Μm Sections Research Articles

Biochemical characterization of green zinc nanoparticles fabricated by Stevia rebaudiana aqueous extract and its nephroprotective effects against streptozotocin-induced nephropathy in mice: A histopathological study

The development and creation of novel therapeutic supplements and medications with exceptional efficacy in treating nephropathy is a top priority for both developed and developing nations. The antioxidant, nephroprotective, and cytotoxicity effects of green synthesized zinc nanoparticles (ZnNPs) containing Stevia rebaudiana aqueous extract were assessed in this study. The combination of Stevia rebaudiana aqueous extract with Zn nitrate solution resulted in the Zn nanoparticles production. The characteristics of Zn nanoparticles were determined using transmission electron microscopy (TEM), Fourier-transform infrared spectroscopy (FT-IR), X-ray crystallography (XRD), ultraviolet–visible spectroscopy (UV–Vis) and field emission scanning electron microscopy (FESEM). The synthesized nanoparticles typically ranged in size from 20 to 30 nm. These nanoparticles displayed a crystalline structure and maintained a uniform shape. To induce diabetes, 60 mg/kg of streptozotocin was administered intraperitoneally in 50 mice (n = 10). The treatment groups received glibenclamide (0.5 mg/kg) and 100 and 200 μg/kg of Zn nanoparticles through gavage for 25 days. Additionally, there was a positive control group for comparison (treated-diabetic). On the final day, serum levels of creatinine, urea, and blood glucose were determined in the samples. Following tissue processing, 5 μm sections of kidneys were stained with periodic acid Schiff (PAS) for subsequent stereological and histopathological analysis. The groups treated with ZnNPs exhibited a notable reduction (p < 0.05) in urea and blood glucose levels (about 190 mg/dL) when compared to the diabetic group (about 500 mg/dL) that did not receive treatment. Furthermore, the treatment with a high dose of ZnNPs resulted in a significant decrease (p < 0.05) in kidney weight and volume (including vessels, interstitial tissues, collecting ducts, loop of Henle, and distal and proximal tubules), and kidney structures length (including vessels, collecting ducts, loop of Henle, and distal and proximal tubules) (p > 0.05). Based on the findings, ZnNPs can control urea and blood glucose levels while preventing kidney damage in streptozotocin-induced diabetic animals. This research proposes utilizing zinc nanoparticles synthesized from Stevia rebaudiana as a supplementary treatment for diabetes and kidney protection in developing nations.

Fiber-type composition and 3D capillary analysis of the human splenius capitis muscle.

Despite the significance of neck muscles in musculoskeletal disorders, their microscopic anatomy remains poorly characterized. This study examined the splenius capitis muscle, focusing on its fiber-type composition, fiber size, and capillary network characteristics. For comparison and validation, the vastus lateralis muscle was also analyzed. Muscle samples from 13 young male subjects (mean age ± SD: 35.7 ± 8.6 years) were collected within 24-h post-mortem during autopsy. Myosin heavy chain (MyHC) isoform expression was characterized immunohistochemically in 10 μm sections, while the capillary network architecture was assessed in 100 μm sections. Immunofluorescence staining, confocal microscopy, and 3D image analysis were employed to quantify capillary tortuosity, anisotropy, branch density (Br dens), and the length of capillaries per muscle volume (LV), per muscle fiber length (LL), per fiber surface area (LS), and per fiber volume (LVf). Compared to the vastus lateralis muscle, the splenius capitis muscle had a higher percentage of type 1 fibers (51.2% vs 39.7%), fewer type 2a fibers (16.2% vs 31.4%), and smaller fiber diameters (35.5-40.9 μm vs 47-56.1 μm). It also displayed lower Br dens (P = 0.0069), higher anisotropy (P = 0.0004), and lower LL (P < 0.0001) but higher LVf (P = 0.0486). In the splenius capitis muscle, body mass index (BMI) negatively correlated with LV (P = 0.0155), LS (P = 0.0091), LVf (P = 0.0137), and anisotropy (P = 0.0425), and positively correlated with tortuosity (P = 0.0473), indicating a reduction in the capillary network. In the vastus lateralis muscle, only LV (P = 0.0161) decreased with high BMI. This study characterized the fiber-type composition, fiber size, and 3D capillary network of the splenius capitis muscle, establishing a baseline for investigations into pathological muscle alterations.

