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Immunotherapies have become the standard treatment for melanoma. To further improve patient responses, combinations of immunotherapies and radiotherapy (RT) are being studied, since radiotherapies can potentially provide additional immune stimulation, in addition to direct antitumor effects. FLASH-RT is a novel, ultrahigh dose rate, radiation delivery approach, with the potential of at least equivalent tumor control efficacy and reduced damage to healthy tissue. However, the effects of combining FLASH-RT and immunotherapy have not been extensively studied in melanoma. Toll-like receptor (TLR) agonists, such as imiquimod (IMQ), are potent immunostimulatory agents, although their utility is limited due to poor solubility and systemic side effects. We therefore developed a novel combination therapy for melanoma consisting of IMQ delivered to the tumor via a radiopaque and radiation responsive hydrogel combined with FLASH-RT. We found that FLASH was able to effectively stimulate IMQ release from the hydrogel. In addition, we found that the combination of FLASH and released IMQ resulted in synergistic melanoma cell killing in vitro . The combination therapy reduced tumor growth compared to controls, enhanced survival, and resulted in remarkable enhancements in certain tumor cytokine levels. CT imaging allowed the hydrogel to be monitored in vivo . In addition, no adverse effects of the treatment were observed. Overall, this IMQ-gel and FLASH-RT combination may have potential as an improved treatment for melanoma and indicates that the interactions of FLASH-RT and TLR agonists merit further study., Competing Interests: The authors declare no competing financial interest.

Significant work has been done to develop nanoparticle contrast agents for computed tomography (CT), with a focus on identifying safer and more effective formulations. Contrast agents for spectral photon-counting computed tomography (SPCCT), a fast-growing imaging modality derived from conventional CT, have also recently gained considerable attention. In this study, we explored the synthesis of ultrasmall ytterbium nanoparticles (YbNP) and demonstrated that, potentially, they can be used as conventional CT and SPCCT contrast agents. These nanoparticles were tested in vitro for their cytotoxicity and contrast-generating properties with a variety of imaging systems. When scanned with conventional CT and SPCCT at clinically relevant energies, YbNP are significantly more attenuating than gold nanoparticles (AuNP), the contrast agents that have been most well studied. Furthermore, YbNP were studied for their potential application for labeling and monitoring hydrogels. The presence of the YbNP payload in hydrogels allowed for hydrogel localization and tracking in vivo . Additionally, the in vivo imaging results revealed that YbNP generate higher contrast when compared to AuNP used as a label. In summary, this is the first research study to examine ultrasmall YbNP as conventional CT and SPCCT contrast agents, as well as using them in a hydrogel system to make it radiopaque. These findings underscore YbNP's utility as CT and SPCCT contrast agents, as well as their potential for tracking hydrogels in vivo .

The use of nanoparticles in the biomedical field has gained much attention due to their applications in biomedical imaging, drug delivery, and therapeutics. Silver telluride nanoparticles (Ag 2 Te NPs) have been recently shown to be highly effective computed tomography (CT) and dual-energy mammography contrast agents with good stability and biocompatibility, as well as to have potential for many other biomedical purposes. Despite their numerous advantageous properties for diagnosis and treatment of disease, the clinical translation of Ag 2 Te NPs is dependent on achieving high levels of excretion, a limitation for many nanoparticle types. In this work, we have synthesized and characterized a library of Ag 2 Te NPs and identified conditions that led to 3 nm core size and were renally excretable. We found that these nanoparticles have good biocompatibility, strong X-ray contrast generation, and rapid renal clearance. Our CT data suggest that renal elimination of nanoparticles occurred within 2 h of administration. Moreover, biodistribution data indicate that 93% of the injected dose (%ID) has been excreted from the main organs in 24 h, 95% ID in 7 days, and 97% ID in 28 days with no signs of acute toxicity in the tissues studied under histological analysis. To our knowledge, this renal clearance is the best reported for Ag 2 Te NP, while being comparable to the highest renal clearance reported for any type of nanoparticle. Together, the results herein presented suggest the use of GSH-Ag 2 Te NPs as an X-ray contrast agent with the potential to be clinically translated in the future.

