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BACKGROUND: Histopathological transformation between different types of lung cancer cells has been reported following a variety of anti-tumor treatments. Examples include transformation from lung adenocarcinoma to squamous-cell carcinoma (SCC) and transformation from non-small cell lung cancer (NSCLC) to small cell lung cancer (SCLC). CASE REPORT: A patient with intermittent hemoptysis for 2 days underwent a computed tomography (CT) scan that revealed interstitial pneumonia in addition to two enlarged paratracheal lymph nodes: one on the right (4R) and one on the left (4L) measuring 10 and 7 mm in diameter, respectively (Fig. 1). There was no evidence of a lung or bronchial mass. Bronchoscopy identified an endoluminal primary mass in a superior segmental bronchus of the left lower lobe and pathological examination following surgery confirmed it to be SCC. At 15 months post operation, a CT scan detected that the 4R lymph node had increased in size from 10 to 16 mm in diameter. At the next follow-up 7 months later, a CT scan showed that the R4 lymph node had further increased in size from 16 to 40 mm in the short axis, making it difficult for a surgeon to resect it "en bloc" immediately. The maximum standardized uptake value was 7.5 on PET-CT images. One month following completion of one cycle of neoadjuvant chemotherapy with gemcitabine and nedaplatin, a further CT scan indicated that the lymph node had decreased in size from 40 to 30 mm in the short axis. A complete mediastinal lymphadenectomy via open thoracotomy was performed and the lymph node was resected. Histological examination identified a main large cell neuroendocrine carcinoma (LCNEC) component with a small fraction of small cell carcinoma, confirmed by immunohistochemical analysis and genetic evidence. CONCLUSION: Histopathological transformation from SCC to LCNEC with a small fraction of SCLC may have occurred spontaneously without any treatment.

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BACKGROUND/AIM: Recently, an increase in the number of asymptomatic rare benign liver tumors (BLTs) has been reported during health check-ups. It is difficult to determine the nature of partial rare BLTs and not easy to distinguish from malignant liver tumors. This study aimed to analysis clinical features, diagnosis and treatment of rare BLTs to reduce misdiagnosis and provide reference for clinical practice. METHODS: From January 2012 to January 2021, we treated 112 rare BLTs by hepatectomy, including 54 focal nodular hyperplasias, 14 hepatocellular adenomas, 28 hepatic angiomyolipomas, 3 hepatic granulomas, 2 inflammatory pseudotumors of the liver, 2 nodular regenerative hyperplasia, 2 hepatic lipomas, 1 solitary fibrous tumor of the liver, 1 hepatic schwannoma and 1 hepatic myelolipoma. RESULTS: The majority of patients were middle-aged female and asymptomatic. Single tumors were dominant. The diagnostic accuracies of computed tomography (CT) and magnetic resonance imaging (MRI) were 32.5% and 44.2%, respectively. The majority of tumors were likely to be misdiagnosed as hepatocellular carcinoma (HCC) or difficult to distinguish from HCC. All patients underwent surgical treatment. Postoperative pathological and immunohistochemical examination can confirm the diagnosis. No patients without tumor recurrence or metastasis during follow-up period. CONCLUSION: Altogether, the clinical symptoms of rare BLTs lack specificity, and their preoperative diagnosis largely depends on imaging examination, with a low diagnostic accuracy rate and high chances of misdiagnosis as HCC. Diagnosis is confirmed by pathological and immunohistochemical examination. Surgical resection for rare BLT is safe and effective, regular postoperative follow-up is necessary.

