Collagen Matrix to Restore the Tympanic Membrane: Developing a Novel Platform to Treat Perforations.

Modern otology faces challenges in treating tympanic membrane (TM) perforations. Instead of surgical intervention, alternative treatments using biomaterials are emerging. Recently, we developed a robust collagen membrane using semipermeable barrier-assisted electrophoretic deposition (SBA-EPD). In this study, a collagen graft shaped like a sponge through SBA-EPD was used to treat acute and chronic TM perforations in a chinchilla model. A total of 24 ears from 12 adult male chinchillas were used in the study. They were organized into four groups. The first two groups had acute TM perforations and the last two had chronic TM perforations. We used the first and third groups as controls, meaning they did not receive the implant treatment. The second and fourth groups, however, were treated with the collagen graft implant. Otoscopic assessments were conducted on days 14 and 35, with histological evaluations and TM vibrational studies performed on day 35. The groups treated with the collagen graft showed fewer inflammatory changes, improved structural recovery, and nearly normal TM vibrational properties compared to the controls. The porous collagen scaffold successfully enhanced TM regeneration, showing high biocompatibility and biodegradation potential. These findings could pave the way for clinical trials and present a new approach for treating TM perforations.

Vocal fold restoration after scarring: biocompatibility and efficacy of an MSC-based bioequivalent.

BACKGROUND: There is growing interest to application of regenerative medicine approaches in otorhinolaryngological practice, especially in the framework of the therapy of vocal fold (VF) scar lesions. The used conservative and surgical methods, despite the achieved positive outcomes, are frequently unpredictable and do not result in the restoration of the VF's lamina propria's structure, which provides the mechanical properties necessary for vibration. In this connection, the aim of this study was to ascertain the safety and efficacy of a bioequivalent in the treatment of VF scars using a rabbit model of chronic damage. METHODS: The bioequivalent consisted of a hydrogel system based on a PEG-fibrin conjugate and human bone marrow-derived MSC. It was characterized and implanted heterotopically into rats and orthotopically into rabbits after VF scar excision. RESULTS: We showed that the fabricated bioequivalent consisted of viable cells retaining their metabolic and proliferative activity. While being implanted heterotopically, it had induced the low inflammatory reaction in 7 days and was well tolerated. The orthotopic implantation showed that the gel application was characterized by a lower hemorrhage intensity (p = 0.03945). The intensity of stridor and respiratory rate between the groups in total and between separate groups had no statistically significant difference (p = 0.96 and p = 1; p = 0.9593 and p = 0.97…1, respectively). In 3 days post-implantation, MSC were detected only in the tissues closely surrounding the VF defect. The bioequivalent injection caused that the scar collagen fibers were packed looser and more frequently mutually parallel that is inherent in the native tissue (p = 0.018). In all experimental groups, the fibrous tissue's ingrowth in the adjacent exterior muscle tissue was observed; however, in Group 4 (PEG-Fibrin + MSC), it was much less pronounced than it was in Group 1 (normal saline) (p = 0.008). The difference between the thicknesses of the lamina propria in the control group and in Group 4 was not revealed to be statistically significant (p = 0.995). The Young's modulus of the VF after the bioequivalent implantation (1.15 ± 0.25 kPa) did not statistically significantly differ from the intact VF modulus (1.17 ± 0.45 kPa); therefore, the tissue properties in this group more closely resembled the intact VF. CONCLUSIONS: The developed bioequivalent showed to be biocompatible and highly efficient in the restoration of VF's tissue.

Measurement of Patient-Derived Glioblastoma Cell Response to Temozolomide Using Fluorescence Lifetime Imaging of NAD(P)H.

Personalized strategies in glioblastoma treatment are highly necessary. One of the possible approaches is drug screening using patient-derived tumor cells. However, this requires reliable methods for assessment of the response of tumor cells to treatment. Fluorescence lifetime imaging microscopy (FLIM) is a promising instrument to detect early cellular response to chemotherapy using the autofluorescence of metabolic cofactors. Here, we explored FLIM of NAD(P)H to evaluate the sensitivity of patient-derived glioma cells to temozolomide (TMZ) in vitro. Our results demonstrate that the more-responsive cell cultures displayed the longest mean fluorescence lifetime τm after TMZ treatment due to an increase in the protein-bound NAD(P)H fraction α2 associated with a shift to oxidative phosphorylation. The cell cultures that responded poorly to TMZ had generally shorter τm, i.e., were more glycolytic, and showed no or insignificant changes after treatment. The FLIM data correlate well with standard measurements of cellular drug response-cell viability and proliferation index and clinical response in patients. Therefore, FLIM of NAD(P)H provides a highly sensitive, label-free assay of treatment response directly on patient-derived glioblastoma cells and can become an innovative platform for individual drug screening for patients.

