Assessment of tantalum nanoparticle-induced MC3T3-E1 proliferation and underlying mechanisms.

OBJECTIVE: In our previous study, tantalum nanoparticle (Ta-NPs) was demonstrated to promote osteoblast proliferation via autophagy induction, but the specific mechanism remains unclear. In the present study, we will explore the potential mechanism. METHODS: Ta-NPs was characterized by transmission electron microscopy, scanning electron microscopy, dynamic light scattering, and BET specific surface area test. MC3T3-E1 were treated with 0 or 20 µg/mL Ta-NPs with or without pretreatment with 10 µM LY294002, Triciribine, Rapamycin (PI3K/Akt/mTOR pathway inhibitors) for 1 h respectively. Western blotting was used to detect the expressions of pathway proteins and LC3B. CCK-8 assay was used to assess cell viability. Flow cytometry was used to detect apoptosis and cell cycle. RESULTS: After pretreatment with LY294002, Triciribine and Rapamycin, the p-Akt/Akt ratio of pathway protein in Triciribine and Rapamycin groups decreased (P < 0.05), while the autophagy protein LC3-II/LC3-I in the Rapamycin group was upregulated obviously (P < 0.001). In all pretreated groups, apoptosis was increased (LY294002 group was the most obvious), G1 phase cell cycle was arrested (Triciribine and Rapamycin groups were more obvious), and MC3T3-E1 cells were proliferated much more (P < 0.01, P < 0.001, P < 0.05). CONCLUSION: Pretreatment with Triciribine or Rapamycin has a greater effect on pathway protein Akt, cell cycle arrest, autophagy protein, and cell proliferation but with inconsistent magnitude, which may be inferred that the Akt/mTOR pathway, as well as its feedback loop, were more likely involved in these processes.

Quantitative assessment of NFκB transcription factor activity.

The Nuclear Factor Kappa B (NFκB) pathway is an important signalling pathway in the immune system. Single gene defects in the NFκB pathway are described in a number of immunodeficiency diseases. These conditions provide a unique opportunity to investigate the mechanisms of NFκB function and how genetic mutations that disrupt this function lead to human disease. Here we describe a robust method for quantifying small differences in the functional activity of the NFκB pathway. Peripheral blood mononuclear cells from healthy donors were stimulated over several days, with a combination of anti-IgM antibody and multimeric CD40 ligand. Nuclear proteins were thereafter extracted and tested for the ability of activated transcription factors, to bind known NFκB DNA binding motifs. Repeatability experiments showed that the DNA binding Activity can be quantified with an average inter and intra assay coefficient of variation of less than 10% (RelB and p52) and less than 15% (p50 and RelA). In healthy individuals there is a significant increase in the DNA binding activity of NFκB transcription factors in response to stimulation, although the magnitude of this response varies across individuals. The kinetics of the DNA binding activity also differs between the canonical and non-canonical transcription factors. P50 and RelA DNA binding activity responds within hours of stimulation, whilst RelB and p52 response was delayed to more than a day after stimulation. Activation of NFκB signalling in response to B cell specific stimulation, can be precisely measured to distinguish individuals with differences in the functional activity of this pathway. This test may prove to be an important biomarker for investigating the functional impact of genetic variants on NFκB signalling.

Generation of a cost-effective cell line for support of high-throughput isolation of primary human B cells and monoclonal neutralizing antibodies.

The isolation of human monoclonal antibodies (mAbs) arising from natural infection with human pathogens has proven to be a powerful technology, facilitating the understanding of the host response to infection at a molecular level. mAbs can reveal sites of vulnerability on pathogens and illuminate the biological function of the antigenic targets. Moreover, mAbs have the potential to be used directly for therapeutic applications such as passive delivery to prevent infection in susceptible target populations, and as treatment of established infection. The isolation of antigen-specific B cells from vaccine trials can also assist in deciphering whether the desired B cells are being targeted by a given vaccine. Several different processes have been developed to isolate mAbs, but all are generally labor-intensive and result in varying degrees of efficiency. Here, we describe the development of a cost-effective feeder cell line that stably expresses CD40-ligand, interleukin-2 and interleukin-21. Sorting of single B cells onto a layer of irradiated feeder cells sustained antibody production that permits functional screening of secreted antibodies in a manner that enables subsequent recovery of B cells for recombinant antibody cloning. As a proof of concept, we show that this approach can be used to isolate B cells that secrete antibodies that neutralize human papilloma virus (HPV) from participants of an HPV vaccine study.

