Optimal Cell Density Research Articles

Osteogenic differentiation of BMSCs on MoS2 composite nanofibers with different cell seeding densities

Molybdenum disulphide (MoS2) exhibits unique properties that are useful for various biomedical applications. Owing to its distinct characteristics and osteogenic differentiation promotion effect, this material has been studied extensively. However, the effect of cell density on osteogenic differentiation between MoS2-based biomaterials and cells is still unknown. In this study, we used MoS2/polyacrylonitrile (PAN) composite nanofibres as substrates to evaluate the effect of cellular density on the osteogenic differentiation of bone marrow-derived mesenchymal stem cells (BMSCs). We created different experimental groups with increasing cell seeding densities and investigated cellular behaviours, biocompatibility, proliferation and osteogenic properties. The results show that MoS2/PAN composite nanofibres can positively regulate osteogenic differentiation. More importantly, in the presence of standard culture conditions, 1.0 × 104 cells/cm2 is the most efficient and suitable cell seeding density for BMSCs osteogenic differentiation. Our findings suggest that the optimal cell density for osteogenesis is vital for the osteogenic differentiation of BMSCs when these cells are cultured onto MoS2-based biomaterials.

4418 Optimization and Validation of a Silk Scaffold-Based Neural Tissue Construct

OBJECTIVES/GOALS: Our goal is to develop a silk fibroin scaffold-based neural tissue construct and characterize it in a rat model of cortical injury. We aim to optimize the construct for transplantation, test pharmacologic interventions that may enhance its survival, and evaluate its integration with the host brain. METHODS/STUDY POPULATION: To optimize cell density and health, silk fibroin scaffolds varying in porosity and stiffness were seeded with E18 GFP+ rat cortical neurons and imaged at DIV 5. Different seeding methods and loads were similarly tested. Constructs, loaded with an inhibitor of apoptosis (ROCK inhibitor Y-27632) or necroptosis (necrostatin-1) in a fibrin hydrogel, were transplanted into aspiration lesions created in the primary motor cortex of Sprague-Dawley rats, and graft survival was compared to negative control at 2 weeks. Lastly, constructs were transplanted and evaluated via immunohistochemistry at 1, 2, and 4-month time points for survival, differentiation, inflammation, and anatomic integration. RESULTS/ANTICIPATED RESULTS: Scaffolds with smaller pore sizes retained more cells after seeding. Softer scaffolds, which enhance hemostasis at transplantation, did not compromise cell health on live/dead assay. We anticipate that seeding concentrated cell suspensions onto multiple surfaces of the construct will produce the most evenly seeded and cell-dense constructs. Based on a prior pilot study, we anticipate that necrostatin-1 will significantly improve intermediate-term construct survival. We have observed up to 15% cell survival at 1 month with retained neuronal identity and abundant axonal projections into the brain despite evidence of persistent inflammation; we anticipate similar outcomes at later time points. DISCUSSION/SIGNIFICANCE OF IMPACT: Our construct, due to its exceptional longevity in vitro, manipulability, and modularity, is an attractive platform for neural tissue engineering. In the present work, we optimize and validate this technology for transplantation with the goal of addressing the morbidity burden of cortical injury.

Agrobacterium rhizogenes-mediated transformation of grain (Amaranthus hypochondriacus) and leafy (A. hybridus) amaranths.

Transgenic A. hypochondriacus and A. hybridus roots were generated. Further, a distinct plant regeneration program via somatic embryos produced from hairy roots was established. Work was implemented to develop an optimized protocol for root genetic transformation of the three grain amaranth species and A. hybridus, their presumed ancestor. Transformation efficiency was species-specific, being higher in A. hypochondriacus and followed by A. hybridus. Amaranthus cruentus and A. caudatus remained recalcitrant. A reliable and efficient Agrobacteruim rhizogenes-mediated transformation of these species was established using cotyledon explants infected with the previously untested BVG strain. Optimal OD600 bacterial cell densities were 0.4 and 0.8 for A. hypochondriacus and A. hybridus, respectively. Hairy roots of both amaranth species were validated by the amplification of appropriate marker genes and, when pertinent, by monitoring green fluorescent protein emission or β-glucuronidase activity. Embryogenic calli were generated from A. hypochondriacus rhizoclones. Subsequent somatic embryo maturation and germination required the activation of cytokinin signaling, osmotic stress, red light, and calcium incorporation. A crucial step to ensure the differentiation of germinating somatic embryos into plantlets was their individualization and subcultivation in 5/5 media containing 5% sucrose, 5g/L gelrite, and 0.2mg/L 2-isopentenyladenine (2iP) previously acidified to pH 4.0 with phosphoric acid, followed by their transfer to 5/5 + 2iP media supplemented with 100mg/L CaCl2. These steps were strictly red light dependent. This process represents a viable protocol for plant regeneration via somatic embryo germination from grain amaranth transgenic hairy roots. Its capacity to overcome the recalcitrance to genetic transformation characteristic of grain amaranth has the potential to significantly advance the knowledge of several unresolved biological aspects of grain amaranths.

