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Complete sequestration of central nervous system tissue and cerebrospinal fluid by the dural membrane is fundamental to maintaining homeostasis and proper organ function, making reconstruction of this layer an essential step during neurosurgery. Primary closure of the dura by suture repair is the current standard, despite facing technical, microenvironmental, and anatomic challenges. Here, we apply a mechanically tough hydrogel paired with a bioadhesive for intraoperative sealing of the dural membrane in rodent, porcine, and human central nervous system tissue. Tensile testing demonstrated that this dural tough adhesive (DTA) exhibited greater toughness with higher maximum stress and stretch compared with commercial sealants in aqueous environments. To evaluate the performance of DTA in the range of intracranial pressure typical of healthy and disease states, ex vivo burst pressure testing was conducted until failure after DTA or commercial sealant application on ex vivo porcine dura with a punch biopsy injury. In contrast to commercial sealants, DTA remained adhered to the porcine dura through increasing pressure up to 300 millimeters of mercury and achieved a greater maximum burst pressure. Feasibility of DTA to repair cerebrospinal fluid leak in a simulated surgical context was evaluated in postmortem human dural tissue. DTA supported effective sutureless repair of the porcine thecal sac in vivo. Biocompatibility and adhesion of DTA was maintained for up to 4 weeks in rodents after implantation. The findings suggest the potential of DTA to augment or perhaps even supplant suture repair and warrant further exploration.

SARS-CoV-2 receptor binding domains (RBDs) interact with both the ACE2 receptor and heparan sulfate on the surface of host cells to enhance SARS-CoV-2 infection. We show that suramin, a polysulfated synthetic drug, binds to the ACE2 receptor and heparan sulfate binding sites on the RBDs of wild-type, Delta, and Omicron variants. Specifically, heparan sulfate and suramin had enhanced preferential binding for Omicron RBD, and suramin is most potent against the live SARS-CoV-2 Omicron variant (B.1.1.529) when compared to wild type and Delta (B.1.617.2) variants in vitro. These results suggest that inhibition of live virus infection occurs through dual SARS-CoV-2 targets of S-protein binding and previously reported RNA-dependent RNA polymerase inhibition and offers the possibility for this and other polysulfated molecules to be used as potential therapeutic and prophylactic options against COVID-19., (© 2023. The Author(s).)

Objectives: This study aimed to test the effect of photodynamic therapy (PDT) on the inhibition and removal of biofilms containing multidrug-resistant Acinetobacter baumannii., Methods: Using multidrug-resistant A. baumannii strains, an antibiotic susceptibility test was performed using the Gram-negative identification card of the Vitek 2 system (bioMérieux Inc., France), as well as an analysis of resistance genes, the effects of treatment with a light-emitting diode (LED) array using Radachlorin (RADA-PHARMA Co., Ltd., Russia), and transmission and scanning electron microscopy to confirm the biofilm-inhibitory effect of PDT., Results: The antibiotic susceptibility test revealed multiple resistance to the antibiotics imipenem and meropenem in the carbapenem class. A class-D-type β-lactamase was found, and OXA-23 and OXA-51 were found in 100% of 15 A. baumannii strains. After PDT using Radachlorin, morphological observations revealed an abnormal structure due to the loss of the cell membrane and extensive morphological changes, including low intracellular visibility and small vacuoles attached to the cell membrane., Conclusion: PDT involving a combination of LED and Radachlorin significantly eliminated the biofilm of multidrug-resistant A. baumannii. Observations made using electron microscopy showed that PDT combining LED and Radachlorin was effective. Additional studies on the effective elimination of biofilms containing multidrug-resistant bacteria are necessary, and we hope that a treatment method superior to sterilization with antibiotics will be developed in the future.

