schist, and the lowest on granite. Our results suggest that rock type has a significant effect on zygote attachment and persistence. Patterns of attachment were the same on smooth and rough surfaces, indicating that surface roughness is not the predominant factor controlling the difference in successful attachment among rock types. Other properties of bedrock, possibly grain size, surface free energy, or chemical interaction with the adhesives used by the zygotes, directly affect the attachment of these algal propagules. These results suggest that patterns of benthic community structure could be determined in part by the distribution of bedrock types.

Plasmodium falciparum PhIL1-associated complex plays an essential role in merozoite reorientation and invasion of host erythrocytes

The human malaria parasite, Plasmodium falciparum possesses unique gliding machinery referred to as the glideosome that powers its entry into the insect and vertebrate hosts. Several parasite proteins including Photosensitized INA-labelled protein 1 (PhIL1) have been shown to associate with glideosome machinery. Here we describe a novel PhIL1 associated protein complex that co-exists with the glideosome motor complex in the inner membrane complex of the merozoite. Using an experimental genetics approach, we characterized the role(s) of three proteins associated with PhIL1: a glideosome associated protein- PfGAPM2, an IMC structural protein- PfALV5, and an uncharacterized protein—referred here as PfPhIP (PhIL1 Interacting Protein). Parasites lacking PfPhIP or PfGAPM2 were unable to invade host RBCs. Additionally, the downregulation of PfPhIP resulted in significant defects in merozoite segmentation. Furthermore, the PfPhIP and PfGAPM2 depleted parasites showed abrogation of reorientation/gliding. However, initial attachment with host RBCs was not affected in these parasites. Together, the data presented here show that proteins of the PhIL1-associated complex play an important role in the orientation of P. falciparum merozoites following initial attachment, which is crucial for the formation of a tight junction and hence invasion of host erythrocytes.

Impact of Implant Surface Material and Microscale Roughness on the Initial Attachment and Proliferation of Primary Human Gingival Fibroblasts

Due to the rising demand for zirconia (Zr) based implant systems, it is important to understand the impact of Zr and titanium (Ti) implants and particularly their topography on soft tissue healing. As human gingival fibroblasts (hGFs) are the predominant cells in peri-implant soft tissue, we focused on examining the effect of implant material and surface roughness on hGFs’ initial attachment, growth and the expression of proteins involved in the focal adhesion. hGFs isolated from eight healthy donors were cultured on the following surfaces: smooth titanium machined surface (TiM), smooth zirconia machined surface (ZrM), moderately rough titanium surface (SLA), or moderately rough zirconia surface (ZLA) for up to 14 days. The initial attachment of hGFs was evaluated by scanning electron microscopy. Cell proliferation/viability was assessed by cell counting kit 8. Focal adhesion and cytoskeleton were visualized by a focal adhesion staining kit. The gene expression of focal adhesion kinase (FAK), α-smooth muscle actin (α-SMA), and integrin subunits ITG-β1, ITG-β4, ITG-α4, ITG-α5, ITG-α6, was evaluated by qPCR. Cell proliferation/viability was slightly decreased by moderately rough surfaces, whereas no effect of surface material was observed. Cell morphology was strikingly different between differently treated surfaces: on machined surfaces, cells had elongated morphology and were attached along the grooves, whereas on moderately rough surfaces, cells were randomly attached. Surface roughness had a more pronounced effect on the gene expression compared to the surface material. The expression of FAK, α-SMA, ITG-β4, ITG-α5, and ITG-α6 was enhanced by moderately rough surfaces compared to smooth surfaces. Within the limitations of this in vitro study, it can be concluded that the behavior of primary hGFs is primarily affected by surface structure, whereas no apparent advantage of Zr over Ti could be observed.

