Pilotins are mobile T3SS components involved in assembly and substrate specificity of the bacterial type III secretion system.

In animal pathogens, assembly of the type III secretion system injectisome requires the presence of so-called pilotins, small lipoproteins that assist the formation of the secretin ring in the outer membrane. Using a combination of functional assays, interaction studies, proteomics, and live-cell microscopy, we determined the contribution of the pilotin to the assembly, function, and substrate selectivity of the T3SS and identified potential new downstream roles of pilotin proteins. In absence of its pilotin SctG, Yersinia enterocolitica forms few, largely polar injectisome sorting platforms and needles. Accordingly, most export apparatus subcomplexes are mobile in these strains, suggesting the absence of fully assembled injectisomes. Remarkably, while absence of the pilotin all but prevents export of early T3SS substrates, such as the needle subunits, it has little effect on secretion of late T3SS substrates, including the virulence effectors. We found that although pilotins interact with other injectisome components such as the secretin in the outer membrane, they mostly localize in transient mobile clusters in the bacterial membrane. Together, these findings provide a new view on the role of pilotins in the assembly and function of type III secretion injectisomes.

Correction of frameshift mutations in the atpB gene by translational recoding in chloroplasts of Oenothera and tobacco.

Translational recoding, also known as ribosomal frameshifting, is a process that causes ribosome slippage along the messenger RNA, thereby changing the amino acid sequence of the synthesized protein. Whether the chloroplast employs recoding is unknown. I-iota, a plastome mutant of Oenothera (evening primrose), carries a single adenine insertion in an oligoA stretch [11A] of the atpB coding region (encoding the ß-subunit of the ATP synthase). The mutation is expected to cause synthesis of a truncated, nonfunctional protein. We report that a full-length AtpB protein is detectable in I-iota leaves, suggesting operation of a recoding mechanism. To characterize the phenomenon, we generated transplastomic tobacco lines in which the atpB reading frame was altered by insertions or deletions in the oligoA motif. We observed that insertion of two adenines was more efficiently corrected than insertion of a single adenine, or deletion of one or two adenines. We further show that homopolymeric composition of the oligoA stretch is essential for recoding, as an additional replacement of AAA lysine codon by AAG resulted in an albino phenotype. Our work provides evidence for the operation of translational recoding in chloroplasts. Recoding enables correction of frameshift mutations and can restore photoautotrophic growth in the presence of a mutation that otherwise would be lethal.

Targeting the High-Density Lipoprotein Proteome for the Treatment of Post-Acute Sequelae of SARS-CoV-2.

Here, we target the high-density lipoprotein (HDL) proteome in a case series of 16 patients with post-COVID-19 symptoms treated with HMG-Co-A reductase inhibitors (statin) plus angiotensin II type 1 receptor blockers (ARBs) for 6 weeks. Patients suffering from persistent symptoms (post-acute sequelae) after serologically confirmed SARS-CoV-2 infection (post-COVID-19 syndrome, PCS, n = 8) or following SARS-CoV-2 vaccination (PVS, n = 8) were included. Asymptomatic subjects with corresponding serological findings served as healthy controls (n = 8/8). HDL was isolated using dextran sulfate precipitation and the HDL proteome of all study participants was analyzed quantitatively by mass spectrometry. Clinical symptoms were assessed using questionnaires before and after therapy. The inflammatory potential of the patients' HDL proteome was addressed in human endothelial cells. The HDL proteome of patients with PCS and PVS showed no significant differences; however, compared to controls, the HDL from PVS/PCS patients displayed significant alterations involving hemoglobin, cytoskeletal proteins (MYL6, TLN1, PARVB, TPM4, FLNA), and amyloid precursor protein. Gene Ontology Biological Process (GOBP) enrichment analysis identified hemostasis, peptidase, and lipoprotein regulation pathways to be involved. Treatment of PVS/PCS patients with statins plus ARBs improved the patients' clinical symptoms. After therapy, three proteins were significantly increased (FAM3C, AT6AP2, ADAM10; FDR < 0.05) in the HDL proteome from patients with PVS/PCS. Exposure of human endothelial cells with the HDL proteome from treated PVS/PCS patients revealed reduced inflammatory cytokine and adhesion molecule expression. Thus, HDL proteome analysis from PVS/PCS patients enables a deeper insight into the underlying disease mechanisms, pointing to significant involvement in metabolic and signaling disturbances. Treatment with statins plus ARBs improved clinical symptoms and reduced the inflammatory potential of the HDL proteome. These observations may guide future therapeutic strategies for PVS/PCS patients.