Emerging viral diseases can substantially threaten national and global public health. Central to our ability to successfully tackle these diseases is the need to quickly detect the causative virus and neutralize it efficiently. Here we present the rational design of DNA nanostructures to inhibit dengue virus infection. The designer DNA nanostructure (DDN) can bind to complementary epitopes on antigens dispersed across the surface of a viral particle. Since these antigens are arranged in a defined geometric pattern that is unique to each virus, the structure of the DDN is designed to mirror the spatial arrangement of antigens on the viral particle, providing very high viral binding avidity. We describe how available structural data can be used to identify unique spatial patterns of antigens on the surface of a viral particle. We then present a procedure for synthesizing DDNs using a combination of in silico design principles, self-assembly, and characterization using gel electrophoresis, atomic force microscopy and surface plasmon resonance spectroscopy. Finally, we evaluate the efficacy of a DDN in inhibiting dengue virus infection via plaque-forming assays. We expect this protocol to take 2-3 d to complete virus antigen pattern identification from existing cryogenic electron microscopy data, ~2 weeks for DDN design, synthesis, and virus binding characterization, and ~2 weeks for DDN cytotoxicity and antiviral efficacy assays., (© 2022. The Author(s), under exclusive licence to Springer Nature Limited.)

In this study, the potential of pulverized waste tires (PWTs), either on their own or mixed with soil (well graded sand), to act as adsorptive fill materials was evaluated by conducting laboratory tests for accessing their adsorption and geotechnical properties. PWT (0, 5, 10, 15, 25, and 100 wt%) was mixed with soil to evaluate the removal of benzene, toluene, ethylbenzene, and xylene (BTEX) components and two heavy metal ions (Pb 2+ and Cu 2+ ). Adsorption batch tests were performed to determine the equilibrium sorption capacity of each mixture. Subsequently, compaction, direct shear, and consolidation tests were performed to establish their geotechnical properties. The results showed that BTEX had the strongest affinity based on the uptake capacity by the soil-PWT mixtures. The adsorption of BTEX increased for greater PWT content, with pure PWT having the highest adsorption capacity toward BTEX removal: uptake capacities for xylene, ethylbenzene, toluene, and benzene were 526, 377, 207 and 127 μg/g sorbent, respectively. Heavy metal removal was increased by increasing the amount of PWT up to 10 wt%, and then decreased beyond this ratio. Compacted soil-PWT mixtures comprising 5-25 wt% PWT have relatively low dry unit weight, low compressibility, adequate shear capacity for many load-bearing field applications, and satisfactory adsorption of organic/inorganic contaminants, such that they could also be used as adsorptive fill materials., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

DNA, when folded into nanostructures with a specific shape, is capable of spacing and arranging binding sites into a complex geometric pattern with nanometre precision. Here we demonstrate a designer DNA nanostructure that can act as a template to display multiple binding motifs with precise spatial pattern-recognition properties, and that this approach can confer exceptional sensing and potent viral inhibitory capabilities. A star-shaped DNA architecture, carrying five molecular beacon-like motifs, was constructed to display ten dengue envelope protein domain III (ED3)-targeting aptamers into a two-dimensional pattern precisely matching the spatial arrangement of ED3 clusters on the dengue (DENV) viral surface. The resulting multivalent interactions provide high DENV-binding avidity. We show that this structure is a potent viral inhibitor and that it can act as a sensor by including a fluorescent output to report binding. Our molecular-platform design strategy could be adapted to detect and combat other disease-causing pathogens by generating the requisite ligand patterns on customized DNA nanoarchitectures.

