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Analytical studies of nanoparticles (NPs) are frequently based on huge datasets derived from hyperspectral images acquired using scanning transmission electron microscopy. These large datasets require machine learning computational tools to reduce dimensionality and extract relevant information. Principal component analysis (PCA) is a commonly used procedure to reconstruct information and generate a denoised dataset; however, several open questions remain regarding the accuracy and precision of reconstructions. Here, we use experiments and simulations to test the effect of PCA processing on data obtained from AuAg alloy NPs a few nanometers wide with different compositions. This study aims to address the reliability of chemical quantification after PCA processing. Our results show that the PCA treatment mitigates the contribution of Poisson noise and leads to better quantification, indicating that denoised results may be reliable from the point of view of both uncertainty and accuracy for properly planned experiments. However, the initial data need to be of sufficient quality: these results can only be obtained if the signal-to-noise ratio of input data exceeds a minimal value to avoid the occurrence of random noise bias in the PCA reconstructions.
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Microchannels can be used to simulate xylem vessels and investigate phytopathogen colonization under controlled conditions. In this work, we explore surface functionalization strategies for polydimethylsiloxane and glass microchannels to study microenvironment colonization by Xylella fastidiosa subsp. pauca cells. We closely monitored cell initial adhesion, growth, and motility inside microfluidic channels as a function of chemical environments that mimic those found in xylem vessels. Carboxymethylcellulose (CMC), a synthetic cellulose, and an adhesin that is overexpressed during early stages of X. fastidiosa biofilm formation, XadA1 protein, were immobilized on the device's internal surfaces. This latter protocol increased bacterial density as compared with CMC. We quantitatively evaluated the different X. fastidiosa attachment affinities to each type of microchannel surface using a mathematical model and experimental observations acquired under constant flow of culture medium. We thus estimate that bacterial cells present â¼4 and 82% better adhesion rates in CMC- and XadA1-functionalized channels, respectively. Furthermore, variable flow experiments show that bacterial adhesion forces against shear stresses approximately doubled in value for the XadA1-functionalized microchannel as compared with the polydimethylsiloxane and glass pristine channels. These results show the viability of functionalized microchannels to mimic xylem vessels and corroborate the important role of chemical environments, and particularly XadA1 adhesin, for early stages of X. fastidiosa biofilm formation, as well as adhesivity modulation along the pathogen life cycle.
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Biofilmes , Xylella , Aderência Bacteriana , Adesão Celular , XilemaRESUMO
Here we show a new nanowire growth procedure, exploring the thermally activated motion of Au droplets on III-V surfaces. We show that by setting a single growth parameter we can activate the crawling motion of Au droplets in vacuum and locally modify surface composition in order to enhance vapor-solid (VS) growth along oxide-free areas on the trail of the metal particle. Asymmetric VS growth rates are comparable in magnitude to the vapor-liquid-solid growth, producing unconventional wurtzite GaP morphologies, which shows negligible defect density as well as optical signal in the green spectral region. Finally, we demonstrate that this effect can also be explored in different substrate compositions and orientations with the final shape finely tuned by group III flow and nanoparticle size. This distinct morphology for wurtzite GaP nanomaterials can be interesting for the design of nanophotonics devices.
