Quantification of Solid Embryonic Cerebellar Graft Volume in a Degenerative Ataxia Model.

Substitution of lost neurons by neurotransplantation would be a possible management of advanced degenerative cerebellar ataxias in which insufficient cerebellar reserve remains. In this study, we examined the volume and structure of solid embryonic cerebellar grafts in adult Lurcher mice, a model of olivocerebellar degeneration, and their healthy littermates. Grafts taken from enhanced green fluorescent protein (EGFP)-positive embryos were injected into the cerebellum of host mice. Two or six months later, the brains were examined histologically. The grafts were identified according to the EGFP fluorescence in frozen sections and their volumes were estimated using the Cavalieri principle. For gross histological evaluation, graft-containing slices were processed using Nissl and hematoxylin-eosin staining. Adjustment of the volume estimation approach suggested that it is reasonable to use all sections without sampling, but that calculation of values for up to 20% of lost section using linear interpolation does not constitute substantial error. Mean graft volume was smaller in Lurchers than in healthy mice when examined 6 months after the transplantation. We observed almost no signs of graft destruction. In some cases, compact grafts disorganized the structure of the host's cerebellar cortex. In Lurchers, the grafts had a limited contact with the host's cerebellum. Also, graft size was of greater variability in Lurchers than in healthy mice. The results are in compliance with our previous findings that Lurcher phenotype-associated factors have a negative effect on graft development. These factors can hypothetically include cerebellar morphology, local tissue milieu, or systemic factors such as immune system abnormalities.

Dental Implant Placement in Medically Compromised Patients: A Literature Review.

As a discipline of dentistry, oral implantology deals with the diagnosis, design, insertion, restoration, and/or management of alloplastic or autogenous oral structures for the purpose of regaining contour, function, aesthetics, and speech in a partially or completely edentulous patient. The present review aims to provide the currently available knowledge about the impact of certain systemic disorders and the usage of some medications on the survival rate of dental implant therapy and to highlight the importance of patient management under these conditions. Diabetes, osteoporosis, cardiovascular diseases, and the intake of some medications can increase the risk of the failure of a dental implant. Even though there are relatively few medical contraindications to dental implant treatment, certain conditions may increase the risk of failure or complications.

Molecular modelling of fullerene C60 functionalized by nitric oxide for use in biological environment.

The unique potential of fullerene C60 for various biological applications has ignited significant interest. However, its inherent non-polarity poses a critical challenge for its effective integration within biological systems. This study delves into the intricate physicochemical characteristics of the innovative [C60 + NO] complex using density functional theory and time-dependent density functional theory. The computational analyses encompass molecular charge, surface electrostatic potential, and dipole moment evaluations. Impressively, the dipole moment of the [C60 + NO] complex significantly increases to 12.92 D. Meticulous surface analysis reveals a subtle interplay between molecular structures, indicating weak interactions. The analysis of the absorption spectrum unveils a noteworthy red-shift of 200 nm subsequent to complex formation. To elucidate the electron transfer mechanisms, we explore photo-induced electron transfer through CAM-B3LYP. This exploration elucidates intricate pathways governing electron transfer, with complementary insights gleaned from Marcus theory's outputs, especially the Gibbs free energy of electron transfer. Changes in the physicochemical properties of approaching C60 and NO molecules reveal interesting results compared to separate molecules. These findings resonate profoundly in the context of potential biological and pharmaceutical utilization. With implications for the biomedical area, the outcomes linked to the [C60 + NO] complex kindle optimism for pioneering biomedical applications.

Optimizing PCL/PLGA Scaffold Biocompatibility Using Gelatin from Bovine, Porcine, and Fish Origin.

This research introduces a novel approach by incorporating various types of gelatins, including bovine, porcine, and fish skin, into polycaprolactone and poly (lactic-co-glycolic acid) using a solvent casting method. The films are evaluated for morphology, mechanical properties, thermal stability, biodegradability, hemocompatibility, cell adhesion, proliferation, and cytotoxicity. The results show that the incorporation of gelatins into the films alters their mechanical properties, with a decrease in tensile strength but an increase in elongation at break. This indicates that the films become more flexible with the addition of gelatin. Gelatin incorporation has a limited effect on the thermal stability of the films. The composites with the gelatin show higher biodegradability with the highest weight loss in the case of fish gelatin. The films exhibit high hemocompatibility with minimal hemolysis observed. The gelatin has a dynamic effect on cell behavior and promotes long-term cell proliferation. In addition, all composite films reveal exceptionally low levels of cytotoxicity. The combination of the evaluated parameters shows the appropriate level of biocompatibility for gelatin-based samples. These findings provide valuable insights for future studies involving gelatin incorporation in tissue engineering applications.

