Artificial Dermis and Human Recombinant Epidermal Growth Factor Application for the Management of Critical Size Calvarial Defect.

Restoration of the 3-dimensional structure of the facial and calvarial skeleton after trauma or ablative oncologic surgeries serves as a framework for soft tissue reconstruction. In the present study, the authors aimed to evaluate the osteogenic effect of artificial dermis and epidermal growth factor treatment in critical-sized calvarial defects, which cannot be healed spontaneously. 8 mm calvarial defects were created in 28 male rats and filled with the artificial dermis, the artificial dermis and growth factor, growth factor or left untreated. Atomic absorption spectrometry was used to determine the amount of calcium, scanning electron microscopy was used to show the bone tissue in 3 dimensions, and immunohistochemistry was used to assess the bone formation and cell density. Histologic evaluation at 6 weeks showed incomplete bone regeneration in all groups. No statistical differences were found between the groups with regard to their scores for the following: inflammation, new bone formation, osteocyte density, resorption of bone at the edges of the defect, or fibrous tissue formation in the defect area. In conclusion, the predictability of bone formation in critical-size defects is not clear. Contrary to popular belief, the combined use of epidermal growth factor with artificial dermis or alone did not enhance the potential for osseous healing.

FFPE++: Improving the quality of formalin-fixed paraffin-embedded tissue imaging via contrastive unpaired image-to-image translation.

Formalin-fixation and paraffin-embedding (FFPE) is a technique for preparing and preserving tissue specimens that has been utilized in histopathology since the late 19th century. This process is further complicated by FFPE preparation steps such as fixation, processing, embedding, microtomy, staining, and coverslipping, which often results in artifacts due to the complex histological and cytological characteristics of a tissue specimen. The term "artifacts" includes, but is not limited to, staining inconsistencies, tissue folds, chattering, pen marks, blurring, air bubbles, and contamination. The presence of artifacts may interfere with pathological diagnosis in disease detection, subtyping, grading, and choice of therapy. In this study, we propose FFPE++, an unpaired image-to-image translation method based on contrastive learning with a mixed channel-spatial attention module and self-regularization loss that drastically corrects the aforementioned artifacts in FFPE tissue sections. Turing tests were performed by 10 board-certified pathologists with more than 10 years of experience. These tests which were performed for ovarian carcinoma, lung adenocarcinoma, lung squamous cell carcinoma, and papillary thyroid carcinoma, demonstrate the clear superiority of the proposed method in many clinical aspects compared with standard FFPE images. Based on the qualitative experiments and feedback from the Turing tests, we believe that FFPE++ can contribute to substantial diagnostic and prognostic accuracy in clinical pathology in the future and can also improve the performance of AI tools in digital pathology. The code and dataset are publicly available at https://github.com/DeepMIALab/FFPEPlus.

The effect of P53 expression and smoking/alcohol in P16(+) and P16(-) oropharyngeal carcinoma and risk classification: the Turkish Society of Radiation Oncology Head & Neck Study Group 01-002.

OBJECTIVE: The aim of this study is to categorize the risk groups of patients with oropharyngeal carcinoma (OPC) according to p16 and p53 status, smoking/alcohol consumption history, and other prognostic factors. STUDY DESIGN: The immunostaining of p16 and p53 of 290 patients was retrospectively evaluated. The history of smoking/alcohol consumption of each patient was noted. p16 and p53 staining patterns were reviewed. The results were compared with demographic findings and prognostic factors. Risk groups have been classified for the p16 status of patients. RESULTS: The median follow-up was 47 months (range 6-240). Five-year disease-free survival (DFS) rates for patients with p16 (+) and (-) were 76% and 36%, and overall survival rates were 83% vs 40%, respectively (HR = 0.34 [0.21-0.57], P < .0001), HR = 0.22 [0.12-0.40] P < .0001, respectively). p16(-), p53(+), heavy smoking/alcohol consumption, performance status; advanced T and N stages in patients with p16(-), and continuing smoking/alcohol consumption after treatment were found to be unfavorable risk factors. Five-year overall survival rates were 95%, 78%, and 36% for low, intermediate, and high-risk groups, respectively. CONCLUSIONS: The results of our study have shown that p16 negativity in patients with oropharyngeal cancer was found to be an important prognostic factor, especially for those with lower p53 expression and not smoking/consuming alcohol.