Silver chalcogenide (Ag 2 X, where X = S, Se, or Te) nanoparticles have been extensively investigated for their applications in electronics but have only recently been explored for biomedical applications. In the past 10 years, Ag 2 X, primarily silver sulfides at first, have become of great importance as quantum dots, since they not only possess excellent deep tissue imaging properties in the near-infrared regions I and II, but also have low toxicities. Their appealing properties have led to numerous recent developments of Ag 2 X for biomedical applications. Furthermore, Ag 2 X have been discovered in the past 2-3 years to be potent X-ray contrast agents, adding to the numerous biomedical uses of these nanoparticles. In this review, we discuss the most recent advances in silver chalcogenide nanoparticle use in areas such as bio-imaging, theranostics, and biosensors. Moreover, we examine the advances in synthetic approaches for these nanoparticles, which include aqueous and organic syntheses routes. Finally, we discuss the advantages and current limitations in the use of silver chalcogenides for different biomedical applications and their potential for advancement and expansions in use.

Silver sulfide nanoparticles (Ag2S NPs) have gained considerable interest in the biomedical field due to their photothermal ablation enhancement, near-infrared fluorescence properties, low toxicity levels, and multi-imaging capabilities. Silver telluride nanoparticles (Ag2Te NPs) have similar properties to Ag2S NPs, should also be stable due to an extremely low solubility product and should generate greater X-ray contrast since tellurium is significantly more attenuating than sulfur at diagnostic X-ray energies. Despite these attractive properties, Ag2Te NPs have only been studied in vivo once and at a low dose (2 mg Ag per kg). Herein, for the first time, Ag2Te NPs' properties and their application in the biomedical field were studied in vivo in the setting requiring the highest nanoparticle doses of all biomedical applications, i.e. X-ray imaging. Ag2Te NPs were shown to be stable, biocompatible (no acute toxicity observed in the cell lines studied or in vivo), and generated higher contrast, compared to controls, in the two X-ray imaging techniques studied: computed tomography (CT) and dual-energy mammography (DEM). In summary, this is the first study where Ag2Te NPs were explored in vivo at a high dose. Our findings suggest that Ag2Te NPs provide strong X-ray contrast while exhibiting excellent biocompatibility. These results highlight the potential use of Ag2Te NPs in the biomedical field and as X-ray contrast agents for breast cancer screening.

Human dental caries is an intractable biofilm-associated disease caused by microbial interactions and dietary sugars on the host's teeth. Commensal bacteria help control opportunistic pathogens via bioactive products such as hydrogen peroxide (H 2 O 2 ). However, high-sugar consumption disrupts homeostasis and promotes pathogen accumulation in acidic biofilms that cause tooth-decay. Here, we exploit the pathological (sugar-rich/acidic) conditions using a nanohybrid system to increase intrinsic H 2 O 2 production and trigger pH-dependent reactive oxygen species (ROS) generation for efficient biofilm virulence targeting. The nanohybrid contains glucose-oxidase that catalyzes glucose present in biofilms to increase intrinsic H 2 O 2 , which is converted by iron oxide nanoparticles with peroxidase-like activity into ROS in acidic pH. Notably, it selectively kills Streptococcus mutans (pathogen) without affecting Streptococcus oralis (commensal) via preferential pathogen-binding and in situ ROS generation. Furthermore, nanohybrid treatments potently reduced dental caries in a rodent model. Compared to chlorhexidine (positive-control), which disrupted oral microbiota diversity, the nanohybrid had significant higher efficacy without affecting soft-tissues and the oral-gastrointestinal microbiomes, while modulating dental health-associated microbial activity in vivo. The data reveal therapeutic precision of a bi-functional hybrid nanozyme against a biofilm-related disease in a controlled-manner activated by pathological conditions., (Copyright © 2020 The Authors. Published by Elsevier Ltd.. All rights reserved.)

