Synthesis of Zwitterionic Phospholipid-Connected Silane Coupling Agents and Their Hybridization with Graphene Oxide.

The hybridization of lipids with graphene is expected to produce a promising, novel biomaterial. However, there are limited examples of the covalent introduction of lipid molecules, especially the immobilization of lipid molecules, onto graphene on a substrate. Therefore, we investigated the hybridization of a silane coupling agent having phospholipid moieties with graphene oxide on substrates prepared by photo-oxidation using chlorine dioxide. Three silane coupling agents with different carbon chain lengths (C4, C6, C8) were synthesized and phospholipid molecules were introduced onto graphene on a substrate. Phospholipid-immobilized graphene on a grid for TEM (transmission electron microscope) was used for EM analysis of proteins (glyceraldehyde 3-phosphate dehydrogenase and ß-galactosidase), enabling the observation of sufficient particles compared to the conventional graphene grid.

Discovery and characterization of a transient chaetal gland during the development of Capitella teleta (Sedentaria: Annelida).

Chaetae are among the most extensively studied structures in polychaetes, serving as a defining morphological trait for annelids. Capitella teleta stands out as one of the few established annelid models for developmental and morphological studies, thus receiving significant scholarly attention. In this study, we unveil a previously unnoticed glandular structure associated with chaetae within the larvae of C. teleta. Our investigations demonstrate the absence of comparable structures in the chaetal follicles of adults and juveniles (older than 1 week), as well as during active chaetogenesis, underscoring the transient nature of these glands. This indicates that larval chaetal follicles transform into a gland that later disappears. Utilizing histology and transmission electron microscopy, we characterized these glands. Our findings underscore the diversity of chaetal ultrastructure in annelids and show that, even in well-studied species, novel morphological details can be found. We emphasize the importance of examining various life-history stages to capture such transient morphological features. This work lays a crucial morphological foundation and deepens our understanding of chaetae and chaetogenesis in C. teleta, paving the way for more accurate interpretations of future experimental studies on chaetogenesis in this species.

Histology, histochemistry and ultrastructure of cornea of domestic pigs (Sus scrofa domesticus).

A comprehensive light and ultrastructural examination of the cornea in Domestic Pigs (Sus scrofa domesticus) revealed four distinct layers: the anterior epithelium, corneal stroma, Descemet's membrane and endothelium. Although Bowman's layer was not distinctly identified through histology, histochemical analysis indicated the presence of a rudimentary Bowman's layer, possibly vestigial from evolution. Scanning electron microscopy of the outer corneal surface unveiled two cell types, characterized by micro-projections, with light cells exhibiting shorter, thicker projections compared to dark cells. Examination of the inner surface via scanning electron microscopy demonstrated an endothelial layer devoid of cilia and microvilli, yet faint round to oval elevations were observed, potentially representing cell nuclei. Transmission electron microscopy unveiled that basal cells of the anterior epithelium closely adhered to the basement membrane, featuring half desmosomes along the basal surface. These basal cells extensively interconnected through interdigitations and a few desmosomes. The superficial cell layer consisted of a few rows of closely attached flat cells, forming a leak-proof layer with zona occludens. The outermost cells of this layer displayed fine projections to enhance the surface area, facilitating tear film distribution. At lower magnification, Transmission electron microscopy of the corneal stroma revealed alternating light and dark bands, with light bands representing transverse sections of collagen fibril lamellae and dark bands corresponding to longitudinal or oblique sections. Spindle-shaped keratocytes (fibroblasts) were identified as the primary stromal cells, intermingled between the lamellae, and featured long processes in close contact with neighbouring keratocytes. Overall, the histomorphology of the pig cornea resembles that of the human cornea except indistinct Bowman's membrane. This detailed understanding of the normal corneal structure in pigs hold great significance for biomedical research, providing a valuable reference for studies involving this animal model.

Mechanisms of Triton X-100 reducing the Ag+-resistance of Enterococcus faecalis.

