Staining Reaction Research Articles

BRAFV600E mutational status assessment in cutaneous melanocytic neoplasms in a group of the Egyptian population

BackgroundMelanocytic neoplasms range from banal nevi to malignant melanomas. The genetic background has been extensively studied in the Caucasian population. BRAF mutations were reported among the early driver mutations in nevogenesis. Nevertheless, the pathogenesis in the Egyptian population has not been elucidated.Aim and MethodsThe present study was carried out to assess the sensitivity and specificity of immunohistochemistry (IHC) using the RM-08 clone in reference to allele-specific real-time PCR (CAST-PCR) for the detection of the BRAF V600E mutation in 50 formalin-fixed paraffin-embedded blocks of melanocytic neoplasms with prior bleaching using hydrogen peroxide in Tris-HCL and Bovine Serum Albumin respectively.ResultsIHC staining was interpreted using staining reaction (positive versus negative) and staining pattern (negative and heterogeneous versus homogenous). Using the staining pattern, the specificity increased from 73.3 to 88.2%, the negative predictive value increased from 73.3 to 100%, the diagnostic accuracy increased from 71.4 to 90.48% and the overall accuracy increased from 69.9 to 77.3%. The sensitivity and positive predictive value remained unchanged. The K-agreement coefficient increased from 0.364 (fair agreement) to 0.741 (good agreement) and was statistically significant (p = 0.00). Next-generation sequencing was performed in 11 cases, 8 cases with IHC-positive and BRAFwild type in addition to 3 cases that failed PCR analysis and revealed no BRAF V600E. No statistically significant difference was found in the clinicopathological parameters between BRAFV600E and BRAF wild−type melanomas.ConclusionsThese findings suggest that IHC staining homogeneity may be more accurate in predicting BRAFV600E mutational status. However, IHC cannot replace molecular methods.

Aloperine inhibits RANKL-induced osteoclast differentiation via suppressing the MAPK signaling pathways

To figure out the molecular mechanism of aloperine (ALO) on receptor activator of nuclear factor (NF)-kappa-B (κb) ligand (RANKL)-caused osteoclast differentiation. The histopathological analysis and tartrate-resistant acid phosphatase (TRAP) staining assays were applied to check the extent of bone loss of the femur. Then, the protein expressions of nitric oxide synthase 2, glutathione reductase, nuclear erythroid 2-related factor 2, heme oxygenase-1, nicotinamide adenine dinucleotide phosphate (NADPH) oxidase 1, and catalase were checked in RAW264.7, a macrophage cell line, by enzyme-linked-immunosorbent serologic assay and Western blot analysis. Further, the TRAP staining and quantitative polymerase chain reaction assay were applied to characterize the level of RAW264.7 osteoclast differentiation. Besides, Western blot assay was used to check the protein expressions of extracellular regulated protein kinases (ERK), c-Jun N-terminal kinase (JNK), and P38 in RAW264.7. Finally, ERK activation blocker U0126 was used to inhibit mitogen-activated protein kinase (MAPK) signaling pathway to determine the effect of MAPK signaling pathway on RAW264.7 osteoclast differentiation. In this study, the results demonstrated that ALO could inhibit lipopolysaccharide-induced bone loss in vivo in a dose-dependent manner, with significant inhibitory effects at high doses. Further research indicated that ALO could inhibit RANKL-induced oxidative stress and osteoclast differentiation of RAW264.7. Then, ALO could inactivate MAPK signaling pathway. Finally, the results showed that inhibition of MAPK signaling pathway could increase ALO inhibition of RANKL-caused osteoclast differentiation. ALO inhibits RANKL-caused osteoclast differentiation by suppressing the MAPK signaling pathways.

P110 activation protein is strongly expressed in intestinal fibrosis in inflammatory bowel disease patients

