Decoding the endometrial niche of Asherman's Syndrome at single-cell resolution.

Asherman's Syndrome is characterized by intrauterine adhesions or scarring, which cause infertility, menstrual abnormalities, and recurrent pregnancy loss. The pathophysiology of this syndrome remains unknown, with treatment restricted to recurrent surgical removal of intrauterine scarring, which has limited success. Here, we decode the Asherman's Syndrome endometrial cell niche by analyzing data from over 200,000 cells with single-cell RNA-sequencing in patients with this condition and through in vitro analyses of Asherman's Syndrome patient-derived endometrial organoids. Our endometrial atlas highlights the loss of the endometrial epithelium, alterations to epithelial differentiation signaling pathways such as Wnt and Notch, and the appearance of characteristic epithelium expressing secretory leukocyte protease inhibitor during the window of implantation. We describe syndrome-associated alterations in cell-to-cell communication and gene expression profiles that support a dysfunctional pro-fibrotic, pro-inflammatory, and anti-angiogenic environment.

Cyclic ß-(1, 2)-glucan blended poly DL lactic co glycolic acid (PLGA 10:90) nanoparticles for drug delivery.

Our group had previously reported the encapsulation efficiency of cyclic ß-(1, 2)-glucan for various drugs. The current study is aimed at evaluating the use of glucan as a drug carrier system by blending with poly lactic-co- glycolic acid (L:G = 10:90). Nanoparticles of glucan (0.5, 5, 10 and 20 wt %) blended with PLGA and gentamicin were synthesized. Encapsulation efficiency was higher for the blends (93% with 20 wt % of glucan) than the PLGA alone (79.8%). The presence of glucan enhanced both the biodegradability, and biocompatibility of PLGA. Degradation of the nanoparticles in vitro, was autocatalytic with an initial burst release of active drug and the release profile was modeled using the Korsmeyer-Peppas scheme. In vivo studies indicated that the drug released from the blends had high volume of distribution, and greater clearance from the system. Pharmacokinetics of the drug was predicted using a double exponential decay model. Blending with PLGA improved the drug release characteristics of the cyclic ß-(1, 2)-glucan.

Biodegradable polymerized simvastatin stimulates bone formation.

Previous research from our labs demonstrated the synthesis of polymerized simvastatin by ring-opening polymerization and slow degradation with controlled release of simvastatin in vitro. The objective of the present study was to evaluate the degradation and intramembranous bone-forming potential of simvastatin-containing polyprodrugs in vivo using a rat calvarial onlay model. Poly(ethylene glycol)-block-poly(simvastatin) and poly(ethylene glycol)-block-poly(simvastatin)-ran-poly(glycolide) were compared with simvastatin conventionally encapsulated in poly(lactic-co-glycolic acid) (PLGA) and pure PLGA. The rate of degradation was higher for PLGA with and without simvastatin relative to the simvastatin polyprodrugs. Significant new bone growth at the circumference of poly(ethylene glycol)-block-poly(simvastatin) disks was observed beginning at 4 weeks, whereas severe bone resorption (4 weeks) and bone loss (8 weeks) were observed for PLGA loaded with simvastatin. No significant systemic effects were observed for serum total cholesterol and body weight. Increased expression of osteogenic (BMP-2, Runx2, and ALP), angiogenic (VEGF), and inflammatory cytokines (IL-6 and NF-ĸB) genes was seen with all polymers at the end of 8 weeks. Poly(ethylene glycol)-block-poly(simvastatin), with slow degradation and drug release, controlled inflammation, and significant osteogenic effect, is a candidate for use in bone regeneration applications. STATEMENT OF SIGNIFICANCE: Traditional drug delivery systems, e.g., drug encapsulated in poly(lactic-co-glycolic acid) (PLGA), are typically passive and have limited drug payload. As an alternative, we polymerized the drug simvastatin, which has multiple physiological effects, into macromolecules ("polysimvastatin") via ring-opening polymerization. We previously demonstrated that the rate of degradation and drug (simvastatin) release can be adjusted by copolymerizing it with other monomers. The present results demonstrate significant new bone growth around polysimvastatin, whereas severe bone loss occurred for PLGA loaded with simvastatin. This degradable biomaterial with biofunctionality integrated into the polymeric backbone is a useful candidate for bone regeneration applications.

Labeling and analysis of chicken taste buds using molecular markers in oral epithelial sheets.