Expression Levels of Glutathione S-Transferase and Cytochrome P450 Isoenzymes in Nasal Polyp Tissue.

Nasal polyps are benign sinonasal masses composed of eosinophils and extracellular edema. Pathogenesis of the polyp formation is unclear but several studies strongly suggest a correlation with infection, inflammation and allergy conditions. Our aim is to investigate the potential link between allergy and nasal polyp in tissue level. Nasal polyp group included 60 patients whose diagnosis was confirmed with biopsy and the control group included 38 healthy patients. Tissue sample of the control group was taken from inferior turbinate mucosa under local anesthesia and nasal polyp tissue was collected from functional endoscopic sinus surgery. The glutathione S-transferase (GST) and cytochrome P450 (CYP) isoenzyme expressions of the tissue samples were investigated under light microscopy and graded by a senior pathologist. GSTP1 protein expression was significantly higher in tissue samples from nasal polyp group compared to that of control group (p < 0.05). However, CYP1A1, GSTM1 and GSTA1 isoenzymes were not different between the two groups (p > 0.05). We have found that GSTP1 isoenzyme was elevated in nasal polyp tissue compared to the control. The increase in protein expression of GSTP1 might have occured as a tissue response to the increased oxidative stress thus suggesting a role of GSTP1 in polyp formation.

Whole Slide Images in Artificial Intelligence Applications in Digital Pathology: Challenges and Pitfalls.

The use of digitized data in pathology research is rapidly increasing. The whole slide image (WSI) is an indispensable part of the visual examination of slides in digital pathology and artificial intelligence applications; therefore, the acquisition of WSI with the highest quality is essential. Unlike the conventional routine of pathology, the digital conversion of tissue slides and the differences in its use pose difficulties for pathologists. We categorized these challenges into three groups: before, during, and after the WSI acquisition. The problems before WSI acquisition are usually related to the quality of the glass slide and reflect all existing problems in the analytical process in pathology laboratories. WSI acquisition problems are dependent on the device used to produce the final image file. They may be related to the parts of the device that create an optical image or the hardware and software that enable digitization. Post-WSI acquisition issues are related to the final image file itself, which is the final form of this data, or the software and hardware that will use this file. Because of the digital nature of the data, most of the difficulties are related to the capabilities of the hardware or software. Being aware of the challenges and pitfalls of using digital pathology and AI will make pathologists' integration to the new technologies easier in their daily practice or research.

A deep-learning model for transforming the style of tissue images from cryosectioned to formalin-fixed and paraffin-embedded.

Histological artefacts in cryosectioned tissue can hinder rapid diagnostic assessments during surgery. Formalin-fixed and paraffin-embedded (FFPE) tissue provides higher quality slides, but the process for obtaining them is laborious (typically lasting 12-48 h) and hence unsuitable for intra-operative use. Here we report the development and performance of a deep-learning model that improves the quality of cryosectioned whole-slide images by transforming them into the style of whole-slide FFPE tissue within minutes. The model consists of a generative adversarial network incorporating an attention mechanism that rectifies cryosection artefacts and a self-regularization constraint between the cryosectioned and FFPE images for the preservation of clinically relevant features. Transformed FFPE-style images of gliomas and of non-small-cell lung cancers from a dataset independent from that used to train the model improved the rates of accurate tumour subtyping by pathologists.

Label-Free Surface Enhanced Raman Spectroscopy for Cancer Detection.

Blood is a vital reservoir housing numerous disease-related metabolites and cellular components. Thus, it is also of interest for cancer diagnosis. Surface-enhanced Raman spectroscopy (SERS) is widely used for molecular detection due to its very high sensitivity and multiplexing properties. Its real potential for cancer diagnosis is not yet clear. In this study, using silver nanoparticles (AgNPs) as substrates, a number of experimental parameters and scenarios were tested to disclose the potential for this technique for cancer diagnosis. The discrimination of serum samples from cancer patients, healthy individuals and patients with chronic diseases was successfully demonstrated with over 90% diagnostic accuracies. Moreover, the SERS spectra of the blood serum samples obtained from cancer patients before and after tumor removal were compared. It was found that the spectral pattern for serum from cancer patients evolved into the spectral pattern observed with serum from healthy individuals after the removal of tumors. The data strongly suggests that the technique has a tremendous potential for cancer detection and screening bringing the possibility of early detection onto the table.

