Elimination of negative feedback in TLR signalling allows rapid and hypersensitive detection of microbial contaminants

The exquisite specificity of Toll-like receptors (TLRs) to sense microbial molecular signatures is used as a powerful tool to pinpoint microbial contaminants. Various cellular systems, from native human blood cells to transfected cell lines exploit TLRs as pyrogen detectors in biological preparations. However, slow cellular responses and limited sensitivity have hampered the replacement of animal-based tests such as the rabbit pyrogen test or lipopolysaccharide detection by Limulus amoebocyte lysate. Here, we report a novel human cell-based approach to boost detection of microbial contaminants by TLR-expressing cells. By genetic and pharmacologic elimination of negative control circuits, TLR-initiated cellular responses to bacterial molecular patterns were accelerated and significantly elevated. Combining depletion of protein phosphatase PP2ACA and pharmacological inhibition of PP1 in the optimized reporter cells further enhanced the sensitivity to allow detection of bacterial lipoprotein at 30 picogram/ml. Such next-generation cellular monitoring is poised to replace animal-based testing for microbial contaminants.

A unique amphiphilic triblock copolymer, nontoxic to human blood and potential supramolecular drug delivery system for dexamethasone

The drug delivery system (DDS) often causes toxicity, triggering undesired cellular injuries. Thus, developing supramolecules used as DDS with tunable self-assembly and nontoxic behavior is highly desired. To address this, we aimed to develop a tunable amphiphilic ABA-type triblock copolymer that is nontoxic to human blood cells but also capable of self-assembling, binding and releasing the clinically used drug dexamethasone. We synthesized an ABA-type amphiphilic triblock copolymer (P2L) by incorporating tetra(aniline) TANI as a hydrophobic and redox active segment along with monomethoxy end-capped polyethylene glycol (mPEG2k; Mw = 2000 g mol−1) as biocompatible, flexible and hydrophilic part. Cell cytotoxicity was measured in whole human blood in vitro and lung cancer cells. Polymer-drug interactions were investigated by UV–Vis spectroscopy and computational analysis. Our synthesized copolymer P2L exhibited tuned self-assembly behavior with and without external stimuli and showed no toxicity in human blood samples. Computational analysis showed that P2L can encapsulate the clinically used drug dexamethasone and that drug uptake or release can also be triggered under oxidation or low pH conditions. In conclusion, copolymer P2L is nontoxic to human blood cells with the potential to carry and release anticancer/anti-inflammatory drug dexamethasone. These findings may open up further investigations into implantable drug delivery systems/devices with precise drug administration and controlled release at specific locations.

Attenuation of Hg(II)-induced cellular and DNA damage in human blood cells by uric acid

Mercury (Hg) is a widespread environmental pollutant and toxicant which induces multiple organ damage in humans and animals. Hg toxicity is mediated by the induction of oxidative stress in target cells. We have used uric acid (UA), a potent antioxidant found in biological fluids, to protect human red blood cells (RBC) and lymphocytes against Hg-mediated cell, organelle and genotoxicity. RBC were incubated with HgCl2, an Hg(II) compound, either alone or in presence of UA. Incubation of RBC with only HgCl2 increased production of nitrogen and oxygen radical species, enhanced methemoglobin levels, heme degradation, free ferrous iron, oxidation of proteins and membrane lipids and reduced antioxidant capacity of cells. UA enhanced the antioxidant capacity of RBC and restored metabolic, plasma membrane-bound and antioxidant enzyme activities. Scanning electron microscopy showed that UA prevented HgCl2-mediated morphological changes in RBC. HgCl2 dissipated the mitochondrial membrane potential and increased lysosomal membrane damage in lymphocytes, but UA pre-treatment attenuated these effects. Genotoxicity analysis by comet assay showed that UA protected lymphocyte DNA from HgCl2-induced damage. Importantly, UA itself did not exhibit any deleterious effects in either RBC or lymphocytes. Thus, UA protects human blood cells from Hg(II)-mediated oxidative damage reducing the harmful effects of this extremely toxic metal. We suggest that UA performs a similar protective role in the plasma against heavy metal toxicity.