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Electrically active field-effect transistors (FET) based biosensors are of paramount importance in life science applications, as they offer direct, fast, and highly sensitive label-free detection capabilities of several biomolecules of specific interest. In this work, we report a detailed investigation on surface functionalization and covalent immobilization of biomarkers using biocompatible ethanolamine and poly(ethylene glycol) derivate coatings, as compared to the conventional approaches using silica monoliths, in order to substantially increase both the sensitivity and molecular selectivity of nanowire-based FET biosensor platforms. Quantitative fluorescence, atomic and Kelvin probe force microscopy allowed detailed investigation of the homogeneity and density of immobilized biomarkers on different biofunctionalized surfaces. Significantly enhanced binding specificity, biomarker density, and target biomolecule capture efficiency were thus achieved for DNA as well as for proteins from pathogens. This optimized functionalization methodology was applied to InP nanowires that due to their low surface recombination rates were used as new active transducers for biosensors. The developed devices provide ultrahigh label-free detection sensitivities â¼1 fM for specific DNA sequences, measured via the net change in device electrical resistance. Similar levels of ultrasensitive detection of â¼6 fM were achieved for a Chagas Disease protein marker (IBMP8-1). The developed InP nanowire biosensor provides thus a qualified tool for detection of the chronic infection stage of this disease, leading to improved diagnosis and control of spread. These methodological developments are expected to substantially enhance the chemical robustness, diagnostic reliability, detection sensitivity, and biomarker selectivity for current and future biosensing devices.
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Antígenos de Protozoários/análise , Técnicas Biossensoriais/instrumentação , Doença de Chagas/diagnóstico , Nanofios/química , Trypanosoma cruzi/isolamento & purificação , Anticorpos Imobilizados/química , Antígenos de Protozoários/genética , Biomarcadores/análise , Técnicas Biossensoriais/métodos , Doença de Chagas/parasitologia , DNA/análise , DNA/genética , Desenho de Equipamento , Humanos , Índio/química , Modelos Moleculares , Fosfinas/química , Propriedades de Superfície , Transistores Eletrônicos , Trypanosoma cruzi/genéticaRESUMO
Surface attachment of a planktonic bacteria, mediated by adhesins and extracellular polymeric substances (EPS), is a crucial step for biofilm formation. Some pathogens can modulate cell adhesiveness, impacting host colonization and virulence. A framework able to quantify cell-surface interaction forces and their dependence on chemical surface composition may unveil adhesiveness control mechanisms as new targets for intervention and disease control. Here we employed InP nanowire arrays to dissect factors involved in the early stage biofilm formation of the phytopathogen Xylella fastidiosa. Ex vivo experiments demonstrate single-cell adhesion forces up to 45 nN, depending on the cell orientation with respect to the surface. Larger adhesion forces occur at the cell poles; secreted EPS layers and filaments provide additional mechanical support. Significant adhesion force enhancements were observed for single cells anchoring a biofilm and particularly on XadA1 adhesin-coated surfaces, evidencing molecular mechanisms developed by bacterial pathogens to create a stronger holdfast to specific host tissues.
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Adesinas Bacterianas/química , Aderência Bacteriana , Biofilmes , Nanofios , Xylella/fisiologiaRESUMO
The intriguing roles of the bacterial Tol-Pal trans-envelope protein complex range from maintenance of cell envelope integrity to potential participation in the process of cell division. In this study, we report the characterization of the XfTolB and XfPal proteins of the Tol-Pal complex of Xylella fastidiosa. X. fastidiosa is a major plant pathogen that forms biofilms inside xylem vessels, triggering the development of diseases in important cultivable plants around the word. Based on functional complementation experiments in Escherichia coli tolB and pal mutant strains, we confirmed the role of xftolB and xfpal in outer membrane integrity. In addition, we observed a dynamic and coordinated protein expression profile during the X. fastidiosa biofilm development process. Using small-angle X-ray scattering (SAXS), the low-resolution structure of the isolated XfTolB-XfPal complex in solution was solved for the first time. Finally, the localization of the XfTolB and XfPal polar ends was visualized via immunofluorescence labeling in vivo during bacterial cell growth. Our results highlight the major role of the components of the cell envelope, particularly the TolB-Pal complex, during the different phases of bacterial biofilm development.
