Round Vesicles Research Articles

Reconstructing cold climate paleoenvironments from micromorphological analysis of relict slope deposits (Serra da Estrela, Central Portugal)

AbstractThe paper focuses on analysis of macro‐ and micromorphological characteristics of relict slope deposits in Serra da Estrela (Portugal) to understand the significance of different slope processes and paleoenvironmental settings. Micromorphology is a useful sedimentology technique allowing significant advances compared to macroscopic techniques. Results show that different processes are involved in the development of the slope deposits, reflecting different environmental conditions. The main processes responsible for the emplacement of the relict slope deposits are solifluction, debris‐flow and runoff, but postdepositional changes are also present. Solifluction was identified in slope deposits between 650 and 1500 m a.s.l. The common microfeatures identified are circular arrangement of grains with a core grain, rounded vesicles, vertical grains, matrix deformations and fine‐grained deposits. Slope deposits above 1300 m a.s.l have a platy microstructure and coincide with the altitudinal range of the relict rock glaciers, indicating the elevation limit of permafrost. Below this altitude platy microstructures are less frequent. Slope deposits in north‐facing slopes were affected by frost‐induced processes in a seasonal frost regime, followed by a postdeposition illuvial phase. Debris‐flow and runoff were identified in slope deposits between 680 and 1260 m a.s.l. The common microfeatures are oblique grains, grain dumps, and coarse and fine grain lineations, and banded microstructures were identified in runoff processes. Debris‐flow deposits have a circular arrangement of grains without a core grain, identified in sediments in paraglacial and periglacial environments. The slope deposits show evidence of past periods of enhanced periglacial activity since the last glaciation until the Younger Dryas.

Extraction and identification of synovial tissue-derived exosomes by different separation techniques

ObjectiveThe aim of this study is to compare the efficiency of different separation techniques for extracting synovial tissue-derived exosomes.MethodsThe synovial tissue discarded during knee arthroscopy or total knee arthroplasty surgery was collected from the Third Affiliated Hospital of Beijing University of Chinese Medicine. Ultracentrifugation (UC), filtration combined with size exclusion chromatography (SECF), and 8% polyethylene glycol (PEG) were used to extract synovial tissue-derived exosomes. Transmission electron microscopy (TEM), nanoparticle tracer analysis (NTA), and Western Blot (WB) were used to detect the morphology, particle size, and biomarker proteins (CD9, CD63, Flotillin-1, and calnexin) of exosomes.ResultsThe extracts of enriched round and discoid vesicles were successfully extracted with UC, SECF, and PEG. The results of TEM have shown that all three extraction methods can extract circular or elliptical vesicles with disc- and cup-shaped structures from the synovial tissue, with the diameter is about 30–150 nm. NTA suggested the main peaks of three groups of exosomes are around 100–120 nm, and the concentration of the three groups of exosomes was greater than 1 × 1010/ml. The results of WB showed that three positive protein markers (CD9, CD63, and Flotillin-1) were highly expressed in the suspension extracted by the three methods and low in the synovial tissue. However, the negative protein (calnexin) was highly expressed in synovial tissues and PEG group, while low in UC and SECF group.ConclusionMorphology, particle size, and labeled protein marker detection confirmed that UC, SECF, and PEG can extract exosomes derived from synovial tissue; UC and SECF are more recommended for the extraction of synovial tissue-derived exosomes, which provides a methodological basis for studying the function and mechanism of synovial tissue exosomes in the future.

Exosomes Secreted by Young Mesenchymal Stem Cells Promote New Bone Formation During Distraction Osteogenesis in Older Rats.

