Intact Epithelial Cells Research Articles

Decreased SMP30 Expression Is Related With EMT in the Kidneys of Two Siberian Tigers With CKD.

Chronic kidney disease (CKD) is one of the most common causes of mortality in wild non-domestic felidae. The molecular mechanism regulating renal fibrosis in nephropathy is not fully understood especially in the felidae. This study aimed to elucidate senescence marker protein 30 (SMP30) expression patterns and its relationship with epithelial-mesenchymal transition (EMT) by immunostaining in two necropsied Siberian tigers (Panthera tigris altaica) with CKD. Two kidney samples from male Siberian tigers were fixed and tissue sections were stained for histopathological assay. In CKD, renal tubular epithelial cells lost their tubular structures surrounded by severe interstitial fibrosis and were detached from the basement membrane. These damaged cells resembled the morphology of mesenchymal cells and showed much lower SMP30 expression compared with intact tubular epithelial cells. These cells also expressed vimentin, which is specifically expressed by mesenchymal cells, and through double staining, it was observed that vimentin was expressed in the tubular epithelial cells where SMP30 was not expressed. In addition, double-positive expression of pan-cytokeratin (pan-CK) and vimentin was found in damaged epithelial cells with mesenchymal features. We demonstrated possible evidence to understand the role of SMP30 as a new pivotal factor and the possibility of decreased SMP30 as a potential indicator of EMT at the end stage of CKD.

Implications of dietary soybean meal replacement with corn gluten meal on growth performance, antioxidant activities, hepatopancreatic histopathology, and intestinal flora of juvenile Pacific shrimp (Litopenaeus vannamei)

This study aimed to investigate the potential of replacing soybean meal (SBM) with corn gluten meal (CGM) in the diet of Pacific white shrimp (Litopaeneaus vannamei). The study examined the effects of a dietary soybean meal replacement on growth, biochemical indices, antioxidant activities, histology, and intestinal flora. A total of 840 juvenile Pacific shrimp were fed seven diets containing different levels of CGM, ranging from diet 1 (diet1) 0% (control), diet2 (3%), diet3 (7%), diet4 (9%), diet5 (11.8%), diet6 (12.7%), and diet 7 (14.4%). The experiment was conducted over 56 days, with three replicate of each group (initial mean weight = 0.13 0.00 g). The results showed that dietary CGM significantly increased final body weight, average weight gain, and specific growth rate. However, the inclusion of dietary CGM decreased with higher levels. The feed conversion ratio of diets 2 and 3 was considerably lower than that of the control group. The activities of alkaline phosphatase and acid phosphatase increased significantly with the feeding of CGM, while the levels of alanine aminotransferase and aspartate aminotransferase decreased significantly. The histology of the hepatopancreas showed an evident epithelial tubule (E, B, R, and F), and the number of intact epithelial cells increased considerably with the increasing inclusion of CGM (P < 0.05). The intestinal flora in the shrimp intestine responded favorably to the replacement of SBM with CGM in the dietary CGM treatment groups, including a number of beneficial bacteria such as Bacteroidetes and Firmicutes. Moreover, with rising CGM levels, diversity indices and richness estimates rose. The regression model with a broken line was found to describing the relationship between the growth rate and the optimal level of CGM required. The results showed that a CGM dose of 3.31% was the most effective for achieving optimal growth performance in juvenile L. vannamei.

Phagocyte population in the inflammatory pattern of the bronchi of asthma patients regulated by interleukin-17A and interferon-gamma during the airway response to a cold stimulus

