Cellular ROS tolerance determines the effect of plumbagin on osteoclast differentiation.

Plumbagin is used in traditional medicine because of its anti-inflammatory and anti-microbial properties. As a naphthoquinone, plumbagin triggers the production of reactive oxygen species (ROS). In vitro cancer studies showed that plumbagin triggers apoptosis in cancer cells through ROS production. As cancer-mediated chronic inflammation can affect bone density, it was hypothesized that plumbagin might directly inhibit the formation of bone-resorbing osteoclasts. We previously showed that the effect of plumbagin on osteoclastogenesis differed between bone marrow-derived macrophages and the macrophage cell line RAW 264.7. Although RAW 264.7 macrophages are able to initiate the gene program required for osteoclastogenesis, only primary macrophages successfully differentiate into osteoclasts. Here, we show that RAW 264.7 cells are more sensitive toward plumbagin-induced apoptosis. In the presence of plumbagin and the cytokine RANKL, which triggers ROS production to drive osteoclastogenesis, RAW 264.7 macrophages produce increased amounts of ROS and die. Addition of the ROS scavenger N-acetyl cysteine prevented cell death, linking the failure to differentiate to increased ROS levels. RAW 264.7 cells show reduced expression of genes protective against oxidative stress, while primary macrophages have a higher tolerance toward ROS. Our data suggest that it is indispensable to consider cell (line)-intrinsic properties when studying phytochemicals.

Mechanistic insight of interleukin-9 induced osteoclastogenesis.

Interleukin (IL)-9 is an emerging player in the pathogenesis of various chronic inflammatory diseases including bone disorders like rheumatoid arthritis (RA) and psoriatic arthritis. Recently, IL-9 was shown to enhance the osteoclast formation and their function in RA. However, the mechanisms by which IL-9 influences osteoclastogenesis are not known. Therefore, in this study we aimed to unravel the direct and indirect ways by which IL-9 can influence osteoclast formation. We used mouse bone marrow precursor cells for checking the effect of IL-9 on osteoclast differentiation and its function. Next, IL-9 induced signalling pathway were checked in the process of osteoclastogenesis. T cells play an important role in enhancing osteoclastogenesis in inflammatory conditions. We used splenic T cells to understand the impact of IL-9 on the functions of T effector (Teff) and regulatory T (Treg) cells. Furthermore, the effect of IL-9 mediated modulation of the T cell response on osteoclasts was checked using a coculture model of T cells with osteoclast precursors. We showed that IL-9 enhanced osteoclast formation and its function. We found that IL-9 activates STAT3, P38 MAPK, ERK1/2, NFκB and we hypothesize that it mediates the effect on osteoclastogenesis by accelerating mitochondrial biogenesis. Additionally, IL-9 was observed to facilitate the functions of pro-osteoclastogenic IL-17 producing T cells, but inhibits the function of anti-osteoclastogenic Treg cells. Our observations suggest that IL-9 can influence osteoclastogenesis directly by modulating the signalling cascade in the precursor cells; indirectly by enhancing IL-17 producing T cells and by reducing the functions of Treg cells.

Occurrence of glycidyl esters in infant formula products on the Canadian market between 2015 and 2019.

Glycidyl fatty acid esters (GEs) are processing contaminants formed during refining steps of vegetable oils. 'In vivo' hydrolysis of GEs releases potentially carcinogenic and genotoxic glycidol (2,3-epoxy-1-propanol). Occurrence of GEs in vegetable oils used for infant formula manufacturing may pose a potential health concern for formula-fed infants. Refined oils are commonly used as the main fat ingredient in formula manufacturing. For this study, different infant formula products (powders, concentrates and ready-to-feed formula products) were purchased and analysed in 2015 (35 samples) and 2019 (33 samples). Seven individual GEs were analysed by LC-MS/MS via direct approach by stable isotope dilution analysis, and total bound glycidol concentrations were calculated. Concentrations of bound glycidol in reconstituted formula reached maxima of 40.3 ng/g in the 2015 samples and 31.5 ng/g in the samples collected in 2019, with respective means of 8.7 ng/g and 6.7 ng/g. The analysed bound glycidol concentrations are comparable with concentration ranges from other studies, but are higher than observed in studies from the European market. Temporal trend data show a reduction of bound glycidol concentrations in 2019. GE concentrations were compared across different manufacturers.

