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According to contemporary dental standards, the primary goal of endodontic therapy is the chemo-mechanical cleaning of the complex root canal system. Watering root canals with approved solutions and activating them are essential parts of this operation. This review outlines various irrigant activation methods for root canal therapy. Specifically, a comparison among the methods of manual dynamic activation, sonics (subsonic, sonic, and ultrasonic), internal heating, and lasers, was conducted. The results in this work were gathered using Scopus, Web of Science, Google Scholar, and PubMed databases by searching the following keywords: sodium hypochlorite, cleaning, activation, and irrigation methods. The present work concluded that the use of irrigant activation has a greater benefit than its absence. Regardless, it is impossible to point to a single effective activation method.

ABSTRACTPeriodontal diseases include a wide range of pathological conditions, damaging the supporting structures of the teeth. Origin and propagation of periodontal disease is believed to be caused by dysbiosis of the commensal oral microbiota. The aim of this study was to evaluate the presence of bacteria in the pulp cavity of teeth with severe periodontal disease with clinically intact external surface. Periodontal (P) and endodontic (E) tissue samples of root canals from six intact teeth of three patients were sampled for analysis of microbial population using Nanopore technology. Streptococcus was the predominant genus in E samples. Porphyromonas (33.4%, p = 0.047), Tannerella (41.7%, p = 0.042), and Treponema (50.0%, p = 0.0064) were significantly more present in P than in E samples. Some samples (E6 and E1) exhibited a remarkable difference in terms of microbial composition, whilst Streptococcus was a common signature in samples E2 to E5, all which were obtained from the same patient. In conclusion, bacteria were identified on both the root surface and the root canal system, thus demonstrating the possibility of bacteria to spread directly from the periodontal pocket to the root canal system even in the absence of crown’s loss of integrity.

In this study a multidisciplinary approach was applied in order to determine the diffusion of resistant bacteria and selected antibiotic resistance genes in antibiotic-free and conventional broiler farms. Litter samples coming from the two farming types and surface sponges obtained from carcasses at slaughterhouse level were screened by end-point PCR targeting specific resistance for tetracycline, ampicillin, sulfonamide, aminoglycoside, carbapenem, nitrofurantoin, vancomycin, quinupristin-dalfopristin, lincomycin, linezolid, chloramphenicol molecules. Microbiological investigations were conducted from the carcasses to determine phenotypical and genetic resistance patterns from pathogenic and commensal Gram-negative and Gram-positive strains. At farm level, catA1, sul2, blaTEM and aadA2 genes were amplified in all samples, while from carcasses the most representative genes were sul2, blaTEM, along with the vatD, relative to quinupristin-dalfopristin resistance. Gram-negative isolates included Aeromonas, Salmonella, Proteus spp. And Escherichia coli, while the Gram-positive were represented by Enterococcus strains. Phenotypical and genetic analysis revealed multidrug resistance patterns in Salmonella, E. coli and Serratia isolates, followed by the Enterococcus species. The comparison between antibiotic-free and conventional farming systems showed some difference regarding the distribution of resistance genes at farm level but no significance was obtained comparing the phenotypical resistance profiles of bacterial strains from both groups of samples, suggesting a poor influence of farming model on the diffusion of antibiotic resistance in poultry meat production chain.

The Clinics and Practice Editorial Office retracts the article titled “Traditional and Recent Root Canal Irrigation Methods and Their Effectiveness: A Review” [...]

Abstract Hepadnaviruses have been identified in several animal species. The hepadnavirus prototype, human hepatitis B virus (HBV), is a major public health problem associated with chronic liver diseases and hepatocellular carcinoma. Recently, a novel hepadnavirus, similar to HBV, was identified in domestic cats. Since several pathogens can be shared between cats and dogs, we hypothesized that dogs could also harbor hepadnaviruses and we tested a collection of canine sera with multiple molecular strategies. Overall, hepadnavirus DNA was identified in 6.3% (40/635) of canine serum samples, although the viral load in positive sera was low (geometric mean of 2.70 × 102 genome copies per mL, range min 1.36 × 102—max 4.03 × 104 genome copies per mL). On genome sequencing, the canine hepadnaviruses revealed high nucleotide identity (about 98%) and similar organization to the domestic cat hepadnavirus. Altered hepatic markers were found in hepadnavirus-positive dogs, although the role of hepadnavirus in canine health remains to be elucidated.