Improving HIV Outgrowth by Optimizing Cell-Culture Conditions and Supplementing With all-trans Retinoic Acid.

The persistence of replication-competent HIV reservoirs in people living with HIV (PLWH) receiving antiretroviral therapy (ART) is a barrier to cure. Therefore, their accurate quantification is essential for evaluating the efficacy of new therapeutic interventions and orienting the decision to interrupt ART. Quantitative viral outgrowth assays (QVOAs) represent the “gold standard” for measuring the size of replication-competent HIV reservoirs. However, they require large numbers of cells and are technically challenging. This justifies the need for the development of novel simplified methods adapted for small biological samples. Herein, we sought to simplify the viral outgrowth procedure (VOP) by (i) using memory CD4+ T-cells, documented to be enriched in HIV reservoirs (ii) optimizing cell-culture conditions, and (iii) supplementing with all-trans retinoic acid (ATRA), a positive regulator of HIV replication. Memory CD4+ T-cells were sorted from the peripheral blood of ART-treated (HIV+ART; n = 14) and untreated (HIV+; n = 5) PLWH. The VOP was first performed with one original replicate of 1 × 106 cells/well in 48-well plates. Cells were stimulated via CD3/CD28 for 3 days, washed to remove residual CD3/CD28 Abs, split every 3 days for optimal cell density, and cultured in the presence or the absence of ATRA for 12 days. Soluble and intracellular HIV-p24 levels were quantified by ELISA and flow cytometry, respectively. Optimal cell-culture density achieved by splitting improved HIV outgrowth detection. ATRA promoted superior/accelerated detection of replication-competent HIV in all HIV+ART individuals tested, including those with low/undetectable viral outgrowth in the absence of ATRA. Finally, this VOP was used to design a simplified ATRA-based QVOA by including 4 and 6 original replicates of 1 × 106 cells/well in 48-well plates and 2 × 105 cells/well in 96-well plates, respectively. Consistently, the number of infectious units per million cells (IUPM) was significantly increased in the presence of ATRA. In conclusion, we demonstrate that memory CD4+ T-cell splitting for optimal density in culture and ATRA supplementation significantly improved the efficacy of HIV outgrowth in a simplified ATRA-based QVOA performed in the absence of feeder/target cells or indicator cell lines.

Cardiac differentiation of induced pluripotent stem cells derived from mesenchymal stem cells