Numerous chemicals including environmental toxicants and drugs have not been fully evaluated for developmental neurotoxicity. A key gap exists in the ability to predict accurately and robustly in vivo outcomes based on in vitro assays. This is particularly the case for predicting the toxicity of chemicals on the developing human brain. A critical need for such in vitro assays is choice of a suitable model cell type. To that end, we have performed high-throughput in vitro assessment of proliferation and differentiation of human neural stem cells (hNSCs). Conventional in vitro assays typically use immunofluorescence staining to quantify changes in cell morphology and expression of neural cell-specific biomarkers, which is often time-consuming and subject to variable specificities of available antibodies. To alleviate these limitations, we developed a miniaturized, three-dimensional (3D) hNSC culture with ReNcell VM on microarray chip platforms and established a high-throughput promoter-reporter assay system using recombinant lentiviruses on hNSC spheroids to assess cell viability, self-renewal, and differentiation. Optimum cell viability and spheroid formation of 3D ReNcell VM culture were observed on a micropillar chip over a period of 9 days in a mixture of 0.75% (w/v) alginate and 1 mg/mL growth factor reduced (GFR) Matrigel with 25 mM CaCl 2 as a crosslinker for alginate. In addition, 3D ReNcell VM culture exhibited self-renewal and differentiation on the microarray chip platform, which was efficiently monitored by enhanced green fluorescent protein (EGFP) expression of four NSC-specific biomarkers including sex determining region Y-box 2 (SOX2), glial fibrillary acidic protein (GFAP), synapsin1, and myelin basic protein (MBP) with the promoter-reporter assay system., (Published by Elsevier Inc.)

The Institute of Medicine and US Food and Drug Administration (FDA) recognize that activating clinical trials in the United States is lengthy and inefficient. Downstream consequences include increased expense, suboptimal accrual, move of clinical trials overseas, and delayed availability of treatments for patients. An in-tandem processing initiative is here highlighted that transformed the activation of clinical trials (TACT), reduced the activation time by 70%, and offers a paradigm for enhanced translational readiness., (© 2017 American Society for Clinical Pharmacology and Therapeutics.)

To reduce attrition in drug development, it is crucial to consider the development and implementation of translational phenotypic assays as well as decipher diverse molecular mechanisms of action for new molecular entities. High-throughput fluorescence and confocal microscopes with advanced analysis software have simplified the simultaneous identification and quantification of various cellular processes through what is now referred to as highcontent screening (HCS). HCS permits automated identification of modifiers of accessible and biologically relevant targets and can thus be used to detect gene interactions or identify toxic pathways of drug candidates to improve drug discovery and development processes. In this review, we summarize several HCS-compatible, biochemical, and molecular biology-driven assays, including immunohistochemistry, RNAi, reporter gene assay, CRISPR-Cas9 system, and protein-protein interactions to assess a variety of cellular processes, including proliferation, morphological changes, protein expression, localization, post-translational modifications, and protein-protein interactions. These cell-based assay methods can be applied to not only 2D cell culture but also 3D cell culture systems in a high-throughput manner.

Unlabelled: A study was conducted to investigate the effectiveness of three air purification systems in reducing the exposure of children to air contaminants inside nine classrooms of three Southern California schools. Continuous and integrated measurements were conducted to monitor the indoor and outdoor concentrations of ultrafine particles (UFPs), fine and coarse particulate matter (PM2.5 and PM10 , respectively), black carbon (BC), and volatile organic compounds. An heating, ventilating, and air conditioning (HVAC)-based high-performance panel filter (HP-PF), a register-based air purifier (RS), and a stand-alone air cleaning system (SA) were tested alone and in different combinations for their ability to remove the monitored pollutants. The combination of a RS and a HP-PF was the most effective solution for lowering the indoor concentrations of BC, UFPs, and PM2.5 , with study average reductions between 87% and 96%. When using the HP-PF alone, reductions close to 90% were also achieved. In all cases, air quality conditions were improved substantially with respect to the corresponding baseline (preexisting) conditions. Data on the performance of the gas-absorbing media included in the RS and SA unit were inconclusive, and their effectiveness, lifetime, costs, and benefits must be further assessed before conclusions and recommendations can be made., Practical Implications: The installation of effective air filtration devices in classrooms may be an important mitigation measure to help reduce the exposure of school children to indoor pollutants of outdoor origin including ultrafine particles and diesel particulate matter, especially at schools located near highly trafficked freeways, refineries, and other important sources of air toxics., (Published 2012. This article is a US Government work and is in the public domain in the USA.)