Distraction osteogenesis (DO) is a clinically effective procedure to regenerate large bone defects. However, the treatment duration is undesirably lengthy, especially in elderly patients. Exosomes derived from mesenchymal stem cells (MSC-Exos) could exert the beneficial effects while avoiding the possible complications of stem cell transplantation. This study aimed to evaluate the effects of MSC-Exos on bone regeneration during DO in older rats. Exosomes were isolated from the supernatants of young bone marrow mesenchymal stem cells (BMSCs) through ultra-centrifugation, and characterized using transmission electron microscopy, western blot, and tunable resistive pulse sensing analysis. The effects of MSC-Exos on the proliferation and differentiation of older BMSCs were evaluated using CCK-8 assay, ALP and ARS staining, and qRT-PCR. Unilateral tibial DO model was established on older Sprague-Dawley rats and MSC-Exos or phosphate buffer saline was locally injected into the distraction gaps after distraction weekly. Bone regeneration were evaluated using X-ray, Micro-CT, mechanical test, and histological staining. The MSC-Exos were round or cup-shaped vesicles ranging from 60 to 130nm in diameter and expressed markers including CD9, CD63, and TSG101. The in vitro results indicated that MSC-Exos could enhance the proliferation and osteogenic differentiation of older BMSCs. Bone regeneration was markedly accelerated in rats treated with MSC-Exos according to the results of X-ray, micro-CT, and histological analysis. The distracted tibias from the MSC-Exos group also demonstrated better mechanical properties. These results suggest that MSC-Exos promote DO-mediated bone regeneration in older rats through enhancing the proliferation and osteogenic capacity of BMSCs.

Comparative study for optimization of folic acid nanoparticles

The present comparative study aims at studying the effect of several adjuvents on enhancing the stability and sustainability of FA NLCs which previously showed very promising results. The problem was encountered in the high drug release, high skin permeation, low deposition and low stability. In the present study, propylene glycol was added to all preparations as a stabilizer and coemulsifier. Plurol® stearique was utilized as solid lipid and stabilizer instead of apifil®. Soft paraffin was used as a softening additional lipid to Plurol® stearique to ensure the amorphous structure of NLCs. Liquid paraffin was used for its emollient effect instead of capryol ™ 90 with Apifil®. The in vitro drug release, ex vivo drug permeation and skin deposition were analyzed. Selection of the most optimum formulation is achieved to investigate its photostability, long term stability. Also, it was photographed under TEM. The selected formulation was stable after six hours of irradiation. The optimized selected formulation was successfully stable in refrigerator temperature throughout 9 months of the study. The TEM photograph reveals the formation of rounded nano vesicles. The present study was successful in development of more stable, more sustained means for topical delivery of FA for future cosmoceutical benefits.

Identifikasi Aspergillus flavus pada Kue Pia yang Di Jual Di Dusun Warurejo Kabupaten Pasuruan

Warurejo village that located in Pasuruan district is the center of pia cake business expecially green beans pia. Pia cake is a cake that can’t durable cause by microorganism that come from the less clear and wraping in production. It makes contamination of mycotoxins from spesies of the fungus is Aspergillus flavus . Aspergillus flavus can produce a toxin metabolite compund that is aflatoxin. If aflatoxin is continuous consumtion will cause liver cancer. This research aim to determine presence of Aspergillus flavus on pia cake that produce by center of pia cake business in Warurejo village. It will be research with storage time of 0, 5 and 10 days. This study is descriptive, the number of sample taken randomly as many as 30 samples from 33 stores. Research this is obtained by macroscopically and microscopically. In macroscopically colonis that, look yellowish greeen and on the bottom is yellow to brown while microscopically rounded vesicles and conidi are round. 73% of Aspergillus flavus has positive contaminated fungus in 0 days and 5 days. 97% positive was Aspergillus flavus with 10 days storage time. In day 10 found more Aspergillus flavus fungus that on days 0 and 5 days.

Effect of adipose-derived stem cell derived exosomes on angiogenesis after skin flap transplantation in rats