Occurrence of 2- and 3-monochloropropanediol esters (MCPDE) in infant formula products on the Canadian market between 2015 and 2019.

2- and 3-monochloropropanediol esters (MCPDEs) are most commonly formed as process-induced contaminants during the refinement of vegetable oils used for food production. 'In vivo' hydrolysis of 3-MCPDEs releases the potential carcinogen 3-monochloropropanediol (3-MCPD). Levels of MCPDEs in infant formula are of particular concern, as refined oils are commonly used as main fat ingredients. For this study, infant formula samples (powders, liquid concentrates and ready-to-feed infant formula samples) from the Canadian market were purchased and analysed in 2015 (35 samples) and 2019 (33 samples). MCPDE concentrations (expressed as free MCPD equivalents) were examined through an indirect analytical approach, applying acid-catalysed ester cleavage and using cyclohexanone as derivatising agent. Labelled diesters were used as internal standards. 2015 Survey data were analysed by gas chromatography-mass spectrometry (GC-MS) in selected ion monitoring mode (SIM). 2019 Survey data were analysed with an updated method using GC-MS/MS in multiple reaction monitoring modes (MRM). In 2015, levels in reconstituted formula ranging from 3.7 ng/g to 111 ng/g for 3-MCPD and 2.2 ng/g to 56.2 ng/g for 2-MCPD were found. In 2019, levels ranging from 3.9 ng/g to 74.8 ng/g for 3-MCPD and 1.0 ng/g to 33.9 ng/g for 2-MCPD were found. A significantly reduced mean of combined MCPDEs was observed between 2015 and 2019 data (64.5 ng/g, standard deviation (SD) 8.6 ng/g in 2015 to 31.8 ng/g, SD 5.6 ng/g in 2019, p-value = 0.024). For the majority of manufacturers, the data comparison among brand products over time shows decreased levels of MCPDEs. Occurrence data of MCPDEs, including data from previously published surveys (2012/2013), were also compared and a temporal trend was established.

Podoplanin is required for tumor cell invasion in cutaneous squamous cell carcinoma.

The invasiveness of late-stage cutaneous squamous cell carcinoma (cSCC) is associated with poor patients' prognosis and linked to strong upregulation of the glycoprotein Podoplanin (PDPN) in cancer cells. However, the function of PDPN in these processes in cSCC carcinogenesis has not been characterized in detail yet. Employing a CRISPR/Cas9-based loss-of-function approach on murine cSCC cells, we show that the loss of Pdpn results in decreased migration and invasion in vitro. Complementing these in vitro studies, labelled murine control and Pdpn knockout cells were injected orthotopically into the dermis of nude mice to recapitulate the formation of human cSCC displaying a well-differentiated morphology with a PDPN-positive reaction in fibroblasts in the tumor stroma. Smaller tumors were observed upon Pdpn loss, which is associated with reduced tumor cell infiltration into the stroma. Utilizing Pdpn mutants in functional experiments in vitro, we provide evidence that both the intra- and extracellular domains are essential for cancer cell invasion. These findings underline the critical role of PDPN in cSCC progression and highlight potential therapeutic strategies targeting PDPN-dependent cancer cell invasion, especially in late-stage cSCC patients.

Surface conditioning of additively manufactured titanium implants and its influence on materials properties and in vitro biocompatibility.

