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Intra-operative ultrasound has become a relevant imaging modality in neurosurgical procedures. While B-mode, with its intrinsic limitations, is still considered the primary ultrasound modality, intra-operative contrast-enhanced ultrasound (ioCEUS) has more recently emerged as a powerful tool in neurosurgery. Though still not used on a large scale, ioCEUS has proven its utility in defining tumor boundaries, identifying lesion vascular supply and mapping neurovascular architecture. Here we propose a step-by-step procedure for performing ioCEUS analysis of the brain, highlighting its neurosurgical applications. Moreover, we provide practical advice on the use of ultrasound contrast agents and review technical ultrasound parameters influencing ioCEUS imaging.

The World Federation for Ultrasound in Medicine and Biology (WFUMB) is addressing the issue of incidental findings (IFs) with a series of publications entitled "Incidental Imaging Findings-The Role of Medical Ultrasound." IFs are less commonly encountered in the spleen than in many other abdominal organs but remain a frequent dilemma in clinical practice. A histological diagnosis is rarely necessary for patient management. Many IFs, such as secondary spleens and splenic cysts, are harmless and do not require any further investigation. The diagnosis of many other focal splenic lesions is, however, often problematic. The following overview is intended to illustrate a variety of incidentally detected spleen pathologies such as size variants, shape variants, secondary spleens, focal splenic lesions and splenic calcifications. It should aid the examiner in establishing the diagnosis. Moreover, it should help the ultrasound practitioner decide which pathologies need no further investigation, those requiring interval imaging and cases in which immediate further diagnostic procedures are required.

The 2019 outbreak of coronavirus disease (COVID-19) in Wuhan (Hubei province of China) has given rise to a pandemic spread of virus, more than 240 million incidences and a death toll larger than 5 million people. COVID-19 has set off large efforts in research, therapy and patient care, as well as public and private debates in every imaginable form. A number of scientists used the publication platforms provided by the Federation of the European Biochemical Societies (FEBS) to present their research data, reviews, opinions and other contributions relating to COVID-19 and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Here, I highlight the recent COVID-19 papers which have been published and collected in a Virtual Issue in FEBS Letters, and discuss their implications towards understanding the molecular, biochemical and cellular mechanisms of SARS-CoV-2 infections, vaccine development and antiviral discovery strategies.

Rapid and accurate clinical assessment of hemostasis is essential for managing patients who undergo invasive procedures, experience hemorrhages, or receive antithrombotic therapies. Hemostasis encompasses an ensemble of interactions between the cellular and non-cellular blood components, but current devices assess only partial aspects of this complex process. In this work, we describe the development of a new approach to simultaneously evaluate coagulation function, platelet count or function, and hematocrit using a carbon nanotube-paper composite (CPC) capacitance sensor. CPC capacitance response to blood clotting at 1.3 MHz provided three sensing parameters with distinctive sensitivities towards multiple clotting elements. Whole blood-based hemostasis assessments were conducted to demonstrate the potential utility of the developed sensor for various hemostatic conditions, including pathological conditions, such as hemophilia and thrombocytopenia. Results showed good agreements when compared to a conventional thromboelastography. Overall, the presented CPC capacitance sensor is a promising new biomedical device for convenient non-contact whole-blood based comprehensive hemostasis evaluation.

Electrochemical paper-based analytical devices (ePADs) are useful analytical devices that serve as point-of-care testing (POCT) devices for various clinical biomarkers in view of their simplicity, portability, and low-cost format. However, multistep reagent manipulation usually restricts the performance of the device for end users. Herein, we developed a sequential ePAD for sequential immunosensing fluid delivery by integrating dual flow behaviors (fast-flow/delayed) within a single paper platform for the simultaneous detection of hepatitis B surface antigen (HBsAg) and hepatitis C core antigen (HCVcAg). In the present work, a fast-flow channel was used for the automated washing of unbound antigens, while a delayed channel was created to store a redox reagent for further electrochemical analysis with a single buffer loading (the analysis time can be completed within 500 s). Hence, the undesirable complex procedure of multi-step reagent manipulation is scarcely needed by the user. The detection limit of the proposed ePAD was as low as 18.2 pg mL−1 for HBsAg and 1.19 pg mL−1 for HCVcAg. In addition, this proposed ePAD was also proven to be effective in real clinical sera from patients to verify its biological applicability. The ePAD sensor shows high promise as an easy-to-use, portable, and extendable sensor for other multiplex biological assays.

The detection of trypsin and its inhibitor is significantly important for both clinical diagnosis and disease treatment. Herein, we demonstrate a hydrogel-assisted paper-based lateral flow sensor for the detection of trypsin and its inhibitor for the first time. The gelatin hydrogel is hydrolyzed based on the gel-to-sol transition in the presence of trypsin, which results in the release of the trapped water molecules in the gelatin hydrogel. By placing one end of a pH indicator strip onto the hydrolyzed gelatin hydrogel, water is flowing along the pH indicator strip. However, in the absence of trypsin, water cannot flow along the pH indicator strip as the water molecules are trapped in the gelatin hydrogel. The detection limit of the system reaches as low as 1.0 × 10−6 mg/mL, and it is also applied to the quantitative detection of trypsin in human serum. In addition, the detection of a clinical drug aprotinin that is an inhibitor of trypsin is also successfully achieved. Noteworthy, only the gelatin hydrogel, pH indicator strip, and PS substrate are needed to fulfill the detection of trypsin without the need of other chemicals or reagents. Overall, we develop a particularly simple, elegant, robust, competitive, high-throughput, and low-cost approach for the rapid and label-free detection of trypsin and its inhibitor, which is very promising in the development of commercial products for sensing, diagnostic, and pharmaceutical applications. Besides, the hydrogel-assisted paper-based lateral flow sensor can also be employed to detect other analytes of interest by use of different stimuli-responsive hydrogel systems.

Bacteria identification has predominantly been conducted using specific bioreceptors such as antibodies or nucleic acid sequences. This approach may be inappropriate for environmental monitoring when the user does not know the target bacterial species and for screening complex water samples with many unknown bacterial species. In this work, we investigate the supervised machine learning of the bacteria-particle aggregation pattern induced by the peptide sets identified from the biofilm-bacteria interface. Each peptide is covalently conjugated to polystyrene particles and loaded together with bacterial suspensions onto paper microfluidic chips. Each peptide interacts with bacterial species to a different extent, leading to varying sizes of particle aggregation. This aggregation changes the surface tension and viscosity of the liquid flowing through the paper pores, altering the flow velocity at different extents. A smartphone camera captures this flow velocity without being affected by ambient and environmental conditions, towards a low-cost, rapid, and field-ready assay. A collection of such flow velocity data generates a unique fingerprinting profile for each bacterial species. Support vector machine is utilized to classify the species. At optimized conditions, the training model can predict the species at 93.3% accuracy out of five bacteria: Escherichia coli, Staphylococcus aureus, Salmonella Typhimurium, Enterococcus faecium, and Pseudomonas aeruginosa. Flow rates are monitored for less than 6 s and the sample-to-answer assay time is less than 10 min. The demonstrated method can open a new way of analyzing complex biological and environmental samples in a biomimetic manner with machine learning classification.