Deciphering the physiological response of Escherichia coli under high ATP demand.

One long-standing question in microbiology is how microbes buffer perturbations in energy metabolism. In this study, we systematically analyzed the impact of different levels of ATP demand in Escherichia coli under various conditions (aerobic and anaerobic, with and without cell growth). One key finding is that, under all conditions tested, the glucose uptake increases with rising ATP demand, but only to a critical level beyond which it drops markedly, even below wild-type levels. Focusing on anaerobic growth and using metabolomics and proteomics data in combination with a kinetic model, we show that this biphasic behavior is induced by the dual dependency of the phosphofructokinase on ATP (substrate) and ADP (allosteric activator). This mechanism buffers increased ATP demands by a higher glycolytic flux but, as shown herein, it collapses under very low ATP concentrations. Model analysis also revealed two major rate-controlling steps in the glycolysis under high ATP demand, which could be confirmed experimentally. Our results provide new insights on fundamental mechanisms of bacterial energy metabolism and guide the rational engineering of highly productive cell factories.

Root form and canal anatomy of maxillary first premolars: a cone-beam computed tomography study.

The aim of this study was to evaluate the root and canal morphology of permanent maxillary first premolars in a Polish population using cone-beam computed tomography scanning (CBCT) and to compare the classifications by Vertucci and Ahmed et al. Images of 350 maxillary first premolars were analyzed. Scans were obtained from 226 patients: 131 women and 95 men. The root canal configurations were classified according to Vertucci and a new system by Ahmed et al. In addition, the number of roots and the level where roots bifurcated were identified. The results were submitted to statistical analysis. Most maxillary first premolars had two roots (69.1%). Most bifurcations were located in the coronal part of the root (44.2%) and the least in the apical part (15.3%). Bifurcation in the coronal part of the root was observed more often in the teeth of men than women. In turn, bifurcation in the central or apical part was significantly more common in women than in men. The most common canal configuration of the maxillary first premolars was type IV (78.2%) according to Vertucci and 2FPB1P1 (65.4%) according to the new classification. Among the remaining cases, almost all types of canals described by Vertucci, and many combinations of codes given in the new classification were demonstrated. The maxillary first premolars displayed a wide range of root and canal anatomical variations. The new system for classifying canal morphology based on Ahmed et al. is more accurate than the Vertucci classification.

Laser Patterning a Graphene Layer on a Ceramic Substrate for Sensor Applications.

This paper describes a method for patterning the graphene layer and gold electrodes on a ceramic substrate using a Nd:YAG nanosecond fiber laser. The technique enables the processing of both layers and trimming of the sensor parameters. The main aim was to develop a technique for the effective and efficient shaping of both the sensory layer and the metallic electrodes. The laser shaping method is characterized by high speed and very good shape mapping, regardless of the complexity of the processing. Importantly, the technique enables the simultaneous shaping of both the graphene layer and Au electrodes in a direct process that does not require a complex and expensive masking process, and without damaging the ceramic substrate. Our results confirmed the effectiveness of the developed laser technology for shaping a graphene layer and Au electrodes. The ceramic substrate can be used in the construction of various types of sensors operating in a wide temperature range, especially the cryogenic range.

Plasma Membranes Are Subcompartmentalized into a Plethora of Coexisting and Diverse Microdomains in Arabidopsis and Nicotiana benthamiana.

Eukaryotic plasma membranes are highly compartmentalized structures. So far, only a few individual proteins that function in a wide range of cellular processes have been shown to segregate into microdomains. However, the biological roles of most microdomain-associated proteins are unknown. Here, we investigated the heterogeneity of distinct microdomains and the complexity of their coexistence. This diversity was determined in living cells of intact multicellular tissues using 20 different marker proteins from Arabidopsis thaliana, mostly belonging to the Remorin protein family. These proteins associate with microdomains at the cytosolic leaflet of the plasma membrane. We characterized these membrane domains and determined their lateral dynamics by extensive quantitative image analysis. Systematic colocalization experiments with an extended subset of marker proteins tested in 45 different combinations revealed the coexistence of highly distinct membrane domains on individual cell surfaces. These data provide valuable tools to study the lateral segregation of membrane proteins and their biological functions in living plant cells. They also demonstrate that widely used biochemical approaches such as detergent-resistant membranes cannot resolve this biological complexity of membrane compartmentalization in vivo.