In chickens, the sensory organs for taste are the taste buds in the oral cavity, of which there are ~240-360 in total number as estimated by scanning electron microscopy (SEM). There is not an easy way to visualize all taste buds in chickens. Here, we report a highly efficient method for labeling chicken taste buds in oral epithelial sheets using the molecular markers Vimentin and α-Gustducin. Immediate tissue fixation following incubation with sub-epithelially injected proteases enabled us to peel off whole epithelial sheets, leaving the shape and integrity of the tissue intact. In the peeled epithelial sheets, taste buds labeled with antibodies against Vimentin and α-Gustducin were easily identified and counted under a light microscope and many more taste buds, patterned in rosette-like clusters, were found than previously reported with SEM. Broiler-type, female-line males have more taste buds than other groups and continue to increase the number of taste buds over stages after hatch. In addition to ovoid-shaped taste buds, big tube-shaped taste buds were observed in the chicken using 2-photon microscopy. Our protocol for labeling taste buds with molecular markers will factilitate future mechanistic studies on the development of chicken taste buds in association with their feeding behaviors.

Distribution of α-Gustducin and Vimentin in premature and mature taste buds in chickens.

The sensory organs for taste in chickens (Gallus sp.) are taste buds in the oral epithelium of the palate, base of the oral cavity, and posterior tongue. Although there is not a pan-taste cell marker that labels all chicken taste bud cells, α-Gustducin and Vimentin each label a subpopulation of taste bud cells. In the present study, we used both α-Gustducin and Vimentin to further characterize chicken taste buds at the embryonic and post-hatching stages (E17-P5). We found that both α-Gustducin and Vimentin label distinct and overlapping populations of, but not all, taste bud cells. A-Gustducin immunosignals were observed as early as E18 and were consistently distributed in early and mature taste buds in embryos and hatchlings. Vimentin immunoreactivity was initially sparse at the embryonic stages then became apparent in taste buds after hatch. In hatchlings, α-Gustducin and Vimentin immunosignals largely co-localized in taste buds. A small subset of taste bud cells were labeled by either α-Gustducin or Vimentin or were not labeled. Importantly, each of the markers was observed in all of the examined taste buds. Our data suggest that the early onset of α-Gustducin in taste buds might be important for enabling chickens to respond to taste stimuli immediately after hatch and that distinctive population of taste bud cells that are labeled by different molecular markers might represent different cell types or different phases of taste bud cells. Additionally, α-Gustducin and Vimentin can potentially be used as molecular markers of all chicken taste buds in whole mount tissue.

Contribution of Underlying Connective Tissue Cells to Taste Buds in Mouse Tongue and Soft Palate.

Taste buds, the sensory organs for taste, have been described as arising solely from the surrounding epithelium, which is in distinction from other sensory receptors that are known to originate from neural precursors, i.e., neural ectoderm that includes neural crest (NC). Our previous study suggested a potential contribution of NC derived cells to early immature fungiform taste buds in late embryonic (E18.5) and young postnatal (P1-10) mice. In the present study we demonstrated the contribution of the underlying connective tissue (CT) to mature taste buds in mouse tongue and soft palate. Three independent mouse models were used for fate mapping of NC and NC derived connective tissue cells: (1) P0-Cre/R26-tdTomato (RFP) to label NC, NC derived Schwann cells and derivatives; (2) Dermo1-Cre/RFP to label mesenchymal cells and derivatives; and (3) Vimentin-CreER/mGFP to label Vimentin-expressing CT cells and derivatives upon tamoxifen treatment. Both P0-Cre/RFP and Dermo1-Cre/RFP labeled cells were abundant in mature taste buds in lingual taste papillae and soft palate, but not in the surrounding epithelial cells. Concurrently, labeled cells were extensively distributed in the underlying CT. RFP signals were seen in the majority of taste buds and all three types (I, II, III) of differentiated taste bud cells, with the neuronal-like type III cells labeled at a greater proportion. Further, Vimentin-CreER labeled cells were found in the taste buds of 3-month-old mice whereas Vimentin immunoreactivity was only seen in the CT. Taken together, our data demonstrate a previously unrecognized origin of taste bud cells from the underlying CT, a conceptually new finding in our knowledge of taste bud cell derivation, i.e., from both the surrounding epithelium and the underlying CT that is primarily derived from NC.

Taste Bud Labeling in Whole Tongue Epithelial Sheet in Adult Mice.