Abstract Background Intestinal fibrosis is a common complication of inflammatory bowel disease (IBD) with limited diagnostic modalities to detect and determine the degree of fibrosis. Fibroblast activation protein alpha (FAP) is a protein with both dipeptidyl peptidase and endopeptidase properties which is virtually absent in healthy tissues and has increased expression in chronic inflammatory and fibrotic diseases. In this study, we investigated the presence of FAP in intestinal fibrosis caused by Crohn’s disease (CD) and ulcerative colitis (UC). Methods For this study, transmural surgical resection specimens were retrieved from the Tytgat institute IBD biobank located at Amsterdam University Medical Center. IBD patients undergoing an ileocecal resection for stricturing CD or a subtotal colectomy for therapy-refractory disease, and patients undergoing a right-sided hemicolectomy or ileocecal resection for dysplasia or non-metastasized colorectal cancer (CRC), were included. All patients had an established diagnosis of (CD, UC, dysplasia or CRC) by previous endoscopy and biopsies and had provided informed consent prior to surgery. FAP protein and gene expression levels were determined using immunohistochemistry staining and quantitative polymerase chain reaction (qPCR). Mass cytometry was used to identify FAP–expressing cells. Results In total, 59 ileal and 35 colonic samples were stained and quantified to asses FAP expression (figure 1). A 1.7-fold and 1.9-fold increase of FAP protein expression was seen in inflamed (mean 22.3, p=0.0069); and stenotic (mean 25.5 p=0.0002) CD ileum compared to healthy non-IBD tissue (mean 13.1). Inflamed UC colon (mean 26.8, p = 0.0406) showed a 1.4-fold increase compared to healthy colon (mean 19.2). For the gene expression, 66 ileal and 42 colonic samples were processed and used for qPCR analysis. A 5-fold and 6.4-fold increase of FAP was found in inflamed (mean 0.011, p=0.0475) and stenotic (mean 0.014, p=0.0042) CD ileum, and a 1.7-fold increase was seen in inflamed UC colon (mean 0.278, p=0.0195) compared to controls (means: ileum 0.002; colon 0.165). Conclusion This is the first study reporting the potential of FAP as a fibrotic biomarker not only in stricturing CD, but also in therapy-refractory UC. Significantly increased FAP protein and gene expression is found in inflamed and stenotic ileum in CD and inflamed colon in UC. Additionally, CD90+GP38+ cells have been identified as the most present FAP expressing cells.

Long-Term Follow-Up Results of Granulomatous Lymphadenitis Diagnosed by Endobronchial Ultrasound-Guided Fine-Needle Aspiration Biopsy.

Introduction Endobronchial ultrasound-guided fine-needle aspiration biopsy (EBUS-FNAB) is a minimally invasive method used to obtain cytological or histological specimens of masses and lymphadenopathies (LAP) adjacent to the trachea and bronchi. Granulomas, which represent a chronic inflammatory response and occur for a variety of reasons, like a 'sarcoid-like reaction', cause LAPs. In this study, it was aimed to evaluate the long-term follow-up results of granulomatous lymphadenitis diagnosed with EBUS-FNAB and to investigate whether granulomatous lymphadenopathies were precursors of malignancies that occurred during the follow-up period. Material and methods The medical records of 123 patients who underwent EBUS-FNAB and were diagnosed with granulomatous lymphadenitiswere retrospectively reviewed. Age, gender, acid-fast bacilli (ARB) staining, tuberculosis culture and tuberculosis polymerase chain reaction (PCR) culture results were examined by FNAB, and the procedure indications of all patients diagnosed with granulomatous lymphadenitis were recorded. The long-term health records of 52 patients could not be accessed. Data were collected from 71 patients. The progression, regressionor stable conditions of LAPs in the long-term radiological follow-up of at least two years and the treatment conditions of diagnosis after biopsywere examined. Results One hundred twenty-three patients were included in the study. Rapid onset evaluation (ROSE) was performed in 93 (75.6%) patients. In 62 (66.6%) of the 93 patients, the smear results were consistent with granulomatous reaction at baseline. Malignancy was present in seven patients (5.6%) at the time of the procedure. In two patients (1.62%), tuberculous lymphadenitis was diagnosed by a positive tuberculosis culture. The long-term follow-up results were not obtained in 52 (42.7%)patients included in the study. At the long-term follow-up of six patients' LAPs with known malignancies, three of them regressed, one of them progressed, and two of them remained stable after chemoradiotherapy. Methylprednisolone treatment was started in eight patients with the diagnosis of sarcoidosis. While LAP remained stable in five patients, regression was observed in three patients. Idiopathic LAPs remained stable in 24 of 55 patients who received no treatment and regressed spontaneously in 31 of them. One of the patients was diagnosed with lymphoma, and the otherpatient was diagnosed with primary lung cancer in the long-term follow-up. Conclusion In cases where tuberculosis is suspected, not only cytomorphology but also microbiological confirmation is important. Granulomatous lymphadenitis can be detected both in the disease course of patients with a history of malignancy and as a precursor to undiagnosed malignancy. So the diagnosis of granulomatous lymphadenitis is a clinicopathological diagnosis that must be followed up in patients without symptoms or other findings.

Histologic Features of Syphilitic Gastritis: A Rare but Resurging Imitator of Common Diseases.