Deep learning-enabled assessment of cardiac allograft rejection from endomyocardial biopsies.

Endomyocardial biopsy (EMB) screening represents the standard of care for detecting allograft rejections after heart transplant. Manual interpretation of EMBs is affected by substantial interobserver and intraobserver variability, which often leads to inappropriate treatment with immunosuppressive drugs, unnecessary follow-up biopsies and poor transplant outcomes. Here we present a deep learning-based artificial intelligence (AI) system for automated assessment of gigapixel whole-slide images obtained from EMBs, which simultaneously addresses detection, subtyping and grading of allograft rejection. To assess model performance, we curated a large dataset from the United States, as well as independent test cohorts from Turkey and Switzerland, which includes large-scale variability across populations, sample preparations and slide scanning instrumentation. The model detects allograft rejection with an area under the receiver operating characteristic curve (AUC) of 0.962; assesses the cellular and antibody-mediated rejection type with AUCs of 0.958 and 0.874, respectively; detects Quilty B lesions, benign mimics of rejection, with an AUC of 0.939; and differentiates between low-grade and high-grade rejections with an AUC of 0.833. In a human reader study, the AI system showed non-inferior performance to conventional assessment and reduced interobserver variability and assessment time. This robust evaluation of cardiac allograft rejection paves the way for clinical trials to establish the efficacy of AI-assisted EMB assessment and its potential for improving heart transplant outcomes.

Warthin-Like Mucoepidermoid Carcinoma: A Morphological Spectrum - A Report of 3 Cases with Histological and Cytological Findings and Review of the Literature.

BACKGROUND: Mucoepidermoid carcinoma (MEC) showing Warthin's tumor (WT)-like features is a low-grade malignancy which should be differentiated from WT. Morphological features may be distinctly different in each case, causing diagnostic difficulties. CASE PRESENTATION: Three cases were presented and discussed with their morphologies. All cases that presented with a mass in the parotid gland went to parotidectomy, and all had preoperative fine-needle aspirations (FNAs). Case 1 was a 16-year-old female; FNA was suggestive of WT and initially interpreted as WT histologically. Case 2 was a 27-year-old male; FNA was interpreted as noninformative due to the presence of cyst fluid only. Case 3 was a 53-year-old male and cytologically was found to be suspicious for MEC which contained squamous and goblet cells on a mucoid background. On histopathological examination, case 2 and case 3 were morphologically consistent with low-grade MEC with WT-like features. Prominent lymphoid stroma and the cystic pattern were the characters of these tumors. Case 1 had the classical WT appearance with some mucinous and squamous metaplasia which could only be interpreted as MEC after the detection of MAML2 rearrangement by FISH. The other 2 showed either focal or relatively diffuse usual low-grade MEC findings, and case 3 was also confirmed by MAML2 rearrangement. CONCLUSION: Cytological and histopathological features revealed a spectrum. Differentiating WT-like MECs from ordinary WTs may be challenging. On the one end of the spectrum, they may look very much like WT, and on the other end, even though usual MEC features are present, still, WT-like appearance may pose diagnostic difficulty. Showing MAML2 rearrangement in these cases is very helpful. The presence of mucinous and squamous cells in an otherwise WT-like looking tumor should be alarming for MEC, and if possible, each case should be analyzed for MAML2 rearrangement.

Salivary glands of fetuses are adversely affected by artificial food colorings in rats.

OBJECTIVE: Artificial food colorings, as types of food additives, are widely used at present in daily life. We aimed to investigate the effects of exposure to artificial food colorings during the intrauterine period on the salivary glands in adulthood. METHODS: A total of 30 Wistar albino female pregnant rats were included in this study. The treatment group was given a mixture of nine artificial food colorings at no observed adverse-effect-level doses. Neither mothers nor offspring were fed with food colorings after delivery. When the offspring became adults, they were sacrificed, and the possible histopathological and immunohistochemical effects of artificial food colorings on the salivary glands were investigated. In these glands, anti-matrix metallopeptidase 2 (anti-MMP2), anti-MMP9, anti-tissue inhibitor of metalloproteinases (TIMP) metallopeptidase inhibitor 1 (anti-TIMP1), anti-TIMP2, and anti-TIMP3 were examined. RESULTS: The expressions of anti-MMP2, anti-TIMP1, anti-TIMP2, and anti-TIMP3 parameters were found to be higher in treatment groups (p<0.05). CONCLUSION: It was suggested that intrauterine exposure of synthetic food colorings may lead to deterioration of the tissue structure of the salivary glands in adulthood, thereby increasing susceptibility to chronic illnesses including malignancy and chronic inflammation. Therefore, pregnant women should give importance to their nutrition in terms of foods containing synthetic colorings.