Background & Aim Induced pluripotent stem cells (iPSCs) are commonly studied for clinical use due to its flexibility in differentiating into any desirable cellular products. iPSCs are commonly derived from fibroblasts or cells from peripheral blood due to its accessibility. Although not common, we have derived iPSCs from mesenchymal stem cells (MSC) for several purposes. We are developing an allogenic combinatory cellular therapy for heart disease using both MSCs with iPSC derived cardiomyocyte (CM) or cardiac progenitor cells (CPCs). We theorize, by utilizing iPSC derived from MSCs, it provides more compatibility when transplanting the two final cellular products from one donor into patients, thus yielding better therapeutic effects. Also, recent changes in donor qualification increase the financial burden when launching new cellular therapeutics due to the increase of required testing on donor and final cellular product. In this scenario, by reprogramming the MSCs into iPSCs then differentiate into CM, we eliminated the additional cost of qualifying the parent cells of iPSCs. Methods, Results & Conclusion Although cardiac differentiation of iPSC is well studied, the optimal differentiation condition needed to be tested for each cell line used. The initial evaluation must be performed to determine the optimal cell density, the concentration of CHIR99021, and the time point of switching to IWP2. After determining these factors, the cardiac differentiation was performed on both IMR90 (lung fibroblast) and MSC derived iPSCs in order to evaluate the efficiency of differentiation of the latter iPSC. The iPSCs were plated onto Vitronectin coated plates until the desired confluence reached using iPSC culture medium. At day 0 of the cardiac differentiation process, the iPSC culture medium was replaced with CHIR99021in differentiation medium (RPMI-1640 with B-27 without insulin). Two or three days later, IWP2 replaced CHIR99021 in the differentiation medium for another two days. After day 7 of the cardiac differentiation process, cells were maintained with CM culture medium (RPMI-1640 with B-27 with insulin) for another seven days. The medium was changed every three to four days. In summary, only the optimized combination of conditions yield beating CM culture at day 14 from both MSC and IMP90 derived iPSCs. CM derived from both iPSCs stained positive for Troponin T. At day 7 of the cardiac differentiation, the cultures were stained positive for GHRHR, which will be used as a marker for CPC selection. Induced pluripotent stem cells (iPSCs) are commonly studied for clinical use due to its flexibility in differentiating into any desirable cellular products. iPSCs are commonly derived from fibroblasts or cells from peripheral blood due to its accessibility. Although not common, we have derived iPSCs from mesenchymal stem cells (MSC) for several purposes. We are developing an allogenic combinatory cellular therapy for heart disease using both MSCs with iPSC derived cardiomyocyte (CM) or cardiac progenitor cells (CPCs). We theorize, by utilizing iPSC derived from MSCs, it provides more compatibility when transplanting the two final cellular products from one donor into patients, thus yielding better therapeutic effects. Also, recent changes in donor qualification increase the financial burden when launching new cellular therapeutics due to the increase of required testing on donor and final cellular product. In this scenario, by reprogramming the MSCs into iPSCs then differentiate into CM, we eliminated the additional cost of qualifying the parent cells of iPSCs. Although cardiac differentiation of iPSC is well studied, the optimal differentiation condition needed to be tested for each cell line used. The initial evaluation must be performed to determine the optimal cell density, the concentration of CHIR99021, and the time point of switching to IWP2. After determining these factors, the cardiac differentiation was performed on both IMR90 (lung fibroblast) and MSC derived iPSCs in order to evaluate the efficiency of differentiation of the latter iPSC. The iPSCs were plated onto Vitronectin coated plates until the desired confluence reached using iPSC culture medium. At day 0 of the cardiac differentiation process, the iPSC culture medium was replaced with CHIR99021in differentiation medium (RPMI-1640 with B-27 without insulin). Two or three days later, IWP2 replaced CHIR99021 in the differentiation medium for another two days. After day 7 of the cardiac differentiation process, cells were maintained with CM culture medium (RPMI-1640 with B-27 with insulin) for another seven days. The medium was changed every three to four days.

A biosorption-based approach for selective extraction of rare earth elements from coal byproducts

Coal byproducts represent an abundant and untapped potential source of critical rare earth elements (REEs). In this study, we tested the efficacy of a biosorption-based approach to recover REEs from leachate solutions derived from North Dakota lignite coal and Powder River Basin (PRB) coal fly ash. A pairwise comparison was performed using a range of cell densities of two distinct bacteria, including an Escherichia coli strain previously genetically engineered for cell surface display of lanthanide binding tags, and Arthrobacter nicotianae, a native bacterium that exhibits high REE adsorption capacity. At optimal cell densities, we observed a recovery efficiency of 80% for total REEs and > 90% for middle and heavy REEs from the lignite leachate. Higher cell densities were required to achieve a similar total REE recovery efficiency in the PRB leachate due to its higher non-REE content, which negatively impacted light REE recovery. Despite the chemical complexity of both feedstocks, separation factors ≥ 30 were observed for Nd relative to nearly all non-REE metals after a single adsorption/desorption cycle, highlighting the high adsorption selectivity for REEs. Quantification of impurities in the extracted metal solutions revealed that Ca and Mg content in the leachate solutions play a dominant role in both the REE recovery yield and purity. The Mg/Ca impurities can be further reduced by incorporation of a low pH wash step between the adsorption and desorption steps, yielding extracted concentrates with a REE purity (mass fraction REE of total metals) of 80% and 50% for lignite and PRB, respectively. Thermodynamic speciation analysis of the REE-enriched eluents from both feedstocks suggests that at a post-biosorption hydroxide precipitation step can be employed to yield high purity total REE precipitates. Collectively, these results highlight the utility of biosorption for selective REE recovery from coal byproducts and pave the way for a sustainable route to diversify the REE supply chain.