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Proteínas da Membrana Bacteriana Externa/química , Biofilmes/crescimento & desenvolvimento , Proteínas de Escherichia coli/química , Regulação Bacteriana da Expressão Gênica , Lipoproteínas/química , Peptidoglicano/química , Proteínas Periplásmicas/química , Xylella/genética , Proteínas da Membrana Bacteriana Externa/genética , Proteínas da Membrana Bacteriana Externa/metabolismo , Membrana Celular/genética , Membrana Celular/metabolismo , Membrana Celular/ultraestrutura , Parede Celular/genética , Parede Celular/metabolismo , Parede Celular/ultraestrutura , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Teste de Complementação Genética , Lipoproteínas/genética , Lipoproteínas/metabolismo , Modelos Moleculares , Peptidoglicano/genética , Peptidoglicano/metabolismo , Proteínas Periplásmicas/genética , Proteínas Periplásmicas/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Espalhamento a Baixo Ângulo , Homologia de Sequência de Aminoácidos , Difração de Raios X , Xylella/metabolismo , Xylella/ultraestruturaRESUMO
The association of scanning transmission electron microscopy (STEM) and detection of a diffraction pattern at each probe position (so-called 4D-STEM) represents one of the most promising approaches to analyze structural properties of materials with nanometric resolution and low irradiation levels. This is widely used for texture analysis of materials using automated crystal orientation mapping (ACOM). Herein, we perform orientation mapping in InP nanowires exploiting precession electron diffraction (PED) patterns acquired by an axial CMOS camera. Crystal orientation is determined at each probe position by the quantitative analysis of diffracted intensities minimizing a residue comparing experiments and simulations in analogy to x-ray structural refinement. Our simulations are based on the two-beam dynamical diffraction approximation and yield a high angular precision (â¼0.03°), much lower than the traditional ACOM based on pattern matching algorithms (â¼1°). We anticipate that simultaneous exploration of both spot positions and high precision crystal misorientation will allow the exploration of the whole potentiality provided by PED-based 4D-STEM for the characterization of deformation fields in nanomaterials.
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Mammaplasty is a widely performed surgical procedure worldwide, utilized for breast reconstruction, in the context of breast cancer treatment, and aesthetic purposes. To enhance post-operative outcomes and reduce risks (hematoma with required evacuation, capsular contracture, implant-associated infection and others), the controlled release of medicaments can be achieved using drug delivery systems based on cyclodextrins (CDs). In this study, our objective was to functionalize commercially available silicone breast implants with smooth and textured surfaces through in-situ polymerization of two CDs: ß-CD/citric acid and 2-hydroxypropyl-ß-CD/citric acid. This functionalization serves as a local drug delivery system for the controlled release of therapeutic molecules that potentially can be a preventive treatment for post-operative complications in mammaplasty interventions. Initially, we evaluated the pre-treatment of sample surfaces with O2 plasma, followed by chitosan grafting. Subsequently, in-situ polymerization using both types of CDs was performed on implants. The results demonstrated that the proposed pre-treatment significantly increased the polymerization yield. The functionalized samples were characterized using microscopic and physicochemical techniques. To evaluate the efficacy of the proposed system for controlled drug delivery in augmentation mammaplasty, three different molecules were utilized: pirfenidone (PFD) for capsular contracture prevention, Rose Bengal (RB) as anticancer agent, and KR-12 peptide (KR-12) to prevent bacterial infection. The release kinetics of PFD, RB, and KR-12 were analyzed using the Korsmeyer-Peppas and monolithic solution mathematical models to identify the respective delivery mechanisms. The antibacterial effect of KR-12 was assessed against Staphylococcus epidermidis and Pseudomonas aeruginosa, revealing that the antibacterial rate of functionalized samples loaded with KR-12 was dependent on the diffusion coefficients. Finally, due to the immunomodulatory properties of KR-12 peptide on epithelial cells, this type of cells was employed to investigate the cytotoxicity of the functionalized samples. These assays confirmed the superior properties of functionalized samples compared to unprotected implants.