X-ray imaging is the most widely used diagnostic imaging method in modern medicine and several advanced forms of this technology have recently emerged. Iodinated molecules and barium sulfate suspensions are clinically approved X-ray contrast agents and are widely used. However, these existing contrast agents provide limited information, are suboptimal for new X-ray imaging techniques and are developing safety concerns. Thus, over the past 15 years, there has been a rapid growth in the development of nanoparticles as X-ray contrast agents. Nanoparticles have several desirable features such as high contrast payloads, the potential for long circulation times, and tunable physicochemical properties. Nanoparticles have also been used in a range of biomedical applications such as disease treatment, targeted imaging, and cell tracking. In this review, we discuss the principles behind X-ray contrast generation and introduce new types of X-ray imaging modalities, as well as potential elements and chemical compositions that are suitable for novel contrast agent development. We focus on the progress in nanoparticle X-ray contrast agents developed to be renally clearable, long circulating, theranostic, targeted, or for cell tracking. We feature agents that are used in conjunction with the newly developed multi-energy computed tomography and mammographic imaging technologies. Finally, we offer perspectives on current limitations and emerging research topics as well as expectations for the future development of the field. This article is categorized under: Diagnostic Tools > in vivo Nanodiagnostics and Imaging Nanotechnology Approaches to Biology > Nanoscale Systems in Biology., (© 2020 Wiley Periodicals LLC.)

Efficient microbial conversion of lignocellulose into valuable products is often hindered by the presence of furfural, a dehydration product of pentoses in hemicellulose sugar syrups derived from woody biomass. For a cost-effective lignocellulose microbial conversion, robust biocatalysts are needed that can tolerate toxic inhibitors while maintaining optimal metabolic activities. A comprehensive plasmid-based library encoding native multidrug resistance (MDR) efflux pumps, porins, and select exporters from Escherichia coli was screened for furfural tolerance in an ethanologenic E. coli strain. Small multidrug resistance (SMR) pumps, such as SugE and MdtJI, as well as a lactate/glycolate:H + symporter, LldP, conferred furfural tolerance in liquid culture tests. Expression of the SMR pump potentially increased furfural efflux and cellular viability upon furfural assault, suggesting novel activities for SMR pumps as furfural efflux proteins. Furthermore, induced expression of mdtJI enhanced ethanol fermentative production of LY180 in the presence of furfural or 5-hydroxymethylfurfural, further demonstrating the applications of SMR pumps. This work describes an effective approach to identify useful efflux systems with desired activities for nonnative toxic chemicals and provides a platform to further enhance furfural efflux by protein engineering and mutagenesis. IMPORTANCE Lignocellulosic biomass, especially agricultural residues, represents an important potential feedstock for microbial production of renewable fuels and chemicals. During the deconstruction of hemicellulose by thermochemical processes, side products that inhibit cell growth and production, such as furan aldehydes, are generated, limiting cost-effective lignocellulose conversion. Here, we developed a new approach to increase cellular tolerance by expressing multidrug resistance (MDR) pumps with putative efflux activities for furan aldehydes. The developed plasmid library and screening methods may facilitate new discoveries of MDR pumps for diverse toxic chemicals important for microbial conversion., (Copyright © 2019 American Society for Microbiology.)