Introduction. Based on the common effector functions of polymorphonuclear neutrophils and macrophages as phagocytic cells, their role in the formation of an acute reaction of the respiratory tract to a cold stimulus in patients with asthma is of concern.Aim. The study of the concentration of phagocytes, IL-17A and IFN-γ in the inflammatory pattern of the bronchi of asthma patients depending on the airway reaction to a cold stimulus.Materials and methods. 129 patients with asthma were examined. The design of the study included questioning patients using a validated questionnaire Asthma Control Test (ACT, Quality Metric Inc., 2002), collection of induced and spontaneously produced sputum, exhaled breath condensate (EBC), bronchoprovocation test with a 3-minute isocapnic hyperventilation with cold (-20ºС) air (IHCA) with an assessment of the airway response (ΔFEV1) by spirometry.Results. Group 1 (n=55) included individuals with ΔFEV1 -10% and below, group 2 (n=74) – with ΔFEV1 above -10%: -15 (-21; -11) and -3.7 (-6.1; -0.38)%, respectively (p=0.0002). According to the level of ACT (17 [13; 21.5] and 19 [14; 22] points) and indicators of lung function (FEV1 [93.0±2.4 and 97.1±2.4%] and FEF25-75 [63.5±3.5 and 72.0±3.7%]), the patients had no significant intergroup differences. The pattern of bronchial inflammation in group 1 was mixed (neutrophils ≥40%), in group 2 – eosinophilic. In response to the IHCA test, the number of neutrophils significantly increased in the sputum of patients in group 1, the number of macrophages and the number of structurally intact epithelial cells decreased, in proportion to this, the level of IFN-γ and IFN-γ-inducible protein IP-10 (CXCL10) increased in the EBC in relation to patients of the 2nd group. A direct relationship was found between baseline concentrations of IP-10 and IFN-γ (Rs=0.7; p&lt;0.01) in EBC.Conclusion. The airway response to a cold stimulus of patients with asthma is accompanied by functional activation of phagocytic cells with an escalation of neutrophilic inflammation and a decrease in the number of macrophages infiltrating the bronchi associated with an increase in the concentration of IFN-γ, which stimulates the processes of respiratory burst and triggers cell destruction and cytolysis.

Corneal optical density: Structural basis, measurements, influencing factors, and roles in refractive surgery.

The cornea is the main refractive medium of the human eye, and its clarity is critical to visual acuity. Corneal optical density (COD) is an important index to describe corneal transparency. Intact corneal epithelial and endothelial cells, regular arrangement of collagen fibers in the stroma, and normal substance metabolism are all integral for the cornea to maintain its transparency. In the last two decades, the Pentacam Scheimpflug imaging system has emerged as a breakthrough for the measurement of COD (also called corneal densitometry). It has been found that a wide variety of factors such as age, refractive status, and corneal diseases can affect COD. Different corneal refractive surgery methods also change COD in different corneal regions and layers and affect visual acuity following the surgery. Thus, COD has gradually become a significant indicator to evaluate corneal health, one on which the attention of clinicians has been increasingly focused.

Safety and Efficacy of Double Lamellar Keratoplasty for Corneal Perforation.

To assess the outcomes of double lamellar keratoplasty in the treatment of corneal perforation secondary to various keratopathies. In this prospective non-comparative interventional case series, 15 eyes from 15 consecutive patients with corneal perforation were chosen to undergo double lamellar keratoplasty, which is characterized by two layers of lamellar grafting in the perforated area. The posterior graft was separated from a relatively healthy and thin lamellar graft of the recipient, and the anterior graft was transplanted from the donor lamellar cornea. Preoperative characteristics and postoperative examination and relevant complications throughout the study were recorded. Nine men and six women with an average age of 50.73 ± 19.89 (range, 9-84) years were enrolled. The median follow-up period was 18 (range, 12-30)months. In all postoperative patients, the integrity of the eyeball was successfully rebuilt, and the anterior chambers were formed without aqueous leakage. At the last visit, best-corrected visual acuity improved in 14/15 patients (93.3%). Slit-lamp microscopy showed that all treated eyes remained fully transparent. Anterior segment optical coherence tomography revealed that the double-layer structure of the treated cornea was clear in the early postoperative stage. In vivo confocal microscopy revealed intact epithelial cells, sub-basal nerves, and clear keratocytes in the transplanted cornea. No immune rejection or recurrence was detected during the follow-up period. Double lamellar keratoplasty presents a new therapeutic option for patients undergoing corneal perforation, and it provides improvement in visual acuity and reduces the risk of postoperative adverse events.

Sphingoid Bases Regulate the Sigma-1 Receptor-Sphingosine and N,N'-Dimethylsphingosine Are Endogenous Agonists.

