Basal Chamber Research Articles

A Cell-Based Nasal Model for Screening the Deposition, Biocompatibility, and Transport of Aerosolized PLGA Nanoparticles.

The olfactory region of the nasal cavity directly links the brain to the external environment, presenting a potential direct route to the central nervous system (CNS). However, targeting drugs to the olfactory region is challenging and relies on a combination of drug formulation, delivery device, and administration technique to navigate human nasal anatomy. In addition, in vitro and in vivo models utilized to evaluate the performance of nasal formulations do not accurately reflect deposition and uptake in the human nasal cavity. The current study describes the development of a respirable poly(lactic-co-glycolic acid) nanoparticle (PLGA NP) formulation, delivered via a pressurized metered dose inhaler (pMDI), and a cell-containing three-dimensional (3D) human nasal cast model for deposition assessment of nasal formulations in the olfactory region. Fluorescent PLGA NPs (193 ± 3 nm by dynamic light scattering) were successfully formulated in an HFA134a-based pMDI and were collected intact following aerosolization. RPMI 2650 cells, widely employed as a nasal epithelial model, were grown at the air-liquid interface (ALI) for 14 days to develop a suitable barrier function prior to exposure to the aerosolized PLGA NPs in a glass deposition apparatus. Direct aerosol exposure was shown to have little effect on cell viability. Compared to an aqueous NP suspension, the transport rate of the aerosolized NPs across the RPMI 2650 barrier was higher at all time points indicating the potential advantages of delivery via aerosolization and the importance of employing ALI cellular models for testing respirable formulations. The PLGA NPs were then aerosolized into a 3D-printed human nasal cavity model with an insert of ALI RPMI 2650 cells positioned in the olfactory region. Cells remained highly viable, and there was significant deposition of the fluorescent NPs on the ALI cultures. This study is a proof of concept that pMDI delivery of NPs is a viable means of targeting the olfactory region for nose-to-brain drug delivery (NTBDD). The cell-based model allows not only maintenance under ALI culture conditions but also sampling from the basal chamber compartment; hence, this model could be adapted to assess drug deposition, uptake, and transport kinetics in parallel under real-life settings.

Method for Testing of Drugs Belonging to Substrates and Inhibitors of the Transporter Protein BCRP on Caco-2 Cells

Introduction. Breast cancer resistance protein (BCRP) is an efflux membrane transporter that controls the pharmacokinetics of a large number of drugs. Its activity may change when taking some endo- and exogenous substances, thus making it a link in drug interactions.Aim. The aim of the study was to develop a method for testing of drugs for belonging to BCRP substrates and inhibitors in vitro.Materials and methods. The work was performed on Caco-2 cells overexpressing BCRP, the cultivation was performed in a transwell-system consisting of the apical and basolateral chambers. Cells were seeded at the bottom of the apical chamber, which is a semipermeable membrane. Primarily, the transport of BCRP substrates: methotrexate, mitoxantrone and quercetin was evaluated in the concentration range of 1, 5, 10, and 50 μM in the direction from the basal chamber to the apical one (Papp b-a) and in the opposite direction (Papp a-b). The ratio Papp b-a / Papp a-b more than «2» characterizes the participation of transporter proteins in the transcellular transport of substances. To confirm the participation of BCRP in their transport the experiment was carried out with the addition of a transporter inhibitor, reserpine, to the transport medium at a concentration of 50 μM. The concentration of substrates in the chambers was analyzed by HPLC-MS/MS.Results and their discussion. The addition of methotrexate (1 μM), mitoxantrone (1 μM), and quercetin (1–10 μM) to both the apical or basolateral chambers of the transwell-system, their content in the recipient chamber was not detected. When methotrexate concentration became 5 μM the Papp b-a / Papp a-b ratio was 3.38 ± 0.08, which indicates the involvement of transporters in its transfer. The addition of methotrexate to the donor chamber at concentrations of 10 and 50 μM, Papp b-a / Papp a-b decreased to values below «2». At mitoxantrone concentration of 5 μM Papp b-a / Papp a-b was 2.72 ± 0.16. An increase in the concentration to 10 μM led to an increase in Papp b-a / Papp a-b to 6.18 ± 0.08. With a substance content of 50 μM the indicator decreased but remained above the value «2». In the quercetin concentration of 50 microns, Papp b-a / Papp was below "2". Reserpine reduced Papp b-a / Papp a-b of methotrexate by 3.31 times (p = 0.0002), which indicates the elimination of asymmetry in the transport of the substance. At a mitoxantrone concentration of 10 microns, reserpine reduced its Papp b-a / Papp a-b by 3.36 times (p < 0.0001). The results indicate the participation of BCRP in the control of the transfer of both substances through the cellular monolayer.Conclusion. A method of testing drugs belonging to BCRP substrates and inhibitors using methotrexate (5 μM) and mitoxantrone (10 μM) as marker substrates and reserpine (50 μM) as inhibitor was developed and tested on Caco-2 cells.