Cytoskeletal Components Define Protein Location to Membrane Microdomains.

The plasma membrane is an important compartment that undergoes dynamic changes in composition upon external or internal stimuli. The dynamic subcompartmentation of proteins in ordered low-density (DRM) and disordered high-density (DSM) membrane phases is hypothesized to require interactions with cytoskeletal components. Here, we systematically analyzed the effects of actin or tubulin disruption on the distribution of proteins between membrane density phases. We used a proteomic screen to identify candidate proteins with altered submembrane location, followed by biochemical or cell biological characterization in Arabidopsis thaliana. We found that several proteins, such as plasma membrane ATPases, receptor kinases, or remorins resulted in a differential distribution between membrane density phases upon cytoskeletal disruption. Moreover, in most cases, contrasting effects were observed: Disruption of actin filaments largely led to a redistribution of proteins from DRM to DSM membrane fractions while disruption of tubulins resulted in general depletion of proteins from the membranes. We conclude that actin filaments are necessary for dynamic movement of proteins between different membrane phases and that microtubules are not necessarily important for formation of microdomains as such, but rather they may control the protein amount present in the membrane phases.

A Fully Transparent Flexible Sensor for Cryogenic Temperatures Based on High Strength Metallurgical Graphene.

Low-temperature electronics operating in below zero temperatures or even below the lower limit of the common -65 to 125 °C temperature range are essential in medical diagnostics, in space exploration and aviation, in processing and storage of food and mainly in scientific research, like superconducting materials engineering and their applications-superconducting magnets, superconducting energy storage, and magnetic levitation systems. Such electronic devices demand special approach to the materials used in passive elements and sensors. The main goal of this work was the implementation of a fully transparent, flexible cryogenic temperature sensor with graphene structures as sensing element. Electrodes were made of transparent ITO (Indium Tin Oxide) or ITO/Ag/ITO conductive layers by laser ablation and finally encapsulated in a polymer coating. A helium closed-cycle cryostat has been used in measurements of the electrical properties of these graphene-based temperature sensors under cryogenic conditions. The sensors were repeatedly cooled from room temperature to cryogenic temperature. Graphene structures were characterized using Raman spectroscopy. The observation of the resistance changes as a function of temperature indicates the potential use of graphene layers in the construction of temperature sensors. The temperature characteristics of the analyzed graphene sensors exhibit no clear anomalies or strong non-linearity in the entire studied temperature range (as compared to the typical carbon sensor).

Unraveling sterol-dependent membrane phenotypes by analysis of protein abundance-ratio distributions in different membrane fractions under biochemical and endogenous sterol depletion.

During the last decade, research on plasma membrane focused increasingly on the analysis of so-called microdomains. It has been shown that function of many membrane-associated proteins involved in signaling and transport depends on their conditional segregation within sterol-enriched membrane domains. High throughput proteomic analysis of sterol-protein interactions are often based on analyzing detergent resistant membrane fraction enriched in sterols and associated proteins, which also contain proteins from these microdomain structures. Most studies so far focused exclusively on the characterization of detergent resistant membrane protein composition and abundances. This approach has received some criticism because of its unspecificity and many co-purifying proteins. In this study, by a label-free quantitation approach, we extended the characterization of membrane microdomains by particularly studying distributions of each protein between detergent resistant membrane and detergent-soluble fractions (DSF). This approach allows a more stringent definition of dynamic processes between different membrane phases and provides a means of identification of co-purifying proteins. We developed a random sampling algorithm, called Unicorn, allowing for robust statistical testing of alterations in the protein distribution ratios of the two different fractions. Unicorn was validated on proteomic data from methyl-ß-cyclodextrin treated plasma membranes and the sterol biosynthesis mutant smt1. Both, chemical treatment and sterol-biosynthesis mutation affected similar protein classes in their membrane phase distribution and particularly proteins with signaling and transport functions.

Phosphoproteomics Reveals Selective Regulation of Signaling Pathways by Lysophosphatidic Acid Species in Macrophages.