In vivo safety assessment of a bio-inspired bone adhesive.

A new class of materials, bone adhesives, could revolutionise the treatment of highly fragmented fractures. We present the first biological safety investigation of a bio-inspired bone adhesive. The formulation was based upon a modified calcium phosphate cement that included the amino acid phosphoserine. This material has recently been described as substantially stronger than other bioresorbable calcium phosphate cements. Four adhesive groups with the active substance (phosphoserine) and two control groups without phosphoserine were selected for in vitro and in vivo biocompatibility testing. The test groups were subject for cell viability assay and subcutaneous implantation in rats that was followed by gene expression analysis and histology assessment after 6 and 12 weeks. All adhesive groups supported the same rate of cell proliferation compared to the α-TCP control and had viability between 45-64% when compared to cell control. There was no evidence of an increased immune response or ectopic bone formation in vivo. To conclude, this bio-inspired bone adhesive has been proven to be safe, in the present study, without any harmful effects on the surrounding soft tissue.

Computational Investigation of Drug Phototoxicity: Photosafety Assessment, Photo-Toxophore Identification, and Machine Learning.

One of the most appreciated capabilities of computational toxicology is to support the design of pharmaceuticals with reduced toxicological hazard. To this end, we have strengthened our drug photosafety assessments by applying novel computer models for the anticipation of in vitro phototoxicity and human photosensitization. These models are typically used in pharmaceutical discovery projects as part of the compound toxicity assessments and compound optimization methods. To ensure good data quality and aiming at models with global applicability we separately compiled and curated highly chemically diverse data sets from 3T3 NRU phototoxicity reports (450 compounds) and clinical photosensitization alerts (1419 compounds) which are provided as supplements. The latter data gives rise to a comprehensive list of explanatory fragments for visual guidance, termed phototoxophores, by application of a Bayesian statistics approach. To extend beyond the domain of well sampled fragments we applied machine learning techniques based on explanatory descriptors such as pharmacophoric fingerprints or, more important, accurate electronic energy descriptors. Electronic descriptors were extracted from quantum chemical computations at the density functional theory (DFT) level. Accurate UV/vis spectral absorption descriptors and pharmacophoric fingerprints turned out to be necessary for predictive computer models, which were both derived from Deep Neural Networks but also the simpler Random Decision Forests approach. Model accuracies of 83-85% could typically be reached for diverse test data sets and other company in-house data, while model sensitivity (the capability of correctly detecting toxicants) was even better, reaching 86%-90%. Importantly, a computer model-triggered response-map allowed for graphical/chemical interpretability also in the case of previously unknown phototoxophores. The photosafety models described here are currently applied in a prospective manner for the hazard identification, prioritization, and optimization of newly designed molecules.

Using Sacrificial Cell Spheroids for the Bioprinting of Perfusable 3D Tissue and Organ Constructs: A Computational Study.