Exosomes are involved in iron transport from human blood–brain barrier endothelial cells and are modified by endothelial cell iron status

Iron is essential for normal brain development and function. Hence, understanding the mechanisms of iron efflux at the blood-brain barrier and their regulation are critical for the establishment of brain iron homeostasis. Here, we have investigated the role of exosomes in mediating the transfer of H-ferritin (FTH1)- or transferrin (Tf)-bound iron across the blood-brain barrier endothelial cells (BBBECs). Our study used ECs derived from human-induced pluripotent stem cells that are grown in bicameral chambers. When cells were exposed to 55Fe-Tf or 55Fe-FTH1, the 55Fe activity in the exosome fraction in the basal chamber was significantly higher compared to the supernatant fraction. Furthermore, we determined that the release of endogenous Tf, FTH1, and exosome number is regulated by the iron concentration of the endothelial cells. Moreover, the release of exogenously added Tf or FTH1 to the basal side via exosomes was significantly higher when ECs were iron loaded compared to when they were iron deficient. The release of exosomes containing iron bound to Tf or FTH1 was independent of hepcidin regulation, indicating this mechanism by-passes a major iron regulatory pathway. A potent inhibitor of exosome formation, GW4869, reduced exosomes released from the ECs and also decreased the Tf- and FTH1-bound iron within the exosomes. Collectively, these results indicate that iron transport across the blood-brain barrier is mediated via the exosome pathway and is modified by the iron status of the ECs, providing evidence for a novel alternate mechanism of iron transport into the brain.

Abstract 15675: Mid-Ventricular Hypertrophic Cardiomyopathy Presenting With Minoca

Background: Mid-ventricular hypertrophic obstructive cardiomyopathy (HOCM) is an uncommon form of cardiomyopathy that is linked to an apical aneurysm, increasing the risk of sudden death. Cases: We described four patients who presented with chest pain or SOB. Troponin elevation, acute myocardial infarction on cardiac MRI and absence of obstructive coronary disease confirmed the diagnosis of MINOCA. Echo revealed a pressure gradient between the apical and basal chambers of the left ventricle. Cardiac MRI showed mid-ventricular HOCM and mid-cavity obliteration with akinetic apical chamber LGE revealed areas of sub-endocardial or transmural myocardial infarction at the level of obstruction and at the apical segments distal to the obstruction. The presence of myocardial edema on tissue mapping in the same area suggested acute myocardial injury (Figure). Discussion: The pathogenesis of development of apical myocardial scarring and aneurysm formation in mid-ventricular HOCM remains unknown. Other variant of hypertrophic cardiomyopathy shows mid-myocardial foci of scar inside the hypertrophied segments, indicating fibrosis. Whereas in our patients, the LGE revealed a sub-endocardial or transmural infarct (myocardial infarction pattern) - an uncommon pattern in other hypertrophic cardiomyopathy variants. This pattern and elevation of troponin during the acute phase of illness supports the hypothesis that apical aneurysm is due to apical myocardial ischemia in mid-ventricular HOCM. Tissue characterization and pattern of scar by cardiac MRI in these patients could be useful in understanding the pathophysiology of this type of HOCM.

Alterations in Intracardiac Flow Patterns Affect Mitral Leaflets Dynamics in a Model of Ischemic Mitral Regurgitation.