To investigate the mechanism of Triton X-100 (TX-100) reducing the Ag+-resistance of Enterococcus faecalis (E. faecalis), and evaluate the antibacterial effect of TX-100 + Ag+ against the induced Ag+-resistant E. faecalis (AREf). The minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) of AgNO3 against E. faecalis with/without TX-100 were determined to verify the enhanced antibacterial activity. Transmission electron microscopy (TEM) was used to observe the morphological changes of E. faecalis after treatment. The intra- and extracellular concentration of Ag+ in treated E. faecalis was evaluated using inductively coupled plasma mass spectrometer (ICP-MS). The changes in cell membrane potential and integrity of treated E. faecalis were also observed using the flow cytometer. Moreover, AREf was induced through continuous exposure to sub-MIC of Ag+ and the antibacterial effect of TX-100 + Ag+ on AREf was further evaluated. The addition of 0.04% TX-100 showed maximal enhanced antibacterial effect of Ag+ against E. faecalis. The TEM and ICP-MS results demonstrated that TX-100 could facilitate Ag+ to enter E. faecalis through changing the membrane structure and integrity. Flow cytometry further showed the effect of TX-100 on membrane potential and permeability of E. faecalis. In addition, the enhanced antibacterial effect of TX-100 + Ag+ was also confirmed on induced AREf. TX-100 can facilitate Ag+ to enter E. faecalis through disrupting the membrane structure and changing the membrane potential and permeability, thus reducing the Ag+-resistance of E. faecalis and enhancing the antibacterial effect against either normal E. faecalis or induced AREf.

Investigation of mechanical properties, remineralization, antibacterial effect, and cellular toxicity of composite orthodontic adhesive combined with silver-containing nanostructured bioactive glass.

BACKGROUND: The formation of white spots, which represent early carious lesions, is a major issue with fixed orthodontics. The addition of remineralizing agents to orthodontic adhesives may prevent the formation of white spots. The aim of this study was to produce a composite orthodontic adhesive combined with nano-bioactive glass-silver (nBG@Ag) for bracket bonding to enamel and to investigate its cytotoxicity, antimicrobial activity, remineralization capability, and bond strength. METHODS: nBG@Ag was synthesized using the sol-gel method, and characterized using transmission electron microscopy (TEM), X-ray diffraction (XRD), and Fourier-transform infrared spectroscopy with an attenuated total reflectance attachment (ATR-FTIR). The cytotoxicity test (MTT) and antimicrobial activity of adhesives containing 1%, 3%, and 5% (wt/wt) nBG@Ag were evaluated, and the shear bond strength of the adhesives was measured using a universal testing machine. Remineralization was assessed through microhardness testing with a Vickers microhardness tester and scanning electron microscopy (SEM). Statistical analyses were conducted using the Shapiro-Wilk test, Levene test, one-way ANOVA, Robust-Welch test, Tukey HSD method, and two-way ANOVA. RESULTS: The biocompatibility of the adhesives was found to be high, as confirmed by the lack of significant differences in the cytotoxicity between the sample and control groups. Discs made from composites containing nBG@Ag exhibited a significant reduction in the growth of Streptococcus mutans (p < 0.05), and the antibacterial activity increased with higher percentages of nBG@Ag. The shear bond strength of the adhesives decreased significantly (p < 0.001) after the addition of nanoparticles, but it remained above the recommended value. The addition of nBG@Ag showed improvement in the microhardness of the teeth, although the differences in microhardness between the study groups were not statistically significant. The formation of hydroxyapatite deposits on the tooth surface was confirmed through SEM and energy-dispersive X-ray spectroscopy (EDX). CONCLUSION: Adding nBG@Ag to orthodontic adhesives can be an effective approach to enhance antimicrobial activity and reduce enamel demineralization around the orthodontic brackets, without compromising biocompatibility and bond strength.

Characterization of early-stage lesions and investigation on the role of mucosal trauma in hemorrhagic bowel syndrome in cattle.