Glibenclamide alters the brain protein profile and morphometry of hippocampal regions in mice fed with a high-refined carbohydrate diet

A high-refined carbohydrate diet (HC diet) has shown impacts not only on metabolic disorders, but also on cognitive and behavioral functions. However, its effects at the protein and morphological level in the brain are not known. In order to investigate the differential expression of proteins in the brain of Balb/c mice fed with a HC diet and treated or not with glibenclamide, biochemical and histological assays were performed. The brains of animals fed for eight weeks with a standard rodent diet or HC diet, and treated or not with glibenclamide for four weeks were homogenized and the supernatant was subjected to 12.5% ​​SDS-PAGE. The brains were also blocked and 3 μm sections were stained with Hematoxylin-Eosin and analyzed using light microscopy. The electrophoretic profile of samples from untreated animals revealed protein expression without significant differences, while the group treated with glibenclamide revealed protein bands with differential expression (44.8, 42.2 and 39.8 KDa). The proteins were predicted using a bioinformatic tool and it is suggested that, for the most part, they are related to the energy metabolism of the brain. Histological analysis of the hippocampus demonstrated similarities between animals fed with a standard diet and HC diet, but with differences in the number of neurons in the CA2 and CA3 regions, and a tendency to cell death in the dentate gyrus of animals group HC diet. These results revealed one of the regions of the brain affected by excess carbohydrates, as well as candidate proteins for proteomic analysis to further investigate the effects of dietary sugars on molecular mechanisms and neurogenesis in the hippocampus.

Relationship between Lesion Parameters after Radiofrequency Catheter Ablation in Striated Muscles and Parenchymal Tissue.

Background and Objectives: Radiofrequency catheter ablation (RFCA) is a highly successful intervention. By comparing the lesion changes in prostate parenchymal and striated muscle tissues after RFCA with and without cooling, it was possible to assess the correlation between the shape regularity, area, and perimeter of the thermal lesion, and to predict the geometric shape changes of the lesions. Materials and Methods: A standard prostate and striated muscle RFCA procedure was performed on 13 non-purebred dogs in two sessions: no cooling and cooling with 0.1% NaCl solution. Microtome-cut 2-3 µm sections of tissue samples were stained with haematoxylin and eosin and further examined. The quotient formula was employed to evaluate the geometric shape of the damage zones at the ablation site. Results: The extent of injury following RFCA in striated muscle tissue was comparable to that in prostate parenchymal tissue. Regression analysis indicated a strong and positive relationship between area and perimeter in all experimental groups. In the experimental groups of parenchymal tissues with and without cooling, an increase in the area or perimeter of the damage zone corresponded to an increase in the quotient value. A similar tendency was observed in the striated muscle group with cooling. However, in the striated muscle group without cooling, an increase in lesion area or perimeter lowered the quotient value. Standardised regression coefficients demonstrated that in the striated muscle with cooling, the damage zone shape was more determined by area than perimeter. However, in the parenchymal tissue, the perimeter had a more substantial impact on the damage zone shape than the area. Conclusions: The damage area and perimeter have predictive power on the overall shape regularity of damage zone geometry in both striated muscles and parenchymal tissue. This approach is employed to achieve a balance between the need for tumour eradication and the minimisation of ablation-induced complications to healthy tissue.

On-slide clearing and imaging of 100-µm-thick histological sections using ethyl cinnamate and epifluorescence.

Thick histological samples are difficult to image without proper tissue clearing methods. Among these methods ethyl cinnamate (ECi)-based clearing preserves antigenicity and is compatible with immunofluorescent labeling. In contrast to many other clearing protocols, ECi-based clearing is fast and is done as a final step after standard immunofluorescent labeling protocols.

Ventricular hyperinnervation in a mouse model of catecholaminergic polymorphic ventricular tachycardia