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The ORF XF2234 in the Xylella fastidiosa genome was identified as encoding a small heat-shock protein of 17.9â kDa (HSP17.9). HSP17.9 was found as one of the proteins that are induced during X. fastidiosa proliferation and infection in citrus culture. Recombinant HSP17.9 was crystallized and surface atomic force microscopy experiments were conducted with the aim of better characterizing the HSP17.9 crystals. X-ray diffraction data were collected at 2.7â Å resolution. The crystal belonged to space group P4(3)22, with unit-cell parameters a = 68.90, b = 68.90, c = 72.51 Å, and is the first small heat-shock protein to crystallize in this space group.
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Proteínas de Choque Térmico Pequenas/química , Xylella/química , Cristalografia por Raios X , Proteínas de Choque Térmico Pequenas/ultraestrutura , Microscopia de Força AtômicaRESUMO
InP nanowire polytypic growth was thoroughly studied using electron microscopy techniques as a function of the In precursor flow. The dominant InP crystal structure is wurtzite, and growth parameters determine the density of stacking faults (SF) and zinc blende segments along the nanowires (NWs). Our results show that SF formation in InP NWs cannot be univocally attributed to the droplet supersaturation, if we assume this variable to be proportional to the ex situ In atomic concentration at the catalyst particle. An imbalance between this concentration and the axial growth rate was detected for growth conditions associated with larger SF densities along the NWs, suggesting a different route of precursor incorporation at the triple phase line in that case. The formation of SFs can be further enhanced by varying the In supply during growth and is suppressed for small diameter NWs grown under the same conditions. We attribute the observed behaviors to kinetically driven roughening of the semiconductor/metal interface. The consequent deformation of the triple phase line increases the probability of a phase change at the growth interface in an effort to reach local minima of system interface and surface energy.
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Double-stranded pBS plasmid DNA was irradiated with gamma rays at doses ranging from 1 to 12 kGy and electron beams from 1 to 10 kGy. Fragment-size distributions were determined by direct visualization, using atomic force microscopy with nanometer-resolution operating in non-tapping mode, combined with an improved methodology. The fragment distributions from irradiation with gamma rays revealed discrete-like patterns at all doses, suggesting that these patterns are modulated by the base pair composition of the plasmid. Irradiation with electron beams, at very high dose rates, generated continuous distributions of highly shattered DNA fragments, similar to results at much lower dose rates found in the literature. Altogether, these results indicate that AFM could supplement traditional methods for high-resolution measurements of radiation damage to DNA, while providing new and relevant information.
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SU-8 polymer is an excellent platform for diverse applications due to its high aspect ratio of micro/nanostructure fabrication and exceptional physicochemical and biocompatible properties. Although SU-8 polymer has often been investigated for various biological applications, how its surface properties influence the interaction of bacterial cells with the substrate and its colonization is poorly understood. In this work, we tailor SU-8 nanoscale surface properties to investigate single-cell motility, adhesion, and successive colonization of phytopathogenic bacteria, Xylella fastidiosa. Different surface properties of SU-8 thin films have been prepared using photolithography processing and oxygen plasma treatment. A more significant density of carboxyl groups in hydrophilic plasma-treated SU-8 surfaces promotes faster cell motility in the earlier growth stage. The hydrophobic nature of pristine SU-8 surfaces shows no trackable bacterial motility and 5-10 times more single cells adhered to the surface than its plasma-treated counterpart. In addition, plasma-treated SU-8 samples suppressed bacterial adhesion, with surfaces showing less than 5% coverage. These results not only showcase that SU-8 surface properties can impact the spatiotemporal bacterial behavior but also provide insights into pathogens' prominent ability to evolve and adapt to different surface properties.