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PURPOSE: To investigate the effects of subconjunctival bevacizumab, ranibizumab, and aflibercept in an experimental corneal neovascularization model. MATERIALS AND METHODS: The eyes of 24 rats were chemically cauterized and randomly divided into four groups: bevacizumab group (0.05 mL/1.25 mg bevacizumab), ranibizumab group (0.05 mL/0.5 mg ranibizumab), aflibercept group (0.05 mL/1.25 mg aflibercept), and control group (0.05 mL saline solution). Plasma vascular endothelial growth factor (VEGF) levels were among the major measurement outcomes to assess corneal neovascularization. The collected plasmas were analyzed using the SIGMA RAB0511 Rat VEGF Elisa kit. The PCR technique and VEGF amplification procedures were used for molecular analysis. Each cornea was removed and histologically examined on day 21. Corneal images were evaluated by image analyzer software. RESULTS: In the post-injection period, the number of major corneal arteries decreased significantly in the injection groups when compared to the control group (p = 0.037), but no statistically significant differences were noted among the injection groups (p > 0.05). The aflibercept group had the lowest area of neovascularization. Immunohistochemical staining revealed substantially lower VEGF percentages in neovascularized arteries of the injection groups than the control group (p = 0.015). In TUNEL staining, the mean TUNEL value (number/1hpf) was substantially greater in the control group than in the injection groups, but the mean TUNEL values were similar between the injection groups (p = 0.019, p > 0.05, respectively). No statistically significant differences were observed between the groups in terms of corneal surface area with increased cellularity, edema, and inflammation (p = 0.263). The mean plasma VEGF concentration in the control group was statistically greater than those in the injection groups (p = 0.001). CONCLUSION: Subconjunctival bevacizumab, ranibizumab, and aflibercept crossed the blood and seemed to be effective in inhibiting corneal neovascularization without causing epitheliopathy in an experimental rat model compared to the controls. However, no significant results were noted between these three anti-VEGF molecules.

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BACKGROUND: Autoimmune atrophic gastritis (AAG) is a type of chronic gastritis that mainly affects the gastric corpus. Due to the lack of standard diagnostic criteria and overlaps with the courses of Helicobacter pylori-related atrophic gastritis, reports on the diagnostic strategy of AAG at an early stage are limited. CASE SUMMARY: A 71-year-old woman with severe anemia was diagnosed with AAG. Endoscopic views and pathological findings showed the coexistence of normal mucosa in the gastric antrum and atrophic mucosa in the gastric fundus. Serological tests showed that anti-parietal cell antibodies and anti-intrinsic factor antibodies were both positive. Immunohistochemical results, which showed negative H+-K+ ATPase antibody staining and positive chromogranin A (CgA) staining, confirmed the mechanism of this disease. After vitamin B12 and folic acid supplementation, the patient recovered well. CONCLUSION: Successful diagnosis of AAG includes serological tests, endoscopic characteristics, and immunohistochemistry for H+-K+ ATPase and CgA antibodies.

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OBJECTIVE: To investigate the clinical and morphological features of SARS-CoV-2-related myocarditis, by determining the presence of viral RNA and proteins in myocardial tissue. MATERIAL AND METHODS: The study was conducted to examine the material of 32 autopsies with a confirmed diagnosis of myocarditis. There were data of a morphological study, including a standard histological study, as well as immunohistochemical determination of the surface markers CD45, CD3, CD20, and CD68 cells of an inflammatory infiltrate and virus proteins (SARS-CoV-2 nucleocapsid protein and spike protein). Positive and negative control tests were carried out. In addition, coronavirus RNA was detected in the myocardium using a polymerase chain reaction. RESULTS: Polymerase chain reaction (PCR) revealed viral RNA in myocardial tissue. Viral proteins were identified in the macrophages of an inflammatory infiltrate and cardiomyocytes. CONCLUSION: The findings may suggest that the virus persists in the myocardium and chronic myocarditis develops.

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A 25-year-old woman with fever and epigastric pain was referred to our hospital. Blood examination showed significant liver dysfunction, markedly high C-reactive protein (CRP 19.1 mg/dL) and procalcitonin (48.3 ng/mL) levels. Dynamic computed tomography showed a tumor approximately 120 mm in size in the right lobe of the liver, but with no abscess formation. The patient was hospitalized and started on antibiotics; her CRP level improved, but the procalcitonin level did not decrease. Histopathological examination of the liver tumor biopsy revealed fibrolamellar hepatocellular carcinoma (FLC). Positive staining of the FLC with an anti-procalcitonin antibody suggested the production of procalcitonin.