Abstract Funding Acknowledgements Type of funding sources: Foundation. Main funding source(s): European Society of Cardiology Basic Research Fellowship. Dutch Heart Foundation Dekker Grant Background Inherited arrhythmia syndromes are a leading cause of sudden cardiac death in young and otherwise healthy patients. They are often caused by mutations in genes that encode ion channels or transporters, leading to conditions such as Catecholaminergic Polymorphic Ventricular Tachycardia (CPVT). CPVT is caused by mutations in RYR2 (encoding ryanodine receptor 2, RyR2). In arrhythmia research, investigation into the effects of these mutations have exclusively been performed in cardiac cells. However, RyR2 is also present in neuronal tissue, including intracardiac neurons [1] (which modulate cardiac function). Moreover, patients often present with clinical signs of autonomic nervous system (ANS) dysfunction, and treatment strategies frequently target the ANS. Aim Our novel research line aims to investigate the neuronal phenotype induced by RyR2 mutations, using a CPVT mouse model to assess functional alterations as well as broader autonomic remodelling. Results Using immunocytochemistry, we reveal for the first time that RyR2 is also expressed in mouse stellate ganglia (crucial autonomic modulators of cardiac function). Through analysis of published RNA sequencing datasets, we find that RYR2 is more abundantly expressed in both stellate ganglion and intracardiac neurons than characteristic neuronal markers such as CHAT, SYN1 and SCN8A [2,3]. Functional patch clamp investigation of isolated stellate ganglion neurons have revealed two distinct populations based on their patterns of action potential (AP) firing, identifying both phasic-firing and tonic-firing type neurons. Investigation of an established CPVT mouse model (RyR2-R2474S) has thus far not revealed any differences in the AP firing frequency of isolated stellate ganglion neurons during sustained depolarising stimuli of various amplitudes. Assessment of calcium homeostasis and neurotransmitter release is currently ongoing. We have furthermore investigated broader autonomic remodelling using immunohistochemistry in mouse heart cryosections. Neuronal innervation density was assessed by staining 10 µm sections of mid-ventricular myocardium for the sympathetic neuron marker tyrosine hydroxylase (TH). Quantification of the TH-positive area revealed a significant increase in the percentage of sympathetic neuronal innervation in ventricular tissue of CPVT mice (5.7% wild-type vs 7.2% RyR2-R2474S, p=0.0253, N=6). Conclusions The neurons that modulate heart function (stellate ganglion and intracardiac neurons) abundantly express RyR2. The RyR2-R2474S mutation leads to significant sympathetic hyper-innervation of ventricular myocardium, which may have important consequences for neurotransmitter release and hence cardiac (electrical) function and arrhythmogenesis.

Abstract PO1-03-10: Differential microbial metabolic pathways associated with resistance to neoadjuvant immunotherapy in locally advanced triple negative breast cancer

Abstract Differential microbial metabolic pathways associated with resistance to neoadjuvant immunotherapy in locally advanced triple negative breast cancer Background: It has previously been shown that microbial metabolic pathways are differentially activated in immunotherapy resistance, hence can serve as potential targets for therapeutic intervention. With the advent of Immune Checkpoint Blockers in the neoadjuvant setting of locally advanced (LA) TNBC, we hypothesized that similar microbial metabolite mediated resistance may be at play. We are presenting the real world data for neoadjuvant treatment in early TNBC along with a subset of tumor metabolite analysis. Method: 35 patients who received neoadjuvant pembrolizumab and chemotherapy for LA TNBC were identified at Moffitt Cancer Center between July 2019 to January 2023. These included 22 Caucasian, 5 African American, 2 Hispanic and 6 patients with undisclosed race. The age range was 33-78 years. The weight and Body Mass Index range was 55.3 kg-127.3 kg and 20.96-45.43 kg/m2 respectively. Nine patients had stage III disease while 24 had stage II breast cancer. The response was assessed in 33 patients; 20 patients (60.6%) achieved pathological complete response (pCR) and 13 (39.4%) had residual disease (RD). 1 patient developed severe immune mediated toxicity with colitis, hepatitis, myasthenia gravis leading to death while 1 patient had disease progression leading to drug discontinuation. Six patients developed immune mediated adverse events. We conducted untargeted metabolomic profiling on 24 patients to identify differentially abundant metabolites associated with pCR (ypT0/is ypN0) vs RD; 16 vs 8 patients respectively. Formalin fixed paraffin embedded (FFPE) slides from tumor tissue vs tumor bed were retrieved and histopathological evaluation of tumor cells was performed. 20 μm sections of FFPE block were used for untargeted metabolomics via liquid chromatography/mass spectrometry. Among the 200 metabolites detected, 63 were differentially abundant between pCR and tumor samples with RD. After correcting for false discovery rate, we observed striking differential activation of two microbial metabolic pathways. Samples with RD had significantly elevated kynurenine (OR-3.64 X1034, P-1.94 X 10-74 and kynurenine/Tryptophan ratio). The tryptophan, kynurenine pathway has been known to be overexpressed in patients resistant to immunotherapy and modulated by the microbiome. Similarly, the purine metabolic pathway, specifically microbiota-derived immunostimulatory metabolites- inosine, hypoxanthine, uric acid were significantly depleted in patients with RD (Inosine OR-0.54, p-0.007, Hypoxanthine OR-0.5, p-0.00039, uric acid OR: 0.41, P-0.003). Previously Inosine has been shown to increase the anti-tumor effects of immune checkpoint blockade by enhancing CD8+ effector T-cell function. Hence depletion of inosine, hypoxanthine and uric acid signals lack of an effector CD8 T cell response. Conclusion: Despite being a small study, it corroborates pCR rates noted in KEYNOTE 522 in the real world setting but shows significant concern for serious immune mediated adverse effects. This highlights the importance of stratifying patients who might be resistant to ICB or develop significant toxicities, thus deriving little to no benefit from standard neoadjuvant treatment. This underscores the need to develop precision-based treatment algorithms. The study represents one of the first investigations of differential abundance of microbial metabolites with immunotherapy resistance in LA TNBC. Our results highlight two microbially mediated mechanisms that could be targeted to improve pCR rates in LA TNBC. We are working to identify microbial metabolic pathways associated with immune mediated toxicity. Citation Format: Humaira Sarfraz, Brian Czerniecki, Zena Jameel, Michael Jaglal, Mostafa Eysha, Turkan Ozkul, Jameel Muzaffar, Aditi Saha, Sara Sarfraz, Fatima Zahra, Min Liu, Khushali Jhaveri, Melissa Sur, Adrianna Bagos-Estevez, Shahla Bari. Differential microbial metabolic pathways associated with resistance to neoadjuvant immunotherapy in locally advanced triple negative breast cancer [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO1-03-10.