The range of histopathologic features of gastric syphilis is not well described. Here we describe the clinicopathologic findings of eight patients with syphilitic gastritis. A search of our Pathology Data System (2003-2022) and multiple other institutions identified eight patients with syphilitic gastritis. Clinical information, pathology reports, and available slides were reviewed. Lesions predominated in middle-aged adults (mean age, 47.2 years; range, 23-61 years) with a propensity for men (n = 7). Three patients had a documented history of human immunodeficiency virus. Clinical presentations included weight loss, abdominal pain, hematochezia, fever, dyspepsia, nausea and vomiting, hematemesis, anemia, and early satiety. Endoscopic findings included ulcerations, erosions, abnormal mucosa, and nodularity. All specimens shared an active chronic gastritis pattern with intense lymphohistiocytic infiltrates, variable plasma cells, and gland loss. Prominent lymphoid aggregates were seen in four specimens. The diagnosis was confirmed either by immunostain for Treponema pallidum (n = 7) or by direct immunofluorescence staining and real-time polymerase chain reaction (n = 1). All patients with available follow-up data showed resolution of symptoms after antibiotic therapy (n = 4). Recognition of the histologic pattern of syphilitic gastritis facilitates timely treatment, prevents further transmission, and avoids unnecessarily aggressive treatment.

Density, Distribution and Staining Properties of Eosinophilic Granular Cells in Oscar Fish (Astronotus ocellatus) Intestine

Fish eosinophilic granular cells are found in the connective tissues of structures associated with the external environment. These cells are similar to mammalian mast cells in terms of structural and functional characteristics. The cytoplasmic granules of these cells give different staining reactions depending on fixative type. This study aimed to determine the staining properties and densities of eosinophilic granular cells in Oscar fish (Astronotus ocellatus Agassiz, 1831) intestine using different fixatives and histochemical techniques. Formalin and basic lead acetate fixation-giemsa staining indicated that eosinophilic granular cells were abundant in anterior intestine, localizing around especially the blood vessels and submucosa. Giemsa staining of Bouin’s fixed rather than other fixatives showed that eosinophilic granular cells were higher in posterior intestine. No reaction was observed in eosinophilic granular cells in Thionin and Toluidine Blue staining in any fixative. Eosinophilic granular cells in samples fixed with different fixatives had metachromatic Alcian Blue staining. In conclusion, this study shows that fixatives may have different effects on the distribution and staining properties of eosinophilic granular cells in Oscar fish intestinal regions. Abundance in anterior intestine and around submucosal blood vessels shows that eosinophilic granular cells could play an active role in mucosal immunity against food-borne pathogens.

Cytological and molecular screening of Chlamydia trachomatis in infertile women attending a maternity teaching hospital in Gezira State, Sudan: a cross-sectional study

Background: Chlamydia trachomatis (CT) is a sexually transmitted pathogen that threatens reproductive health worldwide. This study aims to screen CT urogenital infection using cytology and molecular methods in women suffering infertility. Methods: In total, 415 women suffering infertility, attending Wad Madani Maternity Hospital were included in this study and then classified into two groups: primary infertile women and secondary infertile women. Both urine (n= 415) and vaginal swab samples (n= 130) were collected and tested using Giemsa stain and Polymerase Chain Reaction (PCR) for detection of CT. Results: CT was detected in 33.7% (140/415) of urine samples and 73.1% (95/130) of vaginal swab samples using Giemsa stain, compared with 44.6% (185/415) and 84.6% (110/130) using PCR, respectively. In the primary infertile group (n= 265), chlamydia was detected in 35.8% (95/265) of urine and 75% (60/80) of swab samples by Giemsa stain compared with 50.9% (135/265) and 75% (60/80) of the samples by PCR. In the secondary infertile group (n= 150), chlamydia was detected in 30% (45/150) of urine and 70% (35/50) of swab samples by Giemsa stain compared with 33.3% (50/150) and 100% (50/50) of the samples by PCR. The associated risk factors were age, lower abdominal pain, and urethritis (p< 0.05). The sensitivity, specificity, positive predictive value, and negative predictive value of Giemsa stain in detecting chlamydia compared to PCR were 86.4%, 100%, 100%, and 83.6%, respectively. Conclusions: Giemsa stain can be used as a screening test for detection of urogenital chlamydia in urine and vaginal samples in places where PCR is difficult to be performed.

Comparative study of alginate and type I collagen as biomaterials for cartilage stem/progenitor cells to construct tissue-engineered cartilage in vivo.

With the help of biomaterials, cartilage stem/progenitor cells (CSPCs) derived from cartilage tissue present a promising choice for cartilage regeneration. In our previous study, we investigated whether CSPCs could be ideal seeding cells for cartilage tissue regeneration. Biomaterials are fabricated to accelerate tissue regeneration, providing a suitable environment for cell attachment, proliferation, and differentiation. Among the biomaterials used in cartilage regeneration medicine, alginate and collagen are classified as natural biomaterials and are characterized by high biocompatibility, bioactivity, and non-toxic degradation products. However, it is unclear which material would have a competitive advantage in CSPC-based cartilage regeneration in vivo. In the present study, we employed alginate and type Ⅰ collagen as substrates for CSPCs and chondrocytes, which was made control group, to explore a more suitable biomaterials for CSPCs to fabricate tissue-engineered cartilage, in vivo. Hematoxylin and eosin (HE) staining, Safranin O, immunohistochemical assay, and quantitative real-time polymerase chain reaction (qRT-PCR) were used to evaluate the tissue-engineered cartilage in vivo. Compared with the alginate group, collagen enhanced the expression of cartilage-specific genes, such as ACAN, SOX9, and COLII, more markedly. Furthermore, the marker genes of expression, dedifferentiation, and hypertrophy, COLI and COLX, were downregulated in the collagen group. The results demonstrated that collagen as a substrate was superior to alginate in increasing the accumulation of cartilage-like ECM for CSPCs in vivo. In summary, compared with alginate, collagen hydrogel is an effective biomaterial for CSPC-based cartilage regeneration.