Both bioactive sphingolipids and Sigma-1 receptor (S1R) chaperones occur ubiquitously in mammalian cell membranes. Endogenous compounds that regulate the S1R are important for controlling S1R responses to cellular stress. Herein, we interrogated the S1R in intact Retinal Pigment Epithelial cells (ARPE-19) with the bioactive sphingoid base, sphingosine (SPH), or the pain-provoking dimethylated SPH derivative, N,N'-dimethylsphingosine (DMS). As informed by a modified native gel approach, the basal and antagonist (BD-1047)-stabilized S1R oligomers dissociated to protomeric forms in the presence of SPH or DMS (PRE-084 as control). We, thus, posited that SPH and DMS are endogenous S1R agonists. Consistently, in silico docking of SPH and DMS to the S1R protomer showed strong associations with Asp126 and Glu172 in the cupin beta barrel and extensive van der Waals interactions of the C18 alkyl chains with the binding site including residues in helices 4 and 5. Mean docking free energies were 8.73-8.93 kcal/mol for SPH and 8.56-8.15 kcal/mol for DMS, and calculated binding constants were ~40 nM for SPH and ~120 nM for DMS. We hypothesize that SPH, DMS, and similar sphingoid bases access the S1R beta barrel via a membrane bilayer pathway. We further propose that the enzymatic control of ceramide concentrations in intracellular membranes as the primary sources of SPH dictates availability of endogenous SPH and DMS to the S1R and the subsequent control of S1R activity within the same cell and/or in cellular environments.

Tubular epithelial progenitors are excreted in urine during recovery from severe acute kidney injury and are able to expand and differentiate in vitro.

BackgroundAcute kidney injury (AKI) is a serious condition associated with chronic kidney disease, dialysis requirement and a high risk of death. However, there are specialized repair mechanisms for the nephron, and migrated committed progenitor cells are the key players. Previous work has described a positive association between renal recovery and the excretion of tubular progenitor cells in the urine of kidney transplant recipients. The aim of this work was to describe such structures in non-transplanted AKI patients and to focus on their differentiation.MethodsMorning urine was obtained from four patients with AKI stage 3 and need for RRT on a consecutive basis. Urine sediment gene expression was performed to assess which part of the tubular or glomerular segment was affected by injury, along with measurement of neprilysin. Urine output and sediment morphology were monitored, viable hyperplastic tubular epithelial clusters were isolated and characterized by antibody or cultured in vitro. These cells were monitored by phase contrast microscopy, gene, and protein expression over 9 days by qPCR and confocal immunofluorescence. Furthermore, UMOD secretion into the supernatant was quantitatively measured.ResultsUrinary neprilysin decreased rapidly with increasing urinary volume in ischemic, toxic, nephritic, and infection-associated AKI, whereas the decrease in sCr required at least 2 weeks. While urine output increased, dead cells were present in the sediment along with debris followed by hyperplastic agglomerates. Monitoring of urine sediment for tubular cell-specific gene transcript levels NPHS2 (podocyte), AQP1 and AQP6 (proximal tubule), and SLC12A1 (distal tubule) by qPCR revealed different components depending on the cause of AKI. Confocal immunofluorescence staining confirmed the presence of intact nephron-specific epithelial cells, some of which appeared in clusters expressing AQP1 and PAX8 and were 53% positive for the stem cell marker PROM1. Isolated tubule epithelial progenitor cells were grown in vitro, expanded, and reached confluence within 5–7 days, while the expression of AQP1 and UMOD increased, whereas PROM1 and Ki67 decreased. This was accompanied by a change in cell morphology from a disproportionately high nuclear/cytoplasmic ratio at day 2–7 with mitotic figures. In contrast, an apoptotic morphology of approximately 30% was found at day 9 with the appearance of multinucleated cells that were associable with different regions of the nephron tubule by marker proteins. At the same time, UMOD was detected in the culture supernatant.ConclusionDuring renal recovery, a high replicatory potential of tubular epithelial progenitor cells is found in urine. In vitro expansion and gene expression show differentiation into tubular cells with marker proteins specific for different nephron regions.