Salivary glands of fetuses are adversely affected by artificial food colorings in rats

SUMMARY OBJECTIVE: Artificial food colorings, as types of food additives, are widely used at present in daily life. We aimed to investigate the effects of exposure to artificial food colorings during the intrauterine period on the salivary glands in adulthood. METHODS: A total of 30 Wistar albino female pregnant rats were included in this study. The treatment group was given a mixture of nine artificial food colorings at no observed adverse-effect-level doses. Neither mothers nor offspring were fed with food colorings after delivery. When the offspring became adults, they were sacrificed, and the possible histopathological and immunohistochemical effects of artificial food colorings on the salivary glands were investigated. In these glands, anti-matrix metallopeptidase 2 (anti-MMP2), anti-MMP9, anti-tissue inhibitor of metalloproteinases (TIMP) metallopeptidase inhibitor 1 (anti-TIMP1), anti-TIMP2, and anti-TIMP3 were examined. RESULTS: The expressions of anti-MMP2, anti-TIMP1, anti-TIMP2, and anti-TIMP3 parameters were found to be higher in treatment groups (p<0.05). CONCLUSION: It was suggested that intrauterine exposure of synthetic food colorings may lead to deterioration of the tissue structure of the salivary glands in adulthood, thereby increasing susceptibility to chronic illnesses including malignancy and chronic inflammation. Therefore, pregnant women should give importance to their nutrition in terms of foods containing synthetic colorings.

The Effect of Antioxidant Agents on Cisplatin-Induced Laryngeal Histological Alterations in Rats.

The larynx-related adverse effects that depend on cisplatin decrease patient comfort and many antioxidants have been used to eliminate these side effects. We aimed to identify the laryngeal mucosal changes imposed by cisplatin and investigated whether antioxidants, and their healing effects on these changes, may help reduce laryngeal complications in patients resulting from adverse effects in the larynx. A rat model was designed to evaluate the effects of cisplatin on the larynx and the protective role of antioxidants. Single-dose cisplatin was given both intraperitoneally alone and additionally administered with p-coumaric acid, melatonin, resveratrol, vitamin D, and oleic acid over 5 days. Whole larynges were dissected and evaluated histologically, histochemically, and immunohistochemically. Varying degrees of mucosal changes cisplatin group, but neither erosion nor an ulcer was observed. Numerous variable histological effects of antioxidants were observed on cisplatin exposed laryngeal mucosa. The most obvious effects of cisplatin were edema. The results of the study showed that resveratrol was the most preventive antioxidant agent against cisplatin-dependent mucosal changes. The highest increase in the Ki67 index was in the oleic acid group. Vitamin D increased stromal cyclooxygenase-2 expression that may have an effect on increasing mucosal damage.

Investigation of the preventive effects of dehydroepiandrosterone (DHEA) and Caffeic acid phenethyl ester (CAPE) on cisplatin-induced ovarian damage in rats.

To investigate whether Dehydroepiandrosterone (DHEA) and Caffeic acid phenethyl ester (CAPE) had any preventive effect against the ovarian damage caused by cisplatin (CP) (cis-diamminedichloroplatinum) in rats. On the first day ovaries were removed, Anti-Müllerian hormone (AMH) was measured (Group1, n:6), in the other groups 7.5 mg/kg cisplatin was administered intraperitoneally. In Group 2 (n = 6), 0.1 ml saline, in Group 3 (n = 5), 20 umol/kg CAPE, in Group 4 (n = 7), DHEA 6 mg/kg were administered every day. On the 10th day, ovaries were removed, AMH was measured. Ovary reserve (primordial/primary/secondary/tertiary/atretic follicles, AMH), ovarian damage scores (follicular degeneration, congestion, hemorrhage, edema, inflammation) were compared. The number of tertiary follicles were statistically high in the CAPE group (p = .015), the inflammation score in the DHEA group (p = .012), AMH level (p = .009) in the control group. The lowest number of atretic follicles (AF) was in the control group, while the highest number of AF was in the DHEA group (p = .002). Significant decreases in AF were the case in the cisplatin and DHEA groups compared to the control group (p < .008). The AMH values had the highest positive correlation with the number of primordial follicles and the highest negative correlation with the number of AF. The cut off point for AMH was 1.57 ng/ml as an indicator of low ovarian reserve. Cisplatin causes total damage and increased numbers of AF on the ovary. Depending on this, AMH levels fall. These negative effects of cisplatin are not obstructed by CAPE or DHEA, and may even be increased by DHEA.