To investigate the effect of adipose-derived stem cell derived exosomes (ADSC-Exos) on angiogenesis after skin flap transplantation in rats. ADSCs were isolated and cultured by enzymatic digestion from voluntary donated adipose tissue of patients undergoing liposuction. The 3rd generation cells were observed under microscopy and identified by flow cytometry and oil red O staining at 14 days after induction of adipogenesis. After cells were identified as ADSCs, ADSC-Exos was extracted by density gradient centrifugation. And the morphology was observed by transmission electron microscopy, the surface marker proteins (CD63, TSG101) were detected by Western blot, and particle size distribution was measured by nanoparticle size tracking analyzer. Twenty male Sprague Dawley rats, weighing 250-300 g, were randomly divided into ADSC-Exos group and PBS group with 10 rats in each group. ADSC-Exos (ADSC-Exos group) and PBS (PBS group) were injected into the proximal, middle, and distal regions of the dorsal free flaps with an area of 9 cm×3 cm along the long axis in the two groups. The survival rate of the flap was measured on the 7th day, and then the flap tissue was harvested. The tissue morphology was observed by HE staining, and mean blood vessel density (MVD) was measured by CD31 immunohistochemical staining. ADSCs were identified by microscopy, flow cytometry, and adipogenic induction culture. ADSC-Exos was a round or elliptical membrane vesicle with clear edge and uniform size. It has high expression of CD63 and TSG101, and its size distribution was 30-200 nm, which was in accordance with the size range of Exos. The distal necrosis of the flaps in the ADSC-Exos group was milder than that in the PBS group. On the 7th day, the survival rate of the flaps in the ADSC-Exos group was 64.2%±11.5%, which was significantly higher than that in the PBS group (31.0%±6.6%; t=7.945, P=0.000); the skin appendages in the middle region of the flap in the ADSC-Exos group were more complete, the edema in the proximal region was lighter and the vasodilation was more extensive. MVD of the ADSC-Exos group was (103.3±27.0) /field, which was significantly higher than that of the PBS group [(45.3±16.2)/field; t=3.190, P=0.011]. ADSC-Exos can improve the blood supply of skin flaps by promoting the formation of neovascularization after skin flap transplantation, thereby improve the survival rate of skin flaps in rats.

Autologous Exosome Transfer: A New Personalised Treatment Concept to Prevent Colitis in a Murine Model.

Epigenetic information delivered by intestinal exosomes can be useful for diagnosing intestinal diseases, such as ulcerative colitis, but the therapeutic effects of intestinal exosomes have not been fully exploited. We herein developed an autologous exosome therapy that could treat intestinal disease without any risk of inducing a systemic immunological reaction. Intestinal exosomes were isolated and purified from faeces by our newly developed multi-step sucrose gradient ultracentrifugation method. Lipopolysaccharide [LPS]-activated macrophages were employed to test the in vitro anti-inflammatory ability of intestinal exosomes. To evaluate the in vivo anti-inflammatory activity of our system, we gavaged dextran sulphate sodium [DSS]-induced colitic mice with their own healing phase intestinal exosomes. Mouse intestinal exosomes are round extracellular vesicles with a hydrodynamic diameter of ~140 [±20] nm and a surface charge of ~-12 [±3] mV. Among the exosomes obtained at four different stages of DSS-induced ulcerative colitis [1, before treatment; 2, DSS-treated; 3, healing phase; and 4, back to normal], the healing phase exosomes showed the best in vitro anti-inflammatory effects and promotion of wound healing. Moreover, oral co-administration of autologous healing phase exosomes with DSS was found to significantly reduce the risk of a second round of DSS-induced ulcerative colitis in mice. Intestinal exosomes obtained during the healing phase that follows induced intestinal inflammation could strongly promote wound healing in the host. Oral administration of autologous exosomes from the healing phase could be a safe and effective approach for treating the ulcerative colitis of a given patient in the context of personalised medicine.

Ultrastructure of giant thalamic terminals in the auditory cortex.

The auditory system comprises some very large axonal terminals like the endbulb and calyx of Held and "giant" corticothalamic synapses. Previously, we described a hitherto unknown population of giant thalamocortical boutons arising from the medial division of the medial geniculate body (MGm) in the Mongolian gerbil, which terminate over a wide cortical range but in a columnar manner particularly in the extragranular layers of the auditory cortex. As a first step towards an understanding of their potential functional role, we here describe their ultrastructure combining anterograde tract-tracing with biocytin and electron microscopy. Quantitative ultrastructural analyses revealed that biocytin-labelled MGm boutons reach much larger sizes than other, non-labelled boutons. Also, mitochondria occupy more space within labelled boutons whereas synapses are of similar size. Labelled boutons are very heterogeneous in size but homogeneous with respect to their ultrastructural characteristics, with asymmetric synapses containing clear, round vesicles and targeting dendritic spines. Functionally, the ultrastructure of the MGm terminals indicates that they form excitatory contacts, which may transmit their information in a rapid, powerful and high-fidelity manner onto strategically advantageous compartments of their cortical target cells.