We have developed an inexpensive, standardized paper chromogenic array (PCA) integrated with a machine learning approach to accurately identify single pathogens (Listeria monocytogenes, Salmonella Enteritidis, or Escherichia coli O157:H7) or multiple pathogens (either in multiple monocultures, or in a single cocktail culture), in the presence of background microflora on food. Cantaloupe, a commodity with significant volatile organic compound (VOC) emission and large diverse populations of background microflora, was used as the model food. The PCA was fabricated from a paper microarray via photolithography and paper microfluidics, into which 22 chromogenic dye spots were infused and to which three red/green/blue color-standard dots were taped. When exposed to VOCs emitted by pathogens of interest, dye spots exhibited distinguishable color changes and pattern shifts, which were automatically segmented and digitized into a ΔR/ΔG/ΔB database. We developed an advanced deep feedforward neural network with a learning rate scheduler, L2 regularization, and shortcut connections. After training on the ΔR/ΔG/ΔB database, the network demonstrated excellent performance in identifying pathogens in single monocultures, multiple monocultures, and in cocktail culture, and in distinguishing them from the background signal on cantaloupe, providing accuracy of up to 93% and 91% under ambient and refrigerated conditions, respectively. With its combination of speed, reliability, portability, and low cost, this nondestructive approach holds great potential to significantly advance culture-free pathogen detection and identification on food, and is readily extendable to other food commodities with complex microflora.

21 p.-14 fig. In Memoriam Prof. Kilian Muñiz (R.I.P.). Excellent person, great heart., The innate immunity TLR4/MD-2 system is a membrane receptor of paramount importance as therapeutic target. Its assembly, upon binding of Gram-negative bacteria lipopolysaccharide (LPS), and also dependent on the membrane composition, finally triggers the immune response cascade. We here combine ab-initio calculations, molecular docking, all-atom molecular dynamics simulations, and thermodynamics calculations to provide the most realistic and complete 3D models of the active full TLR4 complex embedded into a realistic membrane to date. Our studies reveal functional and structural insights into the transmembrane domain behavior in different membrane environments, the ectodomain bouncing movement, and the dimerization patterns of the intracellular TIR domain. Our work provides TLR4 models as reasonable 3D structures for the (TLR4/MD-2/LPS) 2 architecture accounting for the active (agonist) state of the TLR4, and pointing to a signal transduction mechanism across cell membrane. These observations unveil relevant molecular aspects involved in the TLR4 innate immune pathways and will promote the discovery of new TLR4 modulators., This work was financially supported by the Spanish Ministry for Science and Innovation (grants CTQ2014-57141-R, CTQ2017-88353-R, and PID2020-113588RB-I00; grants BES-2012-053653 for L.P.R., BES-2015-071588 for J.G.C. and PRE2018-086249 for A.M.R.), and the European Commission Marie Sklodowska-Curie actions (H2020-MSCA-ITN 642157 “TOLLerant”).

Dipeptidyl peptidase III (DPP3) is a ubiquitously expressed Zn‐dependent protease, which plays an important role in regulating endogenous peptide hormones, such as enkephalins or angiotensins. In previous biophysical studies, it could be shown that substrate binding is driven by a large entropic contribution due to the release of water molecules from the closing binding cleft. Here, the design, synthesis and biophysical characterization of peptidomimetic inhibitors is reported, using for the first time an hydroxyethylene transition‐state mimetic for a metalloprotease. Efficient routes for the synthesis of both stereoisomers of the pseudopeptide core were developed, which allowed the synthesis of peptidomimetic inhibitors mimicking the VVYPW‐motif of tynorphin. The best inhibitors inhibit DPP3 in the low μM range. Biophysical characterization by means of ITC measurement and X‐ray crystallography confirm the unusual entropy‐driven mode of binding. Stability assays demonstrated the desired stability of these inhibitors, which efficiently inhibited DPP3 in mouse brain homogenate., A transition‐state‐based inhibitor has been developed which efficiently inhibits the metalloprotease dipeptidylpeptidase 3 (DPP3) by releasing structured water molecules from the binding cleft.

Talin is a cell adhesion molecule that is indispensable for the development and function of multicellular organisms. Despite its central role for many cell biological processes, suitable methods to investigate the nanoscale organization of talin in its native environment are missing. Here, we overcome this limitation by combining single‐molecule resolved PAINT (points accumulation in nanoscale topography) imaging with the IRIS (image reconstruction by integrating exchangeable single‐molecule localization) approach, enabling the quantitative analysis of genetically unmodified talin molecules in cells. We demonstrate that a previously reported peptide can be utilized to specifically label the two major talin isoforms expressed in mammalian tissues with a localization precision of, The PIPKIγ‐peptide‐PAINT approach is introduced in this study to enable quantitative investigation of the cell adhesion protein talin with single‐molecule resolution in cells. The technique does not require genetic modifications and allows the analysis of endogenously expressed molecules. The first application reveals atypical adhesion structures in differentiating mesenchymal stem cells.

Intelligent inorganic nanoparticles were designed and produced for use in imaging and annihilating tumour cells by radio‐frequency (RF) hyperthermia. Nanoparticles synthesised to provide RF hyperthermia must have magnetite properties. For this purpose, magnetite nanoparticles were first synthesised by the coprecipitation method (10–15 NM). These superparamagnetic nanoparticles were then covered with gold ions without losing their magnetic properties. In this step, gold ions are reduced around the magnetite nanoparticles. Surface modification of the gold‐coated magnetic nanoparticles was performed in the next step. A self‐assembled monolayer was created using cysteamine (2‐aminoethanethiol) molecules, which have two different end groups (SH and NH2). These molecules react with the gold surface by SH groups. The NH2 groups give a positive charge to the nanoparticles. After that, a monoclonal antibody (Monoclonal Anti‐N‐CAM Clone NCAM‐OB11) was immobilised by the 1‐ethyl‐3‐(3‐dimethylaminopropyl)carbodiimide/N‐hydroxysuccinimide method. Then, the antenna RF system (144.00015 MHz) was created for RF hyperthermia. The antibody‐nanoparticle binding rate and cytotoxicity tests were followed by in vitro and in vivo experiments. As the main result, antibody‐bound gold‐coated magnetic nanoparticles were successfully connected to tumour cells. After RF hyperthermia, the tumour size decreased owing to apoptosis and necrosis of tumour cells.

A reconfigurable hearing aid is a generic type that can be used for various hearing disabilities without modifying the device hardware. This requires several trials to identify the best matching with the impaired person’s audiogram. The objective of this paper is to propose a novel reconfigurable hearing aid of low complexity with auto-adapting capability which makes it suitable for different types of hearing disabilities ranging from mild to severe intensities. The audio spectrum is divided into three regions and for each region, four different schemes are proposed. An automatic selection of the optimum scheme is proposed for all the regions based on hearing thresholds. Octave and fractional interpolation techniques are performed on a Parks-McClellan based prototype filter to generate the various sub-bands in the reconfigurable filter bank structure. The proposed structure uses only 18 coefficient multipliers which save up to 92% of multipliers when compared to other designs. The delay and matching errors are within the globally accepted limits. The hardware implementation executed on Xilinx Kintex-7 FPGA development board has reaffirmed that the structure is compact and power-efficient. The proposed auto-reconfigurable structure can be used for various types of hearing impairments and can avoid the manual interventions for the selection of schemes in audiogram matching. This in turn minimizes the time to establish the best match with the audiogram. Since the proposed structure has minimal complexity, cost-effective implementation of the device is also possible.

Abstract: During the last decades, there has been growing interest in using therapeutic messager RNA (mRNA) together with drug delivery systems. Naked, unformulated mRNA is, however, unable to cross the cell membrane and is susceptible to degradation. Here we use graphene quantum dots (GQDs) functionalized with polyethyleneimine (PEI) as a novel mRNA delivery system. Our results show that these modified GQDs can be used to deliver intact and functional mRNA to Huh‐7 hepatocarcinoma cells at low doses and, that the GQDs are not toxic, although cellular toxicity is a problem for these first‐generation modified particles. Functionalized GQDs represent a potentially interesting delivery system that is easy to manufacture, stable and effective., Message delivered! A novel graphene quantum dots based mRNA drug delivery platform was prepared. The results show that these modified GQDs can deliver intact and functional mRNA to Huh‐7 hepatocarcinoma cells at low doses. The transfection efficiency for FGQDs/mRNA complexes was 25 % with a formulation concentration of 4000 ng mRNA/mL, but comparable transfection efficiencies could be achieved at much lower doses if the ratio between carrier and cargo was optimized. This work describes the first steps towards a potentially interesting preparation method for stable and effective mRNA delivery systems.