Next Generation Sequencing Analysis and its Benefit for Targeted Therapy of Lung Adenocarcinoma.

BACKGROUND/AIM: Targeted therapy has become increasingly important in treating lung adenocarcinoma, the most common subtype of lung cancer. Next-generation sequencing (NGS) enables precise identification of specific genetic alterations in individual tumor tissues, thereby guiding targeted therapy selection. This study aimed to analyze mutations present in adenocarcinoma tissues using NGS, assess the benefit of targeted therapy and evaluate the progress in availability of targeted therapies over last five years. PATIENTS AND METHODS: The study included 237 lung adenocarcinoma patients treated between 2018-2020. The Archer FusionPlex CTL panel was used for NGS analysis. RESULTS: Gene variants covered by the panel were detected in 57% patients and fusion genes in 5.9% patients. At the time of the study, 34 patients (14.3% of patients) were identified with a targetable variant. Twenty-five patients with EGFR variants, 8 patients with EML4-ALK fusion and one patient with CD74-ROS1 fusion received targeted therapy. Prognosis of patients at advanced stages with EGFR variants treated by tyrosine kinase inhibitors and patients with EML4-ALK fusion treated by alectinib was significantly favorable compared to patients without any targetable variant treated by chemotherapy (p=0.0172, p=0.0096, respectively). Based on treatment guidelines applicable in May 2023, the number of patients who could profit from targeted therapy would be 64 (27.0% of patients), this is an increase by 88% in comparison to recommendations valid in 2018-2020. CONCLUSION: As lung adenocarcinoma patients significantly benefit from targeted therapy, the assessment of mutational profiles using NGS could become a crucial approach in the routine management of oncological patients.

Chitosan and cellulose-based composite hydrogels with embedded titanium dioxide nanoparticles as candidates for biomedical applications.

Hydrogel based matrices and titanium dioxide (TiO2) nanoparticles (NPs) are well established materials in bone tissue engineering. Nevertheless, there is still a challenge to design appropriate composites with enhanced mechanical properties and improved cell growth. Progressing in this direction, we synthesized nanocomposite hydrogels by impregnating TiO2 NPs in a chitosan and cellulose-based hydrogel matrix containing polyvinyl alcohol (PVA), to enhance the mechanical stability and swelling capacity. Although, TiO2 has been incorporated into single and double component matrix systems, it has rarely been combined with a tri-component hydrogel matrix system. The doping of NPs was confirmed by Fourier transform infrared spectroscopy, Raman spectroscopy, scanning electron microscopy and small- and wide-angle X-ray scattering. Our results showed that incorporation of TiO2 NPs improved the tensile properties of the hydrogels significantly. Furthermore, we performed biological evaluation of scaffolds, swelling degree, bioactivity assessment, and hemolytic tests to prove that all types of hydrogels were safe for use in the human body. The culturing of human osteoblast-like cells MG-63 on hydrogels showed better adhesion of cells in the presence of TiO2 and showed increasing proliferation with increasing amount of TiO2. Our results showed that the sample with the highest TiO2 concentration, CS/MC/PVA/TiO2 (1 %) had the best biological properties.

Human cells with osteogenic potential in bone tissue research.

Bone regeneration after injury or after surgical bone removal due to disease is a serious medical challenge. A variety of materials are being tested to replace a missing bone or tooth. Regeneration requires cells capable of proliferation and differentiation in bone tissue. Although there are many possible human cell types available for use as a model for each phase of this process, no cell type is ideal for each phase. Osteosarcoma cells are preferred for initial adhesion assays due to their easy cultivation and fast proliferation, but they are not suitable for subsequent differentiation testing due to their cancer origin and genetic differences from normal bone tissue. Mesenchymal stem cells are more suitable for biocompatibility testing, because they mimic natural conditions in healthy bone, but they proliferate more slowly, soon undergo senescence, and some subpopulations may exhibit weak osteodifferentiation. Primary human osteoblasts provide relevant results in evaluating the effect of biomaterials on cellular activity; however, their resources are limited for the same reasons, like for mesenchymal stem cells. This review article provides an overview of cell models for biocompatibility testing of materials used in bone tissue research.