Lysophosphatidic acid (LPA) species, prevalent in the tumor microenvironment (TME), adversely impact various cancers. In ovarian cancer, the 18:0 and 20:4 LPA species are selectively associated with shorter relapse-free survival, indicating distinct effects on cellular signaling networks. Macrophages represent a cell type of high relevance in the TME, but the impact of LPA on these cells remains obscure. Here, we uncovered distinct LPA-species-specific responses in human monocyte-derived macrophages through unbiased phosphoproteomics, with 87 and 161 phosphosites upregulated by 20:4 and 18:0 LPA, respectively, and only 24 shared sites. Specificity was even more pronounced for downregulated phosphosites (163 versus 5 sites). Considering the high levels 20:4 LPA in the TME and its selective association with poor survival, this finding may hold significant implications. Pathway analysis pinpointed RHO/RAC1 GTPase signaling as the predominantly impacted target, including AHRGEF and DOCK guanine exchange factors, ARHGAP GTPase activating proteins, and regulatory protein kinases. Consistent with these findings, exposure to 20:4 resulted in strong alterations to the actin filament network and a consequent enhancement of macrophage migration. Moreover, 20:4 LPA induced p38 phosphorylation, a response not mirrored by 18:0 LPA, whereas the pattern for AKT was reversed. Furthermore, RNA profiling identified genes involved in cholesterol/lipid metabolism as selective targets of 20:4 LPA. These findings imply that the two LPA species cooperatively regulate different pathways to support functions essential for pro-tumorigenic macrophages within the TME. These include cellular survival via AKT activation and migration through RHO/RAC1 and p38 signaling.

BAG6 restricts pancreatic cancer progression by suppressing the release of IL33-presenting extracellular vesicles and the activation of mast cells.

Recent studies reveal a critical role of tumor cell-released extracellular vesicles (EVs) in pancreatic cancer (PC) progression. However, driver genes that direct EV function, the EV-recipient cells, and their cellular response to EV uptake remain to be identified. Therefore, we studied the role of Bcl-2-associated-anthanogene 6 (BAG6), a regulator of EV biogenesis for cancer progression. We used a Cre recombinase/LoxP-based reporter system in combination with single-cell RNA sequencing to monitor in vivo EV uptake and tumor microenvironment (TME) changes in mouse models for pancreatic ductal adenocarcinoma (PDAC) in a Bag6 pro- or deficient background. In vivo data were validated using mouse and human organoids and patient samples. Our data demonstrated that Bag6-deficient subcutaneous and orthotopic PDAC tumors accelerated tumor growth dependent on EV release. Mechanistically, this was attributed to mast cell (MC) activation via EV-associated IL33. Activated MCs promoted tumor cell proliferation and altered the composition of the TME affecting fibroblast polarization and immune cell infiltration. Tumor cell proliferation and fibroblast polarization were mediated via the MC secretome containing high levels of PDGF and CD73. Patients with high BAG6 gene expression and high protein plasma level have a longer overall survival indicating clinical relevance. The current study revealed a so far unknown tumor-suppressing activity of BAG6 in PDAC. Bag6-deficiency allowed the release of EV-associated IL33 which modulate the TME via MC activation promoting aggressive tumor growth. MC depletion using imatinib diminished tumor growth providing a scientific rationale to consider imatinib for patients stratified with low BAG6 expression and high MC infiltration. EVs derived from BAG6-deficient pancreatic cancer cells induce MC activation via IL33/Il1rl1. The secretome of activated MCs induces tumor proliferation and changes in the TME, particularly shifting fibroblasts into an inflammatory cancer-associated fibroblast (iCAF) phenotype. Blocking EVs or depleting MCs restricts tumor growth.

Root and canal morphology of the maxillary second premolars as indicated by cone beam computed tomography.

The aim was to evaluate the morphology of maxillary second premolars. The root canal configurations were classified according to Vertucci and a new system by Ahmed et al. based on CBCT images. In addition, the number of roots and the level where they bifurcated were identified, and morphological measurements were performed based on anatomical landmarks. Single-rooted forms were most common. Bifurcation occurred most often in the middle part of the root. The most common canal configuration was type I according to Vertucci and code 1 SP1 according to the new classification. The measurements showing the results were as follows (mm): palatal cusp/buccal cusp/the lowest point of the occlusal surface to pulp chamber ceiling 6.18/6.54/4.77; the ceiling of the pulp chamber was found at the CEJ in 92.28% of the specimens. The maxillary second premolars tended to be single-rooted and with one root canal.

Removing Fractured Endodontic Files with a Tube Technique-The Strength of the Glued Joint: Tube-Endodontic File Setup.