Pannexin 1 (PANX1) channel is a critical ATP-releasing pathway that modulates tumor immunity, progression, and prognosis. However, the roles of PANX1 in different cancers remain unclear. We analyzed the expression of PANX1 in human pan-cancer in the Oncomine and GEPIA2.0 databases. The prognostic value of PANX1 expression was determined using Kaplan-Meier plotter and OncoLnc tools. The correlation between PANX1 and tumor-infiltrating immune cells was investigated using the TIMER 2.0. In addition, the relationship between PANX1 and immunomodulators was explored using TISIDB. Finally, gene set enrichment analysis (GSEA) was performed utilizing LinkedOmics. The results indicated that PANX1 was overexpressed in most cancers compared to normal tissues. The high expression of PANX1 was associated with poor prognosis in multiple tumors, especially in pancreatic adenocarcinoma (PAAD). In addition, PANX1 was correlated with a variety of immunomodulators, such as CD274, IL10, CD276, IL2RA, TAP1, and TAP2. PANX1 expression level was significantly related to infiltration of multiple immune cells in many cancers, including cancer associated fibroblast, macrophage, and neutrophil cells. Further analysis revealed that PANX1 was significantly associated with T cells CD8+ (rho = 0.524, P = 1.94e-13) and Myeloid dendritic cell (rho = 0.564, P = 9.45e-16). GSEA results showed that PANX1 was closely associated with leukocyte cell-cell adhesion, endoplasmic reticulum lumen, ECM-receptor interaction, and Focal adhesion pathways in PAAD. PANX1 expression was higher in pan-cancer samples than in normal tissues. The high expression of PANX1 was associated with poor outcome and immune infiltration in multiple cancers, especially in PAAD.

Nanoparticle deployment in drug delivery is contingent upon controlled drug loading and a desired release profile, with simultaneous biocompatibility and cellular targeting. Iron oxide nanoparticles (IONPs), being biocompatible, are used as drug carriers. However, to prevent aggregation of bare IONPs, they are coated with stabilizing agents. We hypothesize that, zwitterionic drugs like norfloxacin (NOR, a fluoroquinolone) can manifest dual functionality – nanoparticle stabilization and antibiotic activity, eliminating the need of a separate stabilizing agent. Since these drugs have different charges, depending on the surrounding pH, drug loading enhancement could be pH dependent. Hence, upon synthesizing IONPs, they were coated with NOR, either at pH 5 (predominantly as cationic, NOR+) or at pH 10 (predominantly as anionic, NOR−). We observed that, drug loading at pH 5 exceeded that at pH 10 by 4.7–5.7 times. Furthermore, only the former (pH 5 system) exhibited a desirable slower drug release profile, compared to the free drug. NOR-coated IONPs also enable a 22 times higher drug accumulation in macrophages, compared to identical extracellular concentrations of the free drug. Thus, lowering the drug coating pH to 5 imparts multiple benefits – improved IONP stability, enhanced drug coating, higher drug uptake in macrophages at reduced toxicity and slower drug release.

Super-resolution microscopy revealed that the plant nuclear lamina proteins NMCP1 and NMCP2 organize into filamentous networks with filament bundles., Plant genomes lack genes encoding intermediate filament proteins, including lamins; however, functional lamin analogues are presumed to exist in plants. Plant-specific coiled-coil proteins, that is, nuclear matrix constituent proteins (NMCPs), are the most likely candidates as the structural elements of the nuclear lamina because they exhibit a lamin-like domain arrangement. They are exclusively localized at the nuclear periphery and have functions that are analogous to those of lamins. However, their assembly into filamentous polymers has not yet been confirmed. In this study, we examined the higher-order structure of NMCP1 and NMCP2 in Apium graveolens cells by using stimulated emission depletion microscopy combined with immunofluorescence cell labelling. Our analyses revealed that NMCP1 and NMCP2 form intricate filamentous networks, which include thick segments consisting of filament bundles, forming a dense filamentous layer extending across the nuclear periphery. Furthermore, the outermost chromatin distribution was found to be in the nucleoplasm-facing region of the nuclear lamina. Recombinant Daucus carota NMCP1 with a His-tag produced in Escherichia coli refolded into dimers and self-assembled into filaments and filament bundles. These results suggest that NMCP1 and NMCP2 organize into the nuclear lamina by forming a filamentous network with filament bundles that localize at the nuclear periphery.

Graphic abstract The interaction of silver nanoparticles (AgNPs) with the immune system has not yet been sufficiently elucidated even though they belong to the most investigated and exploited group of nanomaterials. This study aimed to evaluate immunomodulatory effect of four different AgNPs on human peripheral blood mononuclear cells (hPBMCs). Fresh hPBMCs were exposed to the small sized (~ 10 nm) AgNPs immediately after isolation from the whole blood of healthy volunteers. The study considered coating-, time- and dose-dependent response of hPBMSc and stimulation of both early and intermediate activation of lymphocytes and monocytes using flow cytometry. The AgNPs differed in surface charge and were stabilised with polyvinyl pyrrolidone (PVP), poly-L-lysine (PLL), bis(2-ethylhexyl) sulfosuccinate sodium (AOT) or blood serum albumin (BSA). Response of hPBMCs to coating agents and ionic Ag form was evaluated to distinguish their effect from the AgNPs action as they may be released from the nanosurface. There was no significant effect of any tested AgNPs on relative count of hPBMCs subpopulations. The T-cells and monocytes were not activated after treatment with AgNPs, but the highest concentration of PLL- and BSA-AgNPs decreased density of CD4 and CD8 markers on T-helper and T-cytotoxic cells, respectively. The same AgNPs activated B- and NK-cells. Ionic Ag activated T-, B- and NK-cells, but at very higher concentration, whereas only PLL exhibited immunomodulatory activity. This study evidenced immunomodulatory activity of AgNPs that may be fine-tuned by the design of their surface functionalization. Supplementary Information The online version contains supplementary material available at 10.1007/s00775-021-01898-0.

The lymphatics maintain fluid balance by returning interstitial fluid to veins via contraction/compression of vessel segments with check valves. Disruption of lymphatic pumping can result in a condition called lymphedema with interstitial fluid accumulation. Lymphedema treatments are often ineffective, which is partially attributable to insufficient understanding of specialized lymphatic muscle lining the vessels. This muscle exhibits cardiac-like phasic contractions and smooth muscle-like tonic contractions to generate and regulate flow. To understand the relationship between this sub-cellular contractile machinery and organ-level pumping, we have developed a multiscale computational model of phasic and tonic contractions in lymphatic muscle and coupled it to a lymphangion pumping model. Our model uses the sliding filament model (Huxley in Prog Biophys Biophys Chem 7:255–318, 1957) and its adaptation for smooth muscle (Mijailovich in Biophys J 79(5):2667–2681, 2000). Multiple structural arrangements of contractile components and viscoelastic elements were trialed but only one provided physiologic results. We then coupled this model with our previous lumped parameter model of the lymphangion to relate results to experiments. We show that the model produces similar pressure, diameter, and flow tracings to experiments on rat mesenteric lymphatics. This model provides the first estimates of lymphatic muscle contraction energetics and the ability to assess the potential effects of sub-cellular level phenomena such as calcium oscillations on lymphangion outflow. The maximum efficiency value predicted (40%) is at the upper end of estimates for other muscle types. Spontaneous calcium oscillations during diastole were found to increase outflow up to approximately 50% in the range of frequencies and amplitudes tested. Supplementary Information The online version contains supplementary material available at 10.1007/s10237-021-01501-0.