Eosinophilic Otitis Media: Modern Aspects of Pathogenesis, Clinical Features, Diagnosis and Treatment.

Eosinophilic otitis media (EOM) is an inflammatory chronic disease of the middle ear, characterized by the presence of a particularly viscous effusion with a high content of protein toxins of eosinophilic origin in the middle ear cavity. The pathology has relationship with bronchial asthma, allergic rhinitis and chronic rhinosinusitis with nasal polyps. EOM is characterized by a sluggish course, a tendency to relapse, which can lead to a gradual hearing decrease up to complete deafness. In this paper, we reviewed the international literature with special attention to pathogenesis and treatment management.

Stem cell therapy for vocal fold regeneration after scarring: a review of experimental approaches.

This review aims at becoming a guide which will help to plan the experimental design and to choose adequate methods to assess the outcomes when testing cell-based products in the treatment of the damaged vocal folds. The requirements to preclinical trials of cell-based products remain rather hazy and dictated by the country regulations. Most parameters like the way the cells are administered, selection of the cell source, selection of a carrier, and design of in vivo studies are decided upon by each research team and may differ essentially between studies. The review covers the methodological aspects of preclinical studies such as experimental models, characterization of cell products, assessment of the study outcome using molecular, morphological and immunohistochemical analyses, as well as measuring the tissue physical properties. The unified recommendations to perform preclinical trials could significantly facilitate the translation of cell-based products into the clinical practice.

OCT-Guided Surgery for Gliomas: Current Concept and Future Perspectives.

Optical coherence tomography (OCT) has been recently suggested as a promising method to obtain in vivo and real-time high-resolution images of tissue structure in brain tumor surgery. This review focuses on the basics of OCT imaging, types of OCT images and currently suggested OCT scanner devices and the results of their application in neurosurgery. OCT can assist in achieving intraoperative precision identification of tumor infiltration within surrounding brain parenchyma by using qualitative or quantitative OCT image analysis of scanned tissue. OCT is able to identify tumorous tissue and blood vessels detection during stereotactic biopsy procedures. The combination of OCT with traditional imaging such as MRI, ultrasound and 5-ALA fluorescence has the potential to increase the safety and accuracy of the resection. OCT can improve the extent of resection by offering the direct visualization of tumor with cellular resolution when using microscopic OCT contact probes. The theranostic implementation of OCT as a part of intelligent optical diagnosis and automated lesion localization and ablation could achieve high precision, automation and intelligence in brain tumor surgery. We present this review for the increase of knowledge and formation of critical opinion in the field of OCT implementation in brain tumor surgery.

Chronic tympanic membrane perforation: Histopathological evidence of the experimental model.

OBJECTIVES: This study aims to compare the effectiveness of three different models of chronic tympanic membrane perforations. MATERIALS: The experimental study included 18 male chinchillas, divided into 3 equal groups. Group 1 perforations were performed with infolding technique myringotomy. Laser myringotomy was performed for perforation creation in Group 2. Group 3 perforations were performed with infolding myringotomy combined with ventilation tube insertion. At the end of the follow-up period, which lasts 8 weeks, all tympanic membranes with patent perforations were examined histologically. RESULTS: Although, the mean perforation patency in Group 2 was significantly higher than in Group 1 (5 vs. 2.4 weeks, p < 0.01), both of them failed in creation of chronic perforation according to time parameters. Group 3 demonstrated the longest mean perforation patency among investigated models (8 weeks). In Group 3, histological examination of perforations, which were considered to be chronic, revealed, that stratified squamous epithelium continued from the lateral surface around the perforation edge to join with the medial mucosal layer of TM. CONCLUSION: Our findings demonstrated that the combination of infolding technique and ventilation tube insertion seems to be a potential candidate for an effective animal model of tympanic membrane perforation. Further large-scale studies are required to verify our promising results.