Comparison of histological procedures and antigenicity of human post-mortem brains fixed with solutions used in gross anatomy laboratories.

Brain banks provide small tissue samples to researchers, while gross anatomy laboratories could provide larger samples, including complete brains to neuroscientists. However, they are preserved with solutions appropriate for gross-dissection, different from the classic neutral-buffered formalin (NBF) used in brain banks. Our previous work in mice showed that two gross-anatomy laboratory solutions, a saturated-salt-solution (SSS) and an alcohol-formaldehyde-solution (AFS), preserve antigenicity of the main cellular markers (neurons, astrocytes, microglia, and myelin). Our goal is now to compare the quality of histology and antigenicity preservation of human brains fixed with NBF by immersion (practice of brain banks) vs. those fixed with a SSS and an AFS by whole body perfusion, practice of gross-anatomy laboratories. We used a convenience sample of 42 brains (31 males, 11 females; 25-90 years old) fixed with NBF (N = 12), SSS (N = 13), and AFS (N = 17). One cm3 tissue blocks were cut, cryoprotected, frozen and sliced into 40 μm sections. The four cell populations were labeled using immunohistochemistry (Neurons = neuronal-nuclei = NeuN, astrocytes = glial-fibrillary-acidic-protein = GFAP, microglia = ionized-calcium-binding-adaptor-molecule1 = Iba1 and oligodendrocytes = myelin-proteolipid-protein = PLP). We qualitatively assessed antigenicity and cell distribution, and compared the ease of manipulation of the sections, the microscopic tissue quality, and the quality of common histochemical stains (e.g., Cresyl violet, Luxol fast blue, etc.) across solutions. Sections of SSS-fixed brains were more difficult to manipulate and showed poorer tissue quality than those from brains fixed with the other solutions. The four antigens were preserved, and cell labeling was more often homogeneous in AFS-fixed specimens. NeuN and GFAP were not always present in NBF and SSS samples. Some antigens were heterogeneously distributed in some specimens, independently of the fixative, but an antigen retrieval protocol successfully recovered them. Finally, the histochemical stains were of sufficient quality regardless of the fixative, although neurons were more often paler in SSS-fixed specimens. Antigenicity was preserved in human brains fixed with solutions used in human gross-anatomy (albeit the poorer quality of SSS-fixed specimens). For some specific variables, histology quality was superior in AFS-fixed brains. Furthermore, we show the feasibility of frequently used histochemical stains. These results are promising for neuroscientists interested in using brain specimens from anatomy laboratories.

Abstract A092: Ovarian tumor organotypic slices cultures for functional drug screening (TOSCA)

Abstract A092: Ovarian tumor organotypic slices cultures for functional drug screening (TOSCA)

Optimizing the management of thyroid specimens to efficiently generate whole slide images for diagnosis