Abstract Background Coronaviruses (CoVs) are major human and animal pathogens and antiviral drugs are pursued as a complementary strategy, chiefly if vaccines are not available. Feline infectious peritonitis (FIP) is a fatal systemic disease of felids caused by FIP virus (FIPV), a virulent pathotype of feline enteric coronavirus (FeCoV). Some antiviral drugs active on FIPV have been identified, but they are not available in veterinary medicine. ERDRP-0519 (ERDRP) is a non-nucleoside inhibitor, targeting viral RNA polymerase, effective against morbilliviruses in vitro and in vivo. Results The antiviral efficacy of ERDRP against a type II FIPV was evaluated in vitro in Crandell Reese Feline Kidney (CRFK) cells. ERDRP significantly inhibited replication of FIPV in a dose-dependent manner. Viral infectivity was decreased by up to 3.00 logarithms in cell cultures whilst viral load, estimated by quantification of nucleic acids, was reduced by nearly 3.11 logaritms. Conclusions These findings confirm that ERDRP is highly effective against a CoV. Experiments will be necessary to assess whether ERDRP is suitable for treatment of FIPV in vivo.

Salmonellosis is an infectious disease affecting both animals and humans. Antimicrobial resistant (AMR) and biofilm-producing Salmonella spp., frequently detected in reptiles (who can then act as asymptomatic carriers for warm-blooded animals), have developed resistance to biocides; this represents a warning for the emergence of biocide/antimicrobial cross-resistance. The aim of this study was to evaluate the efficacy of Thymus vulgaris L. essential oil (TEO) in inhibiting bacterial growth and biofilm production of Salmonella spp., which had been isolated from wild reptiles housed in a Zoo in Italy. The resistance profile against different classes of antibiotics showed that all the isolates were susceptible to the tested antibiotics, despite the presence of several AMR genes. All the isolates were also tested with aqueous solutions of TEO at different dilutions (5% to 0.039%). Interestingly, TEO proved effective both in inhibiting bacterial growth at low dilutions, with MIC and MBC values ranging between 0.078% and 0.312%, and in inhibiting biofilm production, with values ranging from 0.039% to 0.156%. TEO demonstrated effective bioactivity against the biofilm producer Salmonella spp., proving to be a valid disinfectant for the prevention of salmonellosis from reptiles, a possible source of infection for humans exposed to the reptiles’ environment.

The overuse and misuse of antibiotics can pose the risk of spreading mutant strains that show antimicrobial resistance (AMR), with negative impacts on the management of bacterial infections and economic implications for healthcare systems. The research and development of natural antibacterial agents could be a priority in the next years to improve a number of effective antibacterial molecules and to reduce the AMR phenomenon and its development. The present study identified the most effective concentration and contact time of Thymus vulgaris L. essential oil (TEO) to obtain bactericidal effects in vitro against different Gram-positive and Gram-negative bacterial strains. Six clinically isolated (wild types) bacterial strains, (Citrobacter freundii, Enterococcus feciorum, Proteus mirabilis, Acinetobacter cioffi, Pseudomonas putrefaciens and Klebsiella pneumoniae) and two ATCCs (Staphylococcus aureus and Streptococcus mutans) were tested after 1 min, 3 min and 5 min of contact with TEO. The preliminary results on S. aureus after 24 h of incubation revealed a TEO concentration of 9.28 mg/mL (w/v) that completely inhibited bacteria growth, keeping cell viability. The total suppression of bacterial growth at all tested contact times was observed for all tested bacterial strains, and the results were confirmed after 48 h of incubation. Bacterial growth suppression was confirmed even with the presence of organic components. These preliminary results showed the in vitro antimicrobial efficacy of TEO against different Gram-positive and Gram-negative bacterial strains. Future studies are necessary to confirm the reproducibility of these results even on other strains and to define the exact molecular mechanisms of EOs in order to consider TEO as a valid alternative to classic antibiotic therapies and subsequently to reduce the occurrence of AMR.

