Scanning electron microscopic study of ovary and oviduct of crossbred dairy cows with ovarian hypoplasia

Hypoplasia of ovary is one of the major causes of infertility in dairy cattle, which is characterized by absence of oestrus cycle, affecting livestock productivity and economics to a great extent. This study was conducted on the female genitalia collected from 100 dairy cows/heifers from the Meat Technology Unit, Mannuthy which included six animals culled on account of factors other than infertility with normal genitalia (control group) and remaining animals with a known history of infertility. Two animals of 22 months and 24 months of age showed bilateral ovarian hypoplasia. The history revealed that the animals were healthy, but had not shown oestrus. Grossly ovaries of the first animal appeared as pink-coloured, small, wrinkled, flattened, elongated structures without any follicles or CL. Second animal showed inactive, small, flat, streak-like left ovary without any cyclical structures. But the right ovary showed a single large corpus haemorrhagicum on the caudal end. Then ovarian tissue was fixed in 2 percent gluteraldehyde in 0.1 M phosphate buffer at pH 7.3. Under scanning electron microscopy, the ovary exhibited an uneven surface with clefts and grooves. The surface cells lost their normal appearance without any microvilli. Large round smooth germ cells were also observed on the ovarian surface. In the oviduct, the mucosa was lined by nonciliated cells having rounded surface interspersed among shrunken cells. The epithelial cell surface was covered with bulbous processes. Ciliated cells were not observed in the present study. Ovarian abnormality is reported to be the main cause of altered morphology of the surface epithelial cells and change in the ultrastructure of oviductal mucosa.

Microparticles from human lower airway showed inhibitory activity against respiratory syncytial virus

Airway microparticles (MPs) have been previously shown to inhibit influenza virus by trapping virions on their surface through surface viral receptor. It was hypothesized that airway MPs may carry most of epithelial cell surface molecules including receptors for respiratory viruses and may be able to inhibit various respiratory viruses. We show here that MPs from human bronchoalveolar lavage (BAL) could inhibit respiratory syncytial virus (RSV). Those MPs were stained positive for the RSV receptor, CX3CR1. Furthermore, incubating the MPs with a monoclonal antibody against CX3CR1 reduced the anti-RSV activity. These indicate that MPs can contribute to respiratory innate antiviral defense.

Simultaneous Fluorescent Identification of Odontoblasts and Ameloblasts

The tooth is mainly composed of dentin and enamel. Identification of dentin-producing odontoblasts and enamel-producing ameloblasts using reporter techniques is useful to study tooth development and regeneration with tissue engineering. Ameloblasts express Amelogenin, Ameloblastin, Enamelin, and Amelotin, whereas odontoblasts express Dentin sialophosphoprotein ( Dspp) and Dentin matrix protein1 ( Dmp1). Although there are several transgenic lines using promoter elements or bacterial artificial chromosomes (BACs) to label odontoblasts and ameloblasts, there is a possibility that the expression patterns vary from the endogenous genes. Here, we established 2 lines of mice where tdTomato was knocked into the second exon of X-chromosomal Amelogenin ( Amelx), and green fluorescent protein (GFP) was knocked into the second exon of Dspp. tdTomato and GFP were highly expressed on secretory ameloblasts and secretory and fully differentiated odontoblasts, respectively. In addition, DSPP and AMELX were not produced in the dentin matrix and enamel matrix of Dspp GFP/GFP and Amelx tdTomato male mice (as representative of AmelxtdTomato/Y hemizygous male mice), respectively. Moreover, micro–computed tomography analysis of Amelx tdTomato male mice revealed a notable reduction in enamel volume but increased dentin mineral density. Dspp GFP/GFP mice had reduced dentin mineral density. To identify odontoblasts and ameloblasts from developing tooth, we examined the expression of mesenchymal cell surface molecules CD90, CD166 and epithelial cell surface molecules CD49f, Epcam1 with fluorescence on odontoblasts and ameloblasts in these mice. We found that GFP+ odontoblasts and tdTomato+ ameloblasts in tooth germ from 0.5-d-old DsppGFP/+ mice and Amelx tdTomato male mice were enriched in CD45−/Ter119−/Epcam1−/CD90+/Integrin α4+cell fractions and CD45−/Ter119−/Epcam1+/CD49f+/CD147+ cell fractions, respectively. By using antibodies against mesenchymal and epithelial cell surface molecules and fluorescence, we can easily distinguish odontoblasts from ameloblasts and isolate each cell for further studies. These mice would serve as useful models for tooth development and regeneration as well as provide concurrent observation for the differentiation processes of odontoblasts and ameloblasts in vivo and in vitro.