One recommended technique for removing broken root canal instruments is to glue the fragment into a cannula adapted to it (i.e., the tube technique). The aim of the study was to determine the influence of the adhesive kind and length of the joint on the breaking force. During the investigation, 120 files (60 H-files and 60 K-files) and 120 injection needles were used. Fragments of broken files were glued into the cannula using one of three materials: cyanoacrylate adhesive, composite prosthetic cement, or glass ionomer cement. The lengths of the glued joints were 2 and 4 mm. After the polymerization of adhesives, a tensile test was carried out to find a breaking force. The results were statistically analyzed (p < 0.05). For 4 mm lengths of glued joints, the breaking force was higher than for 2 mm for both file types (K and H). In the case of K-type files, the breaking force was higher for cyanoacrylate and composite adhesives than glass ionomer cement. For H-type files, no significant difference in joint strength was found between binders at 4 mm, while at 2 mm, a much better connection was obtained for cyanoacrylate glue than prosthetic cements.

Bactericidal Activity of Graphene Oxide Tests for Selected Microorganisms.

The aim of this study was to determine the bactericidal potential of graphene oxide (GO) in contact with four species of bacteria: E. coli, S. mutans, S. aureus and E. faecalis. Bacterial cell suspensions of each species were incubated in a medium containing GO, with incubation times of 5, 10, 30 and 60 min, at final concentrations of 50, 100, 200, 300 and 500 µg/mL. The cytotoxicity of GO was evaluated using live/dead staining. The results were recorded using a BD Accuri C6 flow cytofluorimeter. Obtained data were analyzed using BD CSampler software. A significant bacteria viability reduction was noted in all GO-containing samples. The antibacterial properties of GO were strongly influenced by GO concentration and incubation time. The highest bactericidal activity was observed at concentrations of 300 and 500 µg/mL for all incubation times (5, 10, 30 and 60 min). The highest antimicrobial potential was observed for E. coli: after 60 min, the mortality rate was 94% at 300 µg/mL GO and 96% at 500 µg/mL GO; the lowest was found for S. aureus-49% (300 µg/mL) and 55% (500 µg/mL).

Menstrual blood-derived mesenchymal stromal cells: impact of preconditioning on the cargo of extracellular vesicles as potential therapeutics.

BACKGROUND: Mesenchymal stromal cells (MSCs) have been shown to exert their therapeutic effects through the secretion of broad spectrum of paracrine factors, including extracellular vesicles (EVs). Accordingly, EVs are being pursued as a promising alternative to cell-based therapies. Menstrual blood-derived stromal cells (MenSCs) are a type of MSC that, due to their immunomodulatory and regenerative properties, have emerged as an innovative source. Additionally, new strategies of cell priming may potentially alter the concentration and cargo of released EVs, leading to modification of their biological properties. In this study, we aimed to characterize the EVs released by MenSCs and compare their therapeutic potential under three different preconditioning conditions (proinflammatory stimuli, physioxia, and acute hypoxia). METHODS: MenSCs were isolated from five healthy women. Following culturing to 80% confluence, MenSCs were exposed to different priming conditions: basal (21% O2), proinflammatory stimuli (IFNγ and TNFα, 21% O2), physioxia (1-2% O2), and acute hypoxia (< 1% O2) for 48-72 h. Conditioned media from MenSCs was collected after 48 h and EVs were isolated by a combination of ultra-filtration and differential centrifugation. An extensive characterization ranging from nano-flow cytometry (nFC) to quantitative high-throughput shotgun proteomics was performed. Bioinformatics analyses were used to derive hypotheses on their biological properties. RESULTS: No differences in the morphology, size, or number of EVs released were detected between priming conditions. The proteome analysis associated with basal MenSC-EVs prominently revealed their immunomodulatory and regenerative capabilities. Furthermore, quantitative proteomic analysis of differentially produced MenSC-EVs provided sufficient evidence for the utility of the differential preconditioning in purpose-tailoring EVs for their therapeutic application: proinflammatory priming enhanced the anti-inflammatory, regenerative and immunomodulatory capacity in the innate response of EVs, physioxia priming also improves tissue regeneration, angiogenesis and their immunomodulatory capacity targeting on the adaptive response, while acute hypoxia priming, increased hemostasis and apoptotic processes regulation in MenSC-EVs, also by stimulating immunomodulation mainly through the adaptive response. CONCLUSIONS: Priming of MenSCs under proinflammatory and hypoxic conditions affected the cargo proteome of EVs released, resulting in different therapeutic potential, and thus warrants experimental exploration with the aim to generate better-defined MSC-derived bioproducts.