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After an outbreak of classical scrapie in a dairy goat herd with over 1,800 goats, all goats in the herd were culled in 2008, cleaning and disinfection of the premises was implemented, and restocking with goats took place ~4 months after depopulation. Ten years later the new herd population is over 3,000 goats. This study was carried out to determine whether the measures were effective to prevent re-occurrence of scrapie to the 1% prevalence level seen when scrapie was first detected on this farm. A total of 280 goats with a minimum age of 18 months, which were predominantly at the end of their productive life, were euthanized, and brain and retropharyngeal lymph node examined by immunohistochemistry for disease-associated prion protein. Genotyping was done in all euthanized goats and live male goats used or intended for breeding to determine prion protein gene polymorphisms associated with resistance to classical scrapie. None of the goats presented with disease-associated prion protein in the examined tissues, and 34 (12.2%) carried the K222 allele associated with resistance. This allele was also found in four breeding male goats. The study results suggested that classical scrapie was not re-introduced on this goat farm through mass restocking or inadequate cleaning and disinfection procedures. Further scrapie surveillance of goats on this farm is desirable to confirm absence of disease. Breeding with male goats carrying the K222 allele should be encouraged to increase the scrapie-resistant population.

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BACKGROUND: In Egypt, the incidence of hepatocellular carcinoma (HCC) is approximately 4.7% of chronic liver disease patients due to (HCV) infection. Epidermal growth factor (EGF) plays an important role in hepatocyte regeneration. A functional polymorphism in EGF 61A>G was identified; itwas associated with higher risk of HCC. OBJECTIVES: to investigate the correlation between the epidermal growth factor (EGF) polymorphism and the risk of hepatocellular carcinoma (HCC) in hepatitis C viral (HCV) cirrhotic patients as well as its relation to EGF protein expression in HCC tissue. PATIENTS AND METHODS: this casecontrol study was conducted on 75 HCV cirrhotic patients including 50 HCC patients (25 withresectable HCC and 25 with advanced unresectable HCC) and 25 healthy persons were included. EGF genotype was detected by restriction fragment length polymorphism. EGF expression in HCC tissue biopsiesfrom patientswhounderwent surgical resection was done by immunohistochemical examination. RESULTS: The GG genotype was associated with significant increased risk of HCC compared to AA genotypes (P=0.031) in cirrhotic group. The G allele had a highly significant risk of HCC compared to allele Ain recessive model GG vs. AG+AA (P=0.036) rather than in the dominant model GG +AG vs. AA (P=0.66). There was significant increased expression of EGF in tumour tissues in patients with GG genotype compared to AG genotype and AA genotype p= 0.019. CONCLUSION: EGF gene polymorphism (GG genotype) had a significant risk of HCC development in cirrhotic patients. This is confirmed by increased EGF expression in liver tumor tissue from HCC patients.
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INTRODUCTION: Triple negative breast cancer (TNBC) is characterized by a worse prognosis than other breast cancer subtypes. TNBC is defined by lack of expression of estrogen receptor, progesterone receptor and human epidermal growth factor receptor 2. The aim of this analysis was to evaluate the relationship between immunohistochemical expression of novel prognostic markers (erythropoietin (EPO) and erythropoietin receptor (EPO-R)) and clinicopathological features of TNBC and non-TNBC patients. MATERIAL AND METHODS: Our analysis was conducted on a group of 162 patients with breast carcinoma with lymph node metastasis (111 TNBC and 51 non-TNBC). All statistical analyses were performed with SPSS software v 12.0. RESULTS: Histopathologic subtyping of the 111 triple negative breast cancers identified 89.1% invasive ductal carcinomas of no special type and 10.9% other special types of cancers. TNBC more often presented EPO-R and EPO expression (36%; 37.8%) than non-TNBC (23.5%; 29.4%). Non-TNBC subgroup showed statistically significant correlation only between Ki-67 expression and histological grade (G1-G3) (p < 0.001), while TNBC subgroup demonstrated significant correlation between Ki-67 expression and histological grade (G1-G3) and tumor size (pT1-pT4) as well (p = 0.002; p = 0.042), between the EPO-R expression and histological grade (G1-G3) (p < 0.001). CONCLUSIONS: The relationship between the expression of EPO-R and histological malignancy grade in triple negative breast cancer, suggests that the present EPO-R expression in TNBC may constitute an additional prognostic factor.

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Thymomas are tumors originating from the thymus epithelial cells and are the most common tumors of the anterior mediastinum. They have been classified into types A, AB, B1, B2, and B3 by the World Health Organization. Type B3 thymoma is composed of epithelial cell sheets with mild to moderate atypia and scant lymphocytes. An association between thymic carcinoma and neuroendocrine differentiation has been observed by some authors. However, cases of type B3 thymoma with neuroendocrine differentiation are very rarely discussed in the literature. A 68-year-old woman was referred to our hospital with an abnormal shadow on a chest roentgenogram. Chest computed tomography showed that the lesion was located in the anterior mediastinum. She underwent surgery, and the tumor was diagnosed as a type B3 thymoma with neuroendocrine differentiation. An extremely rare case of a type B3 thymoma showing neuroendocrine differentiation is presented herein.