Exposure to enriched environment ameliorated chronic unpredictable mild stress-induced depression-like symptoms in rats via regulating the miR-92a-3p/kruppel-like factor 2 (KLF2) pathway

BackgroundSilencing of miR-92a-3p may be beneficial in relieving depression of chronically stressed rats. The level of kruppel-like factor 2 (KLF2) was increased in the striatum of depressed rats after ketamine treatment. Enriched environment (EE) ameliorated depression-like behaviors in rats. However, the specific mechanism of EE treatment on depression induced by chronic unpredictable mild stress (CUMS) remains unclear. MethodsAfter CUMS-induced male Sprague Dawley rats were treated under EE or/and Adeno-Associated Virus (AAV)-miR-92a-3p, depression-like behaviors, cognitive ability, dendritic spine density, as well as levels of miR-92a-3p and KLF2 were detected by the behavioral tests, morris water maze test, Golgi staining, and quantitative real-time polymerase chain reaction (qRT-PCR) as needed. The body weight of rats was also measured. Next, primary hippocampal neurons were cultivated. The targeting relationship between miR-92a-3p and KLF2 was analyzed by TargetScan v7.2 and dual-luciferase reporter assay. After hippocampal neurons were transfected with miR-92a-3p mimic or/and overexpressed KLF2 vector, the cell viability, and apoptosis, together with the levels of KLF2, brain-derived neurotrophic factor (BDNF), phosphorylated (p)-tropomysin related kinase B (p-TrkB) and TrkB were determined by MTT assay, flow cytometry, qRT-PCR, and western blot as needed. ResultsEE ameliorated CUMS-induced depression-like behaviors and cognitive ability, and elevated the neuronal dendritic spine density and KLF2 level, but reduced miR-92a-3p level in hippocampal tissues, while the above effects were reversed by AAV-miR-92a-3p. MiR-92a-3p mimic restrained cell viability, along with p-TrkB/ TrkB and BDNF levels, but promoted apoptosis in hippocampal neurons, which were reversed by overexpressed KLF2. ConclusionEE ameliorates CUMS-induced depression-like symptoms in rats via regulating the miR-92a-3p/KLF2 pathway.

Ethnobotanical Survey and Some Biological Activities of Ageratum conyzoides Collected in Southern-Benin

Aims: Ageratum conyzoides L. is a small annual herbaceous highly odorous plant use in traditional medicine. The aim of this study is to evaluate in vitro antioxidant potential, toxicity and antimicrobial activity of aerial part extracts of A. conyzoides on strains potentially involved in vaginal infections.
 Methodology: An ethnobotanical survey has been carried out on A. conyzoides among ethnobotanists and traditional therapists in fifteen markets in the communes of Abomey- Calavi, Cotonou, Zogbodomey, Bohicon and Abomey in Southern Benin. The phytochemical screening was a qualitative analysis based on staining and precipitation reactions. Antimicrobial activity of A. conyzoides aqueous and ethanolic extracts was evaluated on reference and clinical strains of Staphylococcus aureus, Candida albicans and Escherichia coli using micro dilutions method in wells from. The toxicity of A. conyzoides extracts was determine using Artemia salina larvae, whereas the antiradical activity was evaluated using the Ferric Reducing Antioxidant Power (FRAP) method.
 Results: The survey showed that the population of Southern-Benin uses A. conyzoides according to different modes of preparation. Also, the administration in the treatment of a variety of pathologies affecting the female reproductive system. The phytochemical screening revealed the presence of flavonoids, tannins, anthocyanins, triterpenes and C- heterosides. The yield of 6.18% for the aqueous extract and 4.32% for the ethanolic extract as recorded. The highest inhibition diameter (24.05 ± 0.5 mm) was obtained using aqueous extract against the clinical S. aureus strain. In contrast, the lowest inhibition diameter (10±0 mm) was obtained against the S. aureus ATCC29213 with the same extract. The Minimum Inhibitory Concentration varied from 2.5 to 5 mg/ml. Both extracts show a bactericidal and fungicidal effect on the different strains studied but the sensitivity of the strains to the aqueous extract is better compared to the ethanolic extract. In addition, the aqueous extracts showed higher antioxidant power compared to the ethanolic extract. No toxicity is revealed for both extracts.
 Conclusion: The results obtained show that the aqueous and ethanolic extracts of the aerial part of A. conyzoides have antioxidant and antimicrobial properties on strains involved in vaginal infections and do not present a toxicity.