Exosomal miR-423-5p mediates the proangiogenic activity of human adipose-derived stem cells by targeting Sufu

BackgroundHuman adipose-derived stem cells (hADSCs) are an important source of cells for regenerative medicine. Evidence of extensive interactions with the surrounding microenvironment has led researchers to focus more on hADSCs as activating agents of regenerative pathways, rather than simply replacing damaged cells. Several studies have found that functional miRNAs can be packaged into exosomes and transferred from donor cells into recipient cells, indicating that transported miRNAs may be a new class of cell-to-cell regulatory species. The aim of the present study was to evaluate whether the exosome-derived miRNAs secreted by hADSCs are capable of influencing angiogenesis, a key step in tissue regeneration.MethodsExosomes were purified from hADSCs followed by the characterization of their phenotype and angiogenic potential in vitro. RNA sequencing was performed to detect the miRNAs that were enriched in the hADSC-derived exosomes. A miRNA-mimic experiment was used to detect the key miRNAs in the proangiogenic activity of hADSC-derived exosomes.ResultsExosomes isolated from hADSCs were characterized as round membrane vesicles with a size of approximately 100 nm and were positive for CD9 and flotillin. The exosomes were internalized by primary human umbilical vein endothelial cells (HUVECs) and stimulated HUVEC proliferation and migration. Remarkably, the exosomes promoted vessel-like formation by HUVECs in a dose-dependent manner, and their maximum activity (10 μg/mL) was comparable with that of 5% FBS. The RNA-seq bioinformatics analysis predicted 1119 gene targets of the top 30 exosomal miRNAs in Gene Ontology (GO) analysis and Kyoto encyclopedia of genes and genomes (KEGG) pathway analysis, and the pathway involved in the angiogenesis was among the top KEGG pathways. Moreover, intact miR-423-5p was further demonstrated to be transferred into HUVECs via exosomes and to exert its angiogenic function by targeting Sufu.ConclusionsExosomal miR-423-5p mediated the proangiogenic activity of hADSCs by targeting Sufu, which may contribute to the exploitation of exosomes from hADSCs as a therapeutic tool for regenerative medicine.

Self-Organization and Force Production by the Branched Actin Cytoskeleton during Mammalian Clathrin-Mediated Endocytosis

During clathrin-mediated endocytosis (CME), the cell's plasma membrane is deformed from a flat sheet into a round vesicle to internalize transmembrane proteins and extracellular cargo. Actin participates in mammalian CME, but the mechanism by which it ensures internalization of endocytic pits under variable physical constraints is not understood. To determine the molecular mechanism by which actin organizes to produce force in CME, we combined multi-scale mathematical modeling, cryo-electron tomography, and quantitative fluorescence imaging of diploid human cells endogenously expressing fluorescent fusion proteins. We developed a fluorescence microscopy-based calibration method that relates fluorescence intensity to numbers of molecules in live cells, to measure the number of Arp2/3 complexes at endocytic sites over time. These measurements constrained a multiscale mathematical model of actin polymerization coupled to the internalization of an endocytic pit. The model accounts for the mechanics of a coated plasma membrane, as well as individual actin filaments that diffuse, polymerize, and bend under force. Branched actin filaments are nucleated by active Arp2/3 complex at the base of the pit and attach to the coat via actin-binding proteins. Stochastic simulations of the model revealed that these branched actin networks self-organize at endocytic sites in a dendritic cone between the base and tip of the endocytic pit, which directs the growth of actin filaments toward the base of the pit. This self-organization was robust over a range of conditions, ensuring pit internalization against physiological membrane tension. Surprisingly, long actin filaments bent between their attachment sites at the coat and the base of the pit, which we confirmed with cryo-electron tomography of intact mammalian cells. We suggest that the self-organization and bending of actin filaments allows the actin network to robustly internalize cellular membranes across a range of physical constraints.

