Formation, collective motion, and merging of macroscopic bacterial aggregates

Chemotactic bacteria form emergent spatial patterns of variable cell density within cultures that are initially spatially uniform. These patterns are the result of chemical gradients that are created from the directed movement and metabolic activity of billions of cells. A recent study on pattern formation in wild bacterial isolates has revealed unique collective behaviors of the bacteria Enterobacter cloacae. As in other bacterial species, Enterobacter cloacae form macroscopic aggregates. Once formed, these bacterial clusters can migrate several millimeters, sometimes resulting in the merging of two or more clusters. To better understand these phenomena, we examine the formation and dynamics of thousands of bacterial clusters that form within a 22 cm square culture dish filled with soft agar over two days. At the macroscale, the aggregates display spatial order at short length scales, and the migration of cell clusters is superdiffusive, with a merging acceleration that is correlated with aggregate size. At the microscale, aggregates are composed of immotile cells surrounded by low density regions of motile cells. The collective movement of the aggregates is the result of an asymmetric flux of bacteria at the boundary. An agent-based model is developed to examine how these phenomena are the result of both chemotactic movement and a change in motility at high cell density. These results identify and characterize a new mechanism for collective bacterial motility driven by a transient, density-dependent change in motility.

Comparative Investigation on Micro-Structural, Morphological, Optical, Magnetic and Anti-Microbial Traits of Undoped and Nickel-Incorporated Zinc Oxide Nanoparticles

The main objective of the present work is to synthesize pure and nickel doped zinc oxide nanoparticles by facile co precipitation technique. The work is confined to study the effect of various weight ratios (0.3, 0.6 ,0.9) % Nickel into Zinc oxide and to witness the drastic changes that occur in its various physical properties such as structural, optical, magnetic from X ray diffraction (XRD), UV visible (ultra violet) spectra, VSM (Vibrating sample magneto meter). XRD analysis reveals the wurtzite hexagonal structure and it is also found that as the doping concentration increases the crystallite size decreases from 4.6 nm to 3.0 nm. SEM results depicts the agglomeration of the particle, the synthesized samples shows both rod and flakes formation when the doping concentration is increased. Morphological changes were analysed TEM (Transmission electron microscope). The enhancement in the optical behaviour were observed and the energy band gap is calculated with the data obtained from UV-Visible spectra and the optical properties shows a tremendous increase as the Ni content increases which proves the sample a suitable candidate for solar cells and photovoltaic devices. Purity of the prepared sample were investigated through EDAX analysis. The hysteresis loop from the VSM analysis elucidate the saturation magnetization and the ferromagnetic behaviour of the sample. X-ray Photoemission Spectroscopy results indicates the presence of several oxygen species adsorbed on the surface. The study is also extended to analyse its anti-microbial effect against Staphylococcus aureus. The cell culture dish of the sample showed a notable resistance against Staphylococcus aureus when the concentration of nickel is increased and could be extended to pharmaceutical applications in treating several skin infections.

Blockade of glutamine-dependent cell survival augments antitumor efficacy of CPI-613 in head and neck cancer

Background Alterations in metabolism are one of the emerging hallmarks of cancer cells and targeting dysregulated cancer metabolism provides a new approach to developing more selective therapeutics. However, insufficient blockade critical metabolic dependencies of cancer allows the development of metabolic bypasses, thus limiting therapeutic benefits. Methods A series of head and neck squamous cell carcinoma (HNSCC) cell lines and animal models were used to determine the efficacy of CPI-613 and CB-839 when given alone or in combination. Glutaminase 1 (GLS1) depletion was achieved by lentiviral shRNAs. Cell viability and apoptosis were determined in HNSCC cells cultured in 2D culture dish and SeedEZ™ 3D scaffold. Molecular alterations were examined by Western blotting and immunohistochemistry. Metabolic changes were assessed by glucose uptake, lactate production, glutathione levels, and oxygen consumption rate. Results We show here that HNSCC cells display strong addiction to glutamine. CPI-613, a novel lipoate analog, redirects cellular activity towards tumor-promoting glutaminolysis, leading to low anticancer efficacy in HNSCC cells. Mechanistically, CPI-613 inhibits the tricarboxylic acid cycle by blocking the enzyme activities of pyruvate dehydrogenase and alpha-ketoglutarate dehydrogenase, which upregulates GLS1 and eventually promotes the compensatory role of glutaminolysis in cancer cell survival. Most importantly, the addition of a GLS1 inhibitor CB-839 to CPI-613 treatment abrogates the metabolic dependency of HNSCC cells on glutamine, achieving a synergistic anticancer effect in glutamine-addicted HNSCC. Conclusions These findings uncover the critical role of GLS1-mediated glutaminolysis in CPI-613 treatment and suggest that the CB-839 and CPI-613 combination may potentiate synergistic anticancer activity for HNSCC therapeutic gain.