Fibroblast growth factor 5 overexpression ameliorated lipopolysaccharide-induced apoptosis of hepatocytes through regulation of the phosphoinositide-3-kinase/protein kinase B pathway.

Sepsis is a systemic inflammatory syndrome induced by several infectious agents. Multiple organs are affected by sepsis, including the liver, which plays an important role in metabolism and immune homeostasis. Fibroblast growth factors (FGFs) participate in several biological processes, although the role of FGF5 in sepsis is unclear. In this study, lipopolysaccharide (LPS) was administrated to mice to establish a sepsis-induced liver injury. A similar in vitro study was conducted using L-02 hepatocytes. Western blot and immunohistochemistry staining were performed to evaluate the FGF5 expression level in liver tissues and cells. Inflammatory cell infiltrations, cleaved-caspase-3 expressions, reactive oxygen species and levels of inflammatory cytokines were detected by immunofluorescence, dihydroethidium staining, and reverse transcription quantitative polymerase chain reaction analysis, respectively. Flow cytometry was used to detect the apoptosis level of cells. In addition, ribonucleic acid (RNA)-sequencing was applied to explore the possible mechanism by which FGF5 exerted effects. LPS administration caused FGF5 down-regulation in the mouse liver as well as in L-02 hepatocytes. Additionally, with FGF5 overexpression, liver injury and the level of hepatocyte apoptosis were ameliorated. Further, RNA sequencing performed in hepatocytes revealed the phosphoinositide-3-kinase/protein kinase B (PI3K/AKT) pathway as a possible pathway regulated by FGF5 . This was supported using an inhibitor of the PI3K/AKT pathway, which abrogated the protective effect of FGF5 in LPS-induced hepatocyte injury. The anti-apoptotic effect of FGF5 on hepatocytes suffering from LPS has been demonstrated and was dependent on the activation of the PI3K/AKT signaling pathway.

Isolation and Determination of Antibacterial Sensitivity Characteristics of Staphylococcus aureus from Lactating Cows in West Shewa Zone, Ethiopia.

Staphylococcus (S.) aureus is one of the etiologies of bovine mastitis, hindering milk production and productivity in dairy farms. This study was aimed at assessing the distribution of bovine mastitis and the isolation rate of S. aureus in milked cows of West Shewa Zone. The clinical mastitis was diagnosed by physical methods including observation and palpation, whereas the subclinical mastitis was tested by the California mastitis test (CMT). All of the cows tested for mastitis were aseptically sampled (teat-milk) for bacteriology. The bacterium was primarily identified based on colony characterization, catalase, coagulase tests, and Gram stain reaction. Finally, MALDI-TOF Biotyper confirmed the species. The antibacterial sensitivity characteristics of the isolates to different antibacterial drugs were tested by the disk diffusion method. The drugs were selected based on the frequent usage in veterinary medicine in the study area. By using particular primers, the presence of the resistance (mecA and blaZ), and thermonuclease (nuc) genes were determined by polymerase chain reaction (PCR). The data were analyzed by R statistical software. The associations between the dependent variables (prevalence of mastitis and S. aureus) and the explanatory variables were analysed by chi-square (χ 2) and logistic regression tests at a 95% confidence interval (CI). Accordingly, 258 lactating cows were examined, of which 97 (37.6%) were mastitis positive. Of these mastitis positive cows, 59 (60.8%) were subclinical and 38 (39.2%) were clinical. Among the 258 milk samples, 43 (16.7%) were positive for S. aureus. According to the results of the current investigation, subclinical mastitis was significantly more prevalent than clinical mastitis (p < 0.05). The disease was found varied with the lactation stage of the animal, milking with washed hand, udder washing before milking, and tick infestation of the teat. In comparison to animals from farms with lower number of lactating cows, the prevalence of the bacteria was significantly higher in animals managed in farms with large (OR = 12.58, 95% CI = 2.33-68.54, and p < 0.05) and medium (OR = 12.58, 95% CI = 2.33-68.54, and p < 0.05) population of lactating cows per herd. The isoation rate of the bacterium was also found significantly higher in tick-infested cows (OR = 27.69, 95% CI = 9.71-93.01, and p < 0.05) than tick free cows. The antibiogram tests revealed that the isolates resisted penicillin G and tetracycline group drugs (oxytetracycline and tetracycline). Moreover, the nuc gene was confirmed to be present in all of the examined isolates. However, they were not found harboring blaZ and mecA genes. We concluded that S. aureus is sustaining as a main causative agent of bovine mastitis, and they were resistant to the frequently used antibiotics in public and veterinary medicines in the study areas. Therefore, research-based interventions need to be taken in action to combat the pathogen.

