Analysis of in vitro expression of virulence genes related to antibiotic and disinfectant resistance in Escherichia coli as an emerging periodontal pathogen.

The collective involvement of virulence markers of Escherichia coli as an emerging pathogen associated with periodontitis remains unexplained. This study aimed to implement an in vitro model of infection using a human epithelial cell line to determine the virulome expression related to the antibiotic and disinfectant resistance genotype and pulse field gel electrophoresis (PFGE) type in E. coli strains isolated from patients with periodontal diseases. We studied 100 strains of E. coli isolated from patients with gingivitis (n = 12), moderate periodontitis (n = 59), and chronic periodontitis (n = 29). The identification of E. coli and antibiotic and disinfectant resistance genes was performed through PCR. To promote the expression of virulence genes in the strains, an in vitro infection model was used in the human epithelial cell line A549. RNA was extracted using the QIAcube robotic equipment and reverse transcription to cDNA was performed using the QuantiTect reverse transcription kit (Qiagen). The determination of virulence gene expression was performed through real-time PCR. Overall, the most frequently expressed adhesion genes among the isolated strains of gingivitis, moderate periodontitis, and chronic periodontitis were fimH (48%), iha (37%), and papA (18%); those for toxins were usp (33%); those for iron acquisition were feoB (84%), fyuA (62%), irp-2 (61%), and iroN (35%); those for protectins were traT (50%), KpsMT (35%), and ompT (28%); and those for pathogenicity islands were malX (45%). The most common antibiotic and disinfectant resistance genes among gingivitis, moderate periodontitis, and chronic periodontitis strains were sul-2 (43%), blaSHV (47%), blaTEM (45%), tet(A) (41%), dfrA1 (32%), marR-marO (57%), and qacEA1 (79%). The findings revealed the existence of a wide distribution of virulome expression profiles related to the antibiotic and disinfectant resistance genotype and PFGE type in periodontal strains of E. coli. These findings may contribute toward improving the prevention and treatment measures for periodontal diseases associated with E. coli.

Periodontitis exacerbation during pregnancy in mice: Role of macrophage migration inhibitory factor as a key inductor.

OBJECTIVE: The present study was designed to investigate the role of macrophage migration inhibitory factor (MIF) in the exacerbation of pregestational periodontal disease (PGPD). BACKGROUND: Periodontitis (PT) is a severe stage of periodontal disease characterized by inflammation of the supporting tissues of the teeth, which usually worsens during pregnancy. MIF is a proinflammatory cytokine that is significantly elevated in periodontitis, both at the beginning and at the end of pregnancy. Although periodontitis usually presents with greater severity during pregnancy, the participation of MIF in the evolution of periodontitis has not been established. METHODS: To analyze the relevance of MIF in the exacerbation of PGPD, we employed a model of PGPD in WT and Mif-/- mice, both with a BALB/c genetic background. PT was induced with nylon suture ligatures placed supramarginally around the second upper right molar. For PGPD, PT was induced 2 weeks before mating. We evaluated histological changes and performed histometric analysis of the clinical attachment loss, relative expression of MMP-2 and MMP-13 by immunofluorescence, and relative expression of the cytokines mif, tnf-α, ifn-γ, and il-17 by quantitative real-time polymerase chain reaction (qRT-PCR). RESULTS: Our data revealed that periodontal tissue from PGPD WT mice produced a twofold increase in MIF compared with PT WT mice. Moreover, the evolution of periodontitis in Mif-/- mice was less severe than in PGDP WT mice. Periodontal tissue from Mif-/- mice with PGPD produced 80% less TNF-α and no IFN-γ, as well as 50% lower expression of matrix metalloproteinase (MMP)-2 and 25% less MMP-13 compared to WT PGDP mice. CONCLUSIONS: Our study suggests that MIF plays an important role in the exacerbation of periodontitis during pregnancy and that MIF is partially responsible for the inflammation associated with the severity of periodontitis during pregnancy.

Frequency and expression of genes involved in adhesion and biofilm formation in Staphylococcus aureus strains isolated from periodontal lesions.

BACKGROUND/PURPOSE: The aim of this study was to characterize the Staphylococcus aureus strains isolated from periodontal lesions of patients, to determine the expression of genes involved in cell adhesion upon their infection of human epithelial cells using an in vitro model, its biofilm formation, and its resistance to antibiotics. METHODS: S. aureus was analysed by PCR, Kirby-Bauer, and pulsed-field gel electrophoresis (PFGE), measuring gene expression by real-time PCR after infection of human cells in vitro. RESULTS: S. aureus was identified in 18.6% (50/268) of the samples. All strains (n = 50) possessed the virulence genes spa (Staphylococcal protein A), coa (coagulase), and icaAB (intercellular adhesin); 96% (n = 48) possessed clfB (clumping factor B), and 88% (n = 44) possessed ebps (elastin-binding protein) and sdrD (serine aspartate repeat protein D). All strains were resistant to methicillin, ampicillin, dicloxacillin, cefotaxime, and penicillin, and were multidrug resistant to 6-12 antibiotics. PFGE analysis showed 37 different pulsed-field types and most strains (60.4%) had a unique pulsed-field type. Twenty-four distinct combinations of virulence genes and antibiotic-resistant phenotypes were identified. CONCLUSION: Although S. aureus has been considered a transient member of the oral microbiota, our results indicate a high-level expression of virulence genes and multidrug resistance in the strains isolated from periodontal lesions. These strains might complicate the successful treatment of the disease.