Hepatocellular carcinoma (HCC) is the most common subtype of liver cancer. Many patients with hepatocellular carcinoma are diagnosed at an advanced stage because the early symptoms are not obvious. For advanced hepatocellular carcinoma, immunotherapy and targeted therapy seem to be a promising direction. Finding a new prognostic marker for hepatocellular carcinoma and exploring its role in the immune microenvironment is of great value. ABCC transporters have previously been associated with drug resistance in hepatocellular tumors, but the exact mechanism remains unclear. Here, we conducted a study on ABCC5 in HCC and found that the expression of ABCC5 was up-regulated in HCC and was associated with poor prognosis. Further exploration revealed that ABCC5 was associated with immune infiltration of hepatocellular carcinoma. Single-cell analysis revealed a potential relationship between ABCC5 and immune cell differentiation. Therefore, it is significant to continue to explore the role of ABCC5 in hepatocellular carcinoma.

Bladder cancer (Bca) is the second most common malignant tumor of the genitourinary system in Chinese male population with high potential of recurrence and progression. The overall prognosis has not been improved significantly for the past 30 years due to the lack of early theranostic technique. Currently the early theranostic technique for bladder cancer is mainly through the intravesical approach, but the clinical outcomes are poor due to the limited tumor-targeting efficiency. Therefore, the targeting peptides for bladder cancer provide possibility to advance intravesical theranostic technique. However, no systematic review has covered the wide use of the targeting peptides for intravesical theranostic techniques in bladder cancer. Herein, a summary of original researches introduces all aspects of the targeting peptides for bladder cancer, including the peptide screening, the targeting mechanism and its preclinical application.

The treatment outcome of a given fractionated radiotherapy scheme is affected by oxygen tension and cell cycle kinetics of the tumor population. Numerous experimental studies have supported the variability of radiosensitivity with cell cycle phase. Oxygen modulates the radiosensitivity through hypoxia-inducible factor (HIF) stabilization and oxygen fixation hypothesis (OFH) mechanism. In this study, an existing mathematical model describing cell cycle kinetics was modified to include the oxygen-dependent G1/S transition rate and radiation inactivation rate. The radiation inactivation rate used was derived from the linear-quadratic (LQ) model with dependence on oxygen enhancement ratio (OER), while the oxygen-dependent correction for the G1/S phase transition was obtained from numerically solving the ODE system of cyclin D-HIF dynamics at different oxygen tensions. The corresponding cell cycle phase fractions of aerated MCF-7 tumor population, and the resulting growth curve obtained from numerically solving the developed mathematical model were found to be comparable to experimental data. Two breast radiotherapy fractionation schemes were investigated using the mathematical model. Results show that hypoxia causes the tumor to be more predominated by the tumor subpopulation in the G1 phase and decrease the fractional contribution of the more radioresistant tumor cells in the S phase. However, the advantage provided by hypoxia in terms of cell cycle phase distribution is largely offset by the radioresistance developed through OFH. The delayed proliferation caused by severe hypoxia slightly improves the radiotherapy efficacy compared to that with mild hypoxia for a high overall treatment duration as demonstrated in the 40-Gy fractionation scheme.

There is growing empirical evidence that certain types of chemotherapy and phototherapy trigger immunogenic cell death and enhance the therapeutic anticancer efficacy of genetic immunotherapy. However, the main challenge is spatiotemporally co-delivering different drugs to maximize the therapeutic index of the combination therapy. In this study, a drug delivery system (HTCP-Au/shPD-L1/DOX) was designed with a polysaccharide-wrapped shell and a condensed DNA core. To construct the HTCP-Au vector, dodecyl side chains with a polyethylenimine (PEI) head were grafted onto hyaluronic acid, and AuNPs were grafted via Au-S bonds. During drug loading, PEI arrested shRNA plasmid DNA targeting programmed cell death ligand 1 (shPD-L1) via electrostatic interactions. It also formed a PEI-DNA core that was automatically enclosed when aliphatic hydrocarbons pulled the hyaluronic acid backbone. A hydrophobic interlayer consisting of dodecyl bridge chains between the PEI-DNA core and the hyaluronic acid shell was required to accommodate hydrophobic doxorubicin. In vitro and in vivo assays demonstrated that this core-shell drug delivery system could efficiently load and transport three different drugs and effectively target tumors. Moreover, it could activate the immune system, thereby providing promising therapeutic efficacy against tumor growth and metastasis., Graphical abstract HTCP-Au/shPD-L1/DOX was composed of gold nanoparticles (AuNPs)-modified hyaluronic acid shell, shPD-L1 plasmid core compressed by cationic polyethyleneimine and doxorubicin (DOX) encapsulated in hydrophobic layer. It could target to tumor cells by the CD44 receptor which is over-expressed in tumor microenvironment, followed by drug releasing responsively at 808 nm laser and the low pH in lysosome. Then the PD-L1 gene silencing induced by shPD-L1 and immunogenic cell death effect triggered by DOX and AuNPs will eliminate the tumor synergisticallyImage, graphical abstract

Most of Solute carrier family-2 (SLC2) members play a key role of facilitative transporters, and glucose transporter (GLUT) proteins encoded by SLC2s can transport hexoses or polyols. However, the function and mechanism of SLC2s remain unclear in human cancers. Here, we explored the dysregulated expression, prognostic values, epigenetic, genetic alterations, and biomolecular network of SLC2s in human cancers. According to the data from public-omicsrepository, SLC2A4 (GLUT4) was found to be significantly downregulated in most cancers, and higher messenger RNA (mRNA) expression of SLC2A4 significantly associated with better prognosis of breast cancer (BRCA) patients. Moreover, DNA hypermethylation in the promoter of SLC2A4 may affect the regulation of its mRNA expression, and SLC2A4 was strongly correlated with pathways, including the translocation of SLC2A4 to the plasma membrane and PID INSULIN PATHWAY. In conclusion, these results provide insight into SLC2s in human cancers and suggest that SLC2A4 could be an unfavorable prognostic biomarker for the survival of BRCA patients.

Osteocytes are vital for regulating bone remodeling by sensing the flow-induced mechanical stimuli applied to their cell processes. In this mechanosensing mechanism, tethering elements (TEs) connecting the osteocyte process with the canalicular wall potentially amplify the strain on the osteocyte processes. The ultrastructure of the osteocyte processes and canaliculi can be visualized at a nanometer scale using high-resolution imaging via ultra-high voltage electron microscopy (UHVEM). Moreover, the irregular shapes of the osteocyte processes and the canaliculi, including the TEs in the canalicular space, should considerably influence the mechanical stimuli applied to the osteocytes. This study aims to characterize the roles of the ultrastructure of osteocyte processes and canaliculi in the mechanism of osteocyte mechanosensing. Thus, we constructed a high-resolution image-based model of an osteocyte process and a canaliculus using UHVEM tomography and investigated the distribution and magnitude of flow-induced local strain on the osteocyte process by performing fluid–structure interaction simulation. The analysis results reveal that local strain concentration in the osteocyte process was induced by a small number of TEs with high tension, which were inclined depending on the irregular shapes of osteocyte processes and canaliculi. Therefore, this study could provide meaningful insights into the effect of ultrastructure of osteocyte processes and canaliculi on the osteocyte mechanosensing mechanism.