Andrographis paniculata ameliorates estrogen deficiency-related osteoporosis by directing bone marrow mesenchymal stem cell fate.

Although Andrographis paniculata (AP) exhibits various biological functions such as anticancer, anti-inflammatory, antimalarial, antimicrobial, antioxidant, cardioprotective and immunomodulatory, its role in estrogen deficiency-related osteoporosis remains unclear. Ovariectomy (OVX)-induced estrogen deficiency-related osteoporotic mouse models and sham mouse models were established using 8-week-old female C57BL/6J mice. Micro-computed tomography (µCT) scanning was performed to assess the skeletal phenotype. The differentiation potential of bone marrow mesenchymal stem cells (BMSCs) from the OVX and sham groups was assessed by osteogenic or adipogenic induction medium in vitro. To verify the effects of AP, alizarin red S (ARS) staining, alkaline phosphatase (ALP) staining and oil red O (ORO) staining, reverse transcription assay and quantitative real-time polymerase chain reaction were applied to detect the lineage differentiation ability of BMSCs. µCT scanning showed that AP treatment attenuated the osteoporotic phenotype in OVX-induced estrogen deficiency-related osteoporotic mice. The results of ARS staining, ALP staining, ORO staining and quantitative real-time polymerase chain reaction indicated that BMSCs from OVX-induced osteoporotic mice displayed a significant reduction in osteogenic differentiation and an increase in adipogenic differentiation, which could be reversed by AP treatment. Our findings suggested that AP regulated the differentiation potential of BMSCs and ameliorated the development of estrogen deficiency-related osteoporosis, which might be an effective therapeutic method for estrogen deficiency-related osteoporosis.

Estrogen Receptor-α Exacerbates EGF-Inducing Airway Remodeling and Mucus Production in Bronchial Epithelium of Asthmatics.

Although estrogen receptors (ERs) signal pathways are involved in the pathogenesis and development of asthma, their expressions and effects remain controversial. This study aimed to investigate the expressions of ERα and ERβ as well as their mechanisms in airway remodeling and mucus production in asthma. The expressions of ERα and ERβ in the airway epithelial cells of bronchial biopsies and induced sputum cells were examined by immunohistochemistry. The associations of ERs expressions with airway inflammation and remodeling were evaluated in asthmatic patients. In vitro, the regulations of ERs expressions in human bronchial epithelial cell lines were examined using western blot analysis. The epidermal growth factor (EGF)-mediated ligand-independent activation of ERα and its effect on epithelial-mesenchymal transitions (EMTs) were investigated in asthmatic epithelial cells by western blot, immunofluorescent staining, and quantitative real-time polymerase chain reaction. ERα and ERβ were expressed on both bronchial epithelial cells and induced sputum cells, and the expressions showed no sex difference. Compared to controls, male asthmatic patients had higher levels of ERα on the bronchial epithelium, and there were cell-specific expressions of ERα and ERβ in induced sputum. The expression of ERα in the airway epithelium was inversely correlated to forced expiratory volume in 1 second (FEV1) % and FEV1/forced vital capacity. Severe asthmatic patients had significantly greater levels of ERα in the airway epithelium than mild-moderate patients. ERα level was positively correlated with the thickness of the subepithelial basement membrane and airway epithelium. In vitro, co-stimulation of interleukin (IL)-4 and EGF increased the expression of ERα and promoted its nuclear translocation. EGF activated the phosphorylation of ERα via extracellular signal-regulated kinase and c-Jun N-terminal kinase pathways. ERα knockdown alleviated EGF-mediated EMTs and mucus production in airway epithelial cells of asthma. ERα contributes to asthmatic airway remodeling and mucus production through the EGF-mediated ligand-independent pathway.

A functional study of zinc-titanium coatings and exploration of the intrinsic correlation between angiogenesis and osteogenesis.

With the development of implant applications, osseointegration has become a criterion for implant success. A good blood supply is essential for successful osseointegration. To improve the pro-angiogenic ability of the implants, in this experiment we introduced zinc into the titanium coating. The physical morphology, biocompatibility, pro-angiogenic ability, and osteogenic effect of the zinc-containing titanium coatings were investigated. The pro-angiogenic effect of zinc ions was determined, and the intrinsic link between angiogenesis and osteogenesis under the effect of zinc ions was investigated. Zinc-containing titanium coating was prepared using a micro-arc oxidation (MAO) technique. The physical properties of the coating materials were determined by analyzing the microstructure, roughness, hydrophilic properties, constituent elements, and ionic release of the coating. The biocompatibility of the coating materials was examined using apoptosis and proliferation assays of human umbilical vein endothelial cells (HUVECs). The pro-angiogenic function and osteogenic ability of the zinc-containing coating were investigated by CD31 immunofluorescence staining and quantitative polymerase chain reaction (q-PCR) assay. The optimal concentration of zinc ions for pro-angiogenesis was screened by ion assay. Conditioned media (CM) were prepared for the experiments. The intrinsic link between angiogenesis and osteogenesis was determined by q-PCR to detect the expression of genes related to HUVECs and BMSCs after culture in CM. The prepared Zn-containing micro-arc oxide coatings were shown to have good physical properties, stable Zn2+ release ability, and biocompatibility, as well as good angiogenic and osteogenic potential. In addition, ion experiments confirmed that 60 μM Zn2+ exhibited the best angiogenic effect; more importantly, a mutual promotion between angiogenesis and osteogenesis regeneration in the Zn2+ microenvironment was also found. The introduction of Zn2+ improved the implants' functionality and laid the foundation for the clinical application of Zn2+ pro-angiogenesis.