COVID-19 is a life-threatening multisistemic infection caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Infection control relies on timely identification and isolation of infected people who can alberg the virus for up to 14 days, providing important opportunities for undetected transmission. This note describes the application of rRT-PCR test for simpler, faster and less invasive monitoring of SARS-CoV-2 infection using pooling strategy of samples. Seventeen positive patients were provided with sterile dry swabs and asked to self-collected 2 nasal specimens (#NS1 and #NS2). The #NS1 was individually placed in a single tube and the #NS2 was placed in another tube together with 19 NSs collected from 19 negative patients. Both tubes were then tested with conventional molecular rRT-PCR and the strength of pooling nasal testing was compared with the molecular test performed on the single NS of each positive patient. The pooling strategy detected SARS-CoV-2 RNA to a similar extent to the single test, even when Ct value is on average high (Ct 37–38), confirming that test sensibility is not substantially affected even if the pool contains only one low viral load positive sample. Furthermore, the pooling strategy have benefits for SARS-CoV-2 routinary monitoring of groups in regions with a low SARS-CoV-2 prevalence.

Parvovirus infections have been well known for around 100 years in domestic carnivores. However, the use of molecular assays and metagenomic approaches for virus discovery and characterization has led to the detection of novel parvovirus species and/or variants in dogs. Although some evidence suggests that these emerging canine parvoviruses may act as primary causative agents or as synergistic pathogens in the diseases of domestic carnivores, several aspects regarding epidemiology and virus–host interaction remain to be elucidated.

Feline calicivirus (FCV) is a common viral pathogen found in domestic cats. FCV is highly contagious and demonstrates a high genetic variability. Upper respiratory tract disease, oral ulcerations, salivation, and gingivitis–stomatitis have been regarded as typical clinical signs of FCV infection. Ulcerative dermatitis, abortion, severe pneumonia, enteritis, chronic stomatitis, and virulent systemic disease have been reported more sporadically. Limping syndrome has been also described either in naturally or experimentally FCV-infected cats. In this study, we monitored a small outbreak of FCV infection in two household cats, in which limping disease was monitored with a 12-day lag time. The complete genome sequence was determined for the viruses isolated from the oropharyngeal and rectal swabs of the two animals, mapping up to 39 synonymous nucleotide mutations. The four isolates were sensitive to low pH conditions and trypsin treatment, a pattern usually associated with viruses isolated from the upper respiratory tract. Overall, the asynchronous pattern of infections and the results of genome sequencing suggest that a virus of respiratory origin was transmitted between the animals and that the FCV strain was able to retain the limping disease pathotype during the transmission chain, as previously observed in experimental studies with FCV strains associated with lameness.

Equine periodontal disease (EPD) is a painful oral inflammatory syndrome characterized by multifactorial pathogenesis. Although it is well known that bacterial proliferation and consequent gingivitis are caused by the decomposition process of food residues, in hypsodont species, the pathogenetic role and the different bacterial species involved in the progression of EPD must be fully clarified. This study aimed to investigate the association of bacteria, including the complex red bacteria (RCB), with EPD, and to evaluate possible EPD risk factors. Bacterial species, including Treponema denticola, Tannerella forsythia, Porphyromonas gingivalis (belonging to the RCB), Fusobacterium nucleatum, Veilonella parvula, and Prevotella intermedia, were investigated in 125 oral swabs from healthy and EPD-affected horses using real-time multiplex PCR. Subsequently, possible risk factors (i.e., age, gender, and breed of the animals and type of feed used) were evaluated using univariate and multivariate analyses. Tannerella spp. and Treponema spp. were detected in a significantly higher proportion of horses affected by EPD than in healthy animals, although pathogens belonging to RCB were detected in low number of horses. At the same time, none of the investigated pathogens was significantly associated with a particular stage of disease severity. Horses aged older than 20 years were at higher risk of EPD. The high rate of coinfection, statistically associated with EPD, supports the hypothesis that EPD is a complex syndrome characterized by the possible simultaneous involvement of several pathogens and an increased risk depending on the animal’s age. Constant oral hygiene is the best prevention to prevent and treat the disease, especially in old animals.