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Biofilmes , Polímeros , Polímeros/farmacologia , Aderência Bacteriana , Propriedades de Superfície , Membrana CelularRESUMO
Graphene oxide (GO) has immense potential for widespread use in diverse in vitro and in vivo biomedical applications owing to its thermal and chemical resistance, excellent electrical properties and solubility, and high surface-to-volume ratio. However, development of GO-based biological nanocomposites and biosensors has been hampered by its poor intrinsic biocompatibility and difficult covalent biofunctionalization across its lattice. Many studies exploit the strategy of chemically modifying GO by noncovalent and reversible attachment of (bio)molecules or sole covalent biofunctionalization of residual moieties at the lattice edges, resulting in a low coating coverage and a largely bioincompatible composite. Here, we address these problems and present a facile yet powerful method for the covalent biofunctionalization of GO using colamine (CA) and the poly(ethylene glycol) cross-linker that results in a vast improvement in the biomolecular coating density and heterogeneity across the entire GO lattice. We further demonstrate that our biofunctionalized GO with CA as the cross-linker provides superior nonspecific biomolecule adhesion suppression with increased biomarker detection sensitivity in a DNA-biosensing assay compared to the (3-aminopropyl)triethoxysilane cross-linker. Our optimized biofunctionalization method will aid the development of GO-based in situ applications including biosensors, tissue nanocomposites, and drug carriers.
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The morphological plasticity of bacteria to form filamentous cells commonly represents an adaptive strategy induced by stresses. In contrast, for diverse human and plant pathogens, filamentous cells have been recently observed during biofilm formation, but their functions and triggering mechanisms remain unclear. To experimentally identify the underlying function and hypothesized cell communication triggers of such cell morphogenesis, spatially controlled cell patterning is pivotal. Here, we demonstrate highly selective cell adhesion of the biofilm-forming phytopathogen Xylella fastidiosa to gold-patterned SiO2 substrates with well-defined geometries and dimensions. The consequent control of both cell density and distances between cell clusters demonstrated that filamentous cell formation depends on cell cluster density, and their ability to interconnect neighboring cell clusters is distance-dependent. This process allows the creation of large interconnected cell clusters that form the structural framework for macroscale biofilms. The addition of diffusible signaling molecules from supernatant extracts provides evidence that cell filamentation is induced by quorum sensing. These findings and our innovative platform could facilitate therapeutic developments targeting biofilm formation mechanisms of X. fastidiosa and other pathogens.
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Dióxido de Silício , Xylella , Biofilmes , Humanos , Percepção de QuorumRESUMO
Definitive evidence for the direct band gap predicted for Wurtzite Gallium Phosphide (WZ GaP) nanowires has remained elusive due to the lack of strong band-to-band luminescence in these materials. In order to circumvent this problem, we successfully obtained large volume WZ GaP structures grown by nanoparticle-crawling assisted Vapor-Liquid-Solid method. With these structures, we were able to observe bound exciton recombination at 2.14 eV with FHWM of approximately 1 meV. In addition, we have measured the optical absorption edges using photoluminescence excitation spectroscopy. Our results show a 10 K band gap at 2.19 eV and indicate a weak oscillator strength for the lowest energy band-to-band absorption edge, which is a characteristic feature of a pseudo-direct band gap semiconductor. Furthermore, the valence band splitting energies are estimated as 110 meV and 30 meV for the three highest bands. Electronic band structure calculations using the HSE06 hybrid density functional agree qualitatively with the valence band splitting energies.
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An amendment to this paper has been published and can be accessed via a link at the top of the paper.
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We have studied the equilibrium electrostatic profile of III-V semiconductor nanowires using Kelvin probe force microscopy. Qualitative agreement of the measured surface potential levels and expected Fermi level variation for pure InP and InAs nanowires is obtained from electrical images with spatial resolution as low as 10 nm. Surface potential mapping for pure and heterostructured nanowires suggests the existence of charge transfer mechanisms and the formation of a metal-semiconductor electrical contact at the nanowire apex.