Inflammation of Bone in Patients with Periprosthetic Joint Infections of the Knee.

In this study, we analyzed 75 human bone and bone-marrow specimens (obtained from 65 patients undergoing revision arthroplasty with cement for the treatment of PJI) for markers of inflammation. Samples were analyzed using hematoxylin and eosin overview staining, fluorescent immunohistochemical staining, flow cytometry, and polymerase chain reaction (PCR). Leukocyte prevalence was significantly elevated at explantation (femur, +218.9%; tibia, +134.2%). While leukocyte prevalence decreased at reimplantation (femur, -49.5%; tibia, -34.2%), the number of cells remained significantly higher compared with the control group (femur, +61.2%; tibia, +54.2%). Expression of inflammatory markers interleukin (IL)-1α (femur, +2,748.7%; tibia, +1,605.9%), IL-6 (femur, +2,062.5%; tibia, +2,385.7%), IL-10 (femur, +913.7%; tibia, +897.5%), IL-12 (femur, +386.1%; tibia, +52.5%), IL-18 (femur, +805.3%; tibia, +547.7%), and tumor necrosis factor (TNF)-α (femur, +296.9%; tibia, +220.9%) was significantly elevated at prosthesis explantation in both femoral and tibial specimens. Expression remained significantly elevated at reimplantation for all inflammatory markers except IL-12 compared with the control group. Conversely, there were only limited inflammatory changes in the bone marrow environment. The present study demonstrated a strong and lasting upregulation of the proinflammatory environment in the joint-surrounding osseous scaffold in patients with PJI. Our data suggest that modulating the inflammatory environment has substantial potential to improve the clinical outcome in affected patients.

Pathoanatomical algorithm for differential diagnosis of Paget's disease of the breast

Paget's disease of the breast is a rare type of cancer that affects the skin of the nipple and usually the areola. At the same time, most patients also have one or more tumors in the immediate vicinity of the focus of mammary Paget's disease. This tumor must be distinguished from normal or atypical Toker cells, and also differentiated from diseases such as Bowen's disease of the nipple and melanocytic lesions of the nipple and areola region, including nipple melanoma and BAP1-inactivated nevus (Wiesner nevus). Currently, there is no routine pathological diagnostic algorithm for these conditions. The aim of the work is to formulate a clear clinical and morphological algorithm for diagnosing Paget's disease of the breast and Toker cells, Bowen's disease of the nipple and areola, as well as melanoma and BAP1-inactivated nevi of the above localizations. Surgical material obtained from patients with Paget's disease of the breast (18), Toker cells of the nipple (2), Bowen's disease of the nipple (6), melanoma of the nipple (1), BAP1-inactivated nevus (1) was studied. The material was examined histologically with hematoxylin and eosin staining, Alcian blue and PAS reaction, as well as immunohistochemically with the following panel of antibodies: CD138, p53, CK8, CK7, HER2/neu, EMA, HMB-45, Melan A, S-100, p63, p16 and BAP1. An easy-to-learn pathoanatomical algorithm for diagnosing Paget's cancer has been developed, which will be especially useful for pathologists who encounter pathology of the nipple and areola in their work.

Oviductal sperm storage in the Chinese pond turtle, Mauremys reevesii, depends on androgen-based promotion of the BCL 2 anti-apoptotic pathway.

Sperm storage is a complex and highly coordinated process that is regulated by a variety of factors. The BCL 2 protein family plays a key role in regulating apoptosis, and determines sperm survival. The objective of this study was to explore the correlation between sperm storage and the BCL 2 protein family in the oviduct of Mauremys reevesii . Hematoxylin-eosin (HE) staining, immunohistochemistry (IHC) and real-time quantitative polymerase chain reaction (RT-qPCR) techniques were used to investigate three parts of the reproductive tract (isthmus, uterus and vagina) of mated and unmated female M. reevesii . Hematoxylin-eosin staining revealed many sperm stored in the oviduct. IHC showed positive immunostaining for the BCL 2 and BAX proteins in epithelial ciliated and glandular cells. RT-qPCR indicated that the mRNA expressions of anti-apoptotic genes (BCL 2 , MCL 1 , BCL- W , BCL-XL ) and the androgen receptor (AR) were significantly higher in mated turtles than unmated turtles. However, the expression of pro-apoptotic genes (BAX , BAD , BID and CASPASE 3 ) showed the opposite relationship. These results suggest that sperm entering the oviduct can promote the synthesis of anti-apoptotic genes to protect themselves from various degradation factors. These findings will help researchers understand the mechanisms of sperm storage.