This study was to evaluate the effects of ischemic mitral regurgitation (IMR) on vortex formation and leaflet dynamics using an established porcine infarct model of IMR. Using direct coronary ligation, five animals were subjected to a posterolateral myocardial infarction (MI) followed by an MRI at 12-weeks post MI. MR imaging consisted of 4D time-resolved left ventricular (LV) flow, full coverage 2D LV cine, and high resolution 2D cine of mitral valve dynamics. Five additional naïve animals underwent identical imaging protocols to serve as controls. Image analysis was performed to obtain mitral transvalvular flows as well as LV volumes throughout the cardiac cycle. In addition, anterior to posterior mid-leaflet tip distances were measured throughout the cardiac cycle for determination of temporal leaflet dynamics. It was found IMR caused asymmetric vortex ring formation with the anterior vortex having a lower vorticity relative to its posterior counterpart. In contrast, normal ventricles create symmetric and tightly curled vortices in the basal chamber just underneath the mitral leaflets which conserve kinetic energy and aid in effective ejection. IMR animals were also evaluated for leaflet separation and were found to have a greater leaflet opening and achieved peak vorticity and peak leaflet opening later than control animals. In conclusion, this study shows the effects that altered vortex formation, due to IMR, can have on ventricular filling and leaflet dynamics. These findings have important implications for understanding blood flow through the dilated heart and how ring annuloplasty and volume reduction interventions may influence mitral valve dynamics.

The “Cribrilina annulata" problem and new species of Juxtacribrilina (Bryozoa: Cheilostomata: Cribrilinidae) from the North Pacific

Originally described from Greenland, Juxtacribrilina annulata (Fabricius, 1780) (previously known as Cribrilina annulata) has long been regarded as having a circumpolar, Arctic-boreal distribution. The genus Juxtacribrilina Yang, Seo, Min, Grischenko Gordon, 2018 accommodated J. annulata and three related North Pacific species formerly in Cribrilina Gray, 1848 that lack avicularia, have a reduced (hood-like, cap-like, or vestigial) ooecium closely associated with modified latero-oral spines to form an ooecial complex, and produce frontally or marginally positioned dwarf ovicellate zooids. While the recently described NW Pacific species J. mutabilis and J. flavomaris, which have a vestigial ooecium like a short, flattened spine, clearly differ from J. annulata, the differences between J. annulata and other Pacific populations remained unclear. Here we provide descriptions for five species from the North Pacific region. We identified a specimen from the Sea of Okhotsk as J. annulata. Among the other four species, J. ezoensis n. sp. has a trans-Pacific distribution (abundant at Akkeshi, Hokkaido, Japan; also detected in the Commander Islands and at Ketchikan, Southeast Alaska); J. pushkini n. sp. was found only at Ketchikan; J. dobrovolskii n. sp. was found only at Shikotan Island in the Lesser Kuril Chain; and J. tumida n. sp. was found only at Kodiak, Gulf of Alaska. These four species all differ from J. annulata in having one or two frontal pore chambers on the proximal gymnocyst of most zooids; in budding frontal dwarf ovicellate zooids from these chambers rather than from basal pore chambers; in producing dwarf zooids more abundantly; and in having ooecia that are somewhat to markedly more reduced (cap-like rather than hood-like) and more closely integrated with the modified latero-oral spines. Furthermore, in the Pacific species, the ooecium in basal zooids arises from the roof of the distal pore chamber of the maternal zooid; ovicellate zooids can thus also bud a distal autozooid and are often arranged in columnar series with other zooids. In J. annulata, the hood-like kenozooidal ooecium budded from the maternal zooid replaces the distal autozooid, and ovicellate zooids are thus usually not embedded in a columnar series.

127 Plasma from Patients with Burn Injury Increases Endothelial Permeability In-Vitro

Abstract Introduction The contribution of endothelial injury to the pathogenesis of burn shock is not well characterized. Human umbilical endothelial cells (HUVECs) have been used to study endotheliopathy in myriad shock states. This work investigates the impact of burn patient plasma on the vascular endothelium and its barrier function. Methods HUVECs were seeded into the apical chambers of transwell plates and cultured over 5–7 days to a confluent monolayer which was confirmed by a transendothelial electrical resistance (TEER) of ≥30Ω. After IRB approval, plasma was collected from burn-injured patients 4 hours after admission. Demographic and injury characteristics were collected from the medical record. Plasma Syndecan-1 (SDC-1) was quantified by ELISA. HUVEC monolayers were exposed to 10% multi-donor pooled healthy human plasma (HHP) or burn patient plasma. Monolayers were subsequently incubated with FIT-C Dextran (40,000 kD). FIT-C diffusion through monolayers was measured in basal chamber supernatants. Monolayer permeability was measured with indices calculated by normalizing values to blank (transwell inserts) and HHP-treated monolayer FIT-C diffusion. HUVECs were also cultured on glass slides and exposed to HHP or burn patient plasma. Cells were fixed with 4% Paraformaldehyde and F-Actin was stained with Texas Red-Phalloidin. Intercellular gap area was calculated using imaging software. Differences between treatment conditions were analyzed with Welch’s t-test and one-way ANOVA, simple linear regression was used to characterize the relationship between plasma SDC-1 and permeability indices, significance was set at p < 0.05. Results Eight burn patient plasma samples were tested. Patients were mostly male (75%) with a mean age of 50±20 years and mean %TBSA burn of 37±34%. Five burn plasma samples significantly increased monolayer permeability. There were no significant differences between patient samples that increased permeability in age, TBSA, gender, or in-hospital mortality. Monolayer permeability indices increased between 7–15% (p< 0.05) among burn plasma treatment conditions (n=6) that increased permeability. There was a strong relationship between monolayer permeability index (%) and plasma SDC-1 (µg/mL) (p=0.03, R2=0.93). Morphological F-actin rearrangement was apparent on microscopy and intercellular gap area was increased in burn plasma treatment conditions (12% vs. 49%, p≤0.0007, n=6). Conclusions Plasma from burn patients induces endothelial damage that increases endothelial cell monolayer permeability. The endothelial biomarker SDC-1 is a reliable indicator of endothelial damage. F-actin rearrangement and an increase in intercellular gap area likely contributes to burn endotheliopathy.