Osteogenic Differentiation Effect of BMP-9 with Phenamil and Simvastatin on Intact Human Amniotic Epithelial Stem Cells

Background: Bone tissue engineering has shown to be a promising strategy for repairing bone defects without causing harmful side effects to the patient. Three main building blocks of tissue engineering, including seeding cells, scaffold, and signaling molecules, are required for adequate bone regeneration. The human amniotic membrane (hAM) is the innermost of the placental membranes. In addition to providing a source of stem cells and growth factors, hAM has several features that make it an appropriate scaffold containing stem cells for use in tissue engineering purposes. The present investigation aimed to assess the effect of bone morphogenetic protein-9 (BMP-9) combined with phenamil and simvastatin on osteogenic induction of hAM with its human amniotic membrane epithelial cells (hAECs). Methods: Using six different osteogenic medium (OMs), we cultured hAM for 14 days. The basic OMs were chosen as the first group and other media were made by adding BMP-9, phenamil, simvastatin, BMP-9 alongside phenamil, and BMP-9 alongside simvastatin to the basic OMs. Finally, viability assay, tissue mineralization, calcium and phosphate content determination, and measurement of lactic acid dehydrogenase (LDH), and alkaline phosphatase (ALP) activity were performed. Results: Among all study groups, groups containing simvastatin showed a significantly lower level of viability. Although all media could induce osteogenic features, the hAECs cultured in media containing BMP-9 and phenamil demonstrated a wider area of mineralization and a significantly higher level of calcium and phosphate content, LDH, and ALP activity. Conclusion: Our findings indicated that the use of phenamil together with BMP-9 could synergistically show in situ osteogenic induction in hAECs, which could be a new insight into translational medicine.

Is Epithelial-Mesenchymal Transition a New Roadway in the Pathogenesis of Oral Submucous Fibrosis: A Comprehensive Review.

Epithelial-mesenchymal transition (EMT) collectively refers to a series of episodes that reshape polarized, intact epithelial cells into discrete motile cells that can conquer the extracellular matrix (ECM). It performs a pivotal role in embryonic development, wound healing, and tissue repair. Surprisingly, the exact mechanism can also lead to the onset of malignancy and organ fibrosis contributing to scar formation and loss of function. transforming growth factor signaling, WNT signaling, Notch signaling, Hedgehog signaling, and receptor tyrosine kinase signaling, as well as non-transcriptional changes in response to extracellular cues, such as growth factors and cytokines, hypoxia, and contact with the surrounding ECM, are responsible for the initiation of EMT. Although the pathogenesis of oral submucous fibrosis (OSMF) is multifactorial, compelling evidence suggests that it results from collagen deregulation. EMT is one of the spotlight events in the pathogenesis of OSMF, with myofibroblasts and keratinocytes being the victim cells. EMT is an essential step in both physiological and pathological events. The importance of EMT in the malignant development of OSMF and the inflammatory reaction preceding fibrosis implies a new upcoming area of research. This review aims to focus on the EMTevents that function as a double-edged sword between wound healing and fibrosis and further discuss the mechanisms along with the molecular pathways that direct changes in gene expression essential for the same in the oral cavity. As OSMF involves a risk of malignant transformation, understanding the cellular and molecular events will open more avenues for therapeutic breakthroughs targeting EMT.

Flow Cytometry-Based Single Cell Analyses of Bacterial Adaptation to Intracellular Environments.

Since decades, flow cytometry (FC) is a powerful technique to perform single cell analyses with high accuracy and throughput. Moreover, FC is the method of choice to study bacterial cell heterogeneity and complements single-cell imaging techniques. The complex experimental approaches for FC sample preparation and the subsequent FC adjustment and gating strategy demand careful considerations to be successful when analyzing complex microbial populations, especially when liberated populations of intracellular bacterial pathogens, or bacterial pathogens inside intact host cells are analyzed. Here, we provide a set of experimental protocols for FC sample preparation of (1) in vitro cultured bacterial cells, (2) liberated intracellular bacteria from host cells, or (3) preparation of intact infected phagocytic or epithelial cells commonly used as host cells in infection biology. Since sample preparation, cytometer adjustment, and gating strategy are essential for experimental success, we aim to provide our expertise to support application of FC by other researchers.

Overexpression of the MSK1 Kinase in Patients With Chronic Lung Allograft Dysfunction and Its Confirmed Role in a Murine Model.