Microbiological evaluation of the disinfection of the root canal system using different irrigation protocol – An interventional study

ABSTRACT Aim: The focus of the present research is to analyze the potential role of irrigants along with the activation system in the disinfection of the root canal space. Methods: Ninety root canals were randomly divided into two experimental groups based on the irrigants: Group I (n = 45) – Sodium hypochlorite irrigant and Group II (n = 45) –Chlorhexidine irrigant. The two groups are further subdivided into three subgroups base on the activation devices, i.e., passive ultrasonic irrigation, endoactivator, and laser. The first sample as a baseline, and the second sample was collected after the disinfection procedure. All the samples were streaked in brain − heart infusion agar plate to analyze the bacterial colony growth. The confirmatory analysis for the presence of Enterococcus faecalis was done using gram staining, biochemical analysis, and polymerase chain reaction. The nonparametric analysis was done using Kruskal–Wallis, Mann–Whitney U, and Wilcoxon signed-rank test and P &lt; 0.05 was considered to be statistically significant. Results: The mean colony-forming unit was significantly reduced and there exhibited a statistically significant difference in pretreatment and posttreatment irrigated with sodium hypochlorite and chlorhexidine activated with passive ultrasonic activation with (P = 0.001), endoactivator with (P = 0.001), and laser with (P = 0.001). Conclusion: In consideration with advantage of the properties of both irrigants, the present study concludes a combined use with sodium hypochlorite during instrumentation followed by laser activation and a final rinse with chlorhexidine for a better eradication of the microbes from the root canal system thus preventing re-infection.

Inhibition of Abelson Tyrosine-Protein Kinase 2 Suppresses the Development of Alcohol-Associated Liver Disease by Decreasing PPARgamma Expression

Alcohol-associated liver disease (ALD) represents a spectrum of alcohol use-related liver diseases. Outside of alcohol abstinence, there are currently no Food and Drug Administration-approved treatments for advanced ALD, necessitating a greater understanding of ALD pathogenesis and potential molecular targets for therapeutic intervention. The ABL-family proteins, including ABL1 and ABL2, are non-receptor tyrosine kinases that participate in a diverse set of cellular functions. We investigated the role of the ABL kinases in alcohol-associated liver disease. We used samples from patients with ALD compared with healthy controls to elucidate a clinical phenotype. We established strains of liver-specific Abl1 and Abl2 knockout mice and subjected them to the National Institute on Alcohol Abuse and Alcoholism acute-on-chronic alcohol feeding regimen. Murine samples were subjected to RNA sequencing, AST, Oil Red O staining, H&E staining, Western blotting, and quantitative polymerase chain reaction to assess phenotypic changes after alcohol feeding. Invitro modeling in HepG2 cells as well as primary hepatocytes from C57BL6/J mice was used to establish this mechanistic link of ALD pathogenesis. We demonstrate that the ABL kinases are highly activated in ALD patient liver samples as well as in liver tissues from mice subjected to an alcohol feeding regimen. We found that the liver-specific knockout of Abl2, but not Abl1, attenuated alcohol-induced steatosis, liver injury, and inflammation. Subsequent RNA sequencing and gene set enrichment analyses of mouse liver tissues revealed that relative to wild-type alcohol-fed mice, Abl2 knockout alcohol-fed mice exhibited numerous pathway changes, including significantly decreased peroxisome proliferator activated receptor (PPAR) signaling. Further examination revealed that PPARγ, a previously identified regulator of ALD pathogenesis, was induced upon alcohol feeding in wild-type mice, but not in Abl2 knockout mice. Invitro analyses revealed that shRNA-mediated knockdown of ABL2 abolished the alcohol-induced accumulation of PPARγ as well as subsequent lipid accumulation. Conversely, forced overexpression of ABL2 resulted in increased PPARγ protein expression. Furthermore, we demonstrated that the regulation of hypoxia inducible factor 1 subunit alpha (HIF1α) by ABL2 is required for alcohol-induced PPARγ expression. Furthermore, treatment with ABL kinase inhibitors attenuated alcohol-induced PPARγ expression, lipid droplet formation, and liver injury. On the basis of our current evidence, we propose that alcohol-induced ABL2 activation promotes ALD through increasing HIF1α and the subsequent PPARγ expression, and ABL2 inhibition may serve as a promising target forthe treatment of ALD.