In vitro evaluation of drug delivery behavior for inhalable amorphous nanoparticle formulations in a human lung epithelial cell model

Respiratory tract infections caused by multidrug-resistant (MDR) Gram-negative bacteria such as Pseudomonas aeruginosa are serious burdens to public health, especially in cystic fibrosis patients. The combination of colistin, a cationic polypeptide antibiotic, and ivacaftor, a cystic fibrosis transmembrane regulator (CFTR) protein modulator, displays a synergistic antibacterial effect against P. aeruginosa. The primary aim of the present study is to investigate the transport, accumulation and toxicity of a novel nanoparticle formulation containing colistin and ivacaftor in lung epithelial Calu-3 cells. The cell viability results demonstrated that ivacaftor alone or in combination with colistin in the physical mixture showed significant toxicity at an ivacaftor concentration of 10 μg/mL or higher. However, the cellular toxicity was significantly reduced in the nanoparticle formulation. Ivacaftor transport into the cells reached a plateau rapidly as compared to colistin. Colistin transport across the Calu-3 cell monolayer was less than ivacaftor. A substantial amount (46–83%) of ivacaftor, independent of dose, was accumulated in the cell monolayer following transport from the apical into the basal chamber, whereas the intracellular accumulation of colistin was relatively low (2–15%). The nanoparticle formulation significantly reduced the toxicity of colistin and ivacaftor to Calu-3 cells by reducing the accumulation of both drugs in the cell and potential protective effects by bovine serum albumin (BSA), which could be a promising safer option for the treatment of respiratory infections caused by MDR P. aeruginosa.

Abstract 2616: RhoC decreases tight junction stabilization in breast cancer cells, revealing a potential therapeutic target

Abstract RhoC, a member of the Rho GTPase family, has been shown to have a role specifically in facilitating metastasis of breast cancer cells via influencing motility, invasion, and chemokine secretion, but as yet there is no integrated model of the precise mechanism of how RhoC promotes metastasis. As these processes require disruptions in cell-cell junctions, we use wild-type and CRISPR RhoC and RhoA knockout lines of the breast cancer cell lines SUM-149 (inflammatory breast cancer, IBC) and MDA-MB-231 (triple-negative breast cancer, TNBC) in order to assess the influence of RhoC and RhoA on their cell-cell junctions. Protein lysates from these cell lines were blotted for the adherens junction markers E-cadherin and β-catenin, as well as the tight junction markers ZO-1 and Occludin. Cells were also fixed and stained for these junction markers to further assess expression and localization. In order to measure junction stability, we conducted FITC-Dextran assays, wherein cells were grown to confluency on Transwell plates, the apical chambers were incubated with FITC-Dextran solution for 24 hours, and the fluorescence of the basal chambers was subsequently measured. We demonstrate that CRISPR-Cas9 knockout of RhoC in SUM-149 IBC and MDA-MB-231 TNBC cells results in increased epithelial morphology, cell-cell connections, and tight junction marker colocalization, as measured via immunofluorescent staining. Furthermore, RhoC knockout cells have increased junctional proteins compared to wild-type cells, as measured by Western blot. In functional assessments of junction stability, RhoC knockout cells have increased barrier integrity compared to wild-type cells, by the FITC-Dextran leakage assay. These results suggest that high expression of RhoC in cancer cells destabilizes tight junctions, although loose clusters of cells are observed depending on cell type; taken together, upregulation of RhoC activation may contribute to increased metastatic potential, in part through destabilization of tight junctions. We will report on further invasion functional assays and RNA-seq data on the RhoC knockout cell lines. Taken together, these data support an important role for RhoC in tight junction modulation, thereby advancing the evidence for a potential highly specific new therapeutic target in RhoC-driven metastases control. Citation Format: Hannah Abraham, Laura Goo, Andrew Little, Joel Yates, Zhifen Wu, Sofia D. Merajver. RhoC decreases tight junction stabilization in breast cancer cells, revealing a potential therapeutic target [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2616.