Hemorrhagic bowel syndrome (HBS) is characterized by a dissecting intramucosal hematoma at the small bowel, causing obstruction and severe hemorrhage in dairy cattle. Recent investigation revealed the presence of early-stage lesions in cows affected by HBS. These are presumed to be the initial stage of the hematoma, as both share unique dissection of the lamina muscularis mucosae (LMM) as histological hallmark. Early-stage lesions of HBS have not been characterized in greater detail, and neither has the hypothesis of mucosal abrasion as etiology been explored. Therefore, the first objective of the present study was to characterize the morphology of early-stage lesions, by gross examination, histochemistry, immunohistochemistry and transmission electron microscopy. The second objective was to determine the effect of mucosal abrasion to the small intestine in an ex vivo model. A total of 86 early-stage lesions from 10 cows with HBS were characterized. No underlying alterations at the LMM were evident which could explain their occurrence. However, degeneration at the ultrastructural level of the LMM smooth muscle cells was present in 3 of 4 lesions, it is however unclear whether this is primary or secondary. Bacteriological examination did not reveal any association with a specific bacterium. Experimental-induced and early-stage lesions were gross and histologically evaluated and scored in three cows with HBS and seven controls. Experimentally induced lesions in both affected cows and controls, were histologically very similar to the naturally occurring early-stage lesions. Altogether, the results are suggestive for mucosal trauma to play a role in the pathogenesis of HBS.

Development and anatomy of petals with specialized nectar holder and pollen container in Fumarioideae (Papaveraceae).

MAIN CONCLUSION: Petal developmental characteristics in Fumarioideae were similar at early stages, and the specialized nectar holder/pollen container formed by the outer/inner petals. The micro-morphology of these two structures, however, shows diversity in seven species. Elaborate petals have been modified to form different types, including petal lobes, ridges, protuberances, and spurs, each with specialized functions. Nectar holder and pollen container presumably have a function in plant-pollinator interactions. In Fumarioideae, four elaborate petals of the disymmetric/zygomorphic flower present architecture forming the "nectar holder" and "pollen container" structure at the bottom and top separately. In the present study, the petals of seven species in Fumarioideae were investigated by scanning electron microscopy, light microscope, and transmission electron microscopes. The results show that petal development could divided into six stages: initiation, enlargement, adaxial/abaxial differentiation, elaborate specializations (sacs, spurs, and lobes formed), extension, and maturation, while the specialized "nectar holder" and "pollen container" structures mainly formed in stage 4. "Nectar holder" is developed from the shallow sac/spur differentiated at the base of the outer petal, eventually forming a multi-organized complex structure, together with staminal nectaries (1-2) with individual sizes. A semi-closed ellipsoidal "pollen container" is developed from the apical part of the 3-lobed inner petals fused by middle lobes and attain different sizes. The adaxial epidermis cells are specialized, with more distinct punctate/dense columnar protrusions or wavy cuticles presented on obviously thickening cell walls. In addition, a large and well-developed cavity appears between the inner and outer epidermis of the petals. As an exception, Hypecoum erectum middle lobes present stamen mimicry. Elaborate petal structure is crucial for comprehending the petal diversity in Fumarioideae and provides more evidence for further exploration of the reproductive study in Papaveraceae.

Photo-catalytic dye degradation potentials of Ag-Pd bimetallic nanoparticles synthesized from Vitex negundo.

Plant extracts are a great alternative to synthesizing nanoparticles of different metals and metal oxides. This green synthesis method has opened up numerous possibilities in various scientific domains. In present study, Leaf extract from Vitex negundo is a non-deciduous, long-lasting shrub from the Verbenaceae family is used as capping and reducing agents for the synthesis of silver and palladium nanoparticles. The characterization study UV-vis spectrophotometer analysis showed absorbance value around 320 nm which confirming that Ag-Pd nanoparticles have been successfully obtained. Further, SEM is used to investigate the morphology of Ag-Pd NPs, which revealing their spherical and rod-like configuration, aggregation, and the size of the particles are obtained between 50 and 100 nm. The successful synthesis of Ag-Pd NPs was further confirmed by the EDAX chart, which displayed the peak of Ag and Pd at bending energies between 0.5 and 1.5 keV. According to the quantitative study, Ag and Pd ions found about 5.24 and 13.28%, respectively. In addition, surface studies with TEM confirming that synthesized Ag-Pd NPs are predominates with spheres structure morphologies, with sizes averaging 11.20 nm and ranging from 10 to 20 nm. Further, Ag-Pd nanoparticles was applied as potential photocatalyst materials to degrade methylene blue dye and found about 85% of the degradation efficiency within 150 min of the sunlight exposure thus could be used as catalyst to removal of hazardous organic dye molecules.

Characterization of Amaranthus species: ability in nanoparticles fabrication and the antimicrobial activity against human pathogenic bacteria.