Chronic lung allograft dysfunction (CLAD) and its obstructive form, the obliterative bronchiolitis (OB), are the main long-term complications related to high mortality rate postlung transplantation. CLAD treatment lacks a significant success in survival. Here, we investigated a new strategy through inhibition of the proinflammatory mitogen- and stress-activated kinase 1 (MSK1) kinase. MSK1 expression was assessed in a mouse OB model after heterotopic tracheal allotransplantation. Pharmacological inhibition of MSK1 (H89, fasudil, PHA767491) was evaluated in the murine model and in a translational model using human lung primary fibroblasts in proinflammatory conditions. MSK1 expression was graded over time in biopsies from a cohort of CLAD patients. MSK1 mRNA progressively increased during OB (6.4-fold at D21 posttransplantation). Inhibition of MSK1 allowed to counteract the damage to the epithelium (56% restoration for H89), and abolished the recruitment of MHCII+ (94%) and T cells (100%) at the early inflammatory phase of OB. In addition, it markedly decreased the late fibroproliferative obstruction in allografts (48%). MSK1 inhibitors decreased production of IL-6 (whose transcription is under the control of MSK1) released from human lung fibroblasts (96%). Finally, we confirmed occurrence of a 2.9-fold increased MSK1 mRNA expression in lung biopsies in patients at 6 months before CLAD diagnosis as compared to recipients with stable lung function. These findings suggest the overall interest of the MSK1 kinase either as a marker or as a potential therapeutic target in lung dysfunction posttransplantation.

Effects of electroacupuncture on urodynamics, intramedullary apoptosis and bidirectional regulation of neurotrophic factors in neurogenic bladder rats after supersacral spinal cord injury: 电针对骶上脊髓损伤后神经源性膀胱大鼠尿流动力学及髓内凋亡,神经营养因子双向调控作用的影响

ObjectiveTo observe the effects of electroacupuncture on urodynamics and intramedullary apoptosis related factor B cell lymphoma gene-2 (Bcl-2), Bcl-2 related protein X (Bax) and brain-derived neurotrophic factor (BDNF) in neurogenic bladder rats after supersacral spinal cord injury. MethodsA total of 60 female SD rats, SPF grade, were randomly selected, 12 rats in the blank group and 12 rats in the sham operation group, and the remaining 36 rats were made models. Of these 36 rats, 24 rats meeting the model standard were randomized into the model group and electroacupuncture group, with 12 rats in each group. The neurogenic bladder models after supracacral spinal cord injury were prepared by the Hassan Shaker spinal cord transection method, via combining the previous model study experience. At the end of spinal shock stage, “Dàzhuī (大椎GV14)” “Zhōngjí (中极CV 3) ” “ Cìliáo (次髎BL 32) ”, and “Sānyīnjiāo (三阴交SP 6) ” were selected for electroacupuncture. Observation on the histomorphological changes of bladder detrusor and spinal cord in rats was conducted by HE staining. The apoptosis rate of spinal cord cells was measured by TUNEL assay. The maximum bladder volume and compliance were measured by urodynamics test. The protein expressions of Bcl-2, Bax, and BDNF in spinal cord tissues were measured by Western blot assay. The expressions of Bcl-2, Bax, and BDNF mRNA were measured by RT-PCR assay. Results① The results of HE staining showed that compared with the blank group and sham operation group, in the model group the bladder epithelial cell structure was destroyed, the detrusor muscle fibers proliferated, and there were severe bleeding changes, the spinal cord tissue was disordered, the structure was destroyed, the cell space was enlarged, and accompanied by a large number of inflammatory cell infiltration and cystic cavity formation. Compared with the model group, in the electroacupuncture group, the intact bladder epithelial cells increased, the detrusor muscle fiber proliferation decreased, the bleeding changes decreased, the complete structural area of spinal cord tissues increased, the cell space decreased, the levels were clearer, and the cystic cavity decreased. ② The results of TUNEL assay indicated that the TUNEL positive rate of spinal cord cells in the model group was significantly higher than that in the blank group and sham group (both P<0.05). Compared with the model group, the TUNEL positive rate of spinal cord cells in the electroacupuncture group was significantly reduced (P<0.05). ③ The results of urodynamics test suggested that compared with the blank group and sham operation group, the maximum bladder volume and compliance of the model group were significantly reduced (all P<0.05). Compared with the model group, the maximum bladder volume and compliance were significantly increased in the electroacupuncture group (both P<0.05). ④ The results of Western blot assay indicated that, compared with the blank group and sham operation group, in the model group the protein contents of Bcl-2 and BDNF were significantly reduced (all P<0.05), while the protein content of Bax in the model group was significantly increased (both P<0.05). ⑤ The results of RT-PCR assay indicated that, compared with blank group and sham operation group, in the model group the intramedullary Bcl-2 and BDNF mRNA were significantly reduced (all P<0.05), and Bax mRNA was significantly increased (both P<0.05). ConclusionElectroacupuncture at “GV14” “CV3” “BL32”, and “SP6” can promote the repair of bladder detrusor, improve the maximum bladder volume and compliance of bladder, promote morphological repair of bladder detrusor, reduce spinal cord cell apoptosis rate, thus to restore the function of bladder. This is maybe related with the electroacupuncture's two-way regulatory mechanism of increased expression of protein and gene of Bcl-2 and BDNF and decreased expression of protein and gene of Bax in spinal cord tissues.