Evidence for communication of peripheral iron status to cerebrospinal fluid: clinical implications for therapeutic strategy

BackgroundIron is crucial for proper functioning of all organs including the brain. Deficiencies and excess of iron are common and contribute to substantial morbidity and mortality. Whereas iron’s involvement in erythropoiesis drives clinical practice, the guidelines informing interventional strategies for iron repletion in neurological disorders are poorly defined. The objective of this study was to determine if peripheral iron status is communicated to the brain.MethodsWe used a bi-chamber cell culture model of the blood–brain-barrier to determine transcytosis of iron delivered by transferrin as a metric of iron transport. In the apical chamber (representative of the blood) we placed transferrin complexed with iron59 and in the basal chamber (representative of the brain) we placed human cerebrospinal fluid. Cerebrospinal fluid (CSF) samples (N = 24) were collected via lumbar puncture. The integrity of the tight junctions were monitored throughout the experiments using RITC-Dextran.ResultsWe demonstrate that iron transport correlates positively with plasma hemoglobin concentrations but not serum ferritin levels.ConclusionsThe clinical ramifications of these findings are several- fold. They suggest that erythropoietic demands for iron take precedence over brain requirements, and that the metric traditionally considered to be the most specific test reflecting total body iron stores and relied upon to inform treatment decisions–i.e., serum ferritin–may not be the preferred peripheral indicator when attempting to promote brain iron uptake. The future direction of this line of investigation is to identify the factor(s) in the CSF that influence iron transport at the level of the BBB.

Polarized hemichannel opening of pannexin 1/connexin 43 contributes to dysregulation of transport function in blood-brain barrier endothelial cells

Dysregulation of blood-brain barrier (BBB) transport exacerbates brain damage in acute ischemic stroke. Here, we aimed to investigate the mechanism of this BBB transport dysregulation by studying the localization and function of pannexin (Px) and connexin (Cx) hemichannels in blood-brain barrier endothelial cells of rat (TR-BBB13 cells) and human (hCMEC/D3 cells) under acute ischemic stroke-mimicking oxygen/glucose deprivation (OGD) and extracellular Ca2+ ([Ca2+]e)-free conditions. TR-BBB13 cells showed increased uptake of hemichannel-permeable sulforhodamine 101, and this increase was markedly inhibited by carbenoxolone, a hemichannel inhibitor. Transcripts of Px1 and Cx43 were detected in TR-BBB13 cells and freshly isolated brain microvascular endothelial cells. The basal compartment-to-cell uptake of hemichannel-permeable propidium iodide was selectively enhanced in hCMEC/D3 cells under [Ca2+]e-free conditions in the basal Transwell chamber. Immunohistochemical analysis revealed the predominant localization of Cx43 on the lateral membranes of hCMEC/D3 cells. [3H]Taurine uptake by hCMEC/D3 cells was significantly reduced in the absence of [Ca2+]e. Functional knock-down of Px1 and Cx43 with mimetic peptides significantly inhibited the increase of ATP release from hCMEC/D3 cells under [Ca2+]e-free conditions. These results suggest that polarized Px1/Cx43 hemichannel opening in brain capillary endothelial cells under acute ischemic stroke-mimicking conditions contributes to dysregulation of BBB transport function, resulting in release of intracellular taurine and ATP.

Red-Osier Dogwood Extracts Prevent Inflammatory Responses in Caco-2 Cells and a Caco-2 BBe1/EA.hy926 Cell Co-Culture Model.