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INTRODUCTION: Arthrofibrosis (AF) is the result of increased cell proliferation and synthesis of matrix proteins (collagen I, III, and VI). Especially after invasive knee surgery, e.g., ligament reconstruction or knee replacement, abnormal fibroblast proliferation with pathological periarticular fibrosis can be observed leading to severely limited joint motion. The pathogenesis of AF is currently not fully understood. The present work aims to determine pathogenic factors. MATERIALS AND METHODS: A descriptive, histological and immunohistochemical comparative study was performed on tissue samples of 14 consecutive patients undergoing arthrolysis for joint stiffness due to AF. Seven human autopsy specimens served as control. Samples were stained for expression of relevant markers such as CD68, α-smooth muscle actin (ASMA), beta-catenin, BMP-2 and examined for the histological grade of AF (cell-rich versus cell-poor) and compared to a control. Furthermore, a microscopic evaluation of the samples for cell differentiation and number was performed. RESULTS: Tissue sections of cell-rich fibrosis showed a significantly higher expression of CD68 compared to the control with less than 10% of CD68 positive cells (p = 0.002). In cell-poor fibrosis no statistically significant difference was obvious (p = 0.228). Expression of ASMA in synovia, vessels, cell-rich and cell-poor fibrosis showed median values of 2.00 in the AF group and 1.75 in the control. Both groups differed significantly (p = 0.003). AF tissue showed a significantly difference in expression of ß-catenin (p < 0.001) compared to the control. The overall difference between AF and control group in expression of BMP-2 was also statistically significant (p = 0.002). CONCLUSIONS: Expression of CD68, ASMA, beta-catenin and BMP-2 is significantly increased in AF tissue samples. Based on presented findings, histological evaluation and immunohistochemical assessment of CD68, ASMA, ß-catenin and BMP-2 expression may proof useful to diagnose AF and to analyze AF activity.

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Pulmonary sclerosing pneumocytoma (PSP) arising from the hilar lesion is extremely rare. We report an asymptomatic 70-year-old female with a thoracic tumor of unknown origin. Contrast-enhanced chest tomography showed a poorly and heterogeneously enhanced 40-mm tumor compressing the left upper lobe, bronchus, and pulmonary arteries. Positron-emission tomography did not detect abnormal integration in the tumor. Surgical resection was planned to confirm diagnosis and avoid further compression on the structures. Intraoperative findings revealed a dark red-colored tumor, projecting from the left upper lobe in the hilar lesion. Left upper lobectomy was performed through video-assisted thoracoscopic surgery to achieve complete resection and avoid contact bleeding. Immunohistochemical examination revealed the presence of PSP.

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A 25-year-old male patient presented with swelling and pain in the mandibular anterior region for the previous 6 months. He had a history of repeated traumatic episodes associated with his lower incisors. A periapical radiograph revealed a radiolucent lesion involving the apices of the lower incisors, whereas cone beam computed tomography revealed a large osteolytic lesion involving all mandibular incisors and loss of labial and lingual cortical plates. Histological, immunohistochemical and bone scan examination of the curetted tissue established a differential diagnosis of spindle cell rhabdomyosarcoma of the anterior mandible. Such rare but life-threatening oral cancers can be correctly diagnosed and treated only through systematic analysis of clinical findings and examining the diseased tissue with histological and other diagnostic procedures.