RNAi-Mediated Silencing of Pgants Shows Core 1 O-Glycans Are Required for Pupation in Tribolium castaneum

Protein glycosylation is one of the most common and most important post-translational modifications. Despite the growing knowledge on N-glycosylation, the research on O-glycosylation is lagging behind. This study investigates the importance of O-glycosylation in the post-embryonic development of insects using the red flour beetle, Tribolium castaneum, as a model. We identified 28 O-glycosylation-related genes (OGRGs) in the genome of the red flour beetle. 14 OGRGs were selected for functional analysis based on their involvement in the initial attachment of the carbohydrate in the different O-glycosylation pathways or the further elongation of the most abundant O-glycans and, in addition, showing severe RNAi-induced phenotypes in Drosophila melanogaster. The expression profile of these OGRGs was mapped throughout the developmental stages of the insect and in the different tissues of the pupa and adult. Subsequently, these genes were silenced using RNA interference (RNAi) to analyze their role in development. A broad spectrum of phenotypes was observed: from subtle effects and disrupted wing formation when silencing the genes involved in O-mannosylation, to blockage of pupation and high mortality after silencing of the genes involved in O-GalNAc and core 1 O-glycan (O-GalNAc-Gal) synthesis. RNAi experiments were also performed to assess the effects of blocking multiple pathways of O-glycosylation. However, the observed phenotypes induced by multiple RNAi were similar to those of the single gene RNAi experiments. The silencing of OGRGs often resulted in high mortality and wing phenotypes, indicating the importance of O-glycosylation for the survival of the insect and the formation of wings during the post-embryonic development of T. castaneum.

Plasmodium falciparum PhIL1 associated complex play an essential role in merozoite reorientation and invasion of host erythrocytes

The human malaria parasite, Plasmodium falciparum possess a unique gliding machinery referred as glideosome that powers its entry into the insect and vertebrate hosts. A number of parasite proteins including Photosensitized INA-labelled protein 1 (PhIL1) have been shown to associate with glideosome machinery. Here we describe a novel PhIL1 associated protein complex that co-exists with glideosome motor complex in the inner membrane complex of the merozoite. Furthermore, using experimental genetics approach we characterized the role(s) of three proteins associated with PhIL1: a glideosome associated protein- PfGAPM2, an IMC structural protein- PfALV5 and a previously uncharacterised protein - referred here as PfPhIP (PhIL1 Interacting Protein). Parasites lacking PfPhIP or PfGAPM2 were unable to invade the host RBCs. Additionally, the down regulation of PfPhIP resulted in significant defects in merozoite segmentation. Furthermore, the PfPhIP and PfGAPM2 depleted parasites revealed abrogation of reorientation/gliding, however initial attachment with host RBCs was not affected in these parasites. Together, the data presented here shows that proteins of the PhIL1 associated complex plays an important role in orientation of P. falciparum merozoites following initial attachment, which is crucial for formation of tight junction and hence invasion of host erythrocytes. The identification and characterization of PhIL1 associated complex opens new avenues for future anti-malarial drug development.

Dynamic swimming pattern of Pseudomonas aeruginosa near a vertical wall during initial attachment stages of biofilm formation

Studying the swimming behaviour of bacteria in 3 dimensions (3D) allows us to understand critical biological processes, such as biofilm formation. It is still unclear how near wall swimming behaviour may regulate the initial attachment and biofilm formation. It is challenging to address this as visualizing the movement of bacteria with reasonable spatial and temporal resolution in a high-throughput manner is technically difficult. Here, we compared the near wall (vertical) swimming behaviour of P. aeruginosa (PAO1) and its mutants ΔdipA (reduced in swarming motility and increased in biofilm formation) and ΔfimX (deficient in twitching motility and reduced in biofilm formation) using our new imaging technique based on light sheet microscopy. We found that P. aeruginosa (PAO1) increases its speed and changes its swimming angle drastically when it gets closer to a wall. In contrast, ΔdipA mutant moves toward the wall with steady speed without changing of swimming angle. The near wall behavior of ΔdipA allows it to be more effective to interact with the wall or wall-attached cells, thus leading to more adhesion events and a larger biofilm volume during initial attachment when compared with PAO1. Furthermore, we found that ΔfimX has a similar near wall swimming behavior as PAO1. However, it has a higher dispersal frequency and smaller biofilm formation when compared with PAO1 which can be explained by its poor twitching motility. Together, we propose that near wall swimming behavior of P. aeruginosa plays an important role in the regulation of initial attachment and biofilm formation.