Development of novel darunavir amorphous solid dispersions with mesoporous carriers.

The aim of this work was to compare mesoporous carriers based on silica and magnesium aluminosilicate in the amorphous solid dispersion production. Darunavir has been selected as an active pharmaceutical ingredient that is classified as a Class 2 BCS substance and exists in two commercially available forms: crystalline ethanolate and amorphous. In the course of the study, the conditions for the preparation of amorphous samples with the selected carriers were evaluated within the framework of the most common methods for obtaining solid dispersions - hot-melt extrusion, solvent wetting, and spray drying. It was determined that the obtained dispersion properties almost completely repeat the properties of the corresponding carriers. The resulting dispersions were examined in a dissolution test and the best ones was used to formulate tablets, which were studied in an in vitro dissolution test with the original Prezista. The proposed tablet formulation showed improved dissolution compared to the original one. It was also found that silica supports have a greater positive contribution to darunavir dissolution - both ethanolate or amorphous forms.

Analysis of sequence divergence in redfin (Cypriniformes, Cyprinidae, Tribolodon) based on mtDNA and nDNA markers with inferences in systematics and genetics of speciation.

To clarify relationship of species of the genus Tribolodon in the Russian part of their distribution ranges, two mitochondrial markers (Co-1 and Cyt-b), a nuclear marker (Rho), and a gene marker of rDNA internal transcribed spacer (ITS-1,2) were used. Depending on the marker, different numbers of species groups were detected by the ABGD method, but in combination with the analysis of phylograms, these data generally support the known species clusters and regional intraspecies groups. A complex analysis of sequences from three redfin species within the area of the study, based on four marker genes and using the methods of molecular phylogenetics, ordination of genetic distances, recombinant analysis, and population genetic approaches, has revealed clusters of three commonly recognized species, regional intraspecific groups or individuals of local populations, and few hybrid individuals. DNA barcoding technique proved to be efficient with the use of two mtDNA markers: Co-1 and Cyt-b. It has been found that analysis of insertions and substitutions within the ITS-1,2 gene marker is also suitable for identification of Tribolodon species. Results of the studies of local groups do not confirm a sufficient level of differences for defining any new taxa of a species rank in the genus Tribolodon.

Rapid detection and strain typing of Chlamydia trachomatis using a highly multiplexed microfluidic PCR assay.

Nucleic acid amplification tests (NAATs) are recommended by the CDC for detection of Chlamydia trachomatis (Ct) urogenital infections. Current commercial NAATs require technical expertise and sophisticated laboratory infrastructure, are time-consuming and expensive, and do not differentiate the lymphogranuloma venereum (LGV) strains that require a longer duration of treatment than non-LGV strains. The multiplexed microfluidic PCR-based assay presented in this work simultaneously interrogates 13 loci to detect Ct and identify LGV and non-LGV strain-types. Based on amplified fragment length polymorphisms, the assay differentiates LGV, ocular, urogenital, and proctocolitis clades, and also serovars L1, L2, and L3 within the LGV group. The assay was evaluated in a blinded fashion using 95 clinical swabs, with 76 previously reported as urogenital Ct-positive samples and typed by ompA genotyping and/or Multi-Locus Sequence Typing. Results of the 13-plex assay showed that 51 samples fell within urogenital clade 2 or 4, 24 samples showed both clade 2 and 4 signatures, indicating possible mixed infection, gene rearrangement, or inter-clade recombination, and one sample was a noninvasive trachoma biovar (either a clade 3 or 4). The remaining 19 blinded samples were correctly identified as LGV clade 1 (3), ocular clade 3 (4), or as negatives (12). To date, no NAAT assay can provide a point-of-care applicable turnaround time for Ct detection while identifying clinically significant Ct strain types to inform appropriate treatment. Coupled with rapid DNA processing of clinical swabs (approximately 60 minutes from swab-in to result-out), the assay has significant potential as a rapid POC diagnostic for Ct infections.

DNA barcoding for squids of the family Gonatidae (Cephalopoda: Teuthida) from the boreal North Pacific.