Variovorax sp. Has an Optimum Cell Density to Fully Function as a Plant Growth Promoter.

Utilization of plant growth-promoting bacteria colonizing roots is environmentally friendly technology instead of using chemicals in agriculture, and understanding of the effects of their colonization modes in promoting plant growth is important for sustainable agriculture. We herein screened the six potential plant growth-promoting bacteria isolated from Beta vulgaris L. (Rhizobium sp. HRRK 005, Polaromonas sp. HRRK 103, Variovorax sp. HRRK 170, Mesorhizobium sp. HRRK 190, Streptomyces sp. HRTK 192, and Novosphingobium sp. HRRK 193) using a series of biochemical tests. Among all strains screened, HRRK 170 had the highest potential for plant growth promotion, given its ability to produce plant growth substances and enzymes such as siderophores and 1-aminocyclopropane-1-carboxylic acid (ACC) deaminase, respectively, concomitantly with active growth in a wider range of temperatures (10–30 °C) and pH (5.0–10.0). HRRK 170 colonized either as spots or widely on the root surface of all vegetable seedlings tested, but significant growth promotion occurred only in two vegetables (Chinese cabbage and green pepper) within a certain cell density range localized in the plant roots. The results indicate that HRRK 170 could function as a plant growth promoter, but has an optimum cell density for efficient use.

A dual neutrophil-T cell purification procedure and methodological considerations in studying the effects of estrogen on human Th17 cell differentiation

New procedures are required to optimize the use of blood samples to study different cell types. The purification of neutrophils and T cells from the same blood sample is not commonly described. We have previously used PolymorphPrep™ (P) or LymphoPrep™ (L) for purifying neutrophils or T cells, respectively. In this study, we describe a new method for purifying both of these cells using P and L from the same sample, and methodological considerations required to obtain consistent Th17 differentiation results. For T cell studies, we first isolated mononuclear cells from peripheral blood of healthy humans using either P alone, L alone or sequential isolation with P and then L (P + L). CD3+ lymphocytes comprise up to 73% of peripheral blood mononuclear cells (PBMCs) obtained by sequential isolation, with 29% and 36% for P and L, respectively. T lymphocyte subsets, Th1, Th17 or double-positive (Th17/1), were then amplified. Four days of amplification culture after isolation by P alone led to over-expression of Th17/1 cells and of Th17 cells in comparison to cells isolated by L or by sequential P + L. Th17/1 cells comprised 11.0 ± 6.8% (P alone) vs 1.2 ± 0.28% (L alone) vs 0.45 ± 0.11% (P + L) and Th17 cells comprised 2.8 ± 0.4% (P alone) 0.88 ± 0.15% (L alone) vs 0.86 ± 0.14% (P + L). As the second step, we examined T cell purification and differentiation. A higher purity of 97.1 ± 0.44% naïve CD4+ T cell was reached after P + L followed by immunomagnetic bead sorting in comparison to 70 ± 9.3% (L) vs 21.0 ± 8.5% (P). These cells grew well in the density range of 25, 000 to 100, 000 cells per well in 96-well plates during Th17 cell differentiation; higher or lower cell density did not support Th17 cell differentiation. Lastly, to investigate the effect of estrogen on Th17 cell differentiation, serum-free AIM V medium without phenol red was chosen to minimize the hormonal effects of the medium. We found that exogenous estrogen (1 nM) inhibited Th17 cell differentiation in this medium. Taken together, we devised a method to isolate both neutrophils and T cells from the same blood sample and show that high PBMC purity, selected culture medium and an optimal cell density of the initial cell culture produced the most robust and consistent results for Th17 differentiation.

