Corrigendum: HSA-nanobinders crafted from bioresponsive prodrugs for combined cancer chemoimmunotherapy-an in vitro exploration.

[This corrects the article DOI: 10.3389/fchem.2024.1378233.].

HSA-nanobinders crafted from bioresponsive prodrugs for combined cancer chemoimmunotherapy-an in vitro exploration.

Introduction: Triple-negative breast cancer (TNBC) is an aggressive subtype of breast cancer still lacking effective treatment options. Chemotherapy in combination with immunotherapy can restrict tumor progression and repolarize the tumor microenvironment towards an anti-tumor milieu, improving clinical outcome in TNBC patients. The chemotherapeutic drug paclitaxel has been shown to induce immunogenic cell death (ICD), whereas inhibitors of the indoleamine 2,3- dioxygenase 1 (IDO1) enzyme, whose expression is shared in immune regulatory and tumor cells, have been revealed to enhance the anti-tumor immune response. However, poor bioavailability and pharmacokinetics, off-target effects and hurdles in achieving therapeutic drug concentrations at the target tissue often limit the effectiveness of combination therapies. Methods: This work describes the development of novel biomimetic and carrier-free nanobinders (NBs) loaded with both paclitaxel and the IDO1 inhibitor NLG919 in the form of bioresponsive and biomimetic prodrugs. A fine tuning of the preparation conditions allowed to identify NB@5 as the most suitable nanoformulation in terms of reproducibility, stability and in vitro effectiveness. Results and discussion: Our data show that NB@5 effectively binds to HSA in cell-free experiments, demonstrating its protective role in the controlled release of drugs and suggesting the potential to exploit the protein as the endogenous vehicle for targeted delivery to the tumor site. Our study successfully proves that the drugs encapsulated within the NBs are preferentially released under the altered redox conditions commonly found in the tumor microenvironment, thereby inducing cell death, promoting ICD, and inhibiting IDO1.

A systematic literature review of randomised controlled trials evaluating colchicine for cardiovascular prevention: There is an elephant in the room.

OBJECTIVES: To review randomised controlled trials (RCT) investigating colchicine (COL) for cardiovascular (CV) prevention in patients at high to very high CV risk aiming to extract data that could be useful in rheumatology practice. METHODS: A systematic search of multiple databases according to the PICO framework was performed from inception to April 3, 2023. Three researchers independently screened abstracts/titles and reviewed full texts reviewed. Data extraction was performed using a pilot-tested data extraction form. RESULTS: A total of 14,096 references were retrieved by the search and 30 articles, describing 28 RCTs, were included in the review (Total number of patients 16,795, of which 8,463 randomised to COL; dose 0.5-2 mg/day, treatment duration 1day-29 months). Only one of the 28 RCTs provided clear information on whether patients with rheumatic diseases (more specifically gout) were enrolled in the study cohorts and previous use of COL was an exclusion criterion only in 14 (50 %) RCTs. Previous therapy with glucocorticoids and/or non-steroidal anti-inflammatory drugs and/or immune suppressive therapies was an exclusion criterion only in 9 (32 %) RCTs. CONCLUSION: Our results highlight the need to redefine the eligibility criteria as well as the reporting of results in future RCTs in order to minimise bias or previous exposure to COL and also obtain data that could be useful in rheumatology practice.

In Vitro Evaluation of Smear Layer and Debris Removal and Antimicrobial Activity of Different Irrigating Solutions.