Analysis of Brain MRI Images Using Improved CornerNet Approach

The brain tumor is a deadly disease that is caused by the abnormal growth of brain cells, which affects the human blood cells and nerves. Timely and precise detection of brain tumors is an important task to avoid complex and painful treatment procedures, as it can assist doctors in surgical planning. Manual brain tumor detection is a time-consuming activity and highly dependent on the availability of area experts. Therefore, it is a need of the hour to design accurate automated systems for the detection and classification of various types of brain tumors. However, the exact localization and categorization of brain tumors is a challenging job due to extensive variations in their size, position, and structure. To deal with the challenges, we have presented a novel approach, namely, DenseNet-41-based CornerNet framework. The proposed solution comprises three steps. Initially, we develop annotations to locate the exact region of interest. In the second step, a custom CornerNet with DenseNet-41 as a base network is introduced to extract the deep features from the suspected samples. In the last step, the one-stage detector CornerNet is employed to locate and classify several brain tumors. To evaluate the proposed method, we have utilized two databases, namely, the Figshare and Brain MRI datasets, and attained an average accuracy of 98.8% and 98.5%, respectively. Both qualitative and quantitative analysis show that our approach is more proficient and consistent with detecting and classifying various types of brain tumors than other latest techniques.

Green synthesis of gold nanoparticles using Tribulus terrestris extract and antibacterial activity against Gram-negative bacteria

Aqueous extract of the plant Tribulus terrestris was used to reduce chloroauric acid for synthesizing gold nanoparticles (AuNPs). In this green synthesis, the reaction proceeded to give a red/purple color that was monitored by UV-vis spectrophotometry, where the formed AuNPs had an absorption band with max of 550 nm. In terms of the highest absorbance at 550 nm, reaction conditions were optimized at a temperature of 75°C, at pH 7 and using a reaction time of 4 h. The integrity of the synthesized AuNPs was confirmed and their physical properties were characterized by Fourier-transform infrared (FTIR) spectroscopy, X-ray diffraction (XRD) and scanning electron microscopy (SEM), which also showed evidence that plant metabolites contributed to capping or stabilization of the AuNPs. XRD spectra suggested a particle size of around 40 nm and SEM images revealed spherical and relatively uniform and disperse particles with a size of less than 0.1 m. In a broth microdilution assay, the AuNPs showed inhibitory effects against Gram-negative Escherichia coli, but not against Gram-positive Enterococcus faecalis. The AuNPs showed no hemagglutination activity or cytotoxic activity against human blood cells, which is important for them to be explored as therapeutic antibacterial agents.

Human Platelet Mitochondrial Function Reflects Systemic Mitochondrial Alterations: A Protocol for Application in Field Studies

Human blood cells may offer a minimally invasive strategy to study systemic alterations of mitochondrial function. Here we tested the reliability of a protocol designed to study mitochondrial respiratory control in human platelets (PLTs) in field studies, using high-resolution respirometry (HRR). Several factors may trigger PLT aggregation during the assay, altering the homogeneity of the cell suspension and distorting the number of cells added to the two chambers (A, B) of the Oroboros Oxygraph-2k (O2k). Thus, inter-chamber variability (∆ab) was calculated by normalizing oxygen consumption to chamber volume (JO2) or to a specific respiratory control state (flux control ratio, FCR) as a reliable parameter of experimental quality. The method’s reliability was tested by comparing the ∆ab of laboratory-performed experiments (LAB, N = 9) to those of an ultramarathon field study (three sampling time-points: before competition (PRE, N = 7), immediately after (POST, N = 10) and 24 h after competition (REC; N = 10)). Our results show that ∆ab JO2 changed PRE-POST, but also for LAB-POST and LAB-REC, while all ∆ab FCR remained unchanged. Thus, we conclude that our method is reliable for assessing PLT mitochondrial function in LAB and field studies and after systemic stress conditions.

Generation of Recombinant Primary Human B Lymphocytes Using Non-Viral Vectors

Although the development of gene delivery systems based on non-viral vectors is advancing, it remains a challenge to deliver plasmid DNA into human blood cells. The current “gold standard”, namely linear polyethyleneimine (l-PEI 25 kDa), in particular, is unable to produce transgene expression levels >5% in primary human B lymphocytes. Here, it is demonstrated that a well-defined 24-armed poly(2-dimethylamino) ethyl methacrylate (PDMAEMA, 755 kDa) nano-star is able to reproducibly elicit high transgene expression (40%) at sufficient residual viability (69%) in primary human B cells derived from tonsillar tissue. Moreover, our results indicate that the length of the mitogenic stimulation prior to transfection is an important parameter that must be established during the development of the transfection protocol. In our hands, four days of stimulation with rhCD40L post-thawing led to the best transfection results in terms of TE and cell survival. Most importantly, our data argue for an impact of the B cell subsets on the transfection outcomes, underlining that the complexity and heterogeneity of a given B cell population pre- and post-transfection is a critical parameter to consider in the multiparametric approach required for the implementation of the transfection protocol.