Adhesion and Proliferation of Human Adipose-Derived Stem Cells on Titania Nanotube Surfaces

When a biomaterial is implanted, the body reacts similar to an injury and stem cells are recruited to the implant site. Since, stem cells play an important role in tissue repair in the body, their interaction with biomaterials is critical for long-term success of medical devices. Surfaces with nanostructured features have been shown to alter cellular functionality in vitro and even improve fixation of the implant to the surrounding bone tissues in vivo. In this study, the effect of titania nanotube (NT) size and cell density on the adhesion and proliferation of human adipose-derived stem cells (ASCs) was evaluated. Although many studies have evaluated mesenchymal stem cells response on nanostructured surfaces, very few studies have explored the response of adipose-derived stem cells on titanium nanotube surfaces and none have explored the effect of cell density concurrent with nanotube size on ASC proliferation. Proliferation behavior of ASCs on NT surfaces and titanium control were investigated for a week using three different cell densities. The optimal cell density was 2500 cells/well and the smaller diameter NT exhibited higher ASC proliferation. This study confirms that NT surfaces promote ASC adhesion and proliferation. By more fully understanding the effect of nanostructure size on adhesion and proliferation of stem cells, implants could be specifically designed to achieve the optimal stem cell response from the tissue in which they are implanted. Additionally, the favorable response of ASC to these NT surfaces and determination of optimal cell density suggests a potential use in orthopedic tissue regeneration. When a biomaterial is implanted, the body reacts similar to an injury and stem cells are recruited to the implant site. Since, stem cells play an important role in tissue repair in the body, their interaction with biomaterials is critical for long-term success of medical devices. This study confirms that nanotube surfaces promote stem cell adhesion and proliferation.

Porous decellularized trachea scaffold prepared by a laser micropore technique

Rapid development of tissue engineering technology provides new methods for tracheal cartilage regeneration. However, the current lack of an ideal scaffold makes engineering of trachea cartilage tissue into a three-dimensional (3-D) tubular structure a great challenge. Although a decellularized trachea matrix (DTM) has become a recognized scaffold for trachea cartilage regeneration, it is difficult for cells to detach from or penetrate the matrix because of its non-porous structure. To tackle these problems, a laser micropore technique (LMT) was applied in the current study to enhance trachea sample porosity, and facilitate decellularizing treatment and cell ingrowth. Furthermore, after optimizing LMT and decellularizing treatment parameters, LMT-treated DTM (LDTM) retained its natural tubular structure with only minor extracellular matrix damage. Moreover, compared with DTM, the current study showed that LDTM significantly improved the adherence rate of cells with perfect cell biocompatibility. Moreover, the optimal implantation cell density for chondrogenesis with LDTM was determined to be 1 × 108 cells/ml. Collectively, the results suggest that the novel LDTM is an ideal scaffold for trachea tissue engineering.

Optimizing cell density of Pichia pastoris for production of recombinant hepatitis B surface antigen via employing short-period continuous operation

Optimizing cell density of Pichia pastoris for production of recombinant hepatitis B surface antigen via employing short-period continuous operation

Targeted Repression of Essential Genes To Arrest Growth and Increase Carbon Partitioning and Biofuel Titers in Cyanobacteria.

Photoautotrophic production of fuels and chemicals by cyanobacteria typically gives lower volumetric productivities and titers than heterotrophic production. Cyanobacteria cultures become light limited above an optimal cell density, so that this substrate is not supplied to all cells sufficiently. Here, we investigate genetic strategies for a two-phase cultivation, where biofuel-producing Synechocystis cultures are limited to an optimal cell density through inducible CRISPR interference (CRISPRi) repression of cell growth. Fixed CO2 is diverted to ethanol or n-butanol. Among the most successful strategies was partial repression of citrate synthase gltA. Strong repression (>90%) of gltA at low culture densities increased carbon partitioning to n-butanol 5-fold relative to a nonrepression strain, but sacrificed volumetric productivity due to severe growth restriction. CO2 fixation continued for at least 3 days after growth was arrested. By targeting sgRNAs to different regions of the gltA gene, we could modulate GltA expression and carbon partitioning between growth and product to increase both specific and volumetric productivity. These growth arrest strategies can be useful for improving performance of other photoautotrophic processes.

Critical factors for lentivirus-mediated PRDX4 gene transfer in the HepG2 cell line.