Cultivation of hierarchical 3D scaffolds inside a perfusion bioreactor: scaffold design and finite-element analysis of fluid flow

The use of porous 3D scaffolds for the repair of bone nonunion and osteoporotic bone is currently an area of great interest. Using a combination of thermally-induced phase separation (TIPS) and 3D-plotting (3DP), we have generated hierarchical 3DP/TIPS scaffolds made of poly(lactic-co-glycolic acid) (PLGA) and nanohydroxyapatite (nHA). A full factorial design of experiments was conducted, in which the PLGA and nHA compositions were varied between 6‒12% w/v and 10‒40% w/w, respectively, totaling 16 scaffold formulations with an overall porosity ranging between 87%‒93%. These formulations included an optimal scaffold design identified in our previous study. The internal structures of the scaffolds were examined using scanning electron microscopy and microcomputed tomography. Our optimal scaffold was seeded with MC3T3-E1 murine preosteoblastic cells and subjected to cell culture inside a tissue culture dish and a perfusion bioreactor. The results were compared to those of a commercial CellCeram™ scaffold with a composition of 40% β-tricalcium phosphate and 60% hydroxyapatite (β-TCP/HA). Media flow within the macrochannels of 3DP/TIPS scaffolds was modeled in COMSOL software in order to fine tune the wall shear stress. CyQUANT DNA assay was performed to assess cell proliferation. The normalized number of cells for the optimal scaffold was more than twofold that of CellCeram™ scaffold after two weeks of culture inside the bioreactor. Despite the substantial variability in the results, the observed improvement in cell proliferation upon culture inside the perfusion bioreactor (vs. static culture) demonstrated the role of macrochannels in making the 3DP/TIPS scaffolds a promising candidate for scaffold-based tissue engineering.

A scalable coaxial bioprinting technology for mesenchymal stem cell microfiber fabrication and high extracellular vesicle yield

Mesenchymal stem cell (MSC)-derived extracellular vesicles (EVs) are promising candidates for regenerative medicine; however, the lack of scalable methods for high quantity EV production limits their application. In addition, signature EV-derived proteins shared in 3D environments and 2D surfaces, remain mostly unknown. Herein, we present a platform combining MSC microfiber culture with ultracentrifugation purification for high EV yield. Within this platform, a high quantity MSC solution (~3x10^8 total cells) is encapsulated in a meter-long hollow hydrogel-microfiber via coaxial bioprinting technology. In this 3D core-shell microfiber environment, MSCs express higher levels of stemness markers (Oct4, Nanog, Sox2) than in 2D culture, and maintain their differentiation capacity. Moreover, this platform enriches particles by ~1009-fold compared to conventional 2D culture, while preserving their pro-angiogenic properties. Liquid chromatography-mass spectrometry characterization results demonstrate that EVs derived from our platform and conventional 2D culturing have unique protein profiles with 3D-EVs having a greater variety of proteins (1023 vs 605), however, they also share certain proteins (536) and signature MSC-EV proteins (10). This platform, therefore, provides a new tool for EV production using microfibers in one culture dish, thereby reducing space, labor, time, and cost.