Engineering a new-to-nature cascade for phosphate-dependent formate to formaldehyde conversion in vitro and in vivo.

Formate can be envisioned at the core of a carbon-neutral bioeconomy, where it is produced from CO2 by (electro-)chemical means and converted into value-added products by enzymatic cascades or engineered microbes. A key step in expanding synthetic formate assimilation is its thermodynamically challenging reduction to formaldehyde. Here, we develop a two-enzyme route in which formate is activated to formyl phosphate and subsequently reduced to formaldehyde. Exploiting the promiscuity of acetate kinase and N-acetyl-γ-glutamyl phosphate reductase, we demonstrate this phosphate (Pi)-based route in vitro and in vivo. We further engineer a formyl phosphate reductase variant with improved formyl phosphate conversion in vivo by suppressing cross-talk with native metabolism and interface the Pi route with a recently developed formaldehyde assimilation pathway to enable C2 compound formation from formate as the sole carbon source in Escherichia coli. The Pi route therefore offers a potent tool in expanding the landscape of synthetic formate assimilation.

Impaired Plakophilin-2 in obesity breaks cell cycle dynamics to breed adipocyte senescence.

Plakophilin-2 (PKP2) is a key component of desmosomes, which, when defective, is known to promote the fibro-fatty infiltration of heart muscle. Less attention has been given to its role in adipose tissue. We report here that levels of PKP2 steadily increase during fat cell differentiation, and are compromised if adipocytes are exposed to a pro-inflammatory milieu. Accordingly, expression of PKP2 in subcutaneous adipose tissue diminishes in patients with obesity, and normalizes upon mild-to-intense weight loss. We further show defective PKP2 in adipocytes to break cell cycle dynamics and yield premature senescence, a key rheostat for stress-induced adipose tissue dysfunction. Conversely, restoring PKP2 in inflamed adipocytes rewires E2F signaling towards the re-activation of cell cycle and decreased senescence. Our findings connect the expression of PKP2 in fat cells to the physiopathology of obesity, as well as uncover a previously unknown defect in cell cycle and adipocyte senescence due to impaired PKP2.

Barrier Diamond-like Carbon Coatings on Polydimethylsiloxane Substrate.

The plasma modification of polydimethylsiloxane (PDMS) substrates is one way to change their surface geometry, which enables the formation of wrinkles. However, these changes are very often accompanied by the process of restoring the hydrophobic properties of the modified material. In this work, the RF PACVD device (radio frequency plasma-assisted chemical vapor deposition) was used, with which the plasma treatment of PDMS substrates was carried out in argon, nitrogen, oxygen, and methane atmospheres at variable negative biases ranging from 100 V to 500 V. The obtained results show the stability of contact angles for deionized water only in the case of surfaces modified by diamond-like carbon (DLC) coatings. The influence of the applied production conditions on the thickness (between 10 and 30 nm) and chemical structure (ID/IG between 0.41 and 0.8) of DLC coatings is discussed. In the case of plasma treatments with other gases introduced into the working chamber, the phenomenon of changing from hydrophilic to hydrophobic properties after the modification processes was observed. The presented results confirm the barrier nature of the DLC coatings produced on the PDMS substrate.

New Class of Polymer Materials-Quasi-Nematic Colloidal Particle Self-Assemblies: The Case of Assemblies of Prolate Spheroidal Poly(Styrene/Polyglycidol) Particles.

Assemblies of colloidal polymer particles find various applications in many advanced technologies. However, for every type of application, assemblies with properly tailored properties are needed. Until now, attention has been concentrated on the assemblies composed of spherical particles arranged into so-called perfect colloidal crystals and on complex materials containing mixtures of crystal and disordered phases. However, new opportunities are opened by using assemblies of spheroidal particles. In such assemblies, the particles, in addition to the three positional have three angular degrees of freedom. Here, the preparation of 3D assemblies of reference microspheres and prolate spheroidal poly(styrene/polyglycidol) microparticles by deposition from water and water/ethanol media on silicon substrates is reported. The particles have the same polystyrene/polyglycidol composition and the same volumes but differ with respect to their aspect ratio (AR) ranged from 1 to 8.5. SEM microphotographs reveal that particles in the assembly top layers are arranged into the quasi-nematic structures and that the quality of their orientation in the same direction increase with increasing AR. Nano- and microindentation studies demonstrate that interactions of sharp and flat tips with arrays of spheroidal particles lead to different types of particle deformations.