Optimization of critical factors affects transduction efficiency and is able to reduce reagent consumption. The present study aimed to determine the optimum transduction conditions of small hairpin (sh)RNA against peroxiredoxin 4 (PRDX4) in the HepG2 cell line. Cell viability assays were conducted based on serum condition, incubation time, polybrene concentration and antibiotic dose selection. Non-targeting control shRNA was transduced into HepG2 cells in a 5-fold serial dilution, and colonies positive for green fluorescent protein were counted using ImageJ software. Reverse transcription-quantitative polymerase chain reaction and western blot analysis were performed to validate PRDX4 expression. The optimum cell density for transduction was 5.0×103 cells/well in 96-well plates to achieve 40 to 50% confluency the following day. The transduction media consisted of 10% fetal bovine serum (FBS) and 12 µg/ml polybrene, and was used to dilute lentiviral particles at a functional titer of 4.9×105 TU/ml for multiplicity of infection (MOI) of 20, 15 and 10, for 24 h of incubation. Selection with 7 µg/ml puromycin was performed in transduced cells. shRNA 3 was revealed to inhibit PRDX4 mRNA and protein expression. In conclusion, PRDX4 was successfully silenced in 5.0×103 HepG2 cells cultured with 10% FBS and 12 µg/ml polybrene, at a 4.9×105 TU/ml functional titer for MOI of 20, 15 and 10.

A phytobeneficial strain Planomicrobium sp. MSSA-10 triggered oxidative stress responsive mechanisms and regulated the growth of pea plants under induced saline environment.

The study was planned to characterize Planomicrobium sp. MSSA-10 for plant-beneficial traits and to evaluate its inoculation impact on physiology of pea plants under different salinity levels. Strain MSSA-10 was isolated from pea rhizosphere and identified by the analysis of 16S rRNA gene sequence. The strain demonstrated phosphate solubilization and auxin production up to 2moll-1 NaCl and exhibited 1-aminocyclopropane-1-carboxylic acid deaminase activity up to 1·5moll-1 salt. In an inoculation experiment under different salinity regimes, a significant increase in growth was observed associated with decreased levels of reactive oxygen species and enhanced antioxidative enzyme activities. The strain also promoted the translocation of nutrients in plants with subsequent increase in chlorophyll and protein contents as compared to noninoculated plants. It has been observed that rifampicin-resistant derivatives of MSSA-10 were able to survive for 30days at optimum cell density with pea rhizosphere. Growth-stimulating effect of MSSA-10 on pea plants may be attributed to its rhizosphere competence, nutrient mobilization and modulation of plant oxidative damage repair mechanisms under saline environment. Planomicrobium sp. MSSA-10 might be used as potent bioinoculant to relieve pea plants from deleterious effects of salinity.

Determining Optimal Cell Density and Culture Medium Volume simultaneously in MTT Cell Proliferation Assay for Adherent Cancer Cell Lines

Determining Optimal Cell Density and Culture Medium Volume simultaneously in MTT Cell Proliferation Assay for Adherent Cancer Cell Lines

Controlling human corneal stromal stem cell contraction to mediate rapid cell and matrix organization of real architecture for 3-dimensional tissue equivalents.

For the first time, we have demonstrated that human CSSC exhibit the phenomenon of cellular self-alignment in tethered collagen gels. We found this mediated rapid co-alignment of collagen fibrils and thus subsequently exploited this property in vitro to improve the architecture of engineered RAFT tissue equivalents of the corneal stroma. Existing techniques are extremely lengthy and carry significant risk and cost for GMP manufacture. This rapid and tunable technique takes just 8 h of culture and is therefore ideal for clinical manufacture, creating biomimetic tissue equivalents with both cellular and ECM organization. Thus, cellular self-alignment can be a useful bioengineering tool for the development of organized tissue equivalents in a variety of applications.

Biosynthesis and Characterization of Recombinant Silk-Like Polypeptides Derived from the Heavy Chain of Silk Fibrion.