OBJECTIVE: The aim of this in vitro study was to compare the smear layer and debris removal and antimicrobial activity of two dual-action irrigating solutions for continuous chelation (Triton; Brasseler, Savannah, USA and Dual Rinse HEDP; Medcem GmbH, Weinfelden, Switzerland) with a dual step irrigation protocol with sodium hypochlorite (NaOCl) followed by ethylenediaminetetraacetic acid (EDTA). METHODS: Thirty single-rooted single-canal teeth were divided into three groups (n=10) and irrigated with Triton, Dual Rinse HEDP mixed with 6% NaOCl and 6% NaOCl/17% EDTA. The teeth were observed under a scanning electron microscope (SEM) to assess the canal wall cleanliness. In addition, 80 dentine discs were contaminated with Candida albicans and 80 discs with Enterococcus faecalis and irrigated with Triton, Dual Rinse HEDP mixed with 6% NaOCl and 6% NaOCl/17% EDTA or not treated (n=20). Fifteen discs were used to evaluate colony-forming units, while 5 discs were analysed by SEM. Data were analysed using the Shapiro- Wilk, Kruskal-Wallis and One-Way ANOVA tests. RESULTS: Triton was statistically more effective than Dual Rinse HEDP and NaOCl/EDTA in removing debris (p<0.05), except with NaOCl/EDTA in the coronal third. Triton was more effective than Dual Rinse HEDP in removing the smear layer from the apical and middle thirds (p<0.05). All the irrigation protocols significantly re- duced the number of E. faecalis. The Triton group showed the lowest number of remaining C. albicans (p<0.05). CONCLUSION: Triton was the most effective irrigation solution in removing debris and as effective as NaOCl/ EDTA in removing the smear layer. Triton showed the highest efficacy against C. albicans. New irrigating solutions that provide continuous chelation may provide an alternative to current irrigation protocols.

Microbial signature of plaque and gut in acute coronary syndrome.

Gut microbiota is an emerging editable cardiovascular risk factor. We aim to investigate gut and coronary plaque microbiota, using fecal samples and angioplasty balloons from patients with acute coronary syndrome (ACS), chronic coronary syndrome (CCS) and control subjects. We examined bacterial communities in gut and coronary plaques by 16S rRNA sequencing and we performed droplet digital PCR analysis to investigate the gut relative abundance of the bacterial genes CutC/CntA involved in trimethylamine N-oxide synthesis. Linear discriminant analysis effect size (LEfSe) at the genus and species levels displayed gut enrichment in Streptococcus, Granulicatella and P. distasonis in ACS compared with CCS and controls; Roseburia, C. aerofaciens and F. prausnitzii were more abundant in controls than in patients. Principal component analysis (PCA) of 41 differentially abundant gut taxa showed a clustering of the three groups. In coronary plaque, LEfSe at the genus level revealed an enrichment of Staphylococcus and Streptococcus in ACS, and Paracoccus in CCS, whereas PCA of 15 differentially abundant plaque taxa exhibited clustering of ACS and CCS patients. CutC and CntA genes were more abundant in ACS and CCS than in controls while no significant difference emerged between ACS and CCS. Our results indicate that ACS and CCS exhibit a different gut and plaque microbial signature, suggesting a possible role of these microbiotas in coronary plaque instability.

The Interplay between Cardiovascular Risk, Cardiovascular Events, and Disease Activity in Primary Sjögren's Syndrome: Is Uric Acid the Missing Link?

(1) Background: Uric acid is a well-known cardiovascular (CV) risk factor in the general population but its role in the setting of rheumatic diseases other than gout is unclear. (2) Methods: This is a retrospective study investigating a cohort of 105 pSS patients recording clinical, serological, and CV-related variables including adherence to the Mediterranean diet. (3) Results: We observed a strong relationship between disease activity, interstitial lung disease (ILD), and CV events. The association between ILD and CV events was dependent on higher SUA levels but independent of other traditional CV risk factors. All three cases of previous non-fatal stroke were reported by females aged <65 years, with higher SUA levels, and two of them also had pSS-ILD. Forty (38%) patients had a 10-year risk of fatal and non-fatal CV disease events beyond the cut-off recommended for their age, and using the correction factor of 1.5 currently applied only to rheumatoid arthritis, we could better identify patient subsets characterized by different CV risk profiles including different SUA levels. (4) Conclusions: This study is the first to investigate in depth the role of SUA in the CV scenario of pSS. Our findings underpin the importance of assessing SUA levels in pSS in addition to the other traditional CV risk factors and to consider applying the correction factor for CV risk assessment tools to achieve a better stratification of CV risk.

Antimicrobial peptides for tackling cystic fibrosis related bacterial infections: A review.

Antibiotic resistance is a serious health and social problem that will have a substantial impact in the coming years on the world health and economy. Thus, the increasing demand for innovative antibiotics, has prompted many researchers in the medical, microbiological, and biochemical fields to exploit the properties of antimicrobial peptides (AMPs). When properly used, designed, and conveyed, AMPs can really represent a valid alternative to conventional drugs especially in situations that are particularly difficult to treat such as chronic infections found in Cystic Fibrosis (CF) patients. In this review we focused on the applications of AMPs in the specific field of CF, illustrating different types of peptides from natural, naturally modified, synthetic as well as the different strategies used to overcome the barriers, and the physiological conditions in which AMPs must operate.