Red-osier dogwood extracts (RDE) contain high levels of phenolic compounds which have been recognized as natural antioxidants. In this study, the potential of RDE to prevent cardiovascular diseases (CVDs) was evaluated using Caco-2 cells and a co-culture model of Caco-2 BBe1/EA.hy926 cells in Transwell® plates. The results showed that RDE supplementation significantly prevented interleukin-8 (IL-8) production and suppressed the gene expression of IL-8, tumor necrosis factor-alpha (TNF-α), interleukin-6 (IL-6), intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), and cyclooxygenase 2 (COX-2) in the TNF-α inflamed Caco-2 cells. Meanwhile, the polyphenols (quercetin-3-glucoside, quercetin-glucuronide, rutin, quercetin-3-O-malonylglucoside, and kaempferol-glucoside) in the RDE were validated to be absorbed by Caco-2 BBe1 cells and transported to the basal chamber where EA.hy926 cells were located during 12 h incubation. The transported polyphenols were able to prevent IL-8 production and suppress the gene expression of proinflammatory mediators (TNF-α, ICAM-1, VCAM-1, and COX-2) in the TNF-α or oxidized low-density lipoprotein (ox-LDL) treated EA.hy926 cells. These novel findings demonstrated that phenolic compounds in RDE can be transported to the cardiovascular system by intestinal absorption and mitigate the inflammatory responses of vascular endothelial cells, indicating that RDE could be a natural resource of polyphenols to prevent inflammation cytokine or oxidized lipid-induced CVDs.

Revision of Fauveliopsidae Hartman, 1971 (Annelida, Sedentaria).

Revision of Fauveliopsidae Hartman, 1971 (Annelida, Sedentaria).

Surfactant Protein D Reverses the Gene Signature of Transepithelial HIV-1 Passage and Restricts the Viral Transfer Across the Vaginal Barrier.

Effective prophylactic strategy against the current epidemic of sexually transmitted HIV-1 infection requires understanding of the innate gatekeeping mechanisms at the genital mucosa. Surfactant protein D (SP-D), a member of the collectin family of proteins naturally present in the vaginal tract, is a potential HIV-1 entry inhibitor at the cellular level. Human EpiVaginal tissues compartmentalized in culture inserts were apically exposed to HIV-1 and/or a recombinant fragment of human SP-D (rfhSP-D) and viral passage was assessed in the basal chamber containing mononuclear leukocytes. To map the gene signature facilitating or resisting the transepithelial viral transfer, microarray analysis of the HIV-1 challenged EpiVaginal tissues was performed in the absence or presence of rfhSP-D. Mucosal biocompatibility of rfhSP-D was assessed ex vivo and in the standard rabbit vaginal irritation model. The passage of virus through the EpiVaginal tissues toward the underlying target cells was associated with a global epithelial gene signature including differential regulation of genes primarily involved in inflammation, tight junctions and cytoskeletal framework. RfhSP-D significantly inhibited HIV-1 transfer across the vaginal tissues and was associated with a significant reversal of virus induced epithelial gene signature. Pro-inflammatory NF-κB and mTOR transcripts were significantly downregulated, while expression of the tight junctions and cytoskeletal genes was upheld. In the absence of virus, rfhSP-D directly interacted with the EpiVaginal tissues and upregulated expression of genes related to structural stability of the cell and epithelial integrity. There was no increment in the viral acquisition by the PBMCs present in basal chambers wherein, the EpiVaginal tissues in apical chambers were treated with rfhSP-D. The effective concentrations of rfhSP-D had no effect on lactobacilli, epithelial barrier integrity and were safe on repeated applications onto the rabbit vaginal mucosa. This pre-clinical safety data, coupled with its efficacy of restricting viral passage via reversal of virus-induced gene expression of the vaginal barrier, make a strong argument for clinical trials of rfhSP-D as a topical anti-HIV microbicide.

Quantitative proteomic analysis of aqueous humor from patients with drusen and reticular pseudodrusen in age-related macular degeneration