In order to investigate the impacts on the structure and biomedical function of typical fragments derived from repetitive and non-repetitive regions of the Bombyx mori silk fibroin heavy chain, several block combination genes (gs16f1, gs16f4, gs16f8, and gs16f12) were designed, cloned into a fusion protein expression vector tagged with glutathione S-transferase (GST), and expressed in Escherichia coli. Fusion proteins GST-GS16F1, GST-GS16F4, and GST-GS16F8 were purified by GST affinity chromatography, and single bands were identified by SDS-PAGE. Under optimal initial cell density, in ducer concentration and induction expression time, the yield of purified GST-GS16F1, GST-GS16F4, and GST-GS16F8 per liter of bacterial culture reached 79, 53, and 28 mg, respectively. Mass spectrometry revealed molecular weights for GST-GS16F1, GST-GS16F4, and GST-GS16F8 of 37.7, 50.0, and 65.7 kDa, respectively, consistent with the theoretical values of 37.4, 49.4, and 65.5 kDa. Similarly, measured values of pI were 5.35, 4.5, and 4.2 for the fusion proteins, consistent with predicted values of 5.34, 4.44, and 4.09. CD spectra showed the molecular conformation of GS16F1 was mainly β-sheet structure, while more stable α-helix structure formed in GS16F4 and GS16F8.

Heterogeneous Cellular Networks With LoS and NLoS Transmissions—The Role of Massive MIMO and Small Cells

We develop a framework for downlink heterogeneous cellular networks with line-of-sight (LoS) and non-LoS transmissions. Using stochastic geometry, we derive tight approximation of average downlink rate that enables us to compare the performance between densifying small cells and expanding base station (BS) antenna arrays. Interestingly, we find that adding small cells into the network improves the downlink rate much faster than expanding antenna arrays at the macro BS. However, when the small cell density exceeds a critical threshold, the spatial densification will lose its benefits and further impair the network capacity. To this end, we provide the optimal small cell density that maximizes the rate via numerical results for practical deployment guidance. In contrast, expanding macro BS antenna array can always benefit the capacity until an upper bound caused by pilot contamination, and this bound also surpasses the peak rate obtained from the deployment of small cells. Furthermore, we find that allocating part of antennas to distributed small cell BSs works better than centralizing all antennas at the macro BS, and the optimal allocation proportion is also given numerically for practical configuration reference. In summary, this paper provides a further understanding on how to leverage small cells and massive MIMO in future heterogeneous cellular networks deployment.

Energy Efficiency in Cache-Enabled Small Cell Networks With Adaptive User Clustering

Using a network of cache enabled small cells, traffic during peak hours can be reduced by proactively fetching the content that is most likely to be requested. In this paper, we aim to explore the impact of proactive caching on an important metric for future generation networks, namely, energy efficiency (EE). We argue that, exploiting the spatial repartitions of users in addition to the correlation in their content popularity profiles, can result in considerable improvement of the achievable EE. In this paper, the optimization of EE is decoupled into two related subproblems. The first one addresses the issue of content popularity modeling. While most existing works assume similar popularity profiles for all users, we consider an alternative framework in which, users are clustered according to their popularity profiles. In order to showcase the utility of the proposed clustering, we use a statistical model selection criterion, namely, Akaike information criterion. Using stochastic geometry, we derive a closed-form expression of the achievable EE and we find the optimal active small cell density vector that maximizes it. The second subproblem investigates the impact of exploiting the spatial repartitions of users. After considering a snapshot of the network, we formulate a combinatorial problem that optimizes content placement in order to minimize the transmission power. Numerical results show that the clustering scheme considerably improves the cache hit probability and consequently the EE, compared with an unclustered approach. Simulations also show that the small base station allocation algorithm improves the energy efficiency and hit probability.

Chondrogenesis of human bone marrow mesenchymal stem cells in 3-dimensional, photocrosslinked hydrogel constructs: Effect of cell seeding density and material stiffness.

Engineering cartilage with physiologically relevant mechanical properties for point-of-care applications represents a major challenge in orthopedics, given the generally low mechanical strengths of traditional hydrogels used in cartilage tissue engineering. In this study, we characterized a new material that possesses high mechanical strength similar to native cartilage, and determined the optimal cell density and scaffold stiffness to achieve the most efficient chondrogenic response from seeded human bone marrow stem cells. Results show robust chondrogenesis and strongly suggest the potential of this material to be applied clinically for point-of-care repair of cartilage defects.