Thoracic endovascular aortic repair (TEVAR) has been accepted as the mainstream treatment for type B aortic dissection, but post-TEVAR biomechanical-related complications are still a major drawback. Unfortunately, the stent-graft (SG) configuration after implantation and biomechanical interactions between the SG and local aorta are usually unknown prior to a TEVAR procedure. The ability to obtain such information via personalised computational simulation would greatly assist clinicians in pre-surgical planning. In this study, a virtual SG deployment simulation framework was developed for the treatment for a complicated aortic dissection case. It incorporates patient-specific anatomical information based on pre-TEVAR CT angiographic images, details of the SG design and the mechanical properties of the stent wire, graft and dissected aorta. Hyperelastic material parameters for the aortic wall were determined based on uniaxial tensile testing performed on aortic tissue samples taken from type B aortic dissection patients. Pre-stress conditions of the aortic wall and the action of blood pressure were also accounted for. The simulated post-TEVAR configuration was compared with follow-up CT scans, demonstrating good agreement with mean deviations of 5.8% in local open area and 4.6 mm in stent strut position. Deployment of the SG increased the maximum principal stress by 24.30 kPa in the narrowed true lumen but reduced the stress by 31.38 kPa in the entry tear region where there was an aneurysmal expansion. Comparisons of simulation results with different levels of model complexity suggested that pre-stress of the aortic wall and blood pressure inside the SG should be included in order to accurately predict the deformation of the deployed SG.

We successfully synthesized {BiW8}, a 10‐nuclear heteroatom cluster modified {BiW8O30}. At 24 h post‐incubation, the IC50 values of {BiW8} against HUVEC, MG63, RD, Hep3B, HepG2, and MCF7 cells were 895.8, 127.3, 344.3, 455.0, 781.3, and 206.3 μM, respectively. The IC50 value of {BiW8} on the MG63 cells was more than 2‐fold lower than that of the other raw materials. Through morphological and functional features, we demonstrated pyroptosis as a newly identified mechanism of cell death induced by {BiW8}. {BiW8} increased 2‐fold reactive oxygen species (ROS) levels in MG63 cells at 24 h post‐incubation. Compared with 0 h, the glutathione (GSH) content decreased by 59, 65, 75, 94, and 97 % at 6, 12, 24, 36 and 48 h post‐incubation, respectively. Furthermore, multiple antitumor mechanisms of {BiW8} were identified via transcriptome analysis and chemical simulation, including activation of pyroptosis, suppression of GSH generation, depletion of GSH, and inhibition of DNA repair., We successfully synthesized {BiW8}, a 10‐nuclear heteroatom cluster modified {BiW8O30}. {BiW8} suppressed the proliferation, migration, and invasion of MG63 cells. We identified POM‐induced pyroptosis for the first time. Decreased GSH content and increased ROS levels were observed in {BiW8}‐treated cells. {BiW8} executed multiple antitumor mechanisms, such as activation of pyroptosis, regulation of oxidative stress, and inhibition of DNA repair.

α-Synuclein (αS) plays a key role in Parkinson's disease (PD). The αS nuclear role, its binding affinity and specificity to histones and dsDNA remains unknown. Here, we have measured the binding affinity (Kd ) between αS wild-type (wt) and PD-specific αS S129-phosphorylation mimicking (S129E) mutant with full-length and flexible tail truncated individual core histones (H2a, H2b, H3, and H4), linker histone (H1), and carried out αS-dsDNA interaction studies. This study revealed that αS(wt) interacts specifically with N-terminal flexible tails of histone H3, H4, and flexible tails of H1. The αS(S129E) mutant recognizes histones similar to αS(wt) but binds with higher affinity. Intriguingly, αS(S129E) showed a binding affinity for control proteins (bovine serum albumin and lysozyme), while no interaction was seen for αS(wt). Based on our above observation, we contemplate that the physio-chemical properties of αS with S129-phosphorylation has changed compared to αS(wt), resulting in interaction for other proteins, which is the basis for Lewy body formation. Besides, this study showed αS binding to dsDNA is weak and non-specific. Overall, αS specificity for histone binding suggests that its nuclear role is possibly driven through histone interaction. This article is protected by copyright. All rights reserved.

Radiation-induced lung injury (RILI) is a potential complication of thoracic radiotherapy that can result in pneumonitis or pulmonary fibrosis and is associated with significant morbidity and mortality. The pathobiology of RILI is complex and includes the generation of free radicals and DNA damage that precipitate oxidative stress, endothelial cell (EC), and epithelial cell injury and inflammation. While the cellular events involved continue to be elucidated and characterized, targeted and effective therapies for RILI remain elusive. Sphingolipids are known to mediate EC function including many of the cell signaling events associated with the elaboration of RILI. Sphingosine-1-phosphate (S1P) and S1P analogs enhance EC barrier function in vitro and have demonstrated significant protective effects in vivo in a variety of acute lung injury models including RILI. Similarly, statin drugs that have pleiotropic effects that include upregulation of EC S1P receptor 1 (S1PR1) have been found to be strongly protective in a small animal RILI model. Thus, targeting of EC sphingosine signaling, either directly or indirectly, to augment EC function and thereby attenuate EC permeability and inflammatory responses, represents a novel and promising therapeutic strategy for the prevention or treatment of RILI.

Estradiol regulates thyroid function, and chloride channels are involved in the regulation of thyroid function. However, little is known about the role of chloride channels in the regulation of thyroid functions by estrogen. In this study, the effects of estrogen on chloride channel activities in human thyroid Nthy-ori3-1 cells were therefore investigated using the whole cell patch-clamp technique. The results showed that the extracellular application of 17β-estradiol (E2) activated Cl− currents, which reversed at a potential close to Cl− equilibrium potential and showed remarkable outward rectification and an anion permeability of I− > Br− > Cl− > gluconate. The Cl− currents were inhibited by the chloride channel blockers, NPPB and tamoxifen. Quantitative Real-time PCR results demonstrated that ClC-3 expression was highest in ClC family member in Nthy-ori3-1 cells. The down-regulation of ClC-3 expression by ClC-3 siRNA inhibited E2-induced Cl− current. The Cl− current was blocked by the estrogen receptor antagonist, ICI 182780 (fulvestrant). Estrogen receptor alpha (ERα) and not estrogen receptor beta was the protein expressed in Nthy-ori3-1 cells, and the knockdown of ERα expression with ERα siRNA abolished E2-induced Cl− currents. Estradiol can promote the accumulation of ClC-3 in cell membrane. ERα and ClC-3 proteins were partially co-localized in the cell membrane of Nthy-ori3-1 cells after estrogen exposure. The results suggest that estrogen activates chloride channels via ERα in normal human thyroid cells, and ClC-3 proteins play a pivotal role in the activation of E2-induced Cl− current.

Melanoma is a type of cancer with a relatively poor prognosis. The development of immunotherapy for the treatment of patients with melanoma has drawn considerable attention in recent years. It is of great clinical significance to identify novel promising prognostic biomarkers and to explore their roles in the immune microenvironment. The solute carrier (SLC) superfamily is a group of transporters predominantly expressed on the cell membrane and are involved in substance transport. SLC16A1 is a member of the SLC family, participating in the transport of lactate, pyruvate, amino acids, ketone bodies, etc. The role of SLC16A1 in tumor immunity has been recently elucidated, while its role in melanoma remains unclear. In this study, bioinformatics analysis was performed to explore the role of SLC16A1 in melanoma. The results showed that high SLC16A1 expression was correlated with decreased overall survival in patients with melanoma. The genes co-expressed with SLC16A1 were significantly enriched in metabolic regulation, protein ubiquitination, and substance localization. Moreover, SLC16A1 was correlated with the infiltration of immune cells. In conclusion, SLC16A1 is a robust prognostic biomarker for melanoma and may be used as a novel target in immunotherapy.