Nasal Congenital Panfollicular Nevus.

Congenital nasal midline lesions are rarely observed pathologies. While relatively frequently encountered lesions are dermoid cyst, glioma, encephalocele, nasal polyp and epidermoid cyst, an other very rare reason is the congenital panfollicular nevus. The treatment of this syndrome appearing with a recurrent discharge in clinic is the surgical excision. We will hereby present the excision of the case of congenital panfollicular nevus with three different fistula openings.

Comparison of the protective effects of sildenafil, vardenafil and tadalafil treatments in ischemia-reperfusion injury in rat ovary.

OBJECTIVES: The aim of this study was to compare the effects of sildenafil, vardenafil and tadalafil in treatment for ischemia/reperfusion injury which is created experimentally in rat ovaries. MATERIAL AND METHODS: For this study, 30 female Wistar albino rats were used, and the rats were separated randomly intofive groups consisting of six rats each: normal, torsion-detorsion, torsion-detorsion + sildenafil 1.4 mg/kg, torsion-detorsion+ vardenafil 1.7 mg/kg and torsion-detorsion + tadalafil 5.0 mg/kg. The agents were given intraperitoneally 30 minutesbefore detorsion. An ovarian torsion procedure was implemented in all other groups for 3 hours with the exception of thenormal group. Then, a detorsion procedure was implemented to the groups for 3 hours. RESULTS: The sildenafil and vardenafil treatments showed protective effect by preventing significant increase in inflammationparameters. (p = 0.058, 0.138). The tadalafil treatment was only protective for cellular degeneration (p = 0.140). Thevardenafil treatment was protective for edema (p = 0.238), vascular congestion (p = 0.111), inflammation (p = 0.138) andcellular degeneration (p = 0.532). Sildenafil, vardenafil and tadalafil inhibited the increase of atretic follicle. AMH levels werestatistically different between torsion and detorsion and vardenafil group (p = 0.004, 0.004), whereas tadalafil and sildenafilgroups were similar to normal group (p = 0.108, 0.108). CONCLUSIONS: PDE inhibitors were found to be effective in reducing ovarian ischemia/reperfusion injury. Sildenafil andtadalafil seem to be more effective than the vardenafil in protecting the ovarian reserve.

The effect of curcumin on healing in an animal nasal septal perforation model.

OBJECTIVES/HYPOTHESIS: We investigated the effect of intranasal topical curcumin on nasal septum mucosa wound healing in a nasal septal perforation model produced in rabbits. STUDY DESIGN: Experimental study. METHODS: Fourteen male New Zealand rabbits were included in the study. For each rabbit, 5-mm-diameter circular perforations were created at 5 mm away from the columella to the nasal septum. Curcumin (study group) and saline (control group) were administered intranasally once daily for 10 days. At the end of the 10th day, the animals were sacrificed and the nasal septum specimens were sent for histological examination. Epithelial regeneration and degeneration, cartilage degeneration and regeneration, presences of fibroblast, eosinophil, acute/chronic inflammatory and giant cells, capillary density, amounts of granulation tissue and collagen, and macroscopic closure rate of perforation parameters were compared in each group. RESULTS: Epithelial and cartilage regeneration, and the amounts of collagen and granulation tissue were significantly higher in the curcumin group compared to the control group (P < .05). No statistically significant difference was found in comparison of other parameters (P > .05). CONCLUSIONS: Topical application of curcumin improves the wound-healing process of nasal septum perforation in the animal model. Therefore, curcumin can be used as a safe and effective medical agent to prevent the development of septal perforation. LEVEL OF EVIDENCE: NA Laryngoscope, 129:E349-E354, 2019.