Using 6 Degrees of Freedom to Systematically Reduce and Fix Distal Radius Fractures.

Six degrees of freedom (6DOF) refers to the freedom of movement of a rigid body in 3-dimensional space. Specifically, the object can move in 3 translations: up/down, left/right, and in/out, and in 3 rotations: pitch, yaw, and roll. In fracture care, the principle of 6DOF can be applied to each individual fracture fragment to help better understand fracture reduction and alignment. In the instance of a distal radius fracture, the 6DOF concept can be utilized to reduce the articular block in a systematic and controlled fashion. The articular block may be displaced in 3 translations-shortened (proximal/distal axis), dorsally translated (volar/dorsal axis), and radially translated (radial/ulnar axis). The articular block may also be displaced in 3 rotations-dorsally tilted (rotated about the radial/ulnar axis or in the sagittal plane), decreased radial inclination (rotated about the volar/dorsal axis or in the coronal plane), and supinated (rotated about the proximal/distal axis or in the axial plane). We present a surgical technique of open reduction and internal fixation of a distal radius fracture through a volar approach where we address the distal segment's instability in 6DOF in a stepwise format.

Role of deubiquitinases in DNA damage response.

DNA damage response (DDR) serves as an integrated cellular network to detect cellular stress and react by activating pathways responsible for halting cell cycle progression, stimulating DNA damage repair, and initiating apoptosis. Efficient DDR protects cells from genomic instability while defective DDR can allow DNA lesions to go unrepaired, causing permanent mutations that will affect future generations of cells and possibly cause disease conditions such as cancer. Therefore, DDR mechanisms must be tightly regulated in order to ensure organismal health and viability. One major way of DDR regulation is ubiquitination, which has been long known to control DDR protein localization, activity, and stability. The reversal of this process, deubiquitination, has more recently come to the forefront of DDR research as an important new angle in ubiquitin-mediated regulation of DDR. As such, deubiquitinases have emerged as key factors in DDR. Importantly, deubiquitinases are attractive small-molecule drug targets due to their well-defined catalytic residues that provide a promising avenue for developing new cancer therapeutics. This review focuses on the emerging roles of deubiquitinases in various DNA repair pathways.

Egg CD9 protein tides correlated with sperm oscillations tune the gamete fusion ability in mammal.

Mammalian fertilization involves membrane events-adhesion, fusion, sperm engulfment, membrane block to polyspermy-whose causes remain largely unknown. Recently, specific oscillations of the sperm in contact with the egg were shown to be necessary for fusion. Using a microfluidic chip to impose the venue for the encounter of two gametes allowed real-time observation of the membrane remodelling occurring at the sperm/egg interface. The spatiotemporal mapping of egg CD9 revealed that this protein concentrates at the egg/sperm interface as a result of sperm oscillations, until a CD9-rich platform is nucleated on which fusion immediately takes place. Within 2-5 min after fusion, most of the CD9 leaves the egg for the external aqueous medium. Then an egg membrane wave engulfs the sperm head in ~25 min. These results show that sperm oscillations initiate the CD9 recruitment that causes gamete fusion after which CD9 and associated proteins leave the membrane in a process likely to contribute to block polyspermy. They highlight that the gamete fusion story in mammals is an unexpected interplay between mechanical constraints and proteins.

Influence of shape and dispersion media of titanium dioxide nanostructures on microvessel network and ossification.

Titanium dioxide nanoparticles (TiO2 NPs) production has been used for pigment, food and cosmetic industry and more recently, shaped as belts for treatment of contaminated water, self-cleaning windows and biomedical applications. However, the toxicological data have demonstrated that TiO2 NPs inhalation induce inflammation in in vivo models and in vitro exposure leads to cytotoxicity and DNA damage. Dermal exposure has limited adverse effects and the possible risks for implants used for tissue regeneration is still under research. Then, it has been difficult to establish a straight statement about TiO2 NPs toxicity since route of exposure and shapes of nanoparticles play an important role in the effects. In this study we aimed to investigate the effect of three different types of TiO2 NPs (industrial, food-grade and belts) dispersed in fetal bovine serum (FBS) and saline solution (SS) on microvessel network, angiogenesis gene expression and femur ossification using a chick embryo model after an acute exposure of NPs on the day 7 after eggs fertilization. Microvascular density of chorioallantoic membrane (CAM) was analyzed after 7days of NPs injection and vehicles induced biological effects per se. NPs dispersed in FBS or SS have slight differences in microvascular density, mainly opposite effect on angiogenesis gene expression and no effects on femur ossification for NPs dispersed in SS. Interestingly, NPs shaped as belts dramatically prevented the alterations in ossification induced by FBS used as vehicle.