HSA-Binding Prodrugs-Based Nanoparticles Endowed with Chemo and Photo-Toxicity against Breast Cancer.

Exploiting the tumor environment features (EPR effect, elevated glutathione, reactive oxygen species levels) might allow attaining a selective and responsive carrier capable of improving the therapeutic outcome. To this purpose, the in situ covalent binding of drugs and nanoparticles to circulating human serum albumin (HSA) might represent a pioneering approach to achieve an effective strategy. This study describes the synthesis, in vitro and in vivo evaluation of bioresponsive HSA-binding nanoparticles (MAL-PTX2S@Pba), co-delivering two different paclitaxel (PTX) prodrugs and the photosensitizer pheophorbide a (Pba), for the combined photo- and chemo-treatment of breast cancer. Stable and reproducible MAL-PTX2S@Pba nanoparticles with an average diameter of 82 nm and a PTX/Pba molar ratio of 2.5 were obtained by nanoprecipitation. The in vitro 2D combination experiments revealed that MAL-PTX2S@Pba treatment induces a strong inhibition of cell viability of MDA-MB-231, MCF7 and 4T1 cell lines, whereas 3D experiments displayed different trends: while MAL-PTX2S@Pba effectiveness was confirmed against MDA-MB-231 spheroids, the 4T1 model exhibited marked resistance. Lastly, despite using a low PTX-PDT regimen (e.g., 8.16 mg/Kg PTX and 2.34 mg/Kg Pba), our formulation showed to foster primary tumor reduction and curb lung metastases growth in 4T1 tumor-bearing mice, thus setting the basis for further preclinical validations.

Inactivation of the Response Regulator AgrA Has a Pleiotropic Effect on Biofilm Formation, Pathogenesis and Stress Response in Staphylococcus lugdunensis.

Staphylococcus lugdunensis is a coagulase-negative Staphylococcus that emerges as an important opportunistic pathogen. However, little is known about the regulation underlying the transition from commensal to virulent state. Based on knowledge of S. aureus virulence, we suspected that the agr quorum sensing system may be an important determinant for the pathogenicity of S. lugdunensis. We investigated the functions of the transcriptional regulator AgrA using the agrA deletion mutant. AgrA played a role in cell pigmentation: ΔargA mutant colonies were white while the parental strains were slightly yellow. Compared with the wild-type strain, the ΔargA mutant was affected in its ability to form biofilm and was less able to survive in mice macrophages. Moreover, the growth of ΔagrA was significantly reduced by the addition of 10% NaCl or 0.4 mM H2O2 and its survival after 2 h in the presence of 1 mM H2O2 was more than 10-fold reduced. To explore the mechanisms involved beyond these phenotypes, the ΔagrA proteome and transcriptome were characterized by mass spectrometry and RNA-Seq. We found that AgrA controlled several virulence factors as well as stress-response factors, which are well correlated with the reduced resistance of the ΔagrA mutant to osmotic and oxidative stresses. These results were not the consequence of the deregulation of RNAIII of the agr system, since no phenotype or alteration of the proteomic profile has been observed for the ΔRNAIII mutant. Altogether, our results highlighted that the AgrA regulator of S. lugdunensis played a key role in its ability to become pathogenic. IMPORTANCE Although belonging to the natural human skin flora, Staphylococcus lugdunensis is recognized as a particularly aggressive and destructive pathogen. This study aimed to characterize the role of the response regulator AgrA, which is a component of the quorum-sensing agr system and known to be a major element in the regulation of pathogenicity and biofilm formation in Staphylococcus aureus. In the present study, we showed that, contrary to S. aureus, the agrA deletion mutant produced less biofilm. Inactivation of agrA conferred a white colony phenotype and impacted S. lugdunensis in its ability to survive in mice macrophages and to cope with osmotic and oxidative stresses. By global proteomic and transcriptomic approaches, we identified the AgrA regulon, bringing molecular bases underlying the observed phenotypes. Together, our data showed the importance of AgrA in the opportunistic pathogenic behavior of S. lugdunensis allowing it to be considered as an interesting therapeutic target.