The present work aimed at differentiating five Amaranthus species from Saudi Arabia according to their morphology and the ability in nanoparticle formulation. Biogenic silver nanoparticles (AgNPs) were synthesized from leaf extracts of the five Amaranthus species and characterized by different techniques. Fourier-transform infrared spectroscopy (FT-IR) was used to identify the phyto-constituents of Amaranthus species. The nanoparticles (NPs) were characterized by UV-visible spectroscopy, dynamic light scattering (DLS), transmission electron microscopy (TEM), and energy-dispersive X-ray spectroscopy (EDX). The antibacterial activity of the synthesized NPs was tested against Staphylococcus aureus, E. coli, Klebsiella pneumoniae and Pseudomonas aeruginosa using the agar well diffusion method. Spherical NPs varying in size and functional groups from the five plant species were demonstrated by TEM, DLS and FTIR analysis, respectively. Variations in NPs characteristics could be related to the phytochemical composition of each Amaranthus species since they play a significant role in the reduction process. EDX confirmed the presence of Ag in plant fabricated AgNPs. Antibacterial activity varied among the species, possibly related to the NPs characteristics. Varied characteristics for the obtained AgNPs may reflect variations in the phytochemical composition type and concentration among Amaranthus species used for their fabrication.

Novel Transmission Electron Microscopic Method to Quantify Protein Clustering on the Cell Surface.

The cell surface distribution patterns (clustering) of membrane proteins have been widely investigated in cell biology. Here we describe a novel transmission electron microscopic (TEM) protocol designed to improve the quality of information obtained about the protein distribution patterns detected. This novel method makes it possible to study the clustering of all transmembrane proteins on one half of the cytoplasmic membrane of a whole cell. To achieve better imaging, we combine various methods, including critical-point drying, fixation of gold beads with a carbon layer, and a newly developed chemical thinning method. In addition, in our image-processing algorithm, we implemented pair correlation and pair cross-correlation functions, providing more details and better quantitative accuracy in characterizing the size and numbers of possible protein clusters. © 2024 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Sample preparation and transmission electron micrography Alternate Protocol: Direct cell labeling for transmission electron micrography Basic Protocol 2: Analysis of TEM images to detect immunogold-labeled proteins.

How to apply the broad toolbox of correlative light and electron microscopy to address a specific biological question.

Correlative light and electron microscopy (CLEM) is an approach that combines the strength of multiple imaging techniques to obtain complementary information about a given specimen. The "toolbox" for CLEM is broad, making it sometimes difficult to choose an appropriate approach for a given biological question. In this chapter, we provide experimental details for three CLEM approaches that can help the interested reader in designing a personalized CLEM strategy for obtaining ultrastructural data by using transmission electron microscopy (TEM). First, we describe chemical fixation of cells grown on a solid support (broadest approach). Second, we apply high-pressure freezing/freeze substitution to describe cellular ultrastructure (cryo-immobilization approach). Third, we give a protocol for a ultrastructural labeling by immuno-electron microscopy (immuno-EM approach). In addition, we also describe how to overlay fluorescence and electron microscopy images, an approach that is applicable to each of the reported different CLEM strategies. Here we provide step-by step descriptions prior to discussing possible technical problems and variations of these three general schemes to suit different models or different biological questions. This chapter is written for electron microscopists that are new to CLEM and unsure how to begin. Therefore, our protocols are meant to provide basic information with further references that should help the reader get started with applying a tailored strategy for a specific CLEM experiment.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV2) culture and sample preparation for correlative light electron microscopy.

Correlative Light Electron Microscopy (CLEM) is a powerful technique to investigate the ultrastructure of specific cells and organelles at sub-cellular resolution. Transmission Electron Microscopy (TEM) is particularly useful to the field of virology, given the small size of the virion, which is below the limit of detection by light microscopy. Furthermore, viral infection results in the rearrangement of host organelles to form spatially defined compartments that facilitate the replication of viruses. With the emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), there has been great interest to study the viral replication complex using CLEM. In this chapter we provide an exemplary workflow describing the safe preparation and processing of cells grown on coverslips and infected with SARS-CoV-2.

Some tips and tricks for a Correlative Light Electron Microscopy workflow using stable expression of fluorescent proteins.