Comprehensive expression analysis of pathogenicity genes in uropathogenic Escherichia coli strains.

In this study, we investigated distinct expression patterns of genes encoding iron-acquisition systems, adhesins, protectins, and toxins in human uroepithelial cells infected with 194 uropathogenic Escherichia coli (UPEC) strains in vitro. We assessed the association of these genes with antibiotic resistance genes in this group of UPEC strains, previously characterised by polymerase chain reaction (PCR). Strains were isolated from patients with urinary tract infections (UTIs) from Unidad Médica Familiar de Salud Pública, located in Estado de México, México. Antibiotic resistance genes were identified by PCR, and the expression of virulence genes was detected by reverse-transcriptase-PCR after in vitro infection of cultured A431 human keratinocytes derived from a vulvar epidermoid carcinoma. The most frequently expressed virulence genotypes among the investigated UPEC strains included usp (68%), iha (64.9%), kpsMT (61.3%), fim (58.2%), irp2 (48.4), papC (33.5%), set (31.4%) and astA (30.9%), whereas the most frequently detected antibiotic resistance genes were tet(A) (34%), sul1 (31.4%) and TEM (26.3%). Furthermore, the most abundant pattern of gene expression (irp2/fim/iha/kpsMT/usp), associated with 8 different combinations of antibiotic resistance genotypes, was exhibited by 28 strains (14.4%). Taken together, these results indicate collective participation of distinct virulence UPEC genotypes during in vitro infection of cultured human epithelial cells, suggesting their potential involvement in UTI pathogenesis.

Virulence factors, antibiotic resistance phenotypes and O-serogroups of Escherichia coli strains isolated from community-acquired urinary tract infection patients in Mexico.

BACKGROUND/PURPOSE: Uropathogenic Escherichia coli (UPEC) strains isolated from patients with community-acquired urinary tract infections (UTIs) were assessed to determine the prevalence of virulence genes, antibiotic resistance, and the O-serogroup of the strains. METHODS: Consenting patients with community-acquired UTI were enrolled at Unidad Médica Familiar Number 64 (Instituto Mexicano del Seguro Social, Estado de Mexico, Mexico) and 321 urine samples were collected. Polymerase chain reaction (PCR) was used to assess 24 virulence genes and 14 O-serogroups. The Kirby-Bauer method was used to evaluate the antibiotic susceptibility of the isolated strains to 12 commonly used antibiotics. RESULTS: A total of 194 strains were identified as E. coli using standard biochemical tests, followed by PCR amplification of 16S ribosomal RNA gene. Only 58.2% of the strains belonged to the assessed 14 O-serogroups. The serogroups O25, O15, O8, and O75 were present in 20.6%, 17%, 6.1%, and 4.6% of strains, respectively. The most frequently occurring virulence genes among UPEC strains included kpsMT (92.2% strains), usp (87.1%), irp2 (79.3%), iha (64.9%), fim (61.3%), set (36%), astA (33.5%), pap (24.7%), and papGII (21.1%). In addition, 97% of the strains were multi-drug resistant (coresistance to 3-11 antibiotics). CONCLUSION: The isolated UPEC strains predominantly belonged to three serogroups (O25, O15, and O8), harboured numerous virulence genes, and are multiresistant to antibiotics. The findings of this study could be used to orient UTI treatment strategies and in epidemiological studies in Mexico.

Binding of sperm protein Izumo1 and its egg receptor Juno drives Cd9 accumulation in the intercellular contact area prior to fusion during mammalian fertilization.

Little is known about the molecular mechanisms that induce gamete fusion during mammalian fertilization. After initial contact, adhesion between gametes only leads to fusion in the presence of three membrane proteins that are necessary, but insufficient, for fusion: Izumo1 on sperm, its receptor Juno on egg and Cd9 on egg. What happens during this adhesion phase is a crucial issue. Here, we demonstrate that the intercellular adhesion that Izumo1 creates with Juno is conserved in mouse and human eggs. We show that, along with Izumo1, egg Cd9 concomitantly accumulates in the adhesion area. Without egg Cd9, the recruitment kinetics of Izumo1 are accelerated. Our results suggest that this process is conserved across species, as the adhesion partners, Izumo1 and its receptor, are interchangeable between mouse and human. Our findings suggest that Cd9 is a partner of Juno, and these discoveries allow us to propose a new model of the molecular mechanisms leading to gamete fusion, in which the adhesion-induced membrane organization assembles all key players of the fusion machinery.