Canine coronavirus (CCoV) is a positive-strand RNA virus generally responsible for mild-to-severe gastroenteritis in dogs. In recent years, new CCoVs with acquired pathogenic characteristics have emerged, turning the spotlight on the evolutionary potential of CCoVs. To date, two genotypes are known, CCoV type I and CCoV type II, sharing up to 96% nucleotide identity in the genome but highly divergent in the spike gene. In 2009, the detection of a novel CCoV type II, which likely originated from a double recombination event with transmissible gastroenteritis virus (TGEV), led to the proposal of a new classification: CCoV type IIa, including classical CCoVs and CCoV type IIb, including TGEV-like CCoV. Recently, a virus strictly correlated to CCoV was isolated from children with pneumonia in Malaysia. The HuPn-2018 strain, classified as a novel canine–feline-like recombinant virus, is supposed to have jumped from dogs into people. A novel CoV of canine origin, HuCCoV_Z19Haiti, closely related to the Malaysian strain was also detected in a man with fever after travel to Haiti, suggesting that infection with Malaysian-like strains may occur. These data and the emergence of highly pathogenic CoVs in humans underscore the significant threat that CoV spillovers pose to humans and how we should mitigate this hazard.

Massive sequencing techniques have allowed us to develop straightforward approaches for the whole genome sequencing of viruses, including influenza viruses, generating information that is useful for improving the levels and dimensions of data analysis, even for archival samples. Using the Nanopore platform, we determined the whole genome sequence of an H3N8 equine influenza virus, identified from a 2005 outbreak in Apulia, Italy, whose origin had remained epidemiologically unexplained. The virus was tightly related (>99% at the nucleotide level) in all the genome segments to viruses identified in Poland in 2005–2008 and it was seemingly introduced locally with horse trading for the meat industry. In the phylogenetic analysis based on the eight genome segments, strain ITA/2005/horse/Bari was found to cluster with sub-lineage Florida 2 in the HA and M genes, whilst in the other genes it clustered with strains of the Eurasian lineage, revealing a multi-reassortant nature.

The liver is a very tolerogenic organ. It is continually exposed to a multitude of antigens and is able to promote an effective immune response against pathogens and simultaneously immune tolerance against self-antigens. In spite of strong peripheral and central tolerogenic mechanisms, loss of tolerance can occur in autoimmune liver diseases, such as autoimmune hepatitis (AIH) through a combination of genetic predisposition, environmental factors, and an imbalance in immunological regulatory mechanisms. The liver hosts several types of conventional resident antigen presenting cells (APCs) such as dendritic cells, B cells and macrophages (Kupffer cells), and unconventional APCs including liver sinusoidal endothelial cells, hepatic stellate cells and hepatocytes. By standard (direct presentation and cross-presentation) and alternative mechanisms (cross-dressing and MHC class II-dressing), liver APCs presents self-antigen to naive T cells in the presence of costimulation leading to an altered immune response that results in liver injury and inflammation. Additionally, the transport of antigens and antigen:MHC complexes by trogocytosis and extracellular vesicles between different cells in the liver contributes to enhance antigen presentation and amplify autoimmune response. Here, we focus on the impact of antigen presentation on the immune response in the liver and on the functional role of the immune cells in the induction of liver inflammation. A better understanding of these key pathogenic aspects could facilitate the establishment of novel therapeutic strategies in AIH.

The indiscriminate use of antimicrobials in poultry farms is linked to the increase in multi-resistant bacteria. Accordingly, based on the antimicrobial properties of Thyme Essential Oil (TEO), the present study evaluated the effects of TEO on the reduction of common microbial contaminants and Salmonella on poultry litter. A litter bulk sample was collected in a broiler farm and qualitative/quantitative investigations identified Escherichia coli and Mammaliicoccus lentus. The experimental contamination with Salmonella Derby wild strain was also performed. All pathogens showed phenotypic and genotypic resistance to different classes of antibiotics. The litter, split in different units, was treated with aqueous solutions of TEO at different concentrations (5% to 1.25%), demonstrating its effectiveness in reducing the total number of bacteria. The strongest antibacterial action was observed at the lowest concentration against Enterobacteriaceae, with a growth reduction compared to the positive control of 73.3% and 77.8% against E. coli and Salmonella Derby, respectively, while towards M. lentus the reduction was 50%. Our data confirm the antimicrobial activity of TEO and suggest its possible application for the treatment of poultry litter as an effective and natural approach for the prevention of diseases caused by the most common bacteria that colonize poultry farms, counteracting the onset of antibiotic resistance.