Targeting of Mammalian Glycans Enhances Phage Predation in the Gastrointestinal Tract

The human mucosal surface consists of a eukaryotic epithelium, a prokaryotic microbiota, and a carbohydrate-rich interface that separates them. Bacteriophage parasitize the prokaryotes but are not known to associate with eukaryotic cells. In the gastrointestinal tract, the interaction of these two domains influences the health of the host, especially colonization with invasive pathobionts. Antibiotics may be used but they also kill protective commensals and lack the physio-chemical properties to be specifically and optimally active in this complex milieu. Here, we report a novel phage whose lytic cycle is enhanced in intestinal environments. The enhanced activity is encoded in its tail fiber gene, whose protein product binds human heparan sulfated proteoglycans and localizes the phage to the epithelial cell surface, thereby positioning it near its bacterial host, a type of locational targeting mechanism. This finding offers the prospect of developing epithelial-targeting phage to selectively remove invasive pathobiont species from mucosal surfaces.Graphical AbstractModel showing (1) mucins from the intestinal mucus layer inhibit phage infection, (2) phage ES17 can bind to mucin and utilize other intestinal glycans as a receptor to infect and kill mucus-coated bacteria, and (3) phages like ES17 can be utilized to coat the intestinal epithelium by binding heparan sulfate glycans to protect from invasive pathogen infection.

Analysis of Single Circulating Tumor Cells in Renal Cell Carcinoma Reveals Phenotypic Heterogeneity and Genomic Alterations Related to Progression

Circulating tumor cells (CTCs) are promising biomarkers for prognosis, therapeutic response prediction, and treatment monitoring in cancer patients. Despite its epithelial origin, renal cell carcinoma (RCC) shows low expression of epithelial markers hindering CTC-enrichment approaches exploiting epithelial cell surface proteins. In 21 blood samples serially collected from 10 patients with metastatic RCC entering the TARIBO trial, we overcame this limitation using the marker-independent Parsortix™ approach for CTC-enrichment coupled with positive and negative selection with the DEPArray™ with single cell recovery and analysis for copy number alterations (CNA) by next generation sequencing NGS. Two CTC subpopulations were identified: epithelial CTC (eCTC) and non-conventional CTC (ncCTC) lacking epithelial and leukocyte markers. With a threshold ≥1CTC/10 mL of blood, the positivity rates were 28% for eCTC, 62% for ncCTCs, and 71% considering both CTC types. In two patients with detectable eCTCs at baseline, progression free survival was less than 5 months. In an index case, hierarchical structure by translational oncology (TRONCO) identified three clones among 14 CTCs collected at progression and at baseline, each containing cells with a 9p21.3loss, a well-known metastasis driving subclonal alteration. CTCs detection in RCC can be increased by marker-independent approaches, and CTC molecular characterization can allow detection of subclonal events possibly related to tumor progression.

Osteopontin Expression in Small Airway Epithelium in Copd is Dependent on Differentiation and Confined to Subsets of Cells

Osteopontin (OPN) plays a role in inflammation via recruitment of neutrophils and tissue remodeling. In this study, we investigated the distribution of OPN-expressing cells in the airway epithelium of normal lung tissue and that from patients with chronic obstructive pulmonary disease (COPD). OPN was detected on the epithelial cell surface of small airways and in scattered cells within the epithelial cell layer. Staining revealed higher OPN concentrations in tissue showing moderate to severe COPD compared to that in controls. In addition, OPN expression was confined to goblet and club cells, and was absent from ciliated and basal cells as detected via immunohistochemistry. However, OPN expression was up-regulated in submerged basal cells cultures exposed to cigarette smoke (CS) extract. Cell fractioning of air-liquid interface cultures revealed increased OPN production from basal compartment cells compared to that in luminal fraction cells. Furthermore, both constitutive and CS-induced expression of OPN decreased during differentiation. In contrast, cultures stimulated with interleukin (IL)-13 to promote goblet cell hyperplasia showed increased OPN production in response to CS exposure. These results indicate that the cellular composition of the airway epithelium plays an important role in OPN expression and that these levels may reflect disease endotypes in COPD.

Widespread use of Lactobacillus OppA, a surface located protein, as an adhesin that recognises epithelial cell surface glycosaminoglycans

Specific adherence is the first requisite that a microorganism has to fulfil to become established onto a mucosal surface. It was previously shown that the OppA surface protein of Lactobacillus salivarius Lv72 bound HeLa cell cultures through interaction with glycosaminoglycans (GAGs). To determine whether this is a peculiarity of that strain or whether it can be extended to other lactobacilli, 12 strains, belonging to six species, were confronted with HeLa-cell cultures in the presence of soluble GAGs. Interference was observed to six of them, heparan sulphate and chondroitin sulphate C being more interfering than chondroitin sulphate A or chondroitin sulphate B. Furthermore, inhibition of the biosynthesis of GAGs or their elimination from the cell surface with specific enzymes also resulted in reduced adherence. Analysis of the surface proteome of Lactobacillus crispatus Lv25 and of Lactobacillus reuteri RC14 revealed single proteins that immunoreacted with antibodies raised against OppA, the main adhesin of L. salivarius Lv72. Upon MALDI-TOF-TOF analysis, they were identified as OppA-like proteins, thus indicating that these proteins participate as adhesins in attachment of diverse lactobacilli to the surface of human epithelial cells.