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OBJECTIVE: To investigate the expression of proliferation and apoptotic factors (Ki-67, Bcl-2), inflammatory factors (NF-kß p65, COX-2), adhesion factors (ß-catenin), estrogen (ER-α) and progesterone receptors (PR-α) in ovarian endometrioma (OE) in patients with recurrent OE by an immunohistochemical assay. SUBJECT AND METHODS: This investigation enrolled 48 reproductive-aged patients with OE. According to the course of the disease during a follow-up period of 1.5 years after surgical treatment, the biomaterial obtained from the examined patients was divided into two groups: 1) an OE capsule from 19 patients with recurrent OE (a study group); 2) an OE capsule from 28 patients without recurrent OE (a comparison group). This investigation used histological and immunohistochemical examinations. The histological analysis of the OE capsule was performed following a standard procedure. Their immunohistochemical analysis was carried out using the Tissue-Tek Quick-Ray kit that allows the preparation of paraffin blocks with a large number of tissue samples (tissue microarrays). Antibodies to Ki-67 (clone 30-9, VENTANA), Bcl-2 (clone 124, VENTANA), NF-kß p65 (clone p65, 'Spring Bioscience Corp.'), COX-2 (clone CX-294, Agilent), ß-catenin (clone 14, VENTANA), ER-α (clone SP1, VENTANA), and PR-α (clone 1E2, VENTANA) were also employed in the investigation. The specimens were prepared according to a standard protocol using a Ventana Ultra immunohistostainer. Positive and negative controls were used to correctly carry out immunohistochemical tests. Statistical analysis was performed using the applied statistical analysis programs Statistica 10.0 and Microsoft Excel. RESULTS: The patients with recurrent OE had a significantly decreased expression of Ki-67 (2.86% vs. 9.69%; р=0.044) in the epithelial component of the OE capsule; a significantly lower expression of NF-kß p65 (2.54 vs. 3.5; р=0.0082) and СОХ-2 (0.231 vs. 1.381; р=0.0025) in the stromal component of the OE capsule, a significantly increased expression of ß-catenin (2.5 vs. 1.59; р=0.017) in the stromal component of the OE capsule; a significantly increased expression of PR-α (188.46 vs. 71.15; р=0.028) in the epithelial component of an OE capsule. The expression of ER-α (stromal component, 266 vs. 256.84; p=0.48; epithelial component, 251.54 vs. 233.85, p=0.82) and Bcl-2 (stromal component, 0.33 vs. 0.25; p=0.85; epithelial component, 0.944 vs. 0.625; p=0.31) in the OE capsule is not statistically significantly different between the study patient groups. CONCLUSION: The immunohistochemical difference in the expression of a number of the markers under study can serve as the basis for a further investigation of these markers as predictors of recurrent OE after surgical treatment. Further investigations of these factors will also be able to examine the molecular mechanisms underlying the pathogenesis of recurrent OE, which will make it possible to affect these mechanisms in order to eliminate the fundamental causes of a recurrence of this disease.

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The paper describes a clinical and anatomical case of partial trisomy of the long arm of 17 chromosome in a baby aged 1 month and 4 days with the karyotype 46,XY, dup(17)(q21q24) from a dichorionic diamniotic twin born after in vitro fertilization. Intrahepatic bile duct hyperplasia was first detected in the patient with this chromosomal abnormality. Histological and immunohistochemical examinations of the liver were conducted using antibodies to Ki-67, CK-pan, CK8, CK 18, Vim, SMA, CD31, CD34, HER-2/neu.