Chondroitin sulfate-mediated albumin corona nanoparticles were readily prepared without any chemical reaction, and their active tumor targeting and therapeutic effects were examined. Negatively charged chondroitin sulfate (CS) and positively charged doxorubicin (DOX) self-assembled into nanoparticles (CS-DOX-NPs) via electrostatic interactions. Bovine serum albumin (BSA) was then adsorbed on the surface of CS-DOX-NPs to form albumin corona nanoparticles (BC-DOX-NPs) protected from endogenous proteins. Due to the dual effect of BSA and CS, BC-DOX-NPs interacted with the gp60, SPARC and CD44 receptors on tumor cells, facilitating their rapid and efficient transcytosis and improving their accumulation and uptake within tumor tissues. The simultaneous presence of BSA and CS also allowed BC-DOX-NPs to target CD44 efficiently, leading to greater cellular uptake and cytotoxicity against 4T1 cells than CS-DOX-NPs or free DOX. Intravenous injection of BC-DOX-NPs into orthotopic 4T1 tumor-bearing mice led to greater drug accumulation at the tumor site than with CS-DOX-NPs or free DOX, resulting in significant inhibition of tumor growth and lower exposure of major organs to the drug., Graphical abstract Albumin corona can enhance the anti-tumor effect of chondroitin sulfate-mediated nanoparticles through BSA-gp60-SPARC pathway, which helps nanoparticles pass through the vascular endothelial barrier faster and accumulate more in tumor tissues.Image, graphical abstract

Jayakumar Perumal,1 Hann Qian Lim,1 Amalina Binte Ebrahim Attia,1 Riazul Raziq,1 David Ian Leavesley,2 Zee Upton,2 US Dinish,1 Malini Olivo1 1Translational Biophotonics Laboratory, Institute of Bioengineering and Bioimaging, Agency for Science Technology and Research (A*STAR), Singapore; 2Skin Research Institute of Singapore, Agency for Science, Technology and Research (A*STAR), SingaporeCorrespondence: Malini OlivoTranslational Biophotonics Laboratory, Institute of Bioengineering and Bioimaging, Agency for Science Technology and Research (A*STAR), SingaporeEmail Malini_Olivo@sbic.a-star.edu.sgBackground: Wound management is stretching the limits of health systems globally, challenging clinicians to evaluate the effectiveness of their treatments and deliver appropriate care to their patients. Visual inspection and manual measurement of wound size are subjective, often inaccurate and inconsistent. Growth factors, such as pro-inflammatory cytokines and proteases, play important roles in cutaneous wound healing. However, little is known about the point-of-care monitoring of the changes in such markers during the healing process. Here, we explore the capability of surface-enhanced Raman spectroscopy (SERS) as a viable point-of-care platform to monitor the changes of these surrogate indicators of healing status in chronic wounds.Methods: We developed a biofunctionalized flexible, cost-effective, scalable and easy-to-fabricate plasmonic SERS substrate using cellulose fibre (CF), which is used for sensing of wound markers based on a modified immunoassay method.Results: We evaluated and selected the reliable silver nano-island thickness that will be sputtered onto the CF-based substrate forthe highest SERS enhancement. Using this biofunctionalized SERS substrate, we detected varying concentrations of MMP-9 (10– 5000 ng/mL) and TNF-α (5– 100 ng/mL) proteins to model the wound exudates. This SERS detection method demonstrates a linear response within biologically relevant concentrations, ranging from 10 to 500 ng/mL for MMP-9 and 5 to 25 ng/mL for TNF-α for these surrogate indicators.Conclusion: Our SERS sensing platform achieved detection limits in the μM to sub-nM range and displayed high sensitivity and selectivity. This could result in a cheap, point-of-care device that provides a non-invasive measure of cutaneous wound healing in real time. We envision that these flexible substrates after activation may be incorporated into wound dressings in future for routine monitoring of wound healing status.Keywords: SERS, cellulose fibres, membrane SERS, point-of-care, paper devices, wound biomarkers, flexible biosensor

Intracellular copper [Cu(I)] has been hypothesized to play role in the differentiation of the neurons. This necessitates understanding the role of Cu(I) not only in the neurons but also in the glia considering their anatomical proximity, contribution towards ion homeostasis, and neurodegeneration. In this study, we did a systematic investigation of the changes in the cellular copper homeostasis during neuronal and glial differentiation and the pathways triggered by them. Our study demonstrates increased mRNA for the plasma membrane copper transporter CTR1 leading to increased Cu(I) during the neuronal (PC-12) differentiation. ATP7A is retained in the trans-Golgi network (TGN) despite high Cu(I) demonstrating its utilization towards the neuronal differentiation. Intracellular copper triggers pathways essential for neurite generation and ERK1/2 activation during the neuronal differentiation. ERK1/2 activation also accompanies the differentiation of the foetal brain derived neuronal progenitor cells. The study demonstrates that ERK1/2 phosphorylation is essential for the viability of the neurons. In contrast, differentiated C-6 (glia) cells contain low intracellular copper and significant downregulation of the ERK1/2 phosphorylation demonstrating that ERK1/2 activation does not regulate the viability of the glia. But ATP7A shows vesicular localization despite low copper in the glia. In addition to the TGN, ATP7A localizes into RAB11 positive recycling endosomes in the glial neurites. Our study demonstrates the role of copper dependent ERK1/2 phosphorylation in the neuronal viability. Whereas glial differentiation largely involves sequestration of Cu(I) into the endosomes potentially (i) for ready release and (ii) rendering cytosolic copper unavailable for pathways like the ERK1/2 activation.

Introduction This study develops assays to quantify collagen subtypes and crosslinks with liquid chromatography-mass spectrometry (LC-MS) and characterizes the cartilages in the Yucatan minipig. Methods For collagen subtyping, liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis was performed on tissues digested in trypsin. For collagen crosslinks, LC-MS analysis was performed on hydrolysates. Samples were also examined histologically and with bottom-up proteomics. Ten cartilages (femoral condyle, femoral head, facet joint, floating rib, true rib, auricular cartilage, annulus fibrosus, 2 meniscus locations, and temporomandibular joint disc) were analyzed. Results The collagen subtyping assay quantified collagen types I and II. The collagen crosslinks assay quantified mature and immature crosslinks. Collagen subtyping revealed that collagen type I predominates in fibrocartilages and collagen type II in hyaline cartilages, as expected. Elastic cartilage and fibrocartilages had more mature collagen crosslink profiles than hyaline cartilages. Bottom-up proteomics revealed a spectrum of ratios between collagen types I and II, and quantified 42 proteins, including 24 collagen alpha-chains and 12 minor collagen types. Discussion The novel assays developed in this work are sensitive, inexpensive, and use a low operator time relative to other collagen analysis methods. Unlike the current collagen assays, these assays quantify collagen subtypes and crosslinks without an antibody-based approach or lengthy chromatography. They apply to any collagenous tissue, with broad applications in tissue characterization and tissue engineering. For example, a novel finding of this work was the presence of a large quantity of collagen type III in the white-white knee meniscus and a spectrum of hyaline and fibrous cartilages.

In this work, a series of numerical simulations have been performed to obtain the steady shapes of red blood cells under a shear force field in the capillary. Two possible classes of steady shapes, the axisymmetric parachute and the non-axisymmetric parachute, are found. If we assume that oxygen diffusion across the red cell membrane is mediated by membrane curvature, it is found that the non-axisymmetric parachute will be more favorable due to its special shape which enables it to have a larger portion of membrane patch capable of releasing oxygen to tissues.

The degeneration or loss of skeletal muscles, which can be caused by traumatic injury or disease, impacts most aspects of human activity. Among various techniques reported to regenerate skeletal muscle tissue, controlling the external cellular environment has been proven effective in guiding muscle differentiation. In this study, we report a nano-sized graphene oxide (sGO)-modified nanopillars on microgroove hybrid polymer array (NMPA) that effectively controls skeletal muscle cell differentiation. sGO-coated NMPA (sG-NMPA) were first fabricated by sequential laser interference lithography and microcontact printing methods. To compensate for the low adhesion property of polydimethylsiloxane (PDMS) used in this study, graphene oxide (GO), a proven cytophilic nanomaterial, was further modified. Among various sizes of GO, sGO (

We present an integrated analysis of urine and serum proteomics and clinical measurements in asymptomatic, mild/moderate, severe and convalescent cases of COVID-19. We identify the pattern of immune response during COVID-19 infection. The immune response is activated in asymptomatic infection, but is dysregulated in mild and severe COVID-19 patients. Our data suggest that the turning point depends on the function of myeloid cells and neutrophils. In addition, immune defects persist into the recovery stage, until 12 months after diagnosis. Moreover, disorders of cholesterol metabolism span the entire progression of the disease, starting from asymptomatic infection and lasting to recovery. Our data suggest that prolonged dysregulation of the immune response and cholesterol metabolism might be the pivotal causative agent of other potential sequelae. Our study provides a comprehensive understanding of COVID-19 immunopathogenesis, which is instructive for the development of early intervention strategies to ameliorate complex disease sequelae.