Customized osteosynthesis materials of titanium alloy can be generated by additive manufacturing replacing the complex adaptation to the patient individual anatomy, especially to the lower jaw bone which shows a highly individual surface area. After printing further conditioning is necessary to adjust surface roughness. The aim of the present study was to analyse the effect of different grinding and polishing procedures on sample surface and composition and in vitro biocompatibility. Ti-6Al-4V ELI samples printed by laser powder bed fusion (LPBF) were post-treated by multi-level procedures to adjust surface roughness using the surface conditioning technologies sandblasting, vibratory finishing, electro polishing or plasma polishing. Topography and chemical composition of the surfaces was analysed. Furthermore, the release of metal ions in contact to cell culture medium was quantified. Human osteoblasts as well as primary human gingiva cells (fibroblasts and epithelial cells) were cultivated in extracts or directly on the surfaces to analyse cytotoxicity, cell adhesion and cell proliferation. Surface roughness of the different materials after final polishing was in between 0.2 and 0.5 µm, which is in the same range as usually found for conventional titanium materials used in maxillofacial surgery. Furthermore, the wettability was comparable for all post-processing techniques. The chemical compositions of the finished surfaces showed a remarkable impact by the applied finishing technique. Extracts of the samples showed low cytotoxicity with exception of the plasma polished samples, which were shown to release significantly higher amounts of vanadium ions. Accordingly, cells showed good adhesion and proliferation on all samples except plasma polished specimens. Customized devices for midline osseodistraction were exemplarily printed with LPBF starting with patient's 3D data. Those devices can be considered for clinical use, since the printed and finished material meets the requirements of ISO 10993-5 for medical devices.

Hybrid MM/CG Webserver: Automatic Set Up of Molecular Mechanics/Coarse-Grained Simulations for Human G Protein-Coupled Receptor/Ligand Complexes.

Hybrid Molecular Mechanics/Coarse-Grained (MM/CG) simulations help predict ligand poses in human G protein-coupled receptors (hGPCRs), the most important protein superfamily for pharmacological applications. This approach allows the description of the ligand, the binding cavity, and the surrounding water molecules at atomistic resolution, while coarse-graining the rest of the receptor. Here, we present the Hybrid MM/CG Webserver (mmcg.grs.kfa-juelich.de) that automatizes and speeds up the MM/CG simulation setup of hGPCR/ligand complexes. Initial structures for such complexes can be easily and efficiently generated with other webservers. The Hybrid MM/CG server also allows for equilibration of the systems, either fully automatically or interactively. The results are visualized online (using both interactive 3D visualizations and analysis plots), helping the user identify possible issues and modify the setup parameters accordingly. Furthermore, the prepared system can be downloaded and the simulation continued locally.

Ligand Pose Predictions for Human G Protein-Coupled Receptors: Insights from the Amber-Based Hybrid Molecular Mechanics/Coarse-Grained Approach.

Human G protein-coupled receptors (hGPCRs) are the most frequent targets of Food and Drug Administration (FDA)-approved drugs. Structural bioinformatics, along with molecular simulation, can support structure-based drug design targeting hGPCRs. In this context, several years ago, we developed a hybrid molecular mechanics (MM)/coarse-grained (CG) approach to predict ligand poses in low-resolution hGPCR models. The approach was based on the GROMOS96 43A1 and PRODRG united-atom force fields for the MM part. Here, we present a new MM/CG implementation using, instead, the Amber 14SB and GAFF all-atom potentials for proteins and ligands, respectively. The new implementation outperforms the previous one, as shown by a variety of applications on models of hGPCR/ligand complexes at different resolutions, and it is also more user-friendly. Thus, it emerges as a useful tool to predict poses in low-resolution models and provides insights into ligand binding similarly to all-atom molecular dynamics, albeit at a lower computational cost.

Wire like diplatinum, triplatinum, and tetraplatinum complexes featuring X[PtC[triple bond, length as m-dash]CC[triple bond, length as m-dash]CC[triple bond, length as m-dash]CC[triple bond, length as m-dash]C]mPtX segments; iterative syntheses and functionalization for measurements of single molecule properties.