Transition from optical to digital observation requires an additional procedure in the pathology laboratory, the scanning of glass slides, leading to increased time and digital archive consumption. Thyroid surgical samples often carry the need to collect several tissue fragments that generate many slides to be scanned. This study evaluated the impact of using different inking colours for the surgical margin, section thickness, and glass slide type, in the consumption of time and archive. The series comprehended 40 nodules from 30 patients, including 34 benign nodules in follicular nodular disease, 1 NIFTP, and 5 papillary carcinomas. In 12 nodules, the dominant pattern was microfollicular/solid and in 28 it was macrofollicular. Scanning times/mm2 were longer in red-inked fragments in comparison to green (p = 0.04) and black ones (p = 0.024), and in blue-inked in comparison to green ones (p = 0.043). File sizes/mm2 were larger in red-inked fragments in comparison to green (p = 0.008) and black ones (p = 0.002). The dominant pattern microfollicular/solid was associated with bigger file size/mm2 in comparison with the macrofollicular one (p < 0.001). All scanner outputs increase significantly with the thickness of the section. All scanning outputs increase with the usage of adhesive glass slides in comparison to non-adhesive ones. Small interventions in thyroid sample management that can help optimizing the digital workflow include to prefer black and green inking colours for the surgical margins and 2 µm section in non-adhesive glass slides for increased efficiency.

Negative prognostic behaviour of PD-L1 expression in tongue and larynx squamous cell carcinoma and its significant predictive power in combination with PD-1 expression on TILs

BackgroundBiomarkers that can predict outcome will improve the efficacy of treatment for HNSCC patients. In this regard, we retrospectively evaluated the prognostic effect of PD1, PD-L1, and CD45RO in tongue and larynx squamous cell carcinomas.MethodsFFPE tissue blocks of 63 larynx and 40 tongue squamous cell carcinoma samples were selected, cut into 3 µm sections, and immunohistochemically stained for PD1, PD-L1, and CD45RO. The slides were evaluated by an expert pathologist, and results were analysed using Chi-square, univariate, and multivariable Cox regression methods.ResultsTC-PD-L1 expression (P = 0.001) and its expression intensity (P = 0.002) were significantly correlated with a higher percentage of PD-1 + tumor infiltrating lymphocytes. In univariate survival analysis, TC-PD-L1 and its expression intensity had a significant impact on both DFS (HR: 0.203; P = 0.003 and HR: 0.320; P = 0.005) and OS (HR: 0.147; P = 0.002 and HR: 0.322; P = 0.005). Based on the multivariate analysis, PD1 (DFS: HR: 3.202; P = 0.011, OS: HR: 2.671; P = 0.027) and TC-PD-L1 (DFS: HR: 0.174; P = 0.006, OS: HR: 0.189; P = 0.009) were found to be independent prognostic markers. In the second part, scoring systems were defined based on the expression status of PD1 and PD-L1. Patients with higher scores were expected to have longer DFS and OS. In multivariate analysis, the PD1/TC-PD-L1 (DFS: P = 0.001, OS: P = 0.003) scoring systems showed superior prognostic effects. Interestingly, at the highest levels of this score, none of the patients experienced recurrence or cancer-caused death.ConclusionCollectively, this study suggests negative prognostic behaviour for TC-PD-L1 protein and introduces the PD-1/TC-PD-L1 scoring system as a strong prognostic marker in OS and DFS prediction of tongue and larynx HNSCC patients.

Assessment of intraepidermal nerve fiber densities in 5 µm sections from arm and leg - a search for normative age-related values.

Background and aims: Normative values are lacking regarding intraepidermal nerve fiber density (IENFD) in thin sections of 5 µm. Thus, we aimed to assess IENFD in thin sections in a healthy adult population as well as to investigate whether IENFD is related to age, sex, and site of excision. Methods: Archival skin biopsies or excisions at the Department of Pathology, Lund, Sweden, from arm and leg were collected, re-sectioned, and immunohistochemically stained for Protein Gene Product 9.5 during 2020-2023. Nerve fibers were manually quantified in the 5 µm thin sections, and IENFD was compared between age groups, sex, and excision sites. Results: IENFDs were evaluated in 602 samples from 591 healthy adults aged 18 to 97 years (295 women, 296 men). Median IENFD values are presented, stratified by age groups, sex, and excision sites. Higher IENFD was observed in the arm compared to the leg, as well as in the proximal compared to the distal leg, however not across all age groups. Levels of IENFD were lower among older adults, compared to all younger groups. Conclusion: We have presented data on IENFD in thin 5 µm sections from a healthy adult population. Despite differences in IENFD observed across age groups, sexes, and excision sites, no strong conclusions regarding affecting factors could be drawn except that individuals > 65 years present with lower IENFD. Additional research and development of the method are warranted.