In Situ Hydroxyapatite Synthesis Enhances Biocompatibility of PVA/HA Hydrogels.

Bone tissue engineering tries to simulate natural behavior of hard tissues. This study aimed to produce scaffolds based on polyvinyl alcohol (PVA) and hyaluronic acid (HA) with hydroxyapatite (HAp) incorporated in two different ways, by in situ synthesis and physical mixing of pre-prepared HAp. In situ synthesis resulted in calcium deficient form of HAp with lower crystallinity. The proliferation of human osteoblast-like cells MG-63 proved to be better in the scaffolds with in situ synthesized HAp compared to those with physically mixed pre-prepared HAp. For scaffolds with PVA/HA/HAp ratio 3:1:2, there was significantly higher initial adhesion (p = 0.0440), as well as the proliferation in the following days (p < 0.001). It seemed to be advantageous improve the properties of the scaffold by in situ synthesizing of HAp directly in the organic matrix.

Complex Interplay of Genes Underlies Invasiveness in Fibrosarcoma Progression Model.

Sarcomas are a heterogeneous group of mesenchymal tumours, with a great variability in their clinical behaviour. While our knowledge of sarcoma initiation has advanced rapidly in recent years, relatively little is known about mechanisms of sarcoma progression. JUN-murine fibrosarcoma progression series consists of four sarcoma cell lines, JUN-1, JUN-2, JUN-2fos-3, and JUN-3. JUN-1 and -2 were established from a single tumour initiated in a H2K/v-jun transgenic mouse, JUN-3 originates from a different tumour in the same animal, and JUN-2fos-3 results from a targeted in vitro transformation of the JUN-2 cell line. The JUN-1, -2, and -3 cell lines represent a linear progression from the least transformed JUN-2 to the most transformed JUN-3, with regard to all the transformation characteristics studied, while the JUN-2fos-3 cell line exhibits a unique transformation mode, with little deregulation of cell growth and proliferation, but pronounced motility and invasiveness. The invasive sarcoma sublines JUN-2fos-3 and JUN-3 show complex metabolic profiles, with activation of both mitochondrial oxidative phosphorylation and glycolysis and a significant increase in spared respiratory capacity. The specific transcriptomic profile of invasive sublines features very complex biological relationships across the identified genes and proteins, with accentuated autocrine control of motility and angiogenesis. Pharmacologic inhibition of one of the autocrine motility factors identified, Ccl8, significantly diminished both motility and invasiveness of the highly transformed fibrosarcoma cell. This progression series could be greatly valuable for deciphering crucial aspects of sarcoma progression and defining new prognostic markers and potential therapeutic targets.

Biological Evaluation of Polyvinyl Alcohol Hydrogels Enriched by Hyaluronic Acid and Hydroxyapatite.

This study aimed to develop polyvinyl alcohol (PVA) -based scaffold enriched with hyaluronic acid (HA) and hydroxyapatite (HAp) using physical crosslinking by freezing-thawing method. We accomplished biological evaluation of scaffolds, swelling degree, bioactivity assessment, and hemolytic test. The results showed that all types of scaffolds should be safe for use in the human body. The culturing of human osteoblast-like cells MG-63 and their proliferation showed better adhesion of cells due to the presence of HA and confirmed better proliferation depending on the amount of HAp. This paper gives the optimal composition of the scaffold and the optimal amount of the particular components of the scaffold. Based on our results we concluded that the best PVA/HA/HAp combination is in the ratio 3:1:2.

Epidemiological and Microbiological Aspects of the Peritonsillar Abscess.