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The pathogenicity of Xylella fastidiosa is associated with its systematic colonization of the plant xylem, forming bacterial biofilms. Mechanisms of bacterial transport among different xylem vessels, however, are not completely understood yet, but are strongly influenced by the presence of extracellular polymeric substances (EPS), which surrounds the assembly of cells forming the biofilm. In this work, we show quantitative measurements on the elastic properties of the system composed by EPS and bacterial cell. In order to investigate the mechanical properties of this system, force spectroscopy and confocal Raman measurements were carried out during Xylella fastidiosa subsp. pauca initial stages of adhesion and cluster formation. We show that stiffness progressively decreases with increasing culture growth time, from two to five days. For early adhesion samples, stiffness values are quite different at the bacterial polar and body regions. Lower stiffness values at the cell pole suggest a flexible mechanical response at this region, associated with first cell adhesion to a surface. These results correlate very well with our observations of cell motion within microchannels, under conditions simulating xylem flow. Both the oscillatory movement of vertically attached single cells, as well as the transport of cell clusters within the biofilm matrix can be explained by the presence of softer materials at the cell pole and EPS matrix. Our results may thus add to a more detailed understanding of mechanisms used by cells to migrate among vessels in plant xylem.
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Biofilmes/efeitos dos fármacos , Xylella/efeitos dos fármacos , Aderência Bacteriana/efeitos dos fármacos , Polímeros/farmacologiaRESUMO
In this work, nanofilms of hyaluronan/chitosan (HA/CHI) assembled layer by layer were synthesized; their application as a potential antimicrobial material was demonstrated for the phytopathogen Xylella fastidiosa, a gram-negative bacterium, here used as a model. For the synthesis, the influence of pH and ionic strength of these natural polymer stem-solutions on final characteristics of the HA/CHI nanofilms was studied in detail. The antibacterial effect was evaluated using widefield fluorescence microscopy. These results were correlated with the chemical properties of the nanofilms, studied by FTIR and Raman spectroscopy, as well as with their morphology and surface properties characterized using SEM and AFM. The present findings can be extended to design and optimize HA/CHI nanofilms with enhanced antimicrobial behavior for other type of phytopathogenic gram-negative bacteria species, such as Xanthomonas citri, Xanthomas campestri and Ralstonia solanacearum.
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Antibacterianos/química , Antibacterianos/farmacologia , Quitosana/química , Ácido Hialurônico/química , Nanoestruturas/química , Xylella/efeitos dos fármacos , Aderência Bacteriana/efeitos dos fármacos , Propriedades de Superfície , Xylella/fisiologiaRESUMO
The Xylella fastidiosa subsp pauca strain 9a5c is a Gram-negative, xylem-limited bacterium that is able to form a biofilm and affects citrus crops in Brazil. Some genes are considered to be involved in biofilm formation, but the specific mechanisms involved in this process remain unknown. This limited understanding of how some bacteria form biofilms is a major barrier to our comprehension of the progression of diseases caused by biofilm-producing bacteria. Several investigations have shown that the toxin-antitoxin (TA) operon is related to biofilm formation. This operon is composed of a toxin with RNAse activity and its cognate antitoxin. Previous reports have indicated that the antitoxin is able to inhibit toxin activity and modulate the expression of the operon as well as other target genes involved in oxidative stress and mobility. In this study, we characterize a toxin-antitoxin system consisting of XfMqsR and XfYgiT, respectively, from X. fastidiosa subsp. pauca strain 9a5c. These proteins display a high similarity to their homologs in X. fastidiosa strain Temecula and a predicted tridimensional structure that is similar to MqsR-YgiT from Escherichia coli. The characterization was performed using in vitro assays such as analytical ultracentrifugation (AUC), size exclusion chromatography, isothermal titration calorimetry, and Western blotting. Using a fluorometric assay to detect RNAses, we demonstrated that XfMqsR is thermostable and can degrade RNA. XfMqsR is inhibited by XfYgiT, which interacts with its own promoter. XfYgiT is known to be localized in the intracellular compartment; however, we provide strong evidence that X. fastidiosa secretes wild-type XfYgiT into the extracellular environment via outer membrane vesicles, as confirmed by Western blotting and specific immunofluorescence labeling visualized by fluorescence microscopy. Taken together, our results characterize the TA system from X. fastidiosa strain 9a5c, and we also discuss the possible influence of wild-type XfYgiT in the cell.