АКТИН ГЛАДКОМЫШЕЧНЫХ КЛЕТОК КАК КРИТЕРИЙ ГРАВИЧУВСТВИТЕЛЬНОСТИ ЖЕЛУДОЧНО-КИШЕЧНОГО ТРАКТА МЫШЕЙ И МОНГОЛЬСКИХ ПЕСЧАНОК

Despite the key role of the smooth muscle tissue (SMT) in the gastrointestinal organs functioning, mechanisms of SMT gravity sensitivity still remain inadequately studied in space biomedicine. Smooth muscle actin (α-SMA) was studied in membranes of the stomach and small gut fundus in mice С57BL/6N (experiment in 30-d BION-M1 mission and synchronous control BIOS-MLZh), Mongolian gerbils Meriones unguiculatus (experiment in 12-d Foton-M3 missions), and after 30-d tail-suspension according to E.R. Morey-Holton (С57BL/6N males). Immunohistochemical detection of α-SMA was conducted in 2 µm sections using the mouse SMA monoclonal antibodies (1:100, Zytomed Systems, Cat. No. MSK030). Documentary photography and analysis of micropreparations were performed with microscope ZEISS Axio Imager А2. Quantitative analysis was performed on the basis of the area of immunopositive sections using ImageJ 1.51J8. Morphometric analysis of α-SMA in muscular coat of the stomach and jejunum of the space-flown mice and gerbils detected a reduction of immunopositive sections which points to weakening of the SMT functional activity. Gravitational sensitivity of the gastrointestinal contraction structures can be conditioned by changes in structural components of actin myofilament in smooth myocytes or myofibroblasts. Absence of changes in the α-SMA expression after tail-suspension confirms that microgravity affects functional properties of the gastrointestinal muscular coats.

SYNTHESIS OF FIRE RETARDANT WITH PHOSPHORUS AND METAL FOR PRESERVATION AND REACH OF REDUCTION OF FLAMMABILITY OF TEXTILE MATERIALS

In this article, the optimal conditions for synthesizing a new flame retardant based on pentaerythritol, phosphoric acid, and magnesium hydroxide are studied. In this case, the molar ratio of the initial substances was 3.64:1, and the synthesis duration was four hours. After the completion of this process, different (1, 3, and 5%) solutions of magnesium hydroxide were added at a temperature of 90 ºC for 3 hours. The yield of this reaction was 85%. Applying this obtained flame retardant to textile materials increases the fire resistance of textile materials. The composition of synthesized antiperine and antiperine applied textile fabrics was studied by IR spectrum, and the thermal decomposition of antiperine applied fabric was investigated by TGA and DTA analysis, and 100 μm sections of flame retardant treated fabrics were measured by scanning electron microscope. The fire resistance and physical mechanical properties of these materials were studied based on the requirements of GOST 50810-95. The oxygen index of the fabric treated with synthesized flame retardant was 35.6%.

A Rapid and Sensitive UPLC-MS/MS Method for the Determination of Bellidifolin and Pharmacokinetics Study of Bellidifolin Nano-microcells

Background: Bellidifolin (BEL) has a decent enemy of myocardial fibrosis impact, and its preparation into nano-micelles can build security and great biocompatibility in vitro and in vivo. The pharmacokinetic assessment of BEL can be utilized as the reason for the security and viability of BEL in clinical use. Objective: This research aimed to establish an effective UPLC-MS/MS strategy for assuring BEL in rodent plasma and concentrating on its pharmacokinetics in vivo. Methods: Luteolin was utilized as an internal standard (IS). Chromatographic separation was accomplished utilizing a UPLC HSS T3 column (2.1 ×100 mm, 1.8 μm) section using a mobile phase of 0.1% acetonitrile (A) and 0.1% formic acid in water (B) with gradient elution. Electrospray ionization (ESI) coupled mass spectrometry was applied in various response checking (MRM) modes with negative ionization. Results: The pharmacokinetic behaviour of bellidifolin nano-micelles in vivo showed that the peak concentration (Cmax) was 1666.19±479.92 μg/L, the time to peak (Tmax) was 0.167 h, and the apparent elimination half-life (t1/2) was 7.60±3.58 h. The plasma clearance rate (CL/F) was 1.15±0.48 L/h/kg, the apparent volume of distribution (V/F) was 14.38±11.04, the area under the curve (AUC) was 8292.57±4193.13 μg/L*h, and the mean retention time (MRT) was 9.70±4.55 h. Conclusion: The method was successfully applied to the plasma pharmacokinetics of bellidifolin nano-micelles after intragastric administration to rats.

Distributions and expressions of Aquaporin-5 and 7 in the testes of developing male chicks.