Synthesis of Nitrogen Containing Biocompatible Non-ionic Surfactants and Investigation for Their Self-Assembly Based Nano-Scale Vesicles

Abstract Nonionic surfactants are increasingly interesting because of the solubility and release of drugs. Here, a synthesis of four nonionic nitrogen-containing surfactants is reported. In the synthesis, sulfonamide was reacted with alkyl halides of different lipophilicity. The synthesized nonionic surfactants were characterized by 1H NMR and mass spectroscopy. Their critical micelle concentration (CMC) was determined with a UV spectrophotometer. The self-assembly of surfactants to form drug-loaded, niosomal vesicles with Simvastatin as model drug was investigated. The resulting niosoaml vesicles were characterized by atomic force microscope (AFM), zeta-sizer, and UV spectrophotometer for shape, size, polydispersity index, zeta potential, and drug inclusion efficiency. Their biocompatibility has been determined by blood hemolysis and cell toxicity tests. The synthesized surfactants showed low CMC values and were able to form nano-sized round niosomal vesicles with a homogeneous population and surface negativity. Depending on the lipophilicity, they absorbed an increased amount of drug. The biocompatibility studies show that the surfactants are hemocompatible and non-toxic. The results of the study confirm that the synthesized nonionic surfactants are suitable for the solubilization and release of hydrophobic drugs as efficient novel biocompatible carriers.

Paleoseismic Slip Records and Uplift of the Longmen Shan, Eastern Tibetan Plateau

AbstractWe document the geological fault zone geometry and internal architecture of the Yingxiu‐Beichuan fault (YBF) in the Longmen Shan, eastern Tibetan Plateau, which hosted the catastrophic 2008 Wenchuan earthquake. The YBF has been repeatedly reactivated during its long‐term activity and contains widespread brittle fault rocks. Pseudotachylytes are recognized in the Pengguan Complex both from the Wenchuan earthquake Fault Scientific Drilling (WFSD) cores and at a surface outcrop. Microstructural evidence of embayed and rounded fragments, flow structures, vesicles, and microlites of various morphologies indicate that the pseudotachylytes were generated by frictional melting during earthquakes. Cataclasite and fault gouge with fragments of angular shapes and varied sizes observed within the fault zones are also considered as evidence of paleoearthquakes. These imply that the main fault zones in the WFSD cores record ancient seismic slip events. Geological field mapping at the surface and in WFSD cores shows that the YBF is a listric reverse fault, with a 155–290‐m thick fault zone that dips 73°–68° at depths <700 m and 30° at depths >700 m. Three faulted sets of alternating Neoproterozoic Pengguan Complex overthrusting upon the Late Triassic Xujiahe Formation in the WFSD‐2 core imply that the main tectonic framework of Longmen Shan consists of a series of imbricated thrust sheets that have accommodated strong crustal shortening. These suggest that the accumulated crustal shortening caused by imbricated thrusting and long‐term seismic activity along the YBF and deep concealed faults is an important process responsible for the rapid uplift of the Longmen Shan.

Smoking Induced Extracellular Vesicles Release and Their Distinct Properties in Non-Small Cell Lung Cancer.