Role of neutrophils and bronchial epithelium in loss of control of bronchial asthma and formation of respiratory tract response to cold stimulus

Introduction . Neutrophils and bronchial epithelium are actively involved in the generation of oxidative stress and the progression of airway inflammation in patients with asthma. Aim . To study the role of neutrophils and bronchial epithelium as factors contributing to the loss of control over asthma and the formation of the airway response to a cold stimulus. Materials and methods . A total of 146 patients with a diagnosis of persistent asthma were examined. A survey was conducted on the Asthma Control Test (ACT) questionnaire; a study of the lung function by spirometry with subsequent collection of induced sputum; the next day, a 3-minute isocapnic hyperventilation by cold (-20ºC) air (IHCA) was performed, followed by collection of spontaneously produced sputum. In the sputum, cytosis, cellular composition, and glycoprotein concentration in the goblet-like epithelium were assessed. Group 1 (n=89) included persons with uncontrolled (ACT˂20 points), group 2 (n=57) – with controlled (ACT&gt;20 points) asthma. Results . Patients of group 1 in comparison with group 2 had a higher increase in FEV 1 to the inhalation of a short-acting β 2 -agonist ‒ 11.0 (5.4; 21.65) vs. 7.55 (4.11; 13.8)%, respectively (p=0.042) and a more pronounced reaction to the IHCA test ‒ -11.0 (-17.6; -2.5) vs. -6.0 (-12.0; - 2.0)% (p=0.031). In patients of group 1, after the IHCA test, an increase in the number of neutrophils was observed from 45.98±2.17 to 52.0±1.78% (p=0.0013) and an increase in the concentration of glycoproteins in mucocytes from 37.9±3.8 to 45.6±4.2% (p=0.002) against the background of a tendency towards an increase in the concentration of goblet cells and a decrease in the number of intact bronchial epithelial cells (from 4.8±0.32 to 3.9±0.30%; p=0.017). Correlation analysis showed the relationship between the level of goblet cell glycoproteins and the initial concentration of neutrophils in sputum (r=-0.49; p=0.039), as well as between the severity of the reaction of the distal bronchi (ΔMEF 50 ) to a cold stimulus and the concentration of neutrophils in sputum after IHCA (r=0.36; p=0.01). Conclusion . The signs of stimulation of the neutrophilic segment of granulocytes and structural rearrangement of the bronchial epithelium in the sputum of patients with uncontrolled asthma after acute cold exposure were determined. Structural changes in the pool of neutrophils and epithelial parenchyma of the bronchi, indicating an escalation of pro-inflammatory activity, may underlie the emergence and maintenance of the airway response to a cold stimulus and loss of asthma control.

Nanoparticles of selenium as high bioavailable and non-toxic supplement alternatives for broiler chickens.