The addition of fruit to the diet is very important, and we can use nutraceutical and functional foods for this supplement. A little-known fruit is a red pitaya (Hylocereus undatus) that has been widely reported to have a high antioxidant potential. In this study, we analyzed the in vitro and in vivo antioxidant capacity of microencapsulated pitaya extract on the behavior, antioxidant, and nervous system of the nematode Caenorhabditis elegans. The worms were treated with fruit extract before and after juglone-induced stress, to determine the protective or curative effects of pitaya. We have been evaluated cholinergic, antioxidant, and behavioral biomarkers. We have evidenced that the pulp of pitaya contains antioxidant compounds and can serve as a potential nutraceutical product. In addition, the fruit extract was effective in preventing and/or reverse the stress-induced damages, even at high levels of chemical stress at all evaluated parameters. PRACTICAL APPLICATIONS: The potential applications and uses aimed by this research are related to the supplementation of foods given the antioxidant effect. Our data suggested that the effect of the pitaya fruit microencapsulated pulp extract was effective to prevent and repair the damage caused by oxidative stress. Besides the use of this microencapsulated extract can be an auxiliary in the treatment of diseases related to oxidative damage as well as promoting senescent aging. Another important use is the application of this extract as a dietary supplement to fortify the antioxidant system.

A bacteria-capturing platform is a critical function of accurate, quantitative, and sensitive identification of bacterial pathogens for potential usage in the detection of foodborne diseases. Despite the development of various nanostructures and their surface chemical modification strategies, relative to the principal physical contact propagation of bacterial infections, mechanically robust and nanostructured platforms that are available to capture bacteria remain a significant problem. Here, a three-dimensional (3D) hierarchically structured polyaniline nanoweb film is developed for the efficient capture of bacterial pathogens by hand-touching. This unique nanostructure ensures sufficient mechanical resistance when exposed to compression and shear forces and facilitates the 3D interfacial interactions between bacterial extracellular organelles and polyaniline surfaces. The bacterial pathogens (Escherichia coli O157:H7, Salmonella enteritidis, and Staphylococcus aureus) are efficiently captured through finger-touching, as verified by the polymerase chain reaction (PCR) analysis. Moreover, the real-time PCR results of finger-touched cells on a 3D nanoweb film show a highly sensitive detection of bacteria, which is similar to those of the real-time PCR using cultured cells without the capturing step without any interfering of fluorescence signal and structural deformation during thermal cycling. Graphic Abstract

The beta-trefoil protein architecture is characterized by three repeating "trefoil" motifs related by rotational symmetry and postulated to have evolved via gene duplication and fusion events. Despite this apparent structural symmetry, the primary and secondary structural elements typically exhibit pronounced asymmetric features. A survey of this family of proteins has revealed that among the most conserved symmetric structural elements is a ubiquitous buried solvent which participates in a bridging H-bond with three different beta-strands in each of the trefoil motifs. A computational analysis reported that these waters are likely associated with a substantial enthalpic contribution to overall stability. In this report, a Pro mutation is used to disrupt one of the water H-bond interactions to a main chain amide, and the effects upon stability and folding kinetics are determined. Combined with Ala mutations, the separate effects upon side chain truncation and H-bond deletion are analyzed in terms of stability and folding kinetics. The results show that these buried waters act to assemble a central folding nucleus, and are responsible for ~20% of the overall favorable enthalpy of folding.

Photothermal agents (PTAs) based on organic small-molecule dyes emerge as promising theranostic strategy in imaging and photothermal therapy (PTT). However, hydrophobicity, photodegradation, and low signal-to-noise ratio impede their transformation from bench to bedside. In this study, a novel supramolecular PTT formulation by a stimuli-responsive macrocyclic host is prepared to overcome these obstacles of organic small-molecule PTAs. Methods: Sulfonated azocalix[4]arene (SAC4A) was synthesized as a hypoxia-responsive macrocyclic host. Taking IR780 as an example, the supramolecular nanoformulation IR780@SAC4A was constructed by grinding method, and its solubility, photostability, and photothermal conversion were evaluated. The hypoxia tumor-selective imaging and supramolecular PTT of IR780@SAC4A were further evaluated in vitro and in vivo. Results: IR780@SAC4A is capable of enhancing the solubility, photostability, and photothermal conversion of IR780 significantly, which achieve this supramolecular formulation with good imaging-guided PTT efficacy in vitro and in vivo. Conclusions: This study demonstrates that the supramolecular PTT strategy is a promising cancer theranostic method. Moreover, this supramolecular approach is applicative to construct kinds of supramolecular PTAs, opening a general avenue for extending smart PTT formulations.

The cellular trafficking pathways that conduct zinc to its sites of binding in functional proteins remain largely unspecified. In this study, the hypothesis was investigated that nonspecific proteomic binding sites serve as intermediates in zinc trafficking. Proteome from pig kidney LLC-PK1 cells contains a large concentration of such sites, displaying an average conditional stability constant of 1010-11, that are dependent on sulfhydryl ligands to achieve high-affinity binding of zinc. As a result, the proteome competes effectively with induced metallothionein for Zn2+ upon exposure of cells to extracellular Zn2+ or during in vitro direct competition. The reaction of added Zn2+ bound to proteome with apo-carbonic anhydrase was examined as a potential model for intracellular zinc trafficking. The extent of this reaction was inversely dependent upon proteome concentration and under cellular conditions thought to be negligible. The rate of reaction was strictly first order in both Zn2+ and apo-carbonic anhydrase, and also considered to be insignificant in cells. Adding the low molecular weight fraction of cell supernatant to the proteome markedly enhanced the speed of this reaction, a phenomenon dependent on the presence of glutathione (GSH). In agreement, inclusion of GSH accelerated the reaction in a concentration-dependent manner. The implications of abundant high-affinity binding sites for Zn2+ within the proteome are considered in relation to their interaction with GSH in the efficient delivery of Zn2+ to functional binding sites and in the operation of fluorescent zinc sensors as a tool to observe zinc trafficking.

Significance: We introduce a model for better calibration of the trapping force using an equal but oppositely directed drag force acting on a trapped red blood cell (RBC). We demonstrate this approach by studying RBCs’ elastic properties from deidentified sickle cell anemia (SCA) and sickle cell trait (SCT) blood samples. Aim: A laser trapping (LT) force was formulated and analytically calculated in a cylindrical model. Using this trapping force relative percent difference, the maximum (longitudinal) and minimum (transverse) radius rate and stiffness were used to study the elasticity. Approach: The elastic property of SCA and SCT RBCs was analyzed using LT technique with computer controlled piezo-driven stage, in order to trap and stretch the RBCs. Results: For all parameters, the results show that the SCT RBC samples have higher elastic property than the SCA RBCs. The higher rigidity in the SCA cell may be due to the lipid composition of the membrane, which was affected by the cholesterol concentration. Conclusions: By developing a theoretical model for different trapping forces, we have also studied the elasticity of RBCs in SCT (with hemoglobin type HbAS) and in SCA (with hemoglobin type HbSS). The results for the quantities describing the elasticity of the cells consistently showed that the RBCs in the SCT display lower rigidity and higher deformability than the RBCs with SCA.