We present the design, synthesis, spectroscopic properties, and biological evaluation of a single galactose-appended naphthalimide (1). Probe 1 is a multifunctional molecule that incorporates a thiol-specific cleavable disulfide bond, a masked phthalamide fluorophore, and a single galactose moiety as a hepatocyte-targeting unit. It constitutes a new type of targetable ligand for hepatic thiol imaging in living cells and animals. Confocal microscopic imaging experiments reveal that 1, but not the galactose-free control system 2, is preferentially taken up by HepG2 cells through galactose-targeted, ASGP-R-mediated endocytosis. Probe 1 displays a fluorescence emission feature at 540 nm that is induced by exposure to free endogenous thiols, most notably GSH. The liver-specificity of 1 was confirmed in vivo via use of a rat model. The potential utility of this probe in indicating pathogenic states and as a possible screening tool for agents that can manipulate oxidative stress was demonstrated in experiments wherein palmitate was used to induce lipotoxicity in HepG2 cells., (© 2011 American Chemical Society)

A rationally designed, coumarin-based fluorescent sensor imino-coumarin (IC) displays high selectivity for Cu(2+) over a variety of competing metal ions in aqueous solution with a significant fluorescence increase. DFT/TDDFT calculations support that the fluorescence 'turn-on' of IC originates from blocking the electron transfer of the nitrogen lone pair upon complexation with Cu(2+). IC was successfully applied to microscopic imaging for detection of Cu(2+) in LLC-MK2 cells (in vitro) and several living organs (in vivo).

Objectives/hypothesis: The aim of this study was to evaluate the antibacterial effects of photodynamic therapy (PDT) on common bacteria causing otitis media with effusion (OME)., Methods: An in vitro study was carried out using a hematoporphyrin derivative sensitizer (Photogem; Lemonosov Institute of Fine Chemical, Moscow, Russia) and a 632-nm diode laser on Haemophilus influenzae, Moraxella catarrhalis, and Streptococcus pneumoniae. The presence of colony-forming units of the bacteria was examined, the microscopic structures of the bacteria were examined by transmission electron microscopy (TEM), and flow cytometry of the bacteria was performed. An in vivo PDT study was performed using gerbils. S. pneumoniae or H. influenzae were injected into bullae. The Photogem was injected into the bullae 2 days later when OME developed, and transcanal irradiation with the 632-nm diode laser (90 J) was performed. Middle ear and bulla were washed with Dulbecco's phosphate buffered saline (DPBS) and the washed DPBS was cultured. The presence of bacterial colonies was examined., Results: The PDT was effective in killing all three kinds of bacteria. TEM showed damaged bacterial cell membranes and cytoplasmic structures, and the flow cytometry showed a lower number of viable bacteria in the PDT group compared to the control group. PDT was effective in killing S. pneumoniae in 87% of the infected bullae with OME, whereas it was effective in eradicating H. influenzae in 50% of the infected bullae with OME., Conclusions: The results of these studies demonstrated that PDT may be effective to treat otitis media. PDT may have clinical implications in the treatment of otitis media that is resistant to antibiotic therapy.

A novel coumarin-based fluorogenic probe bearing the 2-picolyl unit (1) was developed as a fluorescent chemosensor with high selectivity and suitable affinity in biological systems toward Cu(2+) over other cations tested. The fluorescence on-off mechanism was studied by femtosecond time-resolved fluorescence (TRF) upconversion technique and ab initio calculations. The receptor can be applied to the monitoring of Cu(2+) ion in aqueous solution with a pH span 4-10. To confirm the suitability of 1 for biological applications, we also employed it for the fluorescence detection of the changes of intracellular Cu(2+) in cultured cells. The results indicate that 1 should be useful for the fluorescence microscopic imaging and the study on the biological functions of Cu(2+).

The fluorescence of NABQ increases remarkably in the presence of Zn(2+) and is quenched by Hg(2+). As shown by confocal imaging, NABQ-Zn(2+) can penetrate cells, where the bound Zn(2+) is exchanged for Hg(2+). This results in the concomitant export of Hg(2+) from the cells, showing that NABQ can act as a Zn(2+) carrier and as a Hg(2+) extracting agent in living cells.