Selenium is commonly used in the poultry industry as an additive in broiler feed to improve immunity and overall health. The selenium comes in different forms, inorganic and organic selenium, as sodium selenite and selenomethionine, respectively. This study proposes the use of nanoparticles of selenium (nanoSe) for improved delivery and absorption of the trace element while causing no toxicity. Previous studies have shown the success in utilizing nanoSe in broiler feed, with increased absorption and diffusion of material into organs and tissues, and increased antioxidant capacity. However, the mechanism of nanoSe conversion remains unknown, and the gut microbiota is believed to play a significant role in the process. The use of inorganic selenium in poultry feed demonstrated a lower bioavailability in breast (P ≤ 0.01) and duodenum tissue (P ≤ 0.05), and increased accumulation in organs involved in detoxification processes as compared to organic selenium and selenium nanoparticle supplementation. Histopathological analysis showed that nanoSe did not cause any damaging effects to the tissues analysed, revealing intact epithelial cells in the digestive system and neuronal bodies in brain tissue. The results indicate that nanoparticles of selenium operate a similar way to organic selenium and could potentially be used in poultry feed as a trace element additive.

Ductal tree ablation by local delivery of ethanol prevents tumor formation in an aggressive mouse model of breast cancer

BackgroundProphylactic mastectomy is the most effective intervention to prevent breast cancer. However, this major surgery has life-changing consequences at the physical, emotional, psychological, and social levels. Therefore, only high-risk individuals consider this aggressive procedure, which completely removes the mammary epithelial cells from which breast cancer arises along with surrounding tissue. Here, we seek to develop a minimally invasive procedure as an alternative to prophylactic mastectomy by intraductal (ID) delivery of a cell-killing solution that locally ablates the mammary epithelial cells before they become malignant.MethodsAfter ID injection of a 70% ethanol-containing solution in FVB/NJ female animals, ex vivo dual stained whole-mount tissue analysis and in vivo X-ray microcomputed tomography imaging were used to visualize ductal tree filling, and histological and multiplex immunohistochemical assays were used to characterize ablative effects and quantitate the number of intact epithelial cells and stroma. After ID injection of 70% ethanol or other solutions in cancer-prone FVB-Tg-C3(1)-TAg female animals, mammary glands were palpated weekly to establish tumor latency and examined after necropsy to record tumor incidence. Statistical difference in median tumor latency and tumor incidence between experimental groups was analyzed by log-rank test and logistic mixed-effects model, respectively.ResultsWe report that ID injection of 70% ethanol effectively ablates the mammary epithelia with limited collateral damage to surrounding stroma and vasculature in the murine ductal tree. ID injection of 70% ethanol into the mammary glands of the C3(1)-TAg multifocal breast cancer model significantly delayed tumor formation (median latency of 150 days in the untreated control group [n = 25] vs. 217 days in the ethanol-treated group [n = 13], p value < 0.0001) and reduced tumor incidence (34% of glands with tumors [85 of 250] in the untreated control group vs. 7.3% of glands with tumor [7 of 95] in the ethanol-treated group, risk ratio = 4.76 [95% CI 1.89 to 11.97, p value < 0.0001]).ConclusionsThis preclinical study demonstrates the feasibility of local ductal tree ablation as a novel strategy for primary prevention of breast cancer. Given the existing clinical uses of ethanol, ethanol-based ablation protocols could be readily implemented in first-in-human clinical trials for high-risk individuals.

Whole genome amplification of single epithelial cells dissociated from snap-frozen tissue samples in microfluidic platform.

Single cell sequencing is a technology capable of analyzing the genome of a single cell within a population. This technology is mostly integrated with microfluidics for precise cell manipulation and fluid handling. So far, most of the microfluidic-based single cell genomic studies have been focused on lab-cultured species or cell lines that are relatively easy to handle following standard microfluidic-based protocols without additional adjustments. The major challenges for performing single cell sequencing on clinical samples is the complex nature of the samples which requires additional sample processing steps to obtain intact single cells of interest without using amplification-inhibitive agents. Fluorescent-activated cell sorting is a common option to obtain single cells from clinical samples for single cell applications but requires >100 000 viable cells in suspension and the need for specialized laboratory and personnel. In this work, we present a protocol that can be used to obtain intact epithelial cells from snap-frozen postsurgical human endometrial tissues for single cell whole genome amplification. Our protocol includes sample thawing, cell dissociation, and labeling for genome amplification of targeted cells. Between 80% and 100% of single cell replicates lead to >25 ng of DNA after amplification with no measurable contamination, sufficient for downstream sequencing.