Peritonsillar abscess (PTA) is the most common complication of tonsillitis. Cultivation usually reveals a wide spectrum of aerobic and anaerobic microbiota. This retrospective study compared PTA incidence and the spectrum of individual microbial findings in groups of patients divided by gender, age, and season. Of the 966 samples cultivated, a positive cultivation finding was detected in 606 patients (62.73%). Cultivation findings were negative in 360 (37.27%), meaning no pathogen was present or only common microbiota was cultivated. The highest incidence of PTA was found in group I patients (19-50 years) (p ≤ 0.0001) and the most frequently cultured pathogens was Streptococcus pyogenes (36.23%). Gender seemed to have an influence on the results, with higher incidence found in males (p ≤ 0.0001). The analysis of correlation between PTA incidence and season did not yield statistically significant results (p = 0.4396) and no statistically significant differences were observed in individual pathogen frequency. PTA had a higher incidence in adult males and a slightly higher incidence in girls in childhood. The following findings are clinically significant and have implications for antibiotic treatment strategy: (1) the most frequently cultivated pathogen was Streptococcus pyogenes; (2) an increased incidence of anaerobes was proven in the oldest group (>50 years).

Sonic Hedgehog and Triiodothyronine Pathway Interact in Mouse Embryonic Neural Stem Cells.

Neural stem cells are fundamental to development of the central nervous system (CNS)-as well as its plasticity and regeneration-and represent a potential tool for neuro transplantation therapy and research. This study is focused on examination of the proliferation dynamic and fate of embryonic neural stem cells (eNSCs) under differentiating conditions. In this work, we analyzed eNSCs differentiating alone and in the presence of sonic hedgehog (SHH) or triiodothyronine (T3) which play an important role in the development of the CNS. We found that inhibition of the SHH pathway and activation of the T3 pathway increased cellular health and survival of differentiating eNSCs. In addition, T3 was able to increase the expression of the gene for the receptor smoothened (Smo), which is part of the SHH signaling cascade, while SHH increased the expression of the T3 receptor beta gene (Thrb). This might be the reason why the combination of SHH and T3 increased the expression of the thyroxine 5-deiodinase type III gene (Dio3), which inhibits T3 activity, which in turn affects cellular health and proliferation activity of eNSCs.

Repeated Exposure of Nanostructured Titanium to Osteoblasts with Respect to Peri-Implantitis.

Titanium offers excellent biocompatibility and extraordinary mechanical properties. As a result, it is used as a material for dental implants. Implants infected by peri-implantitis can be cleaned for successful re-osseointegration. Optimal surface properties, such as roughness and wettability, have a significant impact on cell adhesion. The aim of this study was to evaluate the adhesion and proliferation of osteoblasts on the surface of repeatedly cleaned nanostructured titanium samples. Human osteoblast-like cells MG-63 were seeded on nanostructured titanium specimens manufactured from rods produced by the equal channel angular pressing. For surface characterization, roughness and wettability were measured. Cell adhesion after 2 h as well as cell proliferation after 48 h from plating was assessed. We have found that this repeated cleaning of titanium surface reduced cell adhesion as well as proliferation. These events depend on interplay of surface properties, such as wettability, roughness and topography. It is difficult to distinguish which factors are responsible for these events and further investigations will be required. However, even after the several rounds of repeated cleaning, there was a certain rate of adhesion and proliferation recorded. Therefore the attempts to save failing implants by using in situ cleaning are promising.

Fabrication of Scaffolds for Bone-Tissue Regeneration.

The present article describes the state of the art in the rapidly developing field of bone tissue engineering, where many disciplines, such as material science, mechanical engineering, clinical medicine and genetics, are interconnected. The main objective is to restore and improve the function of bone tissue by scaffolds, providing a suitable environment for tissue regeneration and repair. Strategies and materials used in oral regenerative therapies correspond to techniques generally used in bone tissue engineering. Researchers are focusing on developing and improving new materials to imitate the native biological neighborhood as authentically as possible. The most promising is a combination of cells and matrices (scaffolds) that can be fabricated from different kinds of materials. This review summarizes currently available materials and manufacturing technologies of scaffolds for bone-tissue regeneration.

Proliferation of Osteoblasts on Laser-Modified Nanostructured Titanium Surfaces.