A long-standing problem in tissue engineering is the biofabrication of perfusable tissue constructs that can be readily connected to the patient's vasculature. It was partially solved by three-dimensional (3D) printing of sacrificial material (e.g., hydrogel) strands: upon incorporation in another cell-laden hydrogel, the strands were removed, leaving behind perfusable channels. Their complexity, however, did not match that of the native vasculature. Here, we propose to use multicellular spheroids as a sacrificial material and investigate their potential benefits in the context of 3D bioprinting of cell aggregates and/or cell-laden hydrogels. Our study is based on computer simulations of postprinting cellular rearrangements. The computational model of the biological system is built on a cubic lattice, whereas its evolution is simulated using the Metropolis Monte Carlo algorithm. The simulations describe structural changes in three types of tissue constructs: a tube made of a single cell type, a tube made of two cell types, and a cell-laden hydrogel slab that incorporates a branching tube. In all three constructs, the lumen is obtained after the elimination of the sacrificial cell population. Our study suggests that sacrificial cell spheroids (sacrospheres) enable one to print tissue constructs outfitted with a finer and more complex network of channels than the ones obtained so far. Moreover, cellular interactions might give rise to a tissue microarchitecture that lies beyond the bioprinter's resolution. Although more expensive than inert materials, sacrificial cells have the potential to bring further progress towards the biofabrication of fully vascularized tissue substitutes.

Efficacy and safety assessment of two enterococci phages in an in vitro biofilm wound model.

Chronic wounds affect thousands of people worldwide, causing pain and discomfort to patients and represent significant economical burdens to health care systems. The treatment of chronic wounds is very difficult and complex, particularly when wounds are colonized by bacterial biofilms which are highly tolerant to antibiotics. Enterococcus faecium and Enterococcus faecalis are within the most frequent bacteria present in chronic wounds. Bacteriophages (phages) have been proposed as an efficient and alternative against antibiotic-resistant infections, as those found in chronic wounds. We have isolated and characterized two novel enterococci phages, the siphovirus vB_EfaS-Zip (Zip) and the podovirus vB_EfaP-Max (Max) to be applied during wound treatment. Both phages demonstrated lytic behavior against E. faecalis and E. faecium. Genome analysis of both phages suggests the absence of genes associated with lysogeny. A phage cocktail containing both phages was tested against biofilms formed in wound simulated conditions at a multiplicity of infection of 1.0 and a 2.5 log CFU.mL-1 reduction in the bacterial load after at 3 h of treatment was observed. Phages were also tested in epithelial cells colonized by these bacterial species and a 3 log CFU.mL-1 reduction was observed using both phages. The high efficacy of these new isolated phages against multi-species biofilms, their stability at different temperatures and pH ranges, short latent periods and non-cytotoxicity to epithelial cells suggest their therapeutic use to control infectious biofilms present in chronic wounds.

In vitro and In vivo toxicity assessment of phytofabricated ZnO nanoparticles showing bacteriostatic effect and larvicidal efficacy against Culex quinquefasciatus.

Murraya koenigii berry extract based zinc oxide nanoparticles (Mk-ZnO NPs) were synthesized by simple co-precipitation method and examined for bacteriostatic and larvicidal efficiency. Synthesized Mk-ZnO NPs were characterized by UV-Vis spectroscopy at 336 nm. X-Ray diffraction (XRD) showed crystalline nature as hexagonal. Fourier transform infrared spectroscopy (FTIR) spectrum exhibited strong peak at 3442.80 cm-1. Field emission scanning electron microscopy (FE-SEM) showed hexagonal shape of the particle. Transmission electron microscopy (TEM) measured 10-15 nm sized Mk-ZnO NPs. EDX peaks confirm 71.99% of zinc and 11.42% of oxide in Mk-ZnO NPs. Minimum inhibitory concentration (MIC) analysis reveals Mk-ZnO NPs inhibit growth of Gram positive (Staphylococcus aureus, Lysinibacillus fusiformis) and Gram negative (Proteus vulgaris, Providencia vermicola) bacteria at 40 and 50 µg mL-1 respectively. Live & dead assay confirms that Mk-ZnO NPs inhibits bacterial growth at 50 µg mL-1. Bacterial biofilm thickness significantly reduced by Mk-ZnO NPs at 50 µg mL-1. In vitro toxicity of Mk-ZnO NPs on RAW 264.7 macrophages determines 90-50% cell viability at concentrations of 10-100 µg mL-1. In vivo toxicity assay results indicate the lethal concentration of Artemia nauplii were LC50-78.73 µg mL-1 and LC90-130.03 µg mL-1. Larvicidal activity of Mk-ZnO NPs towards mosquito larvae of Culex quinquefasciatus were observed at LC50-2.1 µg mL-1 and LC90-12.1 µg mL-1. Finally the study discloses, potential bacteriostatic effect and mosquito larvae controlling capacity of Mk-ZnO NPs.