Molecular labeling in whole-mount tissues provides an efficient way to obtain general information about the formation, maintenance, degeneration, and regeneration of many organs and tissues. However, labeling of lingual taste buds in whole tongue tissues in adult mice has been problematic because of the strong permeability barrier of the tongue epithelium. In this study, we present a simple method for labeling taste buds in the intact tongue epithelial sheet of an adult mouse. Following intralingual protease injection and incubation, immediate fixation of the tongue on mandible in 4% paraformaldehyde enabled the in situ shape of the tongue epithelium to be well maintained after peeling. The peeled epithelium was accessible to taste bud labeling with a pan-taste cell marker, keratin 8, and a type II taste cell marker, α-gustducin, in all three types of taste papillae, that is, fungiform, foliate, and circumvallate. Overnight incubation of tongue epithelial sheets with primary and secondary antibodies was sufficient for intense labeling of taste buds with both fluorescent and DAB visualizations. Labeled individual taste buds were easy to identify and quantify. This protocol provides an efficient way for phenotypic analyses of taste buds, especially regarding distribution pattern and number.

Bacterial resistance in biofilm-associated bacteria.

Biofilms are structured groups of different bacterial species that are responsible for most chronic and recurrent infections. Biofilm-related infections reoccur in approximately 65-80% of cases. Bacteria associated with the biofilm are highly resistant to antibiotics. Knowledge of biofilm formation, its propagation and the resistance associated with it is scant and a multidisciplinary approach is followed to understand the science and develop strategies to address this problem. This article discusses the role of various biochemical factors, molecular mechanisms and altered host environment causes associated with bacterial resistance in biofilm. It also reveals the target sites and different multidisciplinary strategies adapted for destroying or preventing the formation of biofilms.

Water dispersible Ag@polyaniline-pectin as supercapacitor electrode for physiological environment.

Designing the supercapacitor electrode material for implantable electronic medical devices (IEMDs) requires careful consideration because of the need for materials which are inherently high in capacitance, biocompatibility, and antibacterial activity and are able to work in physiological environment. For the first time, we report the synthesis of a nanocomposite which has the aforementioned properties and demonstrate the nanocomposite as a supercapacitor electrode material operating in physiological fluids. In the first step, water dispersible polyaniline-pectin (PANI-PEC) nanoparticles were synthesized using biopolymer pectin (PEC) as the stabilizer. In the second step, the synthesized PANI-PEC was treated with a silver nitrate solution to afford silver nanoparticles (Ag NPs) decorated PANI-PEC nanocomposite (Ag@PANI-PEC). PANI-PEC acted as a reducing agent to convert silver ions to Ag NPs, thus eliminating the need of an exogenous reducing agent. Ag@PANI-PEC displays a specific capacitance of 140, 290, 144 and 121 F g-1 in phosphate buffer saline, blood, urine and serum, respectively, which are all physiological fluids. Furthermore, due to the use of biopolymer PEC, PANI-PEC and Ag@PANI-PEC exhibited biocompatibility and the presence of silver on Ag@PANI-PEC rendered antibacterial properties to the latter, thus making them an ideal material for in vivo implants. These findings establish the feasibility of using the nanocomposite as a potential material for energy storage device in IEMDs.

Effect of uropathogens on in vitro encrustation of polyurethane double J ureteral stents.

Polyurethane stents are used when there is an obstruction to the flow of urine. A majority of the patients with such stents are at the risk of urinary tract infection and salt encrustation. The present study is aimed at analyzing the in vitro encrustation of calcium oxalate and other salts in the presence of common uropathogens (E. coli and P. mirabilis) on films made from Tecoflex(®), a commercial grade polyurethane. In the absence of uropathogens, sodium ions and ammonia favor calcium adsorption whereas magnesium ions greatly depress it, resulting in increased hydrophillicity of the stent. With E. coli, Mg ions enhance the encrustation of calcium, whereas the other salts decrease its deposition. In case of P. mirabilis, irrespective of the type of salt, it enhances calcium encrustation except in the presence of sodium ions. Adhesion of uropathogens to the stent surface was higher in the presence of bovine serum albumin. Understanding the dynamics between various salts and microorganism in the urine, and urine-stent interface would aid in designing stents that are inert, resist encrustation and biofilm formation.

Polymers as ureteral stents.

Ureteral stents find wide application in urology. The majority of patients with indwelling ureteral stents are at an increased risk of urinary tract infection. Stent encrustation and its associated complications lead to significant morbidity. This review critically evaluates various polymers that find their application as ureteral stents with regard to various issues such as encrustation, bacterial colonization, urinary tract infections, and related clinical issues. A complete literature survey was performed, and all the relevant articles were scrutinized thoroughly. We discuss issues of encrustation/biofilm formation, new approaches to their testing, polymers currently available for use, new biomaterials, coatings, and novel ureteral stent designs, thereby providing a complete update on recent advances in the development of stents. Finally, we discuss the future of biomaterial use in the urinary tract.