Correlative Light Electron Microscopy (CLEM) encompasses a wide range of experimental approaches with different degrees of complexity and technical challenges where the attributes of both light and electron microscopy are combined in a single experiment. Although the biological question always determines what technology is the most appropriate, we generally set out to apply the simplest workflow possible. For 2D cell cultures expressing fluorescently tagged molecules, we report on a simple and very powerful CLEM approach by using gridded finder imaging dishes. We first determine the gross localization of the fluorescence using light microscopy and subsequently we retrace the origin/localization of the fluorescence by projecting it onto the ultrastructural reference space obtained by transmission electron microscopy (TEM). Here we describe this workflow and highlight some basic principles of the sample preparation for such a simple CLEM experiment. We will specifically focus on the steps following the resin embedding for TEM and the introduction of the sample in the electron microscope.

The organisation of the digestive, excretory and reproductive systems in cysts of the freshwater tardigrade Thulinius ruffoi (Parachela, Isohypsibioidea: Doryphoribiidae).

Tardigrades are invertebrates known to science for over 250 years. Although the ability of some species of tardigrades to form cysts has been reported, little is known about the encystment and internal organisation of the cysts. During cyst formation, contraction of the body affects the internal organs' morphology. The organs are compressed and have a compact appearance. The organisation of the digestive system, associated structures, and the reproductive system are analysed in cysts on indefinite and well-defined encystment periods - up to eleven months. The digestive system of encysted animals was organised into three main parts - a foregut, a midgut, and a hindgut. The presence of digestive system-associated structures, such as buccal glands or muscles, was noted and described. The excretory organs, called Malpighian tubules, open into the zone between the midgut and the hindgut. Furthermore, the oviduct opens into the hindgut. The first analysis of the reproductive system of cysts at the ultrastructural level is presented here, revealing interesting and undescribed aspects related to the physiology. Besides the anatomical and histological examination, the morphology and changes that occur during cyst formation are described.

Thermosensitive and mucoadhesive gels containing solid lipid nanoparticles loaded with fluconazole and niosomes loaded with clindamycin for the treatment of periodontal diseases: a laboratory experiment.

BACKGROUND: Periodontal diseases may benefit more from topical treatments with nanoparticles rather than systemic treatments due to advantages such as higher stability and controlled release profile. This study investigated the preparation and characterization of thermosensitive gel formulations containing clindamycin-loaded niosomes and solid lipid nanoparticles (SLNs) loaded with fluconazole (FLZ), as well as their in vitro antibacterial and antifungal effects in the treatment of common microorganisms that cause periodontal diseases. METHODS: This study loaded niosomes and SLNs with clindamycin and FLZ, respectively, and assessed their loading efficiency, particle size, and zeta potential. The particles were characterized using a variety of methods such as differential scanning calorimetry (DSC), dynamic light scattering (DLS), and Transmission Electron Microscopy (TEM). Thermosensitive gels were formulated by combining these particles and their viscosity, gelation temperature, in-vitro release profile, as well as antibacterial and antifungal effects were evaluated. RESULTS: Both types of these nanoparticles were found to be spherical (TEM) with a mean particle size of 243.03 nm in niosomes and 171.97 nm in SLNs (DLS), and respective zeta potentials of -23.3 and -15. The loading rate was 98% in niosomes and 51% in SLNs. The release profiles of niosomal formulations were slower than those of the SLNs. Both formulations allowed the release of the drug by first-order kinetic. Additionally, the gel formulation presented a slower release of both drugs compared to niosomes and SLNs suspensions. CONCLUSION: Thermosensitive gels containing clindamycin-loaded niosomes and/or FLZ-SLNs were found to effectively fight the periodontitis-causing bacteria and fungi.

Histology, histochemistry and fine structure of the lacrimal gland in the one-humped camel (Camelus dromedarius).