The "Race for the Surface" experimentally studied: In vitro assessment of Staphylococcus spp. adhesion and preosteoblastic cells integration to doped Ti-6Al-4V alloys.

OBJECTIVE: Implant-related infection is a devastating complication in orthopedic surgery. Aiming to minimize this problem, many material modifications have been developed. Here we report a study of a surface modification of Ti-6 Al-4 V alloy using a methodology that enables the study of interactions between bacteria and the material in the presence of eukaryotic cells. METHODS: We mixed different concentrations of collection or clinical strains of staphylococci isolated from implant-related infections with preosteoblastic cells using a previously published methodology, analyzing the minimal concentration of bacteria able to colonize the surface of the material through image analysis. Ti-6 Al-4 V alloy was modified by anodization to obtain two F-doped nanostructured surfaces that have been previously described to have antibacterial properties. RESULTS: Our results show similar bacterial adhesion results to nanoporous and nanotubular F-doped surfaces. The presence of preosteoblastic cells increases the adherence of all bacterial strains to both structures. No effect of the surface on eukaryotic cells adherence was detected. CONCLUSION: To our knowledge, this is the first time that anin vitro study emulating the race for the surface evaluates and compares the osseointegration and antibacterial properties between two nanostructured- modified titanium alloy surfaces. Clinical strains show different behavior from collection ones in bacterial adherence. The presence of cells increased bacterial adherence. NP and NT surface modifications didn´t show significant differences in bacterial adhesion and preosteoblastic cells integration.

DenTimol as A Dendrimeric Timolol Analogue for Glaucoma Therapy: Synthesis and Preliminary Efficacy and Safety Assessment.

In this work, we report the synthesis and characterization of DenTimol, a dendrimer-based polymeric timolol analog, as a glaucoma medication. A timolol precursor ( S)-4-[4-(oxiranylmethoxy)-1,2,5-thiadiazol-3-yl]morpholine (OTM) was reacted with the heterobifunctional amine polyethylene glycol acetic acid (amine-PEG-acetic acid, Mn = 2000 g/mol) via a ring opening reaction of an epoxide by an amine to form the OTM-PEG conjugate. OTM-PEG was then coupled to an ethylenediamine (EDA) core polyamidoamine (PAMAM) dendrimer G3 to generate DenTimol using the N-(3-(dimethylamino)propyl)- N'-ethylcarbodiimide hydrochloride (EDC)/ N-hydroxysuccinimide (NHS) coupling reaction. MALDI mass spectrometry, 1H NMR spectroscopy, and HPLC were applied to characterize the intermediate and final products. Ex vivo corneal permeation of DenTimol was assessed using the Franz diffusion cell system mounted with freshly extracted rabbit cornea. The cytotoxicity of DenTimol was assessed using the WST-1 assay. Our results show that DenTimol is nontoxic up to an OTM equivalent concentration of 100 µM. DenTimol is efficient at crossing the cornea. About 8% of the dendrimeric drug permeated through the cornea in 4 h. Its IOP-lowering effect was observed in normotensive adult Brown Norway male rats. Compared to the undosed eye, an IOP reduction by an average of 7.3 mmHg (∼30% reduction from baseline) was observed in the eye topically treated with DenTimol (2 × 5 µL, 0.5% w/v timolol equivalent) in less than 30 min. Daily dosing of DenTimol for a week did not cause any irritation or toxicity as confirmed by the histological examination of ocular tissues, including the cornea, ciliary body, and retina.

Analogs of the Scorpion Venom Peptide Stigmurin: Structural Assessment, Toxicity, and Increased Antimicrobial Activity.