Nitric Oxide Photo-Donor Hybrids of Ciprofloxacin and Norfloxacin: A Shift in Activity from Antimicrobial to Anticancer Agents.

The potential anticancer effect of fluoroquinolone antibiotics has been recently unveiled and related to their ability to interfere with DNA topoisomerase II. We herein envisioned the design and synthesis of novel Ciprofloxacin and Norfloxacin nitric oxide (NO) photo-donor hybrids to explore the potential synergistic antitumor effect exerted by the fluoroquinolone scaffold and NO eventually produced upon light irradiation. Anticancer activity, evaluated on a panel of tumor cell lines, showed encouraging results with IC50 values in the low micromolar range. Some compounds displayed intense antiproliferative activity on triple-negative and doxorubicin-resistant breast cancer cell lines, paving the way for their potential use to treat aggressive, refractory and multidrug-resistant breast cancer. No significant additive effect was observed on PC3 and DU145 cells following NO release. Conversely, antimicrobial photodynamic experiments on both Gram-negative and Gram-positive microorganisms displayed a significant killing rate in Staphylococcus aureus, accounting for their potential effectiveness as selective antimicrobial photosensitizers.

Is aromatherapy effective in obstetrics? A systematic review and meta-analysis.

The aim of this systematic review is to collect clinical trials conducted using essential oils (EOs) in obstetric symptoms by evaluating if and in which context the aromatherapy practice is effective in obstetrics. The research was conducted by using the databases of EMBASE, Medline, Biosis and Toxcenter, PubMed, and Google Scholar search engine, selecting articles from January 2004 to July 2020. This study was performed according to the MOOSE and PRISMA guidelines. Only the randomized clinical trials were considered, and in cases of multiple publications, it was considered the most up to date information. Biases were highlighted. In the presence of homogeneous data, pooling statistics and meta-analysis were applied. The research led to 71 articles, 17 of which were eligible. Among the trials selected, eight investigated the effectiveness of EOs on anxiety, depression, and stress. Two concerned the treatment of nausea and vomiting, six evaluated the application of EOs on labor for pain treatment, and two showed the effectiveness in the treatment of episiotomy. The heterogeneity of works carried out so far has made it possible to develop a meta-analysis only in the field of pain treatment during childbirth, identifying the effectiveness of the EOs Lavandula spp. and Rosa damascena.

Origanum vulgare Essential Oil vs. a Commercial Mixture of Essential Oils: In Vitro Effectiveness on Salmonella spp. from Poultry and Swine Intensive Livestock.

Salmonella spp. represent a public health concern for humans and animals due to the increase of antibiotic resistances. In this scenario, the use of essential oils (EOs) could be a valid tool against Salmonella contamination of meat. This work compares the in vitro effectiveness of an Italian mixture of feed additives based on EOs (GR-OLI) with EO of Origanum vulgare L., recently admitted by European Food Safety Authority (EFSA) for animal use. Twenty-nine Salmonella serotypes isolated from poultry and pig farms were used to assess GR-OLI and O. vulgare EO antimicrobial propeties. O. vulgare EO was active on the disaggregation of mature biofilm, while GR-OLI was capable of inhibiting biofilm formation and disaggregating preformed biofilm. Furthermore, GR-OLI inhibited bacterial adhesion to Caco-2 cells in a dose-dependent manner. Both products showed inhibition of bacterial growth at all time points tested. Finally, the synergistic action of GR-OLI with commonly used antibiotics against resistant strains was investigated. In conclusion, the mixture could be used both to reduce the meat contamination of Salmonella spp. before slaughter, and in synergy with low doses of ciprofloxacin against resistant strains. Although EOs as feed additives are already used in animal husbandry, no scientific study has ever highlighted their real antimicrobial potential.

Cardiospermum halicacabum in atopic dermatitis: Clinical evidence based on phytotherapic approach.