Background: Tuberculosis (TB) remains an important cause of morbidity and mortality throughout the world. The surge of TB has been accompanied by an increase in multi-drug-resistant tuberculosis (MDR-TB). In this study, we developed a denaturing HPLC (DHPLC) method for detecting rpoB gene mutation as a rifampin resistance based on sequence., Methods: In this study, we used 99 mycobacterial isolates grown in Ogawa media. At first, we used a PCR method that can amplify the 235 bp and 136 bp rpoB DNAs of Mycobacterium tuberculosis complex (MTB) and Non-tuberculous mycobacteria (NTM). And then, PCR-restriction fragment length polymorphism (RFLP) of rpoB DNA (342 bp), which comprises the Rif(T) region, was used for the differential identification of Mycobacteria. Finally, we detected these amplicons by DHPLC, compared to PCR-RFLP results, and performed sequencing., Results: Among 99 mycobacterial isolates, 80 (81%) were MTB and 19 (19%) were NTM. NTM were identified to 7 different species by DHPLC and PCR-RFLP. rpoB mutation was detected in 9 (11%) of the MTB specimens. These results were confirmed by using sequencing., Conclusions: DHPLC provided a rapid, simple, and automatable performance for detection of rifampin resistant Mycobacterium tuberculosis complex and would be helpful as a supplemental method in high-throughput clinical laboratories.

Objective: To assess the safety/tolerability and perform a preliminary efficacy evaluation of a multiple-dosing regimen of recombinant human vascular endothelial growth factor (VEGF165 or rhVEGF; telbermin) applied topically to chronic diabetic neuropathic foot ulcers., Method: Subjects with type 1 or 2 diabetes mellitus were randomised to receive either topical applied telbermin (72 microg/cm2) (n=29) or placebo (n=26) treatment to the foot ulcer surface in conjunction with standard ulcer care. Subjects received treatment every 48 hours (maximum three doses per week) for up to six weeks. Weekly 35mm photography, quantitative planimetry and physical examinations documented the ulcer appearance, surface area and stage. Safety endpoints included incidence of clinically significant hypotension, adverse events and ulcer infection. Exploratory efficacy endpoints included percentage reduction in total ulcer surface area, incidence of complete ulcer healing and time to complete ulcer healing., Results: Incidence of adverse events was comparable in the two treatment groups. None of the adverse events were attributed to study drug, and no hypotension was observed as a result of telbermin treatment. Occurrence of infected study ulcers appeared to be balanced between the treatment groups. Positive trends suggestive of potential signals of biological activity were observed for incidence of complete ulcer healing (41.4% telbermin versus 26.9% placebo at day 43 [P=0.39]) and time to complete ulcer healing (25th percentile of 32.5 days telbermin versus 43.0 days placebo [log-rank P=0.13])., Conclusion: The topical application of telbermin 72 microg/cm2 three times a week for up to six weeks appeared to be well tolerated. Further studies are required to characterise the safety/efficacy of telbermin more completely.

Manipulations capable of breaking host tolerance to induce tissue-specific T cell-mediated inflammation are of central importance to tumor immunotherapy and our understanding of autoimmunity. We demonstrate that androgen ablative therapy induces profuse T cell infiltration of benign glands and tumors in human prostates. T cell infiltration is readily apparent after 7-28 days of therapy and is comprised predominantly of a response by CD4+ T cells and comparatively fewer CD8+ T cells. Also, T cells within the treated prostate exhibit restricted TCR Vbeta gene usage, consistent with a local oligoclonal response. Recruitment/activation of antigen-presenting cells in treated prostate tissues may contribute to local T cell activation. The induction of T cell infiltration in prostate tissues treated with androgen ablation may have implications for the immunotherapeutic treatment of prostate cancer as well as other hormone-sensitive malignancies, including breast carcinoma.