Scorpion venom is a rich source of biologically active components and various peptides with high-potential therapeutic use that have been characterized for their antimicrobial and antiproliferative activities. Stigmurin is a peptide identified from the Tityus stigmurus venom gland with high antibacterial and antiproliferative activities and low toxicity. Amino acid substitutions in peptides without a disulfide bridge sequence have been made with the aim of reducing their toxicity and increasing their biological activities. The purpose of this study was to evaluate the structural conformation and structural stability, as well as antimicrobial, antiproliferative, and hemolytic activities of two peptide analogs to Stigmurin, denominated StigA6 and StigA16. In silico analysis revealed the α-helix structure for both analog peptides, which was confirmed by circular dichroism. Data showed that the net charge and hydrophobic moment of the analog peptides were higher than those for Stigmurin, which can explain the increase in antimicrobial activity presented by them. Both analog peptides exhibited activity on cancerous cells similar to the native peptide; however, they were less toxic when tested on the normal cell line. These results reveal a potential biotechnological application of the analog peptides StigA6 and StigA16 as prototypes to new therapeutic agents.

Matching material and cellular timescales maximizes cell spreading on viscoelastic substrates.

Recent evidence has shown that, in addition to rigidity, the viscous response of the extracellular matrix (ECM) significantly affects the behavior and function of cells. However, the mechanism behind such mechanosensitivity toward viscoelasticity remains unclear. In this study, we systematically examined the dynamics of motor clutches (i.e., focal adhesions) formed between the cell and a viscoelastic substrate using analytical methods and direct Monte Carlo simulation. Interestingly, we observe that, for low ECM rigidity, maximum cell spreading is achieved at an optimal level of viscosity in which the substrate relaxation time falls between the timescale for clutch binding and its characteristic binding lifetime. That is, viscosity serves to stiffen soft substrates on a timescale faster than the clutch off-rate, which enhances cell-ECM adhesion and cell spreading. On the other hand, for substrates that are stiff, our model predicts that viscosity will not influence cell spreading, since the bound clutches are saturated by the elevated stiffness. The model was tested and validated using experimental measurements on three different material systems and explained the different observed effects of viscosity on each substrate. By capturing the mechanism by which substrate viscoelasticity affects cell spreading across a wide range of material parameters, our analytical model provides a useful tool for designing biomaterials that optimize cellular adhesion and mechanosensing.

The novel piperazine-containing compound LQFM018: Necroptosis cell death mechanisms, dopamine D4 receptor binding and toxicological assessment.

Piperazine is a promising scaffold for drug development due to its broad spectrum of biological activities. Based on this, the new piperazine-containing compound LQFM018 (2) [ethyl 4-((1-(4-chlorophenyl)-1H-pyrazol-4-yl)methyl)piperazine-1-carboxylate] was synthetized and some biological activities investigated. In this work, we described its ability to bind aminergic receptors, antiproliferative effects as well as the LQFM018 (2)-triggered cell death mechanisms, in K562 leukemic cells, by flow cytometric analyses. Furthermore, acute oral systemic toxicity and potential myelotoxicity assessments of LQFM018 (2) were carried out. LQFM018 (2) was originally obtained by molecular simplification from LASSBio579 (1), an analogue compound of clozapine, with 33% of global yield. Binding profile assay to aminergic receptors showed that LQFM018 (2) has affinity for the dopamine D4 receptor (Ki = 0.26 µM). Moreover, it showed cytotoxicity in K562 cells, in a concentration and time-dependent manner; IC50 values obtained were 399, 242 and 119 µM for trypan blue assay and 427, 259 and 50 µM for MTT method at 24, 48 or 72 h, respectively. This compound (427 µM) also promoted increase in LDH release and cell cycle arrest in G2/M phase. Furthermore, it triggered necrotic morphologies in K562 cells associated with intense cell membrane rupture as confirmed by Annexin V/propidium iodide double-staining. LQFM018 (2) also triggered mitochondrial disturb through loss of ΔΨm associated with increase of ROS production. No significant accumulation of cytosolic cytochrome c was verified in treated cells. Furthermore, it was verified an increase of expression of TNF-R1 and mRNA levels of CYLD with no involviment in caspase-3 and -8 activation and NF-κB in K562 cells. LQFM018 (2) showed in vitro myelotoxicity potential, but it was orally well tolerated and classified as UN GHS category 5 (LD50 > 2000-5000 mg/Kg). Thus, LQFM018 (2) seems to have a non-selective action considering hematopoietic cells. In conclusion, it is suggested LQFM018 (2) promotes cell death in K562 cells via necroptotic signaling, probably with involvement of dopamine D4 receptor. These findings open new perspectives in cancer therapy by use of necroptosis inducing agents as a strategy of reverse cancer cell chemoresistance.