Our research aimed to provide complete histological, histochemical and ultrastructural features of the lacrimal gland of the one-humped camel (Camelus dromedarius) as well as novel insights into its adaptability to the Egyptian desert. Our study was applied to 20 fresh lacrimal glands collected from 10 camels instantly after their slaughtering. The results revealed that the gland was a compound tubulo-acinar gland, and its acini were enclosed by a thick connective tissue capsule that was very rich in elastic and collagen fibres. The gland acini had irregular lumens and were composed of conical to pyramidal cells. The nuclei of secretory cells were found in the basal part, and the cytoplasm was eosinophilic and granular. The glandular tissue consisted of serous and mucous acini and seromucous secretory cells. Histochemically, there was a significant amount of neutral mucopolysaccharides in the acini in which mucous cells had a significant periodic acid-Schiff (PAS)-positive reaction, whereas seromucous cells had a mild PAS-positive reaction. Ultrastructurally, the lacrimal cells had numerous secretory vesicles with contents of moderately to highly electron-dense cytoplasm. The nuclear envelope consisted of two prominent membranes surrounding the peri-nuclear cisterna. The acinar cells had numerous electron-lucent and moderately electron-dense secretory granules, mainly situated on the apical surface, and secreted their contents into the lumen. The luminal surface of the mucous secretory cells represents the remains of secretory granules discharged by the merocrine mechanism. In conclusion, the mucous secretion is believed to aid in the washing and moistening of the eyeball, particularly in dry, hot and dusty environments.

Exploring the neuroprotective role of artesunate in mouse models of anti-NMDAR encephalitis: insights from molecular mechanisms and transmission electron microscopy.

BACKGROUND: The pathway involving PTEN-induced putative kinase 1 (PINK1) and PARKIN plays a crucial role in mitophagy, a process activated by artesunate (ART). We propose that patients with anti-N-methyl-D-aspartate receptor (NMDAR) encephalitis exhibit insufficient mitophagy, and ART enhances mitophagy via the PINK1/PARKIN pathway, thereby providing neuroprotection. METHODS: Adult female mice aged 8-10 weeks were selected to create a passive transfer model of anti-NMDAR encephalitis. We conducted behavioral tests on these mice within a set timeframe. Techniques such as immunohistochemistry, immunofluorescence, and western blotting were employed to assess markers including PINK1, PARKIN, LC3B, p62, caspase3, and cleaved caspase3. The TUNEL assay was utilized to detect neuronal apoptosis, while transmission electron microscopy (TEM) was used to examine mitochondrial autophagosomes. Primary hippocampal neurons were cultured, treated, and then analyzed through immunofluorescence for mtDNA, mtROS, TMRM. RESULTS: In comparison to the control group, mitophagy levels in the experimental group were not significantly altered, yet there was a notable increase in apoptotic neurons. Furthermore, markers indicative of mitochondrial leakage and damage were found to be elevated in the experimental group compared to the control group, but these markers showed improvement following ART treatment. ART was effective in activating the PINK1/PARKIN pathway, enhancing mitophagy, and diminishing neuronal apoptosis. Behavioral assessments revealed that ART ameliorated symptoms in mice with anti-NMDAR encephalitis in the passive transfer model (PTM). The knockdown of PINK1 led to a reduction in mitophagy levels, and subsequent ART intervention did not alleviate symptoms in the anti-NMDAR encephalitis PTM mice, indicating that ART's therapeutic efficacy is mediated through the activation of the PINK1/PARKIN pathway. CONCLUSIONS: At the onset of anti-NMDAR encephalitis, mitochondrial damage is observed; however, this damage is mitigated by the activation of mitophagy via the PINK1/PARKIN pathway. This regulatory feedback mechanism facilitates the removal of damaged mitochondria, prevents neuronal apoptosis, and consequently safeguards neural tissue. ART activates the PINK1/PARKIN pathway to enhance mitophagy, thereby exerting neuroprotective effects and may achieve therapeutic goals in treating anti-NMDAR encephalitis.

A Streamlined and Standardized Procedure for Generating High-Titer, High-Quality Adeno-Associated Virus Vectors Utilizing a Cell Factory Platform.

Preclinical gene therapy research, particularly in rodent and large animal models, necessitates the production of AAV vectors with high yield and purity. Traditional approaches in research laboratories often involve extensive use of cell culture dishes to cultivate HEK293T cells, a process that can be both laborious and problematic. Here, a unique in-house method is presented, which simplifies this process with a specific cell factory (or cell stacks, CF10) platform. An integration of polyethylene glycol/aqueous two-phase partitioning with iodixanol gradient ultracentrifugation improves both the yield and purity of the generated AAV vectors. The purity of the AAV vectors is verified through SDS-PAGE and silver staining, while the ratio of full to empty particles is determined using transmission electron microscopy (TEM). This approach offers an efficient cell factory platform for the production of AAV vectors at high yields, coupled with an improved purification method to meet the quality demands for in vivo studies.