This study evaluated sealing properties of zinc oxide/calcium sulphate filling material (Plastor©) to prevent bacterial infiltration in simulated Class 1 cavities with and without the presence of a spacer apically the restoration. Twenty-eight experimental Eppendorf tubes containing Tryptic Soy Agar were prepared and divided into seven groups: group A (Plastor©), group B (Plastor© + cotton pellet), group C (Plastor© + cotton pellet soaked of m-cresyl acetate), group D (Plastor© + cotton pellet soaked of eugenol), group E (Plastor© + PTFE pellet), group F (positive control), and group G (negative control). All prepared vials were individually immersed in tubes containing 5 mL of a suspension of Streptococcus mutans (105 CFU/mL) previously prepared and then incubated at 37 °C for 5 and 10 days. Subsequently, agar was collected and tested for S.mutans using real-time PCR. Aliquots of each agar samples were separately seeded on TSA for S. mutans isolation. The real-time PCR tests were negative for S. mutans on all the vials at both T5 and T10, except for positive control resulted positive at both T5 and T10. The isolation tests showed bacterial growth of S. mutans only with the agar samples collected from the vials of positive control, confirming real-time PCR tests. This in vitro study evidenced complete sealing ability of zinc oxide/calcium sulphate in temporary restorations of Class 1 cavities without mechanical loading at 10 days and complete immersion in a bacterial suspension. Moreover, sealing ability is not influenced by presence of spacers.

Aim of the study: to evaluate root filling remnants after secondary root canal treatments (SRCTs) of oval-shaped canals with X-rays and cone beam computed tomography (CBCT). The SRCTs were performed using reciprocating NiTi instruments. Methods: Single-rooted teeth (N = 64) were randomly treated with Reciproc Blue (RB) and filled with AH Plus/single cone (SC group) or AH Plus/Guttafusion (GF group). After seven days of storage in HBSS (Hanks balanced salt solution), Gates Glidden burs #2/3 and RB #25 and #40 were used for the SRCTs. The time to complete the procedure was measured. X-rays and CBCT were used to calculate, respectively, the area and the volume occupied by the remnants in the coronal, middle, and apical thirds of each canal. Environmental scanning electron microscopy (ESEM) and energy dispersive X-ray spectroscopy (EDX) were used for qualitative evaluation and morphology composition of the remnants in sectioned roots. A statistical analysis was performed using Sigma Plot (version 13, IBM, Armonk, NY, USA). The study was designed according to PRILE guidelines. Results: After the SRCTs, the middle thirds of the root canals showed the presence of remnants in both groups, as demonstrated by X-rays and CBCT. The GF group showed a statistically significant higher volume of remnants than the SC Group only in the middle third. The ESEM supported by the EDX revealed the remnant composition by the detection of trace elements of sealer and gutta-percha in all root canals. Conclusion: The study demonstrated that the middle third of root canals is a critical region where remnants were packed and spread in the buccal-lingual sides of canals. ESEM-EDX detected a fine layer of filling remnants in all root thirds, suggesting a larger canal contamination than the X-rays and CBCT examinations revealed.

A massive periodontal destruction can affect the root canal (RC) system and potentially expose the pulpo-dentinal complex to opportunistic bacteria. Streptococcus mutans is a major pathogen of human caries and periodontal diseases, and its virulence mostly resides in the ability to adhere to collagen and form biofilms, due to collagen-binding proteins (CBPs) Cnm and Cbm. Seventeen patients affected by severe endo-periodontal lesions without caries and/or exposure of pulpal tissue were subjected to tooth extraction and samples for microbiological investigation were collected from the root surface (RS) and RC. The collected swabs were cultivated and subjected to the quantitative real-time PCR (qPCR) for the detection of S. mutans and to the PCR for the cnm/cbm genes investigation, followed by next-generation sequencing (NGS). S. mutans DNA was detected in 12/17 (70.5%) RS samples and in 8/17 (47.0%) RC samples. In the CBPs screening of positive samples, the cnm gene was detected in 4/12 (33.3%) RS and in 1/8 (12.5%) RC samples, whilst all the samples tested negative for the cbm gene. The presence of the cnm gene could enhance the local virulence of the pathogens. Therefore, S. mutans have to be included as potential periodontopathogen bacterium in severe or refractory forms of periodontal diseases.