Meiotic Chromosome Synapsis and XY-Body Formation In Vitro

To achieve spermatogenesis in vitro, one of the most challenging processes to mimic is meiosis. Meiotic problems, like incomplete synapsis of the homologous chromosomes, or impaired homologous recombination, can cause failure of crossover formation and subsequent chromosome nondisjunction, eventually leading to aneuploid sperm. These meiotic events are therefore strictly monitored by meiotic checkpoints that initiate apoptosis of aberrant spermatocytes and lead to spermatogenic arrest. However, we recently found that, in vitro derived meiotic cells proceeded to the first meiotic division (MI) stage, despite displaying incomplete chromosome synapsis, no discernible XY-body and lack of crossover formation. We therefore optimized our in vitro culture system of meiosis from male germline stem cells (mGSCs) in order to achieve full chromosome synapsis, XY-body formation and meiotic crossovers. In comparison to previous culture system, the in vitro-generated spermatocytes were transferred after meiotic initiation to a second culture dish. This dish already contained a freshly plated monolayer of proliferatively inactivated immortalized Sertoli cells supporting undifferentiated mGSCs. In this way we aimed to simulate the multiple layers of germ cell types that support spermatogenesis in vivo in the testis. We found that in this optimized culture system, although independent of the undifferentiated mGSCs, meiotic chromosome synapsis was complete and XY body appeared normal. However, meiotic recombination still occurred insufficiently and only few meiotic crossovers were formed, leading to MI-spermatocytes displaying univalent chromosomes (paired sister chromatids). Therefore, considering that meiotic checkpoints are not necessarily fully functional in vitro, meiotic crossover formation should be closely monitored when mimicking gametogenesis in vitro to prevent generation of aneuploid gametes.

Ultrasonic control of neurite outgrowth direction

This study investigated a method to control neurite outgrowth direction using ultrasound vibration. An ultrasound cell culture dish comprising a glass-bottom culture surface and a glass disc with an ultrasound transducer was fabricated, and undifferentiated neuron-like PC12 cells were grown on the dish as an adherent culture. The 78 kHz resonant concentric flexural vibration mode of the dish was used to quantitatively evaluate the neurite outgrowth direction and length. Time-lapse imaging of cells was performed for 72 h under ultrasound excitation. Unsonicated neurites grew in random directions, whereas neurite outgrowth was circumferentially oriented during ultrasonication in a power-dependent manner. The neurite orientation correlated with the spatial gradient of the ultrasound vibration, implying that neurites tend to grow in directions along which the vibrational amplitude does not change. Ultrasonication with 30 Vpp for 72 h increased the neurite length by 99.7% compared with that observed in unsonicated cells.

Strategies to Improve the Quality and Freshness of Human Bone Marrow-Derived Mesenchymal Stem Cells for Neurological Diseases

Human bone marrow-derived mesenchymal stem cells (hBM-MSCs) have been studied for their application to manage various neurological diseases, owing to their anti-inflammatory, immunomodulatory, paracrine, and antiapoptotic ability, as well as their homing capacity to specific regions of brain injury. Among mesenchymal stem cells, such as BM-MSCs, adipose-derived MSCs, and umbilical cord MSCs, BM-MSCs have many merits as cell therapeutic agents based on their widespread availability and relatively easy attainability and in vitro handling. For stem cell-based therapy with BM-MSCs, it is essential to perform ex vivo expansion as low numbers of MSCs are obtained in bone marrow aspirates. Depending on timing, before hBM-MSC transplantation into patients, after detaching them from the culture dish, cell viability, deformability, cell size, and membrane fluidity are decreased, whereas reactive oxygen species generation, lipid peroxidation, and cytosolic vacuoles are increased. Thus, the quality and freshness of hBM-MSCs decrease over time after detachment from the culture dish. Especially, for neurological disease cell therapy, the deformability of BM-MSCs is particularly important in the brain for the development of microvessels. As studies on the traditional characteristics of hBM-MSCs before transplantation into the brain are very limited, omics and machine learning approaches are needed to evaluate cell conditions with indepth and comprehensive analyses. Here, we provide an overview of hBM-MSCs, the application of these cells to various neurological diseases, and improvements in their quality and freshness based on integrated omics after detachment from the culture dish for successful cell therapy.