Aquaporins (AQPs) are integral membrane proteins that act as water channels for which a total of 13 orthologs of AQP genes in birds have been reported. Tissue expression and cellular or subcellular localization of AQPs have been poorly investigated in the male reproductive system of birds. We aimed to determine the distribution and localization of AQP5 and AQP7 proteins by immunocytochemistry in testicular tissues obtained from developing chicks (14, 21, 28, 35 and 42 days old). Totally 175 male chicks (Ross308) were used in the study from which testicular tissue was removed, fixed in 10% formaldehyde solution, then embedded in paraffin blocks. Five μm sections were cut, mounted on poly-L-lysine slides, dried in an oven, then dehydrated using standard immunohistochemistry staining protocol. The sections were imaged with a Nikon Eclipse 50i trinocular light microscope. Immunohistochemical evaluation of the immune reactivity of AQP5 revealed a positive immune reaction in spermatocytes and interstitial areas of the testes in 14-day-old chicks. Testicular tissue AQP5 immune reactivity was observed in the tubule and the interstitial regions of 21-, 28-, 35- and 42-day-old chicks. AQP7 immune reactions were determined in the tubule and interstitial areas testes of developing chicks' testis tissue, with increasing positivity corresponding to older age. The expression of AQP5 and AQP7 appears to be species-specific due to differences in localization and expression in male chicks compared with studies of other mammals, which is likely to play an important role in regulating fluid and sperm volume. This research can serve as a base for future studies that will contribute to the understanding of the male genital system of AQPs.

Severe preeclampsia complicated by HELLP syndrome alters the structure of Hofbauer and syncytiotrophoblast cells: ultrastructural and immunohistochemical study.

Hemolysis, elevated liver enzymes, low platelet count (HELLP) syndrome is generally considered to be a variant or complication of preeclampsia. It is a life-threatening obstetric complication. To evaluate the immunohistochemistry and ultrastructural of syncytiotrophoblastand Hoffbauer cells in placental villi of patients with HELLP syndrome. Two groups of patients with a total of 50 full-term human placentas (n = 25 in each group) were assigned as the control (normotensive) and HELLP syndrome. Placental tissue samples were fixed in 10% neutral formalin and paraffin-embedding protocol was performed. We prepared 5 μm sections for histological and immunohistochemical staining. Sections were immunostained with Hoffbauer cell marker CD68. For transmission electron microscopy (TEM), placental tissue samples were fixed in 2.5% buffered glutaraldehyde and then, in 1% osmium tetroxide for routine ultrastructural examinations. When the HELLP group fetal placental sections were examined, intracytoplasmic edema in syncytiotrophoblast, degenerative vacuoles, and degenerative findings on cell surface membranes were observed. Moreover, villous edema was remarkable. The number of CD68-positive Hoffbauer cells per villus control group sections was 0.23 ± 0.02 and the number of CD68-positive cells per villus in HELLP group placenta sections was 0.83 ± 0.12. The increase in the number of Hoffbauer cells per villus in the HELLP group was significant (P < 0.001). Compared with the control group, there was a significant increase in the number of Hoffbauer cells and syncytiotrophoblasts in the HELLP group, and degenerative changes were also observed in the ultrastructure of these cells. Pathology of the HELLP syndrome is in relation to CD68-positive placental macrophages.

Evaluation of the nephroprotective properties of silver nanoparticles formulated by Pistacia atlantica aqueous extract under ultrasonic condition in streptozotocin-induced diabetic nephropathy in mice

Green formulated silver nanoparticles offer a great promise in biomedicine. Administration of streptozotocin causes severe hepatotoxicity and nephrotoxicity by increasing the blood glucose. The development of nephroprotective drugs through eco-friendly production routes is a major challenge for current pharmacology and nanotechnology. The creation of cutting-edge technology for the synthesis of effective nephroprotective agents is crucial on a global scale. So in the current study, we report herein the bio-inspired proficient preparation of silver NPs over Pistacia atlantica (P.a.) aqueous extract as natural reducing/capping and stabilizing agent (P.a./Ag NPs) under ultrasonic irradiation. The final hybrid nanocomposite was thereafter characterized by a range advanced analytical methods like, FE-SEM, ICP-OES, EDX, TEM, and elemental mapping. Diabetes was induced by administration of 60 mg/kg of streptozotocin (STZ) intraperitoneally in 100 mature male mice and they were randomly divided into 5 groups. The negative control group received normal saline and treatment groups received glibenclamide with dose 0.5 mg/kg and 10 and 40 μg/kg of P.a./Ag nanocomposite through gavage for 50 days. On the last day, serum levels of samples blood glucose, urea and creatinine were measured. After tissue processing, 5 μm sections of the kidneys were prepared and they were stained by periodic acid Schiff (PAS) and used for stereological analysis. The kidney weight, kidney volume (Volume of cortex, medulla, glomerulus, proximal and distal tubules, collecting ducts, loop of Henle, interstitial tissues, and vessels) and kidney structures length (length of proximal and distal tubules, collecting ducts, loop of Henle, and vessels) decreased significantly (p ≤ 0.05) after treatment with high dose of P.a./Ag NPs nanocomposite (p > 0.05). The increased levels of blood glucose and urea were decreased (p ≤ 0.05) significantly in P.a./Ag nanocomposite-treated groups as compared to the untreated diabetic. This study suggested using from P.a./Ag nanocomposite as an antidiabetic and nephroprotective drug in the developing countries.