Nanostructured titanium has become a useful material for biomedical applications such as dental implants. Certain surface properties (grain size, roughness, wettability) are highly expected to promote cell adhesion and osseointegration. The aim of this study was to compare the biocompatibilities of several titanium materials using human osteoblast cell line hFOB 1.19. Eight different types of specimens were examined: machined commercially pure grade 2 (cpTi2) and 4 (cpTi4) titanium, nanostructured titanium of the same grades (nTi2, nTi4), and corresponding specimens with laser-treated surfaces (cpTi2L, cpTi4L, nTi2L, nTi4L). Their surface topography was evaluated by means of scanning electron microscopy. Surface roughness was measured using a mechanical contact profilometer. Specimens with laser-treated surfaces had significantly higher surface roughness. Wettability was measured by the drop contact angle method. Nanostructured samples had significantly higher wettability. Cell proliferation after 48 hours from plating was assessed by viability and proliferation assay. The highest proliferation of osteoblasts was found in nTi4 specimens. The analysis of cell proliferation revealed a difference between machined and laser-treated specimens. The mean proliferation was lower on the laser-treated titanium materials. Although plain laser treatment increases surface roughness and wettability, it does not seem to lead to improved biocompatibility.

Follicular fluid levels of anti-Müllerian hormone, insulin-like growth factor 1 and leptin in women with fertility disorders.

Anti-Müllerian hormone (AMH), insulin-like growth factor 1 (IGF1) and leptin are produced in the granulosa cells of follicles and play an important role in the growth and maturation of follicles. The aim of our study was to monitor AMH, IGF1 and leptin levels in a group of healthy women and compare them to a group of women with fertility disorders. The second aim was the evaluation of biomarker levels in relation to the identified cause of infertility. Totally, 146 females were enrolled into our study. Seventy-two healthy controls and seventy-four females with fertility disorders were divided into four subgroups: anovulation, endometriosis, fallopian tube damage, unknown reason. IGF1 was the only biomarker with significantly lower levels throughout the entire group with fertility disorders. We did not identify any statistically significant differences for AMH and leptin. Regarding subgroups, significant differences were only observed in the group of anovulatory women. AMH and leptin showed higher levels while IGF1 showed lower levels. In conclusion, levels of AMH, IGF1 and leptin found in follicular fluid are sensitive markers for anovulatory fertility disorders. AMH, IGF1 and leptin levels in follicular fluid have no relation to the fertility disorders caused by endometriosis, fallopian tube damage or disorders with unknown etiology. ABBREVIATIONS: AMH: anti-Müllerian hormone; IGF1: insulin-like growth factor 1; PCOS: polycystic ovary syndrome.

Predictive relevance of miR-34a, miR-224 and miR-342 in patients with advanced squamous cell carcinoma of the lung undergoing palliative chemotherapy.

Attributing to their pathophysiological role and stability in biological samples, microRNAs (miRNAs) have the potential to become valuable predictive markers for non-small cell lung cancer (NSCLC). Samples of biopsy tissue constitute suitable material for miRNA profiling with the aim of predicting the effect of palliative chemotherapy. The present study group included 81 patients (74 males, 7 females, all smokers or former smokers) with the squamous cell carcinoma (SCC) histological subtype of NSCLC at a late stage (3B or 4). All patients received palliative chemotherapy based on platinum derivatives in combination with paclitaxel or gemcitabine. The expression of 17 selected miRNAs was measured by reverse transcription-quantitative polymerase chain reaction in tumor tissue macrodissected from formalin-fixed paraffin-embedded (FFPE) tissue samples. To predict the effect of palliative chemotherapy, the association between gene expression levels and overall survival (OS) time was analyzed. From the 17 miRNAs of interest, low expression levels of miR-342 and high expression levels of miR-34a and miR-224 were associated with a reduced OS time in subgroups of patients based on smoking status and treatment modality. Using cluster analysis, associations between combinations of miR-34a, -224 and -342 expression levels with patient survival were identified. The present study revealed that patients with the simultaneous high expression of miR-224 and -342 had a similar prognostic outcome to those with the low expression of miR-224 and -342, which was significantly reduced, compared with patients exhibiting high expression of either miR-224 or miR-342 with low expression of the other. We hypothesize that the effect of a particular miRNA is dependent on the expression level of other members of the miRNA network. This finding appears to complicate survival analyses based on individual miRNAs as markers. In conclusion, the present study provides evidence that specific miRNAs were associated with OS time, which may be candidate predictors for the effectiveness of palliative treatment in SCC lung cancer patients. This objective can be better achieved by combining more markers together than by using individual miRNAs.

Predictive Significance of Thymidylate Synthase Expression in Non-small Cell Lung Cancer.