BackgroundTo identify novel biomarkers related to the pathogenesis of dry age-related macular degeneration (AMD), we adopted a human retinal pigment epithelial (RPE) cell culture model that mimics some features of dry AMD including the accumulation of intra- and sub-RPE deposits. Then, we investigated the aqueous humor (AH) proteome using a data-independent acquisition method (sequential window acquisition of all theoretical fragment ion mass spectrometry) for dry AMD patients and controls.MethodsAfter uniformly pigmented polarized monolayers of human fetal primary RPE (hfRPE) cells were established, the cells were exposed to 4-hydroxy-2-nonenal (4-HNE), followed by Western blotting, immunofluorescence analysis and ELISA of cells or conditioned media for several proteins of interest. Data-dependent acquisition for identification of the AH proteome and SWATH-based mass spectrometry were performed for 11 dry AMD patients according to their phenotypes (including soft drusen and reticular pseudodrusen [RPD]) and 2 controls (3 groups).ResultsIncreased intra- and sub-RPE deposits were observed in 4-HNE-treated hfRPE cells compared with control cultures based on APOA1, cathepsin D, and clusterin immunoreactivity. Additionally, the differential abundance of proteins in apical and basal chambers with or without 4-HNE treatment confirmed the polarized secretion of proteins from hfRPE cells. A total of 119 proteins were quantified in dry AMD patients and controls by SWATH-MS. Sixty-five proteins exhibited significantly altered abundance among the three groups. A two-dimensional principal component analysis plot was generated to identify typical proteins related to the pathogenesis of dry AMD. Among the identified proteins, eight proteins, including APOA1, CFHR2, and CLUS, were previously considered major components or regulators of drusen. Three proteins (SERPINA4, LUM, and KERA proteins) have not been previously described as components of drusen or as being related to dry AMD. Interestingly, the LUM and KERA proteins, which are related to extracellular matrix organization, were upregulated in both RPD and soft drusen.ConclusionsDifferential protein expression in the AH between patients with drusen and RPD was quantified using SWATH-MS in the present study. Detailed proteomic analyses of dry AMD patients might provide insights into the in vivo biology of drusen and RPD.

Effects of Monolaurin on Oral Microbe-Host Transcriptome and Metabolome.

The aim of this in vitro study was to evaluate the effects of monolaurin against Aggregatibacter actinomycetemcomitans (Aa) and determine their effects on the host transcriptome and metabolome, using an oral cell/bacteria co-culture dual-chamber model to mimic the human periodontium. For this, the Aa, was applied to cross the monolayer of epithelial keratinocytes (OBA-9) to reach the fibroblasts layer (HGF-1) in the basal chamber. The Monolaurin treatments (25 or 50 μM) were added immediately after the inoculation of the dual-chamber with Aa. After 24 h, the transcriptional factors and metabolites produced were quantified in the remaining cell layers (insert and basal chamber) and in supernatant released from the cells. The genes IL-1α, IL-6, IL-18, and TNF analyzed in HGF-1 concentrations showed a decreased expression when treated with both concentration of Monolaurin. In keratinocytes, the genes IL-6, IL-18, and TNF presented a higher expression and the expression of IL-1α decreased when treated with the two cited concentrations. The production of glycerol and pyruvic acid increased, and the 2-deoxytetronic acid NIST, 4-aminobutyric acid, pinitol and glyceric acid, presented lower concentrations because of the treatment with 25 and/or 50 μM of Monolaurin. Use of monolaurin modulated the immune response and metabolite production when administered for 24 h in a dual-chamber model inoculated with A. actinomycetemcomitans. In summary, this study indicates that monolaurin had antimicrobial activity and modulated the host immune response and metabolite production when administered for 24 h in a dual-chamber model inoculated with A. actinomycetemcomitans.

Medium throughput breathing human primary cell alveolus-on-chip model

Organs-on-chips have the potential to improve drug development efficiency and decrease the need for animal testing. For the successful integration of these devices in research and industry, they must reproduce in vivo contexts as closely as possible and be easy to use. Here, we describe a ‘breathing’ lung-on-chip array equipped with a passive medium exchange mechanism that provide an in vivo-like environment to primary human lung alveolar cells (hAEpCs) and primary lung endothelial cells. This configuration allows the preservation of the phenotype and the function of hAEpCs for several days, the conservation of the epithelial barrier functionality, while enabling simple sampling of the supernatant from the basal chamber. In addition, the chip design increases experimental throughput and enables trans-epithelial electrical resistance measurements using standard equipment. Biological validation revealed that human primary alveolar type I (ATI) and type II-like (ATII) epithelial cells could be successfully cultured on the chip over multiple days. Moreover, the effect of the physiological cyclic strain showed that the epithelial barrier permeability was significantly affected. Long-term co-culture of primary human lung epithelial and endothelial cells demonstrated the potential of the lung-on-chip array for reproducible cell culture under physiological conditions. Thus, this breathing lung-on-chip array, in combination with patients’ primary ATI, ATII, and lung endothelial cells, has the potential to become a valuable tool for lung research, drug discovery and precision medicine.