Two mechanisms are involved in the immune escape of cancer cells: the immunoediting of tumor cells and the suppression of the immune system. Both processes have been revealed in multiple myeloma (MM). Complex interactions between tumor plasma cells and the bone marrow (BM) microenvironment contribute to generate an immunosuppressive milieu characterized by high concentration of immunosuppressive factors, loss of effective antigen presentation, effector cell dysfunction, and expansion of immunosuppressive cell populations, such as myeloid-derived suppressor cells, regulatory T cells and T cells expressing checkpoint molecules such as programmed cell death 1. Considering the great immunosuppressive impact of BM myeloma microenvironment, many strategies to overcome it and restore myeloma immunosurveillance have been elaborated. The most successful ones are combined approaches such as checkpoint inhibitors in combination with immunomodulatory drugs, anti-monoclonal antibodies, and proteasome inhibitors as well as chimeric antigen receptor (CAR) T cell therapy. How best to combine anti-MM therapies and what is the optimal timing to treat the patient are important questions to be addressed in future trials. Moreover, intratumor MM heterogeneity suggests the crucial importance of tailored therapies to identify patients who might benefit the most from immunotherapy, reaching deeper and more durable responses.

A patient with an unusual endo-periodontal lesion, without coronal decay or damage, likely caused by a deep periodontal lesion with subsequent endodontic bacterial migration, required medical care. Next-generation sequencing (NGS) was used to assess the endodontic microbiota in vestibular and palatal canals after tooth extraction, evidencing a predominant population (Fusobacterium nucleatum) in one endodontic canal, and a mixed bacterial population with six major populations almost equally distributed in the other endodontic canal (F. nucleatum, Porphyromonas gingivalis, P. endodontis, Parvimonas, Peptostreptococcus stomatis, Prevotella multiformis). These data could suggest different, separated ecologic niches in the same endodontic system, with potentially different pathogenicity levels, clinical manifestations and prognoses for every single canal of the same tooth.

The antimicrobial resistance (AMR) phenomenon is an emerging global problem and is induced by overuse and misuse of antibiotics in medical practice. In total, 10% of antibiotic prescriptions are from dentists, usually to manage oro-dental pains and avoid postsurgical complications. Recent research and clinical evaluations highlight new therapeutical approaches with a reduction in dosages and number of antibiotic prescriptions and recommend focusing on an accurate diagnosis and improvement of oral health before dental treatments and in patients’ daily lives. In this article, the most common clinical and operative situations in dental practice, such as endodontics, management of acute alveolar abscesses, extractive oral surgery, parodontology and implantology, are recognized and summarized, suggesting possible guidelines to reduce antibiotic prescription and consumption, maintaining high success rates and low complications rates. Additionally, the categories of patients requiring antibiotic administration for pre-existing conditions are recapitulated. To reduce AMR threat, it is important to establish protocols for treatment with antibiotics, to be used only in specific situations. Recent reviews demonstrate that, in dentistry, it is possible to minimize the use of antibiotics, thoroughly assessing patient’s conditions and type of intervention, thus improving their efficacy and reducing the adverse effects and enhancing the modern concept of personalized medicine.

Hepatocellular carcinoma (HCC) is one of most common cancers and the fourth leading cause of death worldwide. Commonly, HCC development occurs in a liver that is severely compromised by chronic injury or inflammation. Liver transplantation, hepatic resection, radiofrequency ablation (RFA), transcatheter arterial chemoembolization (TACE), and targeted therapies based on tyrosine protein kinase inhibitors are the most common treatments. The latter group have been used as the primary choice for a decade. However, tumor microenvironment in HCC is strongly immunosuppressive; thus, new treatment approaches for HCC remain necessary. The great expression of immune checkpoint molecules, such as programmed death-1 (PD-1), cytotoxic T-lymphocyte antigen 4 (CTLA-4), lymphocyte activating gene 3 protein (LAG-3), and mucin domain molecule 3 (TIM-3), on tumor and immune cells and the high levels of immunosuppressive cytokines induce T cell inhibition and represent one of the major mechanisms of HCC immune escape. Recently, immunotherapy based on the use of immune checkpoint inhibitors (ICIs), as single agents or in combination with kinase inhibitors, anti-angiogenic drugs, chemotherapeutic agents, and locoregional therapies, offers great promise in the treatment of HCC. This review summarizes the recent clinical studies, as well as ongoing and upcoming trials.