Reaction of (p-tol3P)2PtCl2 and Me3Sn(C[triple bond, length as m-dash]C)2SiMe3 (1 : 1/THF/reflux) gives monosubstituted trans-Cl(p-tol3P)2Pt(C[triple bond, length as m-dash]C)2SiMe3 (63%), which with wet n-Bu4N+ F- yields trans-Cl(p-tol3P)2Pt(C[triple bond, length as m-dash]C)2H (2, 96%). Hay oxidative homocoupling (O2/CuCl/TMEDA) gives all-trans-Cl(p-tol3P)2Pt(C[triple bond, length as m-dash]C)4Pt(Pp-tol3)2Cl (3, 68%). Reaction of 3 and Me3Sn(C[triple bond, length as m-dash]C)2SiMe3 (1 : 1/rt) affords monosubstituted all-trans-Cl(p-tol3P)2Pt(C[triple bond, length as m-dash]C)4Pt(Pp-tol3)2(C[triple bond, length as m-dash]C)2SiMe3 (46%), which is converted by a similar desilylation/homocoupling sequence to all-trans-Cl[(p-tol3P)2Pt(C[triple bond, length as m-dash]C)4]3Pt(Pp-tol3)2Cl (7; 79%). Reaction of (p-tol3P)2PtCl2 and excess H(C[triple bond, length as m-dash]C)2SiMe3 (HNEt2/cat. CuI) gives trans-Me3Si(C[triple bond, length as m-dash]C)2Pt(Pp-tol3)2(C[triple bond, length as m-dash]C)2SiMe3 (78%), which with wet n-Bu4N+ F- affords trans-H(C[triple bond, length as m-dash]C)2Pt(Pp-tol3)2(C[triple bond, length as m-dash]C)2H (96%). Hay oxidative cross coupling with 2 (1 : 4) gives all-trans-Cl[(p-tol3P)2Pt(C[triple bond, length as m-dash]C)4]2Pt(Pp-tol3)2Cl (10, 36%) along with homocoupling product 3 (33%). Reaction of 3 and Me3Sn(C[triple bond, length as m-dash]C)2SiMe3 (1 : 2/rt) yields all-trans-Me3Si(C[triple bond, length as m-dash]C)2(p-tol3P)2Pt(C[triple bond, length as m-dash]C)4Pt(Pp-tol3)2(C[triple bond, length as m-dash]C)2SiMe3 (17, 77%), which with wet n-Bu4N+ F- gives all-trans-H(C[triple bond, length as m-dash]C)2(p-tol3P)2Pt(C[triple bond, length as m-dash]C)4Pt(Pp-tol3)2(C[triple bond, length as m-dash]C)2H (96%). Reaction of 3 and excess Me3P gives all-trans-Cl(Me3P)2Pt(C[triple bond, length as m-dash]C)4Pt(PMe3)2Cl (4, 86%). A model reaction of trans-(p-tol)(p-tol3P)2PtCl and KSAc yields trans-(p-tol)(p-tol3P)2PtSAc (12, 75%). Similar reactions of 3, 7, 10, and 4 give all-trans-AcS[(R3P)2Pt(C[triple bond, length as m-dash]C)4]nPt(PR3)2SAc (76-91%). The crystal structures of 3, 17, and 12 are determined. The first exhibits a chlorine-chlorine distance of 17.42 Å; those in 10 and 7 are estimated as 30.3 Å and 43.1 Å.

Predicting ligand binding poses for low-resolution membrane protein models: Perspectives from multiscale simulations.

Membrane receptors constitute major targets for pharmaceutical intervention. Drug design efforts rely on the identification of ligand binding poses. However, the limited experimental structural information available may make this extremely challenging, especially when only low-resolution homology models are accessible. In these cases, the predictions may be improved by molecular dynamics simulation approaches. Here we review recent developments of multiscale, hybrid molecular mechanics/coarse-grained (MM/CG) methods applied to membrane proteins. In particular, we focus on our in-house MM/CG approach. It is especially tailored for G-protein coupled receptors, the largest membrane receptor family in humans. We show that our MM/CG approach is able to capture the atomistic details of the receptor/ligand binding interactions, while keeping the computational cost low by representing the protein frame and the membrane environment in a highly simplified manner. We close this review by discussing ongoing improvements and challenges of the current implementation of our MM/CG code.

THREE SIBLINGS WITH ANDROGEN INSENSITIVITY SYNDROME.