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AIM: to investigate the myocardial expression of some structural proteins and markers of cellular proliferation and innate immunity for assessing their possible diagnostic and prognostic role in patients with chronic myocarditis. SUBJECTS AND METHODS: The investigation enrolled 23 patients (16 men; mean age, 52.0±12.4 years (range, 27 to 73) with various forms of noncoronarogenic myocardial injury who underwent right ventricular endomyocardial biopsy (n=4), intraoperative left ventricular biopsy (n=17) or autopsy (n=2). Prior to their morphological examination, the patients were divided into two groups: 1) 10 patients with dilated cardiomyopathy and presumptive myocarditis; 2) 13 patients with valvular heart disease, hypertrophic cardiomyopathy, myxoma, and chronic pulmonary thromboembolism, presumptively without myocarditis. Along with myocardial histological and immunohistochemical (IHC) examinations, the expression of vimentin, desmin, c-kit, Ki-67, and Toll-like receptors (TLR) 2 and 9 was determined. Polymerase chain reaction was used to identify whether herpes viruses of and parvovirus B19 genomes were present in the blood and myocardial samples; indirect ELISA was applied to estimate the blood level of antibodies against various cardiac antigens. RESULTS: According to the histological findings, active/borderline lymphocytic myocarditis was diagnosed in all the patients (Group 1) and in 6 patients (Group 2) in conjunction with the underlying disease (only in 9 and 7 patients, respectively), viral genome was detected in the myocardium of 15 patients, including in 5 without morphological signs of myocarditis (parvovirus B19 (n=11), herpesvirus 6 (n=4), herpes simplex virus types 1 and 2 (n=1), Epstein-Barr virus (n=2), and cytomegalovirus (n=1)), and in the blood (n=4). A marked correlation was found between TLR2 and TLR9 expressions and the morphological pattern of active myocarditis in the absence of this correlation with the expression level of other studied markers. The expression level of TLR2 in patients with and without borderline myocarditis was 0 [0; 0,75] and in those with active myocarditis was 1.5 [1; 1,5] points; that of TLR9 was 2 [2; 2] and 4 [3; 4] points, respectively (p<0.001). The expression of TLR2 and TLR9 in patients with borderline myocarditis was lower than in those without myocarditis (0 [0; 0] versus 0 [0; 1] and 2 [1,5; 2] versus 2 [2; 3] points), which can reflect cardiomyocyte destruction/depletion at later stages of the disease. There was also a close correlation between the expression level of TLR2 and that of TLR9 (r=0.824; p<0.001) and with Ki-67 levels (r=-0.531 and r=-0.702; p<0.01). There was also a correlation of the expression of the studied markers with viral persistence (desmin), the degree of myocardial dysfunction and cardiosclerosis (c-kit), which calls for further investigations. CONCLUSION: Determination of the myocardial expression level of TLR2 and TLR9 may serve as an immunohistochemical marker for myocarditis and preservation of its activity, which is especially valuable in patients with borderline forms. The marked expression of these markers for innate immunity may reflect both one of the mechanisms of genetic predisposition to myocarditis and its severe course and their secondary activation in the pathogenesis of the disease and is a potential target of therapy.

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AIM: to investigate the proliferative activity and distribution of pluripotent stem cells in various histological types of amelobrastoma. MATERIAL AND METHODS: The surgical and archival materials of 76 patients with ameloblastoma from the Central Research Institute of Dentistry and Oral Surgery, Ministry of Health of the Russian Federation, were used. Tissue antigens were determined using a rabbit monoclonal antibody against to Ki-67 and SOX2. RESULTS: Proliferating (Ki-67) and pluripotent (SOX2) cells were identified in various histological types of ameloblastoma. A weak significant correlation was found between tumor size, pluripotent stem cells, and cellular proliferative activity. Enhanced cell proliferative activity and extensive bone tissue destruction significantly correlated with the development of recurrent ameloblastoma. CONCLUSION: The presence of pluripotent stem cells, the proliferative activity of ameloblastoma cells, and the extensiveness of bone tissue destruction allow basal-cell and plexiform options to be considered as the most aggressive indicators.

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PURPOSE: To immunohistochemically examine the lacrimal sac walls harvested during dacryocystorhinostomy using an immunostain for immunoglobulin G4 (IgG4). METHODS: Forty-four lacrimal sac walls were evaluated. We determined "intensively positive," "sparsely positive," or "negative" staining when the specimens showed IgG4-positive lymphoplasmacytic staining with or without >50 IgG4-positive cells/high-power field, or the absence of stained IgG4-positive cells. RESULTS: Intensively positive, sparsely positive, or negative staining was observed in 8 (18.2%), 14 (31.8%), and 22 specimens (50.0%), respectively. Stained cells infiltrated the subepithelial layer in all specimens with positive staining. Four of the 8 specimens demonstrated partial epithelial denudation with loss of goblet cells. CONCLUSIONS: IgG4-positive lymphoplasmacytic infiltration was observed in the subepithelial layer in specimens with intensively positive staining, of which some showed a partially denuded epithelium with loss of goblet cells. These may lead to narrowing of the lacrimal sac lumen and adhesions of the sac walls.

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Automated image analysis methods are highly important for biotechnology research. The authors developed and tested a program for the morphometric analysis of photomicrographs of the sections processed using the standard immunohistochemical examination protocols. The color deconvolution method used in the algorithm was proven to be effective in mapping the distribution of DAB chromogen in the sample containing multiple dyes. The experiment demonstrated that the level of extracellular matrix proteins could be comparatively quantified in different groups of samples. The effective methods for the quantitative analysis of the Ki-67 labelling index were also tested using the same algorithms. The developed program was published under free GPL 3.0.

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