Short chain fatty acids induced the type 1 and type 2 fimbrillin-dependent and fimbrillin-independent initial attachment and colonization of Actinomyces oris monoculture but not coculture with streptococci

Background Actinomyces oris is an early colonizer and has two types of fimbriae on its cell surface, type 1 fimbriae (FimP and FimQ) and type 2 fimbriae (FimA and FimB), which contribute to the attachment and coaggregation with other bacteria and the formation of biofilm on the tooth surface, respectively. Short-chain fatty acids (SCFAs) are metabolic products of oral bacteria including A. oris and regulate pH in dental plaques. To clarify the relationship between SCFAs and fimbrillins, effects of SCFAs on the initial attachment and colonization (INAC) assay using A. oris wild type and fimbriae mutants was investigated. INAC assays using A. oris MG1 strain cells were performed with SCFAs (acetic, butyric, propionic, valeric and lactic acids) or a mixture of them on human saliva-coated 6-well plates incubated in TSB with 0.25% sucrose for 1 h. The INAC was assessed by staining live and dead cells that were visualized with a confocal microscope. Results Among the SCFAs, acetic, butyric and propionic acids and a mixture of acetic, butyric and propionic acids induced the type 1 and type 2 fimbriae-dependent and independent INAC by live A. oris, but these cells did not interact with streptococci. The main effects might be dependent on the levels of the non-ionized acid forms of the SCFAs in acidic stress conditions. GroEL was also found to be a contributor to the FimA-independent INAC by live A. oris cells stimulated with non-ionized acid. Conclusion SCFAs affect the INAC-associated activities of the A. oris fimbrillins and non-fimbrillins during ionized and non-ionized acid formations in the form of co-culturing with other bacteria in the dental plaque but not impact the interaction of A. oris with streptococci.

Unravelling a PhIL1-glideosome associated complex in Plasmodium falciparum merozoite that is essential for invasion in host erythrocytes

The human malaria parasite, Plasmodium falciparum possess a unique mechanism of gliding motility guided by glideosome that powers its entry into insect and vertebrate hosts to facilitate its invasion and internalization within the targeted host cell. Photosensitized INA-labelled protein 1 (PhIL1) forms a novel protein complex that is associated with glideosome motor complex in the inner membrane complex of invasive merozoite. To establish the role of PfPhIL1 associated novel complex at asexual blood stages, we characterized three proteins associated with PhIL1: a glideosome associated protein- PfGAPM2, an IMC structural protein- PfALV5 and a previously uncharacterised protein - referred here as PfPhIP (PhIL1 interacting protein). GFP targeting and co-immunoprecipitation analysis confirmed that these proteins are part of a PhIL1 associated novel complex, which co-exists with the glideosomal complex. To know the functional significance of PhIL1 associated complex, transgenic parasites were generated for glmS mediated conditional knock-down of each of the three proteins. Parasites lacking PfPhIP or PfGAPM2 were unable to invade the RBCs. PfPhIP deficient parasites also showed defects in merozoite segmentation. PfPhIP and PfGAPM2 depleted parasites revealed abrogation of reorientation/gliding, although initial attachment with human RBCs was not affected in these knock-down parasites. Together, the data presented here shows that proteins of the PhIL1 associated complex play an important role in orientation of P. falciparum merozoites post initial attachment, which is crucial for formation of tight junction and hence invasion of host erythrocytes.