Salivary gland anlage tumour of the nasopharynx: A case report and review for histopathological characteristics

@#Introduction: Congenital salivary gland anlage tumour of the nasopharynx is a lesion which usually presents with nasal and upper respiratory tract obstruction in the neonatal period. Timely diagnosis is essential to prevent the occurrence of respiratory complications in later childhood. Case Report: We present a 8-year-old boy complaining from difficulty in breathing and breastfeeding in the neonatal period due to an adenoid-like nasopharyngeal mass. Histological examination revealed solid and cystic squamous nests and numerous duct-like structures within collagenised stroma. Both epithelial and myoepithelial differentiation were noted in the tubular component. Discussion: A review of the clinical and histopathological features of published cases revealed that ancient lesions showed more prominent and complex epithelial component and more collagen rich stroma. We would like to suggest the possibility of salivary gland anlage tumour to be considered in the differential diagnosis of neonatal respiratory distress cases.

Relationship between irritable bowel syndrome and plasma and tissue ghrelin levels.

BACKGROUND/AIMS: This study aimed to evaluate the relationship between irritable bowel syndrome (IBS) and plasma and tissue ghrelin levels. MATERIALS AND METHODS: Patients who had undergone gastroscopy procedure for any reason previously were enrolled in the study. Among these, patients with IBS symptoms were evaluated according to the Roma III criteria. The healthy control group comprised patients with no IBS symptom and had undergone gastroscopy procedure for another reason. The plasma ghrelin level and tissue ghrelin level obtained by immunohistochemical examination of biopsy specimens taken from the gastric antrum and corpus were evaluated in all participants. RESULTS: The mean age of 90 participants was 43.64}12.64 years. The median value of the plasma ghrelin level was 3.29 (1.2-12.7) in the diarrhea group (IBS-D), 1.49 (0.82-7.08) in the constipation group (IBS-C), and 1.5 (0.2-3.7) in the control group. The plasma ghrelin levels between the groups were found to be significantly higher in IBS-D than in IBS-C and the control groups (p=0.001 and p=0.001, respectively). On comparing antral mucosal gland biopsy outcomes among the groups, staining intensity score was found to be significantly high in IBS-C as compared with the control group, whereas no significant difference was observed between IBS-D and the control groups (p=0.020 and p=0.429, respectively). CONCLUSION: The plasma ghrelin level in IBS-D and the staining intensity in the antral mucosal gland in IBS-C were found to be significantly higher. In addition, there was no difference between the groups in terms of ghrelin staining intensity in the gastric corpus.

The role of an herbal agent in treatment for Escherichia coli induced bacterial cyctitis in rats.

OBJECTIVES: The aim of this study is to evaluate the effects of the herbal agent in the prevention and treatment of bacterial cystitis in a rat model. MATERIAL AND METHODS: A total of twenty-eight male Sprague- Dawley rats were divided into four groups. Group-1 constituted the control group (operated and normal saline injected into the bladder, received only drinking water for 7 days); Group-2 constituted the no-treatment group (operated, E.coli J96 strain injected into the bladder, received only drinking water for 7 days); Group-3 constituted the short-term treatment (operated, E.coli J96 strain injected into the bladder, received the herbal agent added into drinking water for 7 days) and Group-4 constituted the long-term treatment (operated, E. coli J96 strain injected into the bladder, received herbal agent added into drinking water for 14 days). At the end of the pre-defined treatment periods of duration, the rats were sacrificed, urine samples collected from the bladder for culture and bladders were harvested for histopathological evaluation. Urine culture results and histopathological findings were comparatively evaluated between the groups. RESULTS: Urine cultures were positive for implanted E. coli strains in 0%, 85.7%, 42.8% and 0% of rats in Group 1, Group 2, Group 3 and Group 4, respectively (p = 0.001). Although histopathological evaluation revealed increased vascular dilation in the bladder specimens obtained from Group 2 and Group 3 (p = 0.028) no significant difference was noticed in level of inflammation (p = 0.610), edema (p = 0.754) and thickness of uroepithelium (p = 0.138). CONCLUSION: While long term (14 days) treatment with an herbal agent added into the drinking water resulted in complete clearance of urine from E. coli; shorter application of the agent revealed partial clearance. Further clinical studies are needed to support our results.