Purpose: Smoking is a strong relative risk factor for lung cancer. Extracellular vesicles (EVs), particularly exosomes, have been implicated in cancers. In this study, we characterized smoking induced extracellular vesicles in smokers with non-small cell lung cancer (NSCLC).Methods: EVs were isolated from bronchoalveolar lavage (BAL) from smokers and NSCLC patients. EV microRNAs (miRNAs) were analyzed by using a TaqMan microRNA assays. Vesicle mRNAs and long non-coding RNAs (lncRNAs) were measured with quantitative RT-PCR. Tumor associated antigens were examined by Western Blot.Results: Higher levels of local site EVs are found in the lung of smokers and NSCLC patients. Further, over 90% of lung EVs are round vesicles of approximately 50-200 nm, ie., exosomes. There are 21 EV miRNAs up regulated, while 10 miRNAs under regulated, in smokers when compared to controls (relative fold > 2, p < 0.05). These miRNAs were further observed to be dysregulated in NSCLC patients when compared to smokers. Bioinformatic analysis demonstrated that Proteoglycans, Fatty acid biosynthesis, ErbB, Hippo, TGF-beta, Wnt, Rap1, AMPK and Ras pathways were the most prominent pathways enriched in NSCLC EV miRNA signatures. In addition, messenger RNA transcripts including EGFR, KRAS, ALK, MET, LKB1, BRAF, PIK3CA, RET, and ROS1 were significantly higher expressed in lung EVs in smokers and NSCLC patients compared to controls. Long non-coding RNAs, including MALAT1, HOTAIR, HOTTIP, AGAP2-AS1, ATB, TCF7, FOXD2-AS1, HOXA11-AS, PCAF1, and BCAR4, were over expressed in EVs from smokers and NSCLC patients. Furthermore, protein levels of tumor associated antigens including BAGE, PD-L1, MAGE-3, and AKAP4 were significantly dysregulated in EVs of smokers and NSCLC patients compared to healthy controls.Conclusions: In conclusion, these data demonstrated an intrinsic relationship of smoking dysregulated EVs and EVs contained RNA, proteins which may involve in the development of NSCLC.

Ultrastructure of Substance P Immunopositive Axons in the Human Dental Pulp

There exists very little information on the ultrastructure of substance P immunopositive (+) fibers in the human dental pulp, which may help in understanding the mechanism for substance P associated pulpal inflammatory pain. To address this issue, we investigated the presence of substance P+ fibers in the human dental pulp by light- and electron-microscopic immunohistochemistry. Light microscopy revealed that substance P+ fibers ran within neurovascular bundles in the radicular pulp and in the core of coronal pulp. They were also frequently present in the peripheral pulp. Substance P+ fibers showed beads like swellings interconnected by thin axonal strand, in a manner similar to bouton en passants and interconnecting axonal strand in the spinal cord. Electron microscopy revealed that almost all the substance P+ axons were unmyelinated. The axonal swellings of the substance P+ contained numerous clear round vesicles (40-50 nm in diameter) and many large dense-cored vesicles (80-110 nm in diameter) as well as many mitochondria. The vesicles and mitochondria were rarely observed in the thin axonal strand interconnecting the swellings. Intimate interrelationship or synaptic structure between the swellings of substance P+ axon and nearby pulpal cells or axons was not found. These findings suggest co-release of substance P and glutamate from the substance P+ pulpal axons and its action on nearby structures in a paracrine manner.

The expression of plasma exosomal miR-422a in ischaemic stroke and its diagnostic value as biomarker

Objective To explore the potential predictive value of plasma exosomal miR-422a in different time after ischaemic stroke (1, 2, 3, 4 day). Methods Retrospective study. Forty patients diagnosed with ischaemic stroke (IS) and ten age and sex matched people who underwent a standard physical examination were recruited from the First Affiliated Hospital of Guangxi Medical University between May 2016 and December 2016. Plasma exosomes were extracted by use of related kit and the expression level of plasma exosomal miR-422a in both IS patients (1, 2, 3, 4 day) and healthy controls were examined via quantitative real-time polymerase chain reaction (qRT-PCR). The areas under the curve (AUC) of the receiver operating characteristic curve were constructed to evaluate the diagnostic accuracy of the miR-422a in IS, and one-way analysis of variance followed by the Games-Howell post hoc test was used for difference analysis. Results The exosomes isolated from human plasma showed round or oval vesicles, the average particle size was 128.2 nm and with maximum peak distribution of 186.9 nm. Moreover, all of the isolated exosomes were positive for a marker, with the CD63-positive rate at 80.6% and the CD81-positive rate at 91.7%. qRT-PCR confirmed that miR-422a was expressed in human plasma exosomes, and the expression levels of plasma exosomal miR-422a [median relative values, 1.221 (95% CI: 0.640-1.802) for healthy group, 4.418 (95% CI: 2.642-6.193) for group of 1 dayafter IS, 2.912 (95% CI: 2.262-3.562) for 2 day, 2.744 (95% CI: 2.000-3.487) for 3 day and 0.449 (95% CI: 0.170-0.727) for 4 day] were significantly increased in the time after IS 1, 2, 3 day (F=13.57, P<0.05, P<0.01, P<0.05, respectively; and the AUC values were 0.920, 0.945, 0.870, respectively). The expression of plasma exosomal miR-422a on the 4th day after IS were lower than those in other IS groups (F=13.57, P<0.005, P<0.001, P<0.001, respectively), and no statistical significance was found between the expression of plasma exosomal miR-422a on the 4th day after IS than that in the control group[0.449 (95% CI: 0.170-0.727)]. Conclusion Plasma exosomal miR-422a showed high diagnostic value as blood-based biomarker for diagnosing IS in the early phase (1-3 d). (Chin J Lab Med, 2018, 41: 658-663) Key words: Brain ischemia; Stroke; Exosomes; MicroRNAs; Biomarkers