Atopic dermatitis is a chronic inflammatory disease that mainly affects children, causing a strong impact on the quality of life. The most affected subjects live in urban areas or in developed countries, with prevalence increased up to 15%-20% in children and from 1% to 3% in adults during the past 30 years. Most adults had the first signs of illness before the age of 5, many of them before the age of 1. Many therapies in use are associated with side effects and often induce intolerance; therefore, many studies are based on the search for topical products that have an effective benefit, are well tolerated and appreciated by patients. Natural products are expected to have promising prospects in the management of atopy. Cardiospermum halicacabum is a member of the Sapindaceae family that seems to express its cortisone-like anti-inflammatory activity by activating phospholipase A2 while maintaining the stability of cell membranes, an effect due to the presence of phytosterols, having an affinity for lipids in the epidermis and cell membranes. We report our experience during a 15 days observational clinical study aimed to demonstrate efficacy and safety of the treatment with HALICAR: a cream containing C. halicacabum. Clinical data indicates that the treatment with HALICAR leads to positive effects in subjects with dermatitis of various degrees, regardless of which concomitant treatment is considered.

Antimicrobial and Antibiofilm Properties of Graphene Oxide on Enterococcus faecalis.

The aim of this study was to evaluate the antibacterial properties of graphene oxide (GO) against Enterococcus faecalis in vitro conditions and when used to coat dentin surface to prevent E. faecalis adhesion. The ATCC strain of E. faecalis 29212 has been used to perform a viability test. The pellet was suspended in ultrapure water, NaCl, PBS buffer, CaCl2 and MgCl2, Luria-Bertani broth solutions. The viability was evaluated by the colony forming unit counting method. Atomic force microscopy images and the measure of surface zeta potential variation were analyzed. Dentin discs were covered with a film of GO (n = 15) or were not treated (n = 15). Bacterial suspension was added to each sample of dentine discs and microbial counts were calculated. Statistically significant differences between two groups were assessed by a two-tailed unpaired t-test. Bacteria cell morphology was investigated with scanning electron microscopy. The highest growth inhibition was obtained in ddH2O and CaCl2 solution while, in PBS and NaCl, GO had poor antibacterial efficacy with a growth enhancing effect in the latter. GO on dentin discs demonstrated high antibacterial activity. GO film has demonstrated acceptable adhesion properties to root dentin and a role in the inhibition of bacterial film proliferation and biofilm formation.

Prevalence and Clonal Distribution of Azole-Resistant Candida parapsilosis Isolates Causing Bloodstream Infections in a Large Italian Hospital.

The most prevalent cause of nosocomial bloodstream infection (BSI) among non-C. albicans Candida species, Candida parapsilosis, may not only be resistant to azole antifungal agents but also disseminate to vulnerable patients. In this survey of BSIs occurring at a large Italian hospital between May 2014 and May 2019, C. parapsilosis accounted for 28.5% (241/844) of all Candida isolates causing BSI episodes. The majority of episodes (151/844) occurred in medical wards. Across the 5 yearly periods, the rates of azole non-susceptibility were 11.8% (4/34), 17.8% (8/45), 28.6% (12/42), 32.8% (19/58), and 17.7% (11/62), respectively, using the Sensititre YeastOne® method. Among azole non-susceptible isolates (54/241; 22.4%), 49 were available for further investigation. Using the CLSI reference method, all 49 isolates were resistant to fluconazole and, except one (susceptible dose-dependent), to voriconazole. Forty (81.6%) isolates harbored the Erg11p Y132F substitution and nine (18.4%) isolates the Y132F in combination with the Erg11p R398I substitution. According to their genotypes, as defined using a microsatellite analysis based on six short tandem repeat markers, 87.7% of isolates (43/49) grouped in two major clusters (II and III), whereas 4.1% of isolates (2/49) belonged to a separate cluster (I). Interestingly, all the isolates from cluster II harbored the Y132F substitution, and those from cluster III harbored both Y132F and R398I substitutions. Of 56 non-Italian isolates included as controls, two Indian isolates with the Y132F substitution had a genotype clearly differing from that of the isolates from clusters II and I. In conclusion, these findings show the dominance of clonal Y132F isolates in our hospital and suggest detection of the Y132F substitution as helpful tool to prevent transmission among hospitalized patients at risk of BSI.

Graphene Oxide Coatings as Tools to Prevent Microbial Biofilm Formation on Medical Device.