Genetic, gonadal, phenotypic and psychological genderis the basis for gender assignment to an individual. Derangement in genetic makeup, under or over exposure to sex hormones and problems related to sex hormone receptors will lead to abnormal development of the external and internal genitalia. Failure to respond for the endogenous androgen, Androgen Insensitivity Syndrome is one of the common causes of genital ambiguity and intersex. In this case series we have presented three girls from a family of seven children visited Tikur Anbassa Specialized Hospital (TASH) with a complaint of primary amenorrhea and diagnosed to have androgen insensitivity syndrome. Their clinical presentation, relevant laboratory and histopathologic findings, karyotype and genetic analysis results are summarized. Potential causes and treatment options are discussed.

Highly enantioselective Simmons-Smith fluorocyclopropanation of allylic alcohols via the halogen scrambling strategy of zinc carbenoids.

Highly enantio- and diastereoenriched monofluorocyclopropanes were accessed via the Simmons-Smith fluorocyclopropanation of allylic alcohols using difluoroiodomethane and ethylzinc iodide as the substituted carbenoid precursors. The scrambling of halogens at the zinc carbenoid led to the formation of the fluorocyclopropanating agent (fluoroiodomethyl)zinc(II) fluoride. This strategy circumvented the ongoing limitation in Simmons-Smith fluorocyclopropanations relying on the use of the relatively inaccessible and expensive carbenoid precursor fluorodiiodomethane.

Mucin-producing adenocarcinoma arising in an atrial myxoma.

We describe the unique autopsy findings of a patient who died of a metastasizing giant right atrial adenocarcinoma containing few areas of typical myxoma. That no mucin-producing extracardiac tumor was detected pointed to the atrial adenocarcinoma as being the primary. We hypothesize that the adenocarcinoma may have developed from coexistent bland glandular structures within the myxoma.

A case report of celiac disease; a report done at Tikur Anbessa Specialized Hospital, Addis Ababa.

A 23 year old male patient who was diagnosed to have malabsorption syndrome secondary to celiac disease after he presented with 08 months' duration of chronic diarrhea, significant weight loss and marked body weakness with easy fatigability. Celiac disease is one of the rare causes of malabsorption in African countries. The diagnosis requires appropriate clinical history, high index of suspicion, exclusion of common causes of chronic diarrhea and confirmation by histology after the characteristic endoscopy appearance is identified. Supportive care and gluten restriction was advised and the patient has shown marked improvement in symptoms and general wellbeing. Celiac disease needs to be considered in patients who present with chronic diarrhea that doesn't respond to conventional management.

Veno-occlusive liver disease: a case report.

A case of veno-occlusive liver disease (VOLD) in a 12-years old Ethiopian boy is described The salient clinical features and gross and microscopic examination of biopsy material are reviewed. Veno-occlusive disease which occurs in the West Indies, East and West Africa, and India is an acute, subacute or chronic condition that affects the central and sublobular hepatic veins. In the West Indies (1) it is related to the consumption of bush tea made from plants that contain toxic pyrrolizidine alkaloids, such as Crotalaria and Senecio (2). Hepatotoxic compounds in Crotalaria, Senecio, Heliotropium and other composite plants can also enter the diet through the contamination of cereals with weed seeds. For example 28 of 67 patients died with veno-occlusive disease in central India after consuming a local cereal, gondli contaminated with the seeds of Crotalaria (3). Heliotropium Popovii has been implicated in outbreaks in villages in northwestern Afghanistan, with high mortality (4). The primary pathological change of hepatic veno-occlusive disease is sub-endothelial edema followed by intimal growth of connective tissue, with narrowing and occlusion of the central and sub-lobular hepatic veins. Atrophy or necrosis of liver cells, with consequent fibrosis leads to gross changes similar to those seen in cardiac cirrhosis, portal hypertension results. The present report, the first of it kind in Ethiopia describes a case of veno-occlusive liver disease in a 12-year old Ethiopian boy.

Colon cancer cells produce immunoregulatory glucocorticoids.

Expression or release of immunosuppressive molecules may protect tumor cells from the recognition and destruction by the immune system. New findings indicate that colorectal tumors produce immunoregulatory glucocorticoids and thereby suppress immune cell activation. The nuclear receptor LRH-1 plays a critical role in the regulation of colorectal tumor proliferation and glucocorticoid synthesis.