Epithelium segmentation using deep learning in H&E-stained prostate specimens with immunohistochemistry as reference standard

Given the importance of gland morphology in grading prostate cancer (PCa), automatically differentiating between epithelium and other tissues is an important prerequisite for the development of automated methods for detecting PCa. We propose a new deep learning method to segment epithelial tissue in digitised hematoxylin and eosin (H&E) stained prostatectomy slides using immunohistochemistry (IHC) as reference standard. We used IHC to create a precise and objective ground truth compared to manual outlining on H&E slides, especially in areas with high-grade PCa. 102 tissue sections were stained with H&E and subsequently restained with P63 and CK8/18 IHC markers to highlight epithelial structures. Afterwards each pair was co-registered. First, we trained a U-Net to segment epithelial structures in IHC using a subset of the IHC slides that were preprocessed with color deconvolution. Second, this network was applied to the remaining slides to create the reference standard used to train a second U-Net on H&E. Our system accurately segmented both intact glands and individual tumour epithelial cells. The generalisation capacity of our system is shown using an independent external dataset from a different centre. We envision this segmentation as the first part of a fully automated prostate cancer grading pipeline.

Discovery of potent calpain inhibitors based on the azolo-imidazolidenone scaffold

A series of new azolopyrimidine-peptide hybrids and indolomethylideneimidazolones were obtained and evaluated as calpain inhibitors. The hybrid compounds were inactive, whereas some members of the initial azolomethylideneimidazolone series showed interesting calpain inhibitory activity. By using 4b as a hit compound, a new series of analogs were synthesized by an efficient synthetic procedure based on a multicomponent reaction followed by an unprecedented reaction at the methylene position of the molecule. The best inhibitor found for calpain I (IC50 = 20 nM) was about 20 times more potent than the hit compound. Studies on 4b showed that its inhibition is consistent with an uncompetitive inhibition mode. This compound did not exhibit cellular toxicity at any of the doses tested (0.1–10 μM) and further studies indicated that it was capable of blockading chemical ischemia induction of apoptosis by preventing sodium azide-dependent calpain activation in intact human kidney tubular epithelial cells. The results of molecular modeling studies rationalized the inhibitory activity found for this series and account, from a structural point of view, for the most active compound identified (4j).

Polymerase chain reaction assay for the detection of human papillomavirus type 16 genetic material in the intraoperative material from patients with prostate cancer

Background. The high rates of prostate cancer (PC) morbidity and mortality, as well as their high growth rates suggest that investigations of the nature of PC are of importance. The possible association of PC with high-risk human papillomaviruses (HPV) remains open. Objective: to examine surgically removed prostate tissue from patients with PC for HPV 16 type E7 oncogene, the main type of HPV being responsible for cervical cancer. Materials and methods. Polymerase chain reaction was used to test the prostate tissues removed from 17 patients with PC during radical prostatectomy. Cryopreserved (formalin- and paraffin- untreated) tumor samples were employed for better preservation of DNA. The multifocal growth pattern typical of PC was taken into account using microdissection to accumulate homogeneous prostate cancer, dysplastic, and intact epithelial cells. Results. HPV 16 type E7 oncogene DNA was detected in the samples from 7 patients with PC out of the 17 examinees, including all 5 cases where DNA had been isolated from the homogeneous regions of PC. Conclusion. The finding may suggest that HPV 16 is frequently present in the prostate glands of Russian patients with PC.

Forward-scatter and side-scatter dataset for epithelial cells from touch samples analyzed by flow cytometry.

‘Touch’ or trace biological samples submitted to caseworking labs as evidence often contain biological material from multiple individuals which can result in mixed DNA profiles. These mixture profiles are difficult to interpret and may cause analytical bottlenecks for forensic laboratories. The data in this brief reports the variation in the relative abundance of intact epithelial cells deposited by four different donors across nine days. Touch samples were generated each day by rubbing a polypropylene tube with both hands for five minutes. Forward-scatter area (FSC-A) and side-scatter area (SSC-A) data was acquired with the BD FACSCanto™ II Analyzer. The relative abundance of different sub-populations within the FSC-A and SSC-A plots was calculated against the total number of events analyzed in each sample. Mean and standard deviation values were calculated for each donor.