BACKGROUND/AIM: To date, many studies have suggested that thymidylate synthase (TS) could be used as a prognostic and predictive marker in non-small cell lung cancer (NSCLC) patients. However, results have been contradictory. The aim of this study was to evaluate TS mRNA levels in tumor tissue of NSCLC patients who underwent complete surgical resection and to analyze its prognostic and predictive potential. MATERIALS AND METHODS: The study group consisted of 64 patients who underwent curative lung resection. Paired lung tissue samples were taken directly from the tumor tissue and from adjacent, histologically cancer-free lung tissue. The quantitative estimation of TS expression was performed by reverse transcription real-time polymerase chain reaction (RT-qPCR). The relationship between TS expression level and disease-free interval (DFI) and overall survival (OS) was analyzed. RESULTS: There was significantly higher TS expression in NSCLC tumor tissue comparing to normal lung tissue. In the group of patients who received adjuvant chemotherapy based on platinum derivatives in combination with paclitaxel or gemcitabine, we found shorter DFI (p=0.0473) and OS (p=0.0053) in those with high expression of TS. CONCLUSION: Our results demonstrated the relationship of high tumor tissue TS levels to adverse prognosis in patients undergoing adjuvant chemotherapy. TS is a non-specific tumor marker with respect to NSCLC, therefore we think that its best use would be as a member of the panel of predictors of adjuvant treatment efficacy.

Prognostic Significance of TMPRSS2-ERG Fusion Gene in Prostate Cancer.

BACKGROUND/AIM: Current research of prostate cancer (PCa) offers a promising way of identifying patients with adverse prognosis who do benefit from radical treatment that can affect quality of life as resections are associated with numerous side-effects. The aim of our study was to evaluate the relationship of TMPRSS2-ERG fusion gene status, tumor tissue prostate-specific antigen (PSA), prostate cancer antigen 3 (PCA3), miR-23b, miR-26a and miR-221 expression levels in combination with preoperative serum PSA level to the risk of PCa recurrence after radical prostatectomy. PATIENTS AND METHODS: The study group consisted of 108 patients who underwent radical prostatectomy. PSA was measured in peripheral blood collected preoperativelly. The expression of TMPRSS2-ERG transcript and the expression of miR-23b, miR-26a and miR-221 in formalin-fixed, paraffin-embedded (FFPE) tumor tissues was analyzed by reverse transcription (RT) real-time polymerase chain reaction (PCR). RESULTS: Significantly shorter time to recurrence was observed in patients with high expression of TMPRSS2-ERG (p=0.0020). High levels of preoperative PSA (>10.0 ng/ml) proved to be marker of shorter time to recurrence (p=0.0153). The most promising marker of the risk of recurrence after radical prostatectomy was a combination of high level of preoperative serum PSA and high expression of TMPRSS2-ERG fusion transcript in tumor tissue (p=0.0001). CONCLUSION: A combination of high preoperative serum PSA and high expression of TMPRSS2-ERG could be promising in distinguishing those tumors that are aggressive and life-threatening.

Evaluating the osseointegration of nanostructured titanium implants in animal models: Current experimental methods and perspectives (Review).

The aim of this paper is to review the experimental methods currently being used to evaluate the osseointegration of nanostructured titanium implants using animal models. The material modifications are linked to the biocompatibility of various types of oral implants, such as laser-treated, acid-etched, plasma-coated, and sand-blasted surface modifications. The types of implants are reviewed according to their implantation site (endoosseous, subperiosteal, and transosseous implants). The animal species and target bones used in experimental implantology are carefully compared in terms of the ratio of compact to spongy bone. The surgical technique in animal experiments is briefly described, and all phases of the histological evaluation of osseointegration are described in detail, including harvesting tissue samples, processing undemineralized ground sections, and qualitative and quantitative histological assessment of the bone-implant interface. The results of histological staining methods used in implantology are illustrated and compared. A standardized and reproducible technique for stereological quantification of bone-implant contact is proposed and demonstrated. In conclusion, histological evaluation of the experimental osseointegration of dental implants requires careful selection of the experimental animals, bones, and implantation sites. It is also advisable to use larger animal models and older animals with a slower growth rate rather than small or growing experimental animals. Bones with a similar ratio of compact to spongy bone, such as the human maxilla and mandible, are preferred. A number of practical recommendations for the experimental procedures, harvesting of samples, tissue processing, and quantitative histological evaluations are provided.