Mapping the Mouse Cell Atlas by Microwell-Seq.

Single-cell RNA sequencing (scRNA-seq) technologies are poised to reshape the current cell-type classification system. However, a transcriptome-based single-cell atlas has not been achieved for complex mammalian systems. Here, we developed Microwell-seq, a high-throughput and low-cost scRNA-seq platform using simple, inexpensive devices. Using Microwell-seq, we analyzed more than 400,000 single cells covering all of the major mouse organs and constructed a basic scheme for a mouse cell atlas (MCA). We reveal a single-cell hierarchy for many tissues that have not been well characterized previously. We built a web-based "single-cell MCA analysis" pipeline that accurately defines cell types based on single-cell digital expression. Our study demonstrates the wide applicability of the Microwell-seq technology and MCA resource.

Gpbar1 agonism promotes a Pgc-1α-dependent browning of white adipose tissue and energy expenditure and reverses diet-induced steatohepatitis in mice.

Gpbar1 is a bile acid activated receptor for secondary bile acids. Here we have investigated the mechanistic role of Gpbar1 in the regulation of adipose tissues functionality in a murine model of steatohepatitis (NASH). Feeding wild type and Gpbar1-/- mice with a high fat diet-fructose (HFD-F) lead to development of NASH-like features. Treating HFD-F mice with 6ß-ethyl-3a,7b-dihydroxy-5b-cholan-24-ol (BAR501), a selective Gpbar1-ligand, reversed insulin resistance and histologic features of NASH, increased the weight of epWAT and BAT functionality and promoted energy expenditure and the browning of epWAT as assessed by measuring expression of Ucp1 and Pgc-1α. The beneficial effects of BAR501 were lost in Gpbar1-/- mice. In vitro, BAR501 promoted the browning of 3T3-L1 cells a pre-adipocyte cell line and recruitment of CREB to the promoter of Pgc-1α. In conclusion, Gpbar1 agonism ameliorates liver histology in a rodent model of NASH and promotes the browning of white adipose tissue.

Pre-clinical efficacy assessment of Malva sylvestris on chronic skin inflammation.

In the search for improved quality of life, the treatment of skin diseases like psoriasis (hyperproliferative disease) is valid, since it causes huge social discomfort to the patient. In this context, earlier studies showed that Malva sylvestris L. has anti-inflammatory activity demonstrated by acute animal models of skin inflammation, becoming a promising target for further studies. The present investigation aimed to verify the effect of hydroalcoholic extract of M. sylvestris (HEMS) on the chronic inflammatory and hyperproliferative response caused by multiple applications of 12-O-tetradecanoylphorbol-13-acetate (TPA) on mouse ears. Topical application of HEMS reduced oedema, leukocyte migration (mono- and polymorphonuclear cells) and keratinocyte hyperproliferation, confirmed by histology and proliferating cell nuclear antigen (PCNA) immunostaining. It was found that the anti-inflammatory effects of the extract did not involve the glucocorticoid system, and its incubation with HaCaT keratinocytes caused low toxicity and reduced cell proliferation by apoptosis. Thus, HEMS proved to be effective as an anti-psoriatic therapy, with the ability to prevent keratinocyte hyperproliferation and with low toxicity by topical application.