Oxidation of engineered nanomaterials during application in various industrial sectors can alter their toxicity. Oxidized nanomaterials also have widespread industrial and biomedical applications. In this study, we evaluated the cardiopulmonary hazard posed by these nanomaterials using oxidized carbon black (CB) nanoparticles (CBox) as a model particle. Particle surface chemistry was characterized by X-ray photo electron spectroscopy (XPS) and Fourier-transform infrared spectroscopy (FTIR). Colloidal characterization and in vitro dosimetry modeling (particle kinetics, fate and transport modeling) were performed. Lung inflammation was assessed following oropharyngeal aspiration of CB or oxidized CBox particles (20 μg per mouse) in C57BL/6J mice. Toxicity and functional assays were also performed on murine macrophage (RAW 264.7) and endothelial cell lines (C166) with and without pharmacological inhibitors. Oxidant generation was assessed by electron paramagnetic resonance spectroscopy (EPR) and via flow cytometry. Endothelial toxicity was evaluated by quantifying pro-inflammatory mRNA expression, monolayer permeability, and wound closure. XPS and FTIR spectra indicated surface modifications, the appearance of new functionalities, and greater oxidative potential (both acellular and in vitro) of CBox particles. Treatment with CBox demonstrated greater in vivo inflammatory potentials (lavage neutrophil counts, secreted cytokine, and lung tissue mRNA expression) and air-blood barrier disruption (lavage proteins). Oxidant-dependent pro-inflammatory signaling in macrophages led to the production of CXCR3 ligands (CXCL9,10,11). Conditioned medium from CBox-treated macrophages induced significant elevation in endothelial cell pro-inflammatory mRNA expression, enhanced monolayer permeability and impairment of scratch healing in CXCR3 dependent manner. In summary, this study mechanistically demonstrated an increased biological potency of CBox particles and established the role of macrophage-released chemical mediators in endothelial damage., Graphical abstract Image 1, Highlights • Exposure to ozone oxidizes carbon black nanoparticles. • Oxidized carbon black particles demonstrate enhanced oxidant production. • Oxidized carbon black particles exposed macrophages secreted CXCL9/10/11 activate CXCR3 on endothelial cells. • CXCR3 activation mediate increased endothelial monolayer permeability and migration impairment.

Copper (Cu) is a key transition metal that is involved in many important biological processes in a cell. Cu is also utilized by the immune system to hamper pathogen growth during infection. However, genome-level knowledge on the mechanisms involved in adaptation to Cu stress is limited. Here, we report the results of a genome-wide reverse genetic screen for Cu-responsive phenotypes in Escherichia coli. Our screen has identified novel genes involved in adaptation to Cu stress in E. coli. We detected multiple genes involved in the biosynthesis and uptake of enterobactin, a siderophore utilized for high-affinity TonB-dependent acquisition of iron (Fe), as critical players in survival under Cu intoxication. We demonstrated the specificity of Cu-dependent killing by chelation of Cu and by genetic complementation of tonB. Notably, TonB is involved in protection from Cu in both laboratory and uropathogenic strains of E. coli. Cu stress leads to increased expression of the genes involved in Fe uptake, indicating that Fur regulon is derepressed during exposure to excess Cu. Trace element analyses revealed that Fe homeostasis is dysregulated during Cu stress. Taken together, our data supports a model in which lack of enterobactin-dependent Fe uptake leads to exacerbation of Cu toxicity, and elucidates the intricate connection between the homeostasis of Cu and Fe in a bacterial cell., Graphical Abstract Graphical AbstractCopper Stress and Iron Homeostasis in E. coli.

Pancreatic beta-cells synthesize and secrete insulin maintaining an organism's energy homeostasis. In humans, beta-cell dysfunction and death contribute to the pathogenesis of type 2 diabetes (T2D). Although the causes of beta-cell dysfunction are complex, obesity-induced low-grade systemic inflammation plays a role. For example, obese individuals exhibiting increased levels of proinflammatory cytokines IL-6 and IL-1beta have a higher risk of beta-cell dysfunction and T2D. Interestingly, obesity-induced inflammation changes the expression of several cellular metal regulating genes, prompting this study to examine changes in the beta-cell metallome after exposure to proinflammatory-cytokines. Primary mouse beta-cells were exposed to a combination of IL-6 and IL-1beta for 48 hours, were chemically fixed and imaged by synchrotron X-ray fluorescent microscopy. Quantitative analysis showed a surprising 2.4-fold decrease in the mean total cellular content of zinc from 158 ± 57.7 femtograms (fg) to 65.7 ± 29.7 fg; calcium decreased from 216 ± 67.4 to 154.3 ± 68.7 fg (control vs. cytokines, respectively). The mean total cellular iron content slightly increased from 30.4 ± 12.2 to 47.2 ± 36.4 fg after cytokine treatment; a sub-population of cells (38%) exhibited larger increases of iron density. Changes in the subcellular distributions of zinc and calcium were observed after cytokine exposure. Beta-cells contained numerous iron puncta that accumulated still more iron after exposure to cytokines. These findings provide evidence that exposure to low levels of cytokines is sufficient to cause changes in the total cellular content and/or subcellular distribution of several metals known to be critical for normal beta-cell function.

Knee Osteoarthritis (ΚΟΑ) is a degenerative joint disease of the knee that results from the progressive loss of cartilage. Due to KOA’s multifactorial nature and the poor understanding of its pathophysiology, there is a need for reliable tools that will reduce diagnostic errors made by clinicians. The existence of public databases has facilitated the advent of advanced analytics in KOA research however the heterogeneity of the available data along with the observed high feature dimensionality make this diagnosis task difficult. The objective of the present study is to provide a robust Feature Selection (FS) methodology that could: (i) handle the multidimensional nature of the available datasets and (ii) alleviate the defectiveness of existing feature selection techniques towards the identification of important risk factors which contribute to KOA diagnosis. For this aim, we used multidisciplinary data obtained from the Osteoarthritis Initiative database for individuals without or with KOA. The proposed fuzzy ensemble feature selection methodology aggregates the results of several FS algorithms (filter, wrapper and embedded ones) based on fuzzy logic. The effectiveness of the proposed methodology was evaluated using an extensive experimental setup that involved multiple competing FS algorithms and several well-known ML models. A 73.55 % classification accuracy was achieved by the best performing model (Random Forest classifier) on a group of twenty-one selected risk factors. Explainability analysis was finally performed to quantify the impact of the selected features on the model’s output thus enhancing our understanding of the rationale behind the decision-making mechanism of the best model.

Pulmonary hypertension (PH) is one of the least understood and highly elusive cardiovascular conditions associated with elevated pulmonary arterial pressure. Although the disease mechanisms are not completely understood, evidence has accumulated from human and animal studies that irreversible processes of pulmonary arterial wall damage, compensated by stress-mediated growth, play critical roles in eliciting the mechanisms of disease progression. The aim of this study is to develop a thermodynamic modeling structure of the pulmonary artery to consider coupled plastic-degradation-growth irreversible processes to investigate the mechanical roles of the dissipative phenomena in the disease progression. The proposed model performs a model parameter study of plastic deformation and degradation processes coupled with dissipative growth subjected to elevated pulmonary arterial pressure and computationally generates in silico simulations of PH progression using the clinical features of PH, found in human morphological and mechanical data. The results show that considering plastic deformation can provide a much better fitting of the ex vivo inflation tests than a widely used pure hyperelastic model in higher pressure conditions. In addition, the parameter sensitivity study illustrates that arterial damage and growth cause the increased stiffness, and the full simulation (combining elastic-plastic-degradation-growth models) reveals a key postpathological recovery process of compensating vessel damage by vascular adaptation by reducing the rate of vessel dilation and mediating vascular wall stress. Finally, the simulation results of luminal enlargement, arterial thickening, and arterial stiffness for an anisotropic growth are found to be close to the values from the literature.