Young male patient with advanced breast cancer: a case report.

Breast cancer in male is a rare disease, accounting for less than 1% all breast cancer cases. The incidence of male breast carcinoma increases with advancing patient age. This is the case report of a young man with a breast mass, which turned out to be malignant.

Diagnosis and treatment of microsporidial keratoconjunctivitis: literature review and case series.

PURPOSE: The purpose of this study is to describe the clinical characteristics, microscopic findings, and treatment response to albendazole of microsporidial keratoconjunctivitis among immunocompromised individuals with HIV/AIDS. METHODS: This is a retrospective case series. Diagnosis of microsporidial keratoconjunctivitis was confirmed by subspecialist examination and conjunctival swabs examined by light microscopy. HIV infection was documented, and absolute CD4+ T cell count was determined. Patients were treated with albendazole and followed for clinical response. RESULTS: Light microscopy from the conjunctival swabs demonstrated myriad small, round to oval microsporidial organisms that stained positively with modified acid-fast methods. Two of the patients initially not taking highly active antiretroviral therapy (HAART) and presenting with an absolute CD4+ T cell count less than 100 cells/µL had a more severe form of keratoconjunctivitis than the third patient (receiving HAART, with a CD4+ T cell count of 259 cells/µL). All patients were started or continued on HAART. Two of the patients responded to oral albendazole, with resolution of symptoms and signs. The third patient did not initially respond, perhaps because of an immune recovery inflammatory syndrome, but subsequently had temporary improvement with albendazole. CONCLUSIONS: Microsporidial keratoconjunctivitis is a rare ocular complication of HIV/AIDS. Light microscopic evaluation of conjunctival swabs may be a useful minimally invasive first step toward diagnosis of microsporidial keratoconjunctivis in settings where electron microscopy is not available. Based on the limited available information, albendazole often is effective for this condition, and is widely available in developing countries at low cost.

Gastrointestinal stromal tumors in Addis Ababa, Ethiopia.

BACKGROUND: The last decade brought a deeper understanding of gastrointestinal stromal tumors (GIST). The cell of origin is the pacemaker cell of Cajal which has an intermediate function between the autonomic nervous system and the smooth muscle cells of the gastrointestinal tract. These cells express KIT/CD117 and less consistently CD34. Based on this facts new diagnostic and therapeutic methods have been developed. In this study we applied the current immunohistochemical methods on tumor tissue previously diagnosed as stromal tumor. METHOD: Histological specimens diagnosed as GISTs during 3 years (2005-2007) in the Department of Pathology, Tikur Anbessa Hospital, Addis Ababa, Ethiopia were reviewed and the available blocks were used for immunohistochemical assessment of KIT/CD]17 and CD34 expression. RESULTS: Tumor specimens of 17 patients were analyzed histologically. Most tumors were purely spindle celled (82%), the rest were mixed (spindle cells/epithelioid cells). Blocks of 14 tumors allowed immunohistochemical investigation. KIT/CD117 positivity could be shown in 11 of 14 tumors, CD34 positivity in 9 of 13 tumors. In contrast to the reported cases from Western countries Ethiopian patients with GISTs were younger, their tumors were larger and the most common sites were small and large intestine, not the stomach. CONCLUSIONS: GISTs became probably not more common but gave rise to increased interest during the last decade because of growing understanding of origin, behavior, and molecular mechanism. GISTs seen in Ethiopia differ from those reported in Western countries in age, size and location within the gastrointestinal tract.

Readily available hydrogen bond catalysts for the asymmetric transfer hydrogenation of nitroolefins.

This paper focuses on readily accessible thiourea hydrogen bond catalysts derived from amino acids, whose steric and electronic features are modulated by their degree of substitution at the carbinol carbon center. These catalysts were applied in the asymmetric transfer hydrogenation of nitroolefins furnishing the chiral products in up to 99% yield and 86% enantiomeric excess. The proposed catalyst's mode of action is supported by mechanistic investigations.