A fragment of cytochrome c oxidase I was used to assess whether species of the squid family Gonatidae from the North Pacific could be identified using DNA barcoding approach. Pairwise intra- and interspecific p-distances were assessed, and systematic relationships among species were estimated by NJ analysis. Examined species formed well-differentiated species-specific clades on the neighbor-joining and Bayesian trees. Multiple taxa formed clades supported by both tree topologies and species hypothesis-free ABGD method. Species morphologically identified as Gonatus tinro and Gonatopsis okutanii demonstrated intraspecific level of molecular genetic divergence (0.2-0.3%) indicating that they are conspecific. Genetic differences between the G. berryi clade and a squid morphologically close to that species may indicate a new cryptic species. High levels (>6.2%) of genetic differentiation within B. borealis suggested the existence of two cryptic species. This study confirms the usefulness of DNA barcoding for identifying species as well as discovering cryptic diversity in the gonatid squids, and indicates the need for further deeper insights into the phylogeny of the Gonatidae.

Rapid focused sequencing: a multiplexed assay for simultaneous detection and strain typing of Bacillus anthracis, Francisella tularensis, and Yersinia pestis.

BACKGROUND: The intentional release of Bacillus anthracis in the United States in 2001 has heightened concern about the use of pathogenic microorganisms in bioterrorism attacks. Many of the deadliest bacteria, including the Class A Select Agents Bacillus anthracis, Francisella tularensis, and Yersinia pestis, are highly infectious via the pulmonary route when released in aerosolized form. Hence, rapid, sensitive, and reliable methods for detection of these biothreats and characterization of their potential impact on the exposed population are of critical importance to initiate and support rapid military, public health, and clinical responses. METHODOLOGY/PRINCIPAL FINDINGS: We have developed microfluidic multiplexed PCR and sequencing assays based on the simultaneous interrogation of three pathogens per assay and ten loci per pathogen. Microfluidic separation of amplified fluorescently labeled fragments generated characteristic electrophoretic signatures for identification of each agent. The three sets of primers allowed significant strain typing and discrimination from non-pathogenic closely-related species and environmental background strains based on amplicon sizes alone. Furthermore, sequencing of the 10 amplicons per pathogen, termed "Rapid Focused Sequencing," allowed an even greater degree of strain discrimination and, in some cases, can be used to determine virulence. Both amplification and sequencing assays were performed in microfluidic biochips developed for fast thermal cycling and requiring 7 µL per reaction. The 30-plex sequencing assay resulted in genotypic resolution of 84 representative strains belonging to each of the three biothreat species. CONCLUSIONS/SIGNIFICANCE: The microfluidic multiplexed assays allowed identification and strain differentiation of the biothreat agents Bacillus anthracis, Francisella tularensis, and Yersinia pestis and clear discrimination from closely-related species and several environmental background strains. The assays may be extended to detect a large number of pathogens, are applicable to the evaluation of both environmental and clinical samples, and have the potential to be applied in military, public health, and clinical diagnostic settings.

A multiplexed microfluidic PCR assay for sensitive and specific point-of-care detection of Chlamydia trachomatis.

BACKGROUND: Chlamydia trachomatis (Ct) is the most common cause of bacterial sexually transmitted diseases (STD) worldwide. While commercial nucleic acid amplification tests (NAAT) are available for Ct, none are rapid or inexpensive enough to be used at the point-of-care (POC). Towards the first Ct POC NAAT, we developed a microfluidic assay that simultaneously interrogates nine Ct loci in 20 minutes. METHODOLOGY AND PRINCIPAL FINDINGS: Endocervical samples were selected from 263 women at high risk for Ct STDs (∼35% prevalence). A head-to-head comparison was performed with the Roche-Amplicor NAAT. 129 (49.0%) and 88 (33.5%) samples were positive by multiplex and Amplicor assays, respectively. Sequencing resolved 71 discrepant samples, confirming 53 of 53 positive multiplex samples and 12 of 18 positive Amplicor samples. The sensitivity and specificity were 91.5% and 100%, and 62.4% and 95.9%, respectively, for multiplex and Amplicor assays. Positive and negative predictive values were 100% and 91%, and 94.1% and 68.6%, respectively. CONCLUSIONS: This is the first rapid multiplex approach to Ct detection, and the assay was also found to be superior to a commercial NAAT. In effect, nine simultaneous reactions significantly increased sensitivity and specificity. Our assay can potentially increase Ct detection in globally diverse clinical settings at the POC.