The clinical challenge on surface engineering of medical devices to prevent microorganisms adhesion and biofilm formation, has become an essential aspect for medical implants. Antibacterial properties of Graphene Oxide (GO) have been demonstrated across a broad spectrum of bacteria, and the different mechanisms of action with which this nanomaterial interacts with the microbial surface have been elucidated in detail. Innovative protective coatings based on graphene film and hydrogel could represent an innovative solution for the prevention of nosocomial pathogens colonization on implantable device. This brief review mainly focuses on the applications of graphene in nanomedicine with a particular deepening on the antibacterial properties of GO and GO-based nanomaterials. In order to evaluate the possible future applications of GO as an anti-biofilm coating material for medical devices, studies on the ability of graphene coated surface to prevent microbial adhesion are also discussed. A concise review on in vitro toxicity and in vivo safety is also presented.

In vitro characterization, ADME analysis, and histological and toxicological evaluation of BM1, a macrocyclic amidinourea active against azole-resistant Candida strains.

BACKGROUND: Candida species are one of the most common causes of nosocomial bloodstream infections among the opportunistic fungi. Extensive use of antifungal agents, most of which were launched on the market more than 20 years ago, led to the selection of drug-resistant or even multidrug-resistant fungi. We recently described a novel class of antifungal macrocyclic compounds with an amidinourea moiety that is highly active against azole-resistant Candida strains. OBJECTIVE: A compound from this family, BM1, was investigated in terms of in vitro activity against various Candida species, including C. auris isolates, interaction with the ABC transporter, CDR6, and in vivo distribution and safety. METHODS: In vitro assays (CYP inhibition, microsomal stability, permeability, spot assays) were used to collect chemical and biological data; animal models (rat) paired with LC-MS analysis were utilised to evaluate in vivo toxicology, pharmacokinetics, and distribution. RESULTS: The current research shows BM1 has a low in vivo toxicity profile, affinity for the renal system in rats, and good absorption, distribution, metabolism, and excretion (ADME). BM1 also has potent activity against azole-resistant fungal strains, including C. auris isolates and CDR6-overexpressing strains. CONCLUSIONS: The results confirmed low minimum inhibitory concentrations (MICs) against several Candida species, including preliminary data vs. C. auris. BM1 has good ADME and biochemical characteristics, is suitable and safe for daily administration and is particularly indicated for renal infections. These data indicate BM1 and its derivatives form a novel, promising antifungal class.

Light-Induced Therapies for Prostate Cancer Treatment.

Prostate cancer (PC) is one of the most widespread tumors affecting the urinary system and the fifth-leading cause from cancer death in men worldwide. Despite PC mortality rates have been decreasing during the last years, most likely due to an intensification of early diagnosis, still more than 300,000 men die each year because of this disease. In this view, researchers in all countries are engaged in finding new ways to tackle PC, including the design and synthesis of novel molecular and macromolecular entities able to challenge different PC biological targets, while limiting the extent of unwanted side effects that significantly limit men's life quality. Among this field of research, photo-induced therapies, such as photodynamic and photothermal therapies (PDT and PTT), might represent an important advancement in PC treatment due to their extremely localized and controlled cytotoxic effect, as well as their low incidence of side effects and tumor resistance occurrence. Based on these considerations, this review aims to gather and discuss the last 5-years literature reports dealing with the synthesis and biological activity of molecular conjugates and nano-platforms for photo-induced therapies as co-adjuvant or combined therapeutic modalities for the treatment of localized PC.

Antibiofilm Activity of Three Different Irrigation Techniques: An in Vitro Study.

The microbial infection of the endodontic space occurs in a necrotic tooth as a result of dental caries, trauma, periodontal disease, or previous root canal therapy. The disruption of the biofilms and the reduction of the bacterial load inside root canals are crucial for the success of root canal therapy. The aim of this study was to compare, in vitro, the antibiofilm efficacy of a novel passive sonic irrigation (PSI) device with passive ultrasonic irrigation (PUI) and conventional needle irrigation (CNI). Forty-four single-rooted human teeth were inoculated with a culture of E. faecalis for 28 days. The specimens were randomly divided into three groups: PUI, CNI, and PSI (n = 12). The activation protocols were performed using both 17% EDTA and 5.25% NaOCl. Residual bacterial biofilm was taken by means of a canal brush and colony-forming unit (CFU) were counted. The data were analyzed using one-way ANOVA and Games-Howell's post hoc tests. A major reduction in CFU was observed in the PSI and PUI groups, in comparison with the CNI group. No difference was found (p > 0.05) in terms of CFU reduction between PSI and PUI. PSI could be as effective as PUI in the removal of bacterial biofilms from straight root canals.