Effects of Open-Top Chamber on Soil Chemical Properties and Microbial Growth

Global warming is the main concern in today’s century as it comes with numerous side effects on the natural environment. Open Top Chambers (OTC) consist of metal constructions with transparent vertical side-walls and a frustum on top. An opening in the middle of the frustum allows an air exchange to reduce temperature and humidity effects in the chamber. The size of the open top chamber which is located in Universiti Putra Malaysia is slanted 60o, 50cm tall, 2.08m basal diameter hexagon chamber. The Open Top chamber experiments were carried out to determine how much global warming has affected and is still affecting the temperature, pH, the moisture and the growth of the microbes in the tropical soil. The aim of this study is to elucidate the effects of temperature increase on the soil microbes’ population and on the pH of the soil. The study was conducted to observe the effect of heat on soil microbes, and the pH of the soil which was collected on the same day for 6 consecutive months. The microbes values were then used for Colony Forming Unit (CFU) value calculations. The effects of OTCs on mean temperature showed a large range of CFU values throughout the 6 months but did not differ significantly between studies. Increases in mean monthly and diurnal temperature were strongly related, indicating that the presence of warming effect by the OTCs. Such predictive power allows a better mechanistic understanding of observed biotic response to experimental warming. This study will be useful for the understanding of the global warming effect on microbes. The open top chamber has proven to be one of the effective model for global warming research and data collected especially on the growth of soil microbial obtained would be off great use for further experiments.

A fluorescent metal-sensor study provides evidence for iron transport by transcytosis in the intestinal epithelial cells.

Iron transport across the intestinal epithelium is facilitated by the divalent metal transporter 1 (DMT1) on the brush border membrane (BBM). The fluorescent metal sensor calcein, which is hydrophilic, membrane-impermeable and quenched by chelation with iron, was used to test our hypothesis that intestinal iron absorption is through the endocytic processes and is involved in a pathway where BBM-derived vesicles fuse with basolateral membrane (BLM)-derived vesicles. To monitor the flux of iron via transcytosis, Caco-2 cells were employed as a polarized cell layer in Transwell chambers. When calcein was added to the basal chamber along with apo-transferrin (apo-Tf), calcein rapidly underwent endocytosis and co-localized with apo-Tf. Calcein was quenched by adding an iron-ascorbate complex and then restored by adding 2,2'-dipyridyl into the apical chamber. These results were confirmed by live-cell imaging. When hemin from the apical surface and calcein from the basal chamber were added to the Caco-2 cells, internalization of DMT1 and quenching of calcein were not observed until 2 h later. These results indicated that absorbed hemin required processing before hemin-derived iron was available to BLM-derived vesicles. These studies suggest that iron is transported in Caco-2 cells by transcytosis with apical-derived vesicles that are fused to BLM-derived vesicles.

Ontogeny of the jaws of monogonont rotifers: the malleate trophi of Rhinoglena and Proalides (Ploima, Epiphanidae)

AbstractInformation on the embryonic development of the malleate trophi in Epiphanidae (Rotifera, Monogononta, Ploima) is presented, based on scanning electron microscopy observations in Rhinoglena fertoeensis, R. frontalis, R. kutikovae, R. tokioensis, and Proalides tentaculatus, to contribute to the understanding of this structure of high evolutionary and functional relevance in Rotifera. The first observable and distinctly sclerotized structures were a double row of median transversal sclerites along the longitudinal axis, wherein the future unci, rostellar scleropili, cristae rami, and basal apophyses became recognizable. Fulcrum and manubria arose subsequently; the fulcrum sclerites were longitudinally ordered in a double layer. The rami chambers developed last as lamellar structures. Unci appeared as separate thin, elongate elements, the primary uncini, developing to uncus plates by transversal growth and apposition of sclerite material on the shafts of the uncini. The heads of the uncini showed their greatest development after fusion of their shafts into uncus plates. The interjacent spaces between the heads functioned as a mold, organizing bundles of sclerites which developed into the uniseriate, zigzag‐shaped cristae rami. The fulcrum attained its definite shape by elongation of the double layer of rod‐shaped sclerites into appressed sclerofibrillae. Manubria became visible as a proximal ridge of sclerites, whereupon a triangular lamella composed of crisscross‐oriented sclerites developed distally, growing out to the manubrial chambers. Ramus chambers originated from two longitudinal amorphous lamellae incorporating the median rami sclerites and closing from distal to proximal; subbasal chambers were formed before the basal chambers.