MCM-2 Levels as a Potential Biomarker for Predicting High-Risk Breast Cancer Patients According to TAILORx Classification.

The minichromosome maintenance protein-2 (MCM-2) is a more sensitive proliferation marker than Ki-67. This study aimed to evaluate the relationship between MCM-2 and Oncotype DX recurrence score (ODX-RS) and determine an MCM-2 cutoff value in high-risk patients according to TAILORx risk categorization. Hormone receptor (HR) positive HER-2 negative early-stage breast cancer patients (pT1-2, pN0-N1, M0) who had ODX-RS were included in the study. According to the TAILORx trial, patients were divided into two groups with high (ODX-RS ≥26) and low risk (ODX-RS <26) in terms of ODX-RS. Formalin-fixed-paraffin-embedded tissues of patients were re-evaluated, and 3 µm sections were prepared for MCM-2 immuno-histochemical staining. The relationship between ODX-RS and the percentage of MCM-2 staining was evaluated in two groups. The ROC curve analysis was performed to determine the MCM-2 cut-off value for the TAILORx high-risk group (ODX-RS ≥26). The mean MCM-2 value was significantly higher in the high-risk group [(60.2 ± 11.2 vs 34.4 ± 13.8, p < 0.001)]. In the multivariate analysis, MCM-2 (OR: 1.27, 95% CI: 1.08-1.49, p = 0.003) and progesterone receptor (PR) levels ≤10% (OR: 60.9, 95% CI: 4.1-89.7, p = 0.003) were found to be independent factors indicating a high-risk group. A one-unit increase in MCM-2 level increased the likelihood of being in the high-risk group by 1.27 times. In the ROC curve analysis, the optimal MCM-2 cut-off level was 50 (AUC: 0.921, sensitivity: 86.7%, specificity: 96.0%, p < 0.001). Our study is the first study in the literature to investigate the relationship between ODX-RS and MCM-2 levels in HR-positive HER-2 negative early breast-cancer patients. In this study, MCM-2 was an independent risk factor in identifying high-risk patients according to TAILORx risk classification. MCM 2 cut-off value (50) may help the decision on adjuvant chemotherapy in patients where the Oncotype DX test cannot be performed.

Differences in the Tumor Molecular and Microenvironmental Landscape between Early (Non-Metastatic) and De Novo Metastatic Primary Luminal Breast Tumors.

Background: The molecular mechanisms underlying the de novo metastasis of luminal breast cancer (dnMBC) remain largely unknown. Materials and Methods: Newly diagnosed dnMBC patients (grade 2/3, ER+, PR+/-, HER2-), with available core needle biopsy (CNB), collected from the primary tumor, were selected from our clinical-pathological database. Tumors from dnMBC patients were 1:1 pairwise matched (n = 32) to tumors from newly diagnosed patients who had no distant metastases at baseline (eBC group). RNA was extracted from 5 × 10 µm sections of FFPE CNBs. RNA sequencing was performed using the Illumina platform. Differentially expressed genes (DEG)s were assessed using EdgeR; deconvolution was performed using CIBERSORTx to assess immune cell fractions. A paired Wilcoxon test was used to compare dnMBC and eBC groups and corrected for the false discovery rate. Results: Many regulatory DEGs were significantly downregulated in dnMBC compared to eBC. Also, immune-related and hypoxia-related signatures were significantly upregulated. Paired Wilcoxon analysis showed that the CCL17 and neutrophils fraction were significantly upregulated, whereas the memory B-cell fraction was significantly downregulated in the dnMBC group. Conclusions: Primary luminal tumors of dnMBC patients display significant transcriptomic and immunological differences compared to comparable tumors from eBC patients.