Angina Bullosa Hemorrhagica - An Uncommon Oral Lesion

Abstract - Angina Bullosa Hemorrhagica (ABH) is used to describe an acute, benign sub-epithelial oral mucosal blood filled vesicle/vesicles or blisters. Intra-orally a large (2.0 cm diametre) blood filled, painless, raised round vesicle with dark red in colour was found in the posterior to the hard palate, extending towards soft palate of the patient. On the basis of history, clinical appearance and absence of any laboratory findings suggestive of bleeding disorder, a diagnosis of Angina Bullosa Hemorrhagica was established and an excisional biopsy was planned with successful outcome.

Enhancing the Therapeutic Efficacy of Tamoxifen Citrate Loaded Span-Based Nano-Vesicles on Human Breast Adenocarcinoma Cells.

Serious adverse effects and low selectivity to cancer cells are the main obstacles of long term therapy with Tamoxifen (Tmx). This study aimed to develop Tmx-loaded span-based nano-vesicles for delivery to malignant tissues with maximum efficacy. The effect of three variables on vesicle size (Y1), zeta potential (Y2), entrapment efficiency (Y3) and the cumulative percent release after 24h (Y4) were optimized using Box-Behnken design. The optimized formula was prepared and tested for its stability in different storage conditions. The observed values for the optimized formula were 310.2nm, - 42.09mV, 75.45 and 71.70% for Y1, Y2, Y3, and Y4, respectively. The examination using electron microscopy confirmed the formation of rounded vesicles with distinctive bilayer structure. Moreover, the cytotoxic activity of the optimized formula on both breast cancer cells (MCF-7) and normal cells (BHK) showed enhanced selectivity (9.4 folds) on cancerous cells with IC50 values 4.7 ± 1.5 and 44.3 ± 1.3μg/ml on cancer and normal cells, respectively. While, free Tmx exhibited lower selectivity (2.5 folds) than optimized nano-vesicles on cancer cells with IC50 values of 9.0 ± 1.1μg/ml and 22.5 ± 5.3μg/ml on MCF-7 and BHK cells, respectively. The promising prepared vesicular system, with greater efficacy and selectivity, provides a marvelous tool to overcome breast cancer treatment challenges.

Post mortem single-cell labeling with DiI and immunoelectron microscopy unveil the fine structure of kisspeptin neurons in humans