Micropatterned Co-Cultures of Human Hepatocytes and Stromal Cells for the Assessment of Drug Clearance and Drug-Drug Interactions.

Drug clearance rates from the body can determine drug exposure that can affect efficacy or toxicity. Thus, accurate prediction of drug clearance during preclinical development can help guide dose selection in humans, but animal testing is not always predictive of human outcomes. Because hepatic drug metabolism is a rate-limiting step in the overall clearance of many drugs, primary human hepatocytes (PHHs) in suspension cultures or monolayers are used for drug clearance predictions. Yet, the precipitous decline in drug metabolism capacity can lead to significant underestimation of clearance rates, particularly for low turnover compounds that have desirable one-pill-a-day dosing regimens. In contrast, micropatterned co-cultures (MPCCs) of PHHs and fibroblasts display phenotypic stability for several weeks and can help mitigate the limitations of conventional cultures. Here, we describe protocols to create and use MPCCs for drug clearance predictions, and for modeling clinically-relevant drug-drug interactions that can affect drug clearance. © 2017 by John Wiley & Sons, Inc.

Assaying Cellular Viability Using the Neutral Red Uptake Assay.

The neutral red uptake assay is a cell viability assay that allows in vitro quantification of xenobiotic-induced cytotoxicity. The assay relies on the ability of living cells to incorporate and bind neutral red, a weak cationic dye, in lysosomes. As such, cytotoxicity is expressed as a concentration-dependent reduction of the uptake of neutral red after exposure to the xenobiotic under investigation. The neutral red uptake assay is mainly used for hazard assessment in in vitro toxicology applications. This method has also been introduced in regulatory recommendations as part of 3T3-NRU-phototoxicity-assay, which was regulatory accepted in all EU member states in 2000 and in the OECD member states in 2004 as a test guideline (TG 432). The present protocol describes the neutral red uptake assay using the human hepatoma cell line HepG2, which is often employed as an alternative in vitro model for human hepatocytes. As an example, the cytotoxicity of acetaminophen and acetyl salicylic acid is assessed.

Seq-Well: portable, low-cost RNA sequencing of single cells at high throughput.

Single-cell RNA-seq can precisely resolve cellular states, but applying this method to low-input samples is challenging. Here, we present Seq-Well, a portable, low-cost platform for massively parallel single-cell RNA-seq. Barcoded mRNA capture beads and single cells are sealed in an array of subnanoliter wells using a semipermeable membrane, enabling efficient cell lysis and transcript capture. We use Seq-Well to profile thousands of primary human macrophages exposed to Mycobacterium tuberculosis.

In vitro and in vivo assessment of chitosan modified urocanic acid as gene carrier.

Chitosan nanoparticles modified with 10 and 30% urocanic acid (CUA) via carbodiimide crosslinking were examined for an efficient gene delivery carrier. The CUA gene carrier was characterized by FTIR, TEM, SEM and the in vitro transfection efficiency CUA polyplex was tested with HeLa and 3T3 cells. The loading efficiency of CUA complexes with DNA was assessed at different N/P ratio of 1, 2, 4, 6, 8, and 10. The DNA loading efficiency was found be to >85% for chitosan, CUA10 and CUA30% and the DNA protection ability of CUA10 and CUA30 nanoparticle complexes was confirmed upon incubation with NheI and HindIII. The cell toxicity and cell viability results have supported the non-toxic nature of CUA10 and CUA30 nanoparticles. In vitro transfection efficiency of CUA10 and CUA30 polyplex was tested for EGFP expression in 3T3 and HeLa cells and a relative maximum % transfection of about 10% was confirmed by CUA10 and CUA30 after 96h transfection. The feasibility and biocompatibility of CUA gene carrier in transgenic chickens was also demonstrated. The in vitro transfection and in vivo embryonic viability studies further confirmed the CUA as promising gene carrier because of the improved biocompatibility and DNA protection ability.