CD9 tetraspanin generates fusion competent sites on the egg membrane for mammalian fertilization.

CD9 tetraspanin is the only egg membrane protein known to be essential for fertilization. To investigate its role, we have measured, on a unique acrosome reacted sperm brought in contact with an egg, the adhesion probability and strength with a sensitivity of a single molecule attachment. Probing the binding events at different locations of wild-type egg we described different modes of interaction. Here, we show that more gamete adhesion events occur on Cd9 null eggs but that the strongest interaction mode disappears. We propose that sperm-egg fusion is a direct consequence of CD9 controlled sperm-egg adhesion properties. CD9 generates adhesion sites responsible for the strongest of the observed gamete interaction. These strong adhesion sites impose, during the whole interaction lifetime, a tight proximity of the gamete membranes, which is a requirement for fusion to take place. The CD9-induced adhesion sites would be the actual location where fusion occurs.

Spontaneous and reversible switch from amphiphilic to oil-like structures.

Current theories assume that the amphiphilicity of biological membranes is always preserved. We observed that two hydrogen-bonding lipid layers in contact can spontaneously and reversibly lose their amphiphilic structure and turn into an assembly of oily complexes. This result opens a new angle for understanding the reorganization of lipids during membrane fusion, since similar complexes could fill the troubling hydrophobic voids displayed in the current models. The unique tribological properties described here may also find application in the development of novel nanolubricants.

The binding energy of two nitrilotriacetate groups sharing a nickel ion.

Among the various molecular interactions used to construct supramolecular self-assembling systems, homoliganded metallic NTA-Ni-NTA complexes have received little attention despite their considerable potential applications, such as the connection of different biochemical functions. The stability of this complex is investigated here by using two concordant nanotechniques (surface forces apparatus and vesicle micromanipulation) that allow direct measurements of adhesion energies due to the chelation of nickel ions by nitrilotriacetate (NTA) groups grafted on surfaces. We show that two NTA groups can share a nickel ion, and that the association of a Ni-NTA complex with an NTA group has a molecular binding energy of 1.4 kcal/mol. Binding measurements in bulk by isothermal titration calorimetry experiments give the same value and, furthermore, indicate that the Ni-NTA chelation bond is about five times stronger than the NTA-Ni-NTA one. This first direct proof and quantification of the simultaneous chelation of a nickel ion by two NTA groups sheds new light on association dynamics involving chelation processes and offers perspectives for the development of new supramolecular assemblies and anchoring strategies.

Johnson-Kendall-Roberts theory applied to living cells.

Johnson-Kendall-Roberts (JKR) theory is an accurate model for strong adhesion energies of soft slightly deformable material. Little is known about the validity of this theory on complex systems such as living cells. We have addressed this problem using a depletion controlled cell adhesion and measured the force necessary to separate the cells with a micropipette technique. We show that the cytoskeleton can provide the cells with a 3D structure that is sufficiently elastic and has a sufficiently low deformability for JKR theory to be valid. When the cytoskeleton is disrupted, JKR theory is no longer applicable.

Nanocapsules with functionalized surfaces and walls.

With the possibility of creating and handling nanometer-sized objects, it became popular to dream of future miniaturized tools allowing completely new applications in medicine and technology. Mother nature created during evolution many such self-assembled highly sophisticated and robust objects, and it is very tempting to modify them for other purposes: artificial cells as intelligent submarines floating in the bloodstream attacking cancer cells, single-molecule detection and manipulation allowing the bottom-up construction of devices, and so on. Nanobiotechnology became a hot topic and significant budgets have been attributed to it. However, caution has to be paid that unrealistic dreams might have a fairly negative effect on the taxpayer seeing unkept promises. In this paper, we explore the possibilities of using biological substances in material science.

Energy of hydrogen bonds probed by the adhesion of functionalized lipid layers.

It is now well admitted that hydrophobic interactions and hydrogen bonds are the main forces driving protein folding and stability. However, because of the complex structure of a protein, it is still difficult to separate the different energetic contributions and have a reliable estimate of the hydrogen bond part. This energy can be quantified on simpler systems such as surfaces bearing hydrogen-bonding groups. Using the surface force apparatus, we have directly measured the interaction energy between monolayers of lipids whose headgroups can establish hydrogen bonds in water: nitrilotriacetate, adenosine, thymidine, and methylated thymidine lipids. From the adhesion energy between the surfaces, we have deduced the energy of a single hydrogen bond in water. We found in each case an energy of 0.5 kcal/mol. This result is in good agreement with recent experimental and theoretical studies made on protein systems showing that intramolecular hydrogen bonds make a positive contribution to protein stabilization.