Kisspeptin (KP) synthesizing neurons of the hypothalamic infundibular region are critically involved in the central regulation of fertility; these cells regulate pulsatile gonadotropin-releasing hormone (GnRH) secretion and mediate sex steroid feedback signals to GnRH neurons. Fine structural analysis of the human KP system is complicated by the use of post mortem tissues. To gain better insight into the neuroanatomy of the somato-dendritic cellular compartment, we introduced the diolistic labeling of immunohistochemically identified KP neurons using a gene gun loaded with the lipophilic dye, DiI. Confocal microscopic studies of primary dendrites in 100-µm-thick tissue sections established that 79.3% of KP cells were bipolar, 14.1% were tripolar, and 6.6% were unipolar. Primary dendrites branched sparsely, contained numerous appendages (9.1 ± 1.1spines/100µm dendrite), and received rich innervation from GABAergic, glutamatergic, and KP-containing terminals. KP neuron synaptology was analyzed with immunoelectron microscopy on perfusion-fixed specimens. KP axons established frequent contacts and classical synapses on unlabeled, and on KP-immunoreactive somata, dendrites, and spines. Synapses were asymmetric and the presynaptic structures contained round and regular synaptic vesicles, in addition to dense-core granules. Although immunofluorescent studies failed to detect vesicular glutamate transporter isoforms in KP axons, ultrastructural characteristics of synaptic terminals suggested use of glutamatergic, in addition to peptidergic, neurotransmission. In summary, immunofluorescent and DiI labeling of KP neurons in thick hypothalamic sections and immunoelectron microscopic studies of KP-immunoreactive neurons in brains perfusion-fixed shortly post mortem allowed us to identify previously unexplored fine structural features of KP neurons in the mediobasal hypothalamus of humans.

A Novel Secretory Vesicle from Deer Antlerogenic Mesenchymal Stem Cell-Conditioned Media (DaMSC-CM) Promotes Tissue Regeneration.

Multipotent stem cells have the capacity to generate terminally differentiated cell types of each lineage; thus, they have great therapeutic potential for a wide variety of diseases. The most widely available stem cells are derived from human tissues, and their use for therapeutic application is limited by their high cost and low productivity. Herein, we report that conditioned media of mesenchymal stem cells (MSCs) isolated from deer antlers enhanced tissue regeneration through paracrine action via a combination of secreted growth factors and cytokines. Notably, DaMSC-conditioned media (DaMSC-CM) enhanced hair regeneration by activating the Wnt signaling pathway. In addition, DaMSC-CM had regenerative potential in damaged skin tissue through induction of skin regeneration-related genes. Remarkably, we identified round vesicles derived from DaMSC-CM, with an average diameter of ~120 nm that were associated with hair follicle formation, suggesting that secretory vesicles may act as paracrine mediators for modulation of local cellular responses. In addition, these secretory vesicles could regulate the expression of Wnt-3a, Wnt-10b, and lymphoid enhancer-binding factor-1 (LEF-1), which are related to tissue renewal. Thus, our findings demonstrate that the use of DaMSC-CM as a unique natural model for rapid and complete tissue regeneration has possible application for therapeutic development.

Enriched Expression of Neutral Sphingomyelinase 2 in the Striatum is Essential for Regulation of Lipid Raft Content and Motor Coordination

Sphingomyelinases are a family of enzymes that hydrolyze sphingomyelin to generate phosphocholine and ceramide. The brain distribution and function of neutral sphingomyelinase 2 (nSMase2) were elucidated in this study. nSMase2 mRNA expression was greatest in the striatum, followed by the prefrontal cortex, hippocampus, cerebellum, thalamus, brainstem, and olfactory bulb. The striatum had the highest level of nSMase2 protein expression, followed by the prefrontal cortex, thalamus, hippocampus, brainstem, and cerebellum. Dense immunolabeling was observed in the striatum, including the caudate-putamen, while moderately dense staining was found in the olfactory bulb and cerebral neocortex. Electron microscopy of the caudate-putamen showed nSMase2 immunoreaction product was present in small diameter dendrites or dendritic spines, that formed asymmetrical synapses with unlabeled axon terminals containing small round vesicles; and characteristics of glutamatergic axons. Lipidomic analysis of the striatum showed increase in long chain sphingomyelins, SM36:1 and SM38:1 after inhibition of nSMase activity. Quantitative proteomic analysis of striatal lipid raft fraction showed many proteins were downregulated by more than 2-fold after inhibition or antisense knockdown of nSMase; consistent with the notion that nSMase2 activity is important for aggregation or clustering of proteins in lipid rafts. Inhibition or antisense knockdown of nSMase2 in the caudate-putamen resulted in motor deficits in the rotarod and narrow beam tests; as well as decreased acoustic startle and improved prepulse inhibition of the startle reflex. Together, results indicate an important function of nSMase2 in the striatum.