Castaways: the Leeward Antilles endemic spider genus Papiamenta (Araneae: Pholcidae).

Ninetinae is a group of small to tiny short-legged spiders largely restricted to arid habitats. Among daddy-long-legs spiders (Pholcidae) this is by far the least diverse subfamily but this may partly be a result of inadequate collecting, poor representation in collections or scientific neglect. We build on a large recent collection of the ninetine genus Papiamenta Huber, 2000 from the Leeward Antilles and use cytochrome oxidase 1 (COI ) sequences, extensive scanning electron microscopy data, transmission electron microscopy data and karyotyping to analyse this geographically isolated and poorly known island genus. COI sequences support the split between the two morphologically distinct species on Curaçao but genetic distances between these are surprisingly low (7.4-9.8%; mean 8.6%). The type species P. levii (Gertsch, 1982) may include more than one species but COI and morphology suggest conflicting clade limits. A third species, P. bonay Huber sp. nov. is newly described from Bonaire. Our data on sperm ultrastructure and karyology are puzzling as these suggest different phylogenetic affinities of Papiamenta to other genera. Males transfer sperm as individual sperm (cleistosperm), agreeing with the putative closest relatives as suggested by molecular data, the North American genera Pholcophora and Tolteca . The sex chromosome system (X 1 X 2 X 3 Y ) of P. levii , however, is as in the South American Ninetinae genera Gertschiola and Nerudia but different from the putative closest relatives. ZooBank: urn:lsid:zoobank.org:pub:7A6A2E84-3A61-4637-AF6F-0E31A9FA79A8.

Combining analytical techniques to assess the translocation of diesel particles across an alveolar tissue barrier in vitro.

BACKGROUND: During inhalation, airborne particles such as particulate matter ≤ 2.5 µm (PM2.5), can deposit and accumulate on the alveolar epithelial tissue. In vivo studies have shown that fractions of PM2.5 can cross the alveolar epithelium to blood circulation, reaching secondary organs beyond the lungs. However, approaches to quantify the translocation of particles across the alveolar epithelium in vivo and in vitro are still not well established. In this study, methods to assess the translocation of standard diesel exhaust particles (DEPs) across permeable polyethylene terephthalate (PET) inserts at 0.4, 1, and 3 µm pore sizes were first optimized with transmission electron microscopy (TEM), ultraviolet-visible spectroscopy (UV-VIS), and lock-in thermography (LIT), which were then applied to study the translocation of DEPs across human alveolar epithelial type II (A549) cells. A549 cells that grew on the membrane (pore size: 3 µm) in inserts were exposed to DEPs at different concentrations from 0 to 80 µg.mL- 1 ( 0 to 44 µg.cm- 2) for 24 h. After exposure, the basal fraction was collected and then analyzed by combining qualitative (TEM) and quantitative (UV-VIS and LIT) techniques to assess the translocated fraction of the DEPs across the alveolar epithelium in vitro. RESULTS: We could detect the translocated fraction of DEPs across the PET membranes with 3 µm pore sizes and without cells by TEM analysis, and determine the percentage of translocation at approximatively 37% by UV-VIS (LOD: 1.92 µg.mL- 1) and 75% by LIT (LOD: 0.20 µg.cm- 2). In the presence of cells, the percentage of DEPs translocation across the alveolar tissue was determined around 1% at 20 and 40 µg.mL- 1 (11 and 22 µg.cm- 2), and no particles were detected at higher and lower concentrations. Interestingly, simultaneous exposure of A549 cells to DEPs and EDTA can increase the translocation of DEPs in the basal fraction. CONCLUSION: We propose a combination of analytical techniques to assess the translocation of DEPs across lung tissues. Our results reveal a low percentage of translocation of DEPs across alveolar epithelial tissue in vitro and they correspond to in vivo findings. The combination approach can be applied to any traffic-generated particles, thus enabling us to understand their involvement in public health.