Microbial production of fuels and chemicals from lignocellulosic biomass provides promising biorenewable alternatives to the conventional petroleum-based products. However, heterogeneous sugar composition of lignocellulosic biomass hinders efficient microbial conversion due to carbon catabolite repression. The most abundant sugar monomers in lignocellulosic biomass materials are glucose and xylose. Although industrial Escherichia coli strains efficiently use glucose, their ability to use xylose is often repressed in the presence of glucose. Here we independently evolved three E. coli strains from the same ancestor to achieve high efficiency for xylose fermentation. Each evolved strain has a point mutation in a transcriptional activator for xylose catabolic operons, either CRP or XylR, and these mutations are demonstrated to enhance xylose fermentation by allelic replacements. Identified XylR variants (R121C and P363S) have a higher affinity to their DNA binding sites, leading to a xylose catabolic activation independent of catabolite repression control. Upon introducing these amino acid substitutions into the E. coli D-lactate producer TG114, 94% of a glucose-xylose mixture (50 g⋅L -1 each) was used in mineral salt media that led to a 50% increase in product titer after 96 h of fermentation. The two amino acid substitutions in XylR enhance xylose utilization and release glucose-induced repression in different E. coli hosts, including wild type, suggesting its potential wide application in industrial E. coli biocatalysts., Competing Interests: Conflict of interest statement: Results of this study are part of the nonprovisional patent application 15/176,866.

In order for vaccines to induce efficacious immune responses against mucosally transmitted pathogens, such as HIV-1, activated lymphocytes must efficiently migrate to and enter targeted mucosal sites. We have previously shown that all-trans retinoic acid (ATRA) can be used as a vaccine adjuvant to enhance mucosal CD8 + T cell responses during vaccination and improve protection against mucosal viral challenge. However, the ATRA formulation is incompatible with most recombinant vaccines, and the teratogenic potential of ATRA at high doses limits its usage in many clinical settings. We hypothesized that increasing in vivo production of retinoic acid (RA) during vaccination with a DNA vector expressing retinaldehyde dehydrogenase 2 (RALDH2), the rate-limiting enzyme in RA biosynthesis, could similarly provide enhanced programming of mucosal homing to T cell responses while avoiding teratogenic effects. Administration of a RALDH2- expressing plasmid during immunization with a HIVgag DNA vaccine resulted in increased systemic and mucosal CD8 + T cell numbers with an increase in both effector and central memory T cells. Moreover, mice that received RALDH2 plasmid during DNA vaccination were more resistant to intravaginal challenge with a recombinant vaccinia virus expressing the same HIVgag antigen (VACVgag). Thus, RALDH2 can be used as an alternative adjuvant to ATRA during DNA vaccination leading to an increase in both systemic and mucosal T cell immunity and better protection from viral infection at mucosal sites., Competing Interests: Conflict of interest: none, (Copyright © 2016 Elsevier Ltd. All rights reserved.)

The pediatric syndrome characterized by periodic fever, aphthous stomatitis, pharyngitis, and cervical adenitis (PFAPA) and adult Behçet's disease share some clinical manifestations and are both polygenic autoinflammatory disorders with interleukin-1β showing to play a pivotal role. However, the diagnosis is mostly clinical and we hypothesize that specific criteria may be addressed differently by different physicians. To determine the diagnostic variability, we compared the answers of 80 patients with a definite diagnosis of Behçet's disease (age 42.1 ± 13.7 years) obtained by separate telephone interviews conducted by a rheumatologist, a pediatrician, and an internist working largely in the field of autoinflammatory disorders. Questions were related to the age of symptom onset, the occurrence of recurrent fevers during childhood, and the association with oral aphthosis, cervical adenitis and/or pharyngitis, previous treatments, possible growth impairment, the time lapse between PFAPA-like symptoms and the onset of Behçet's disease, and the occurrence of Behçet-related manifestation during childhood. The rheumatologist identified 30 % of patients with Behçet's disease fulfilling PFAPA syndrome diagnostic criteria, compared to the pediatrician and the internist identifying 10 and 7.5 %, respectively. Most of the patients suffered from recurrent oral aphthosis in childhood also without fever (50, 39, and 48 % with each interviewer), yet no patient fulfilled the Behçet's disease diagnostic criteria. Our data suggest that physician awareness and expertise are central to the diagnosis of autoinflammatory disorders through an accurate collection of the medical history.

Osteosarcoma is the most common bone cancer in children and adolescents. Although 70% of patients with localized disease are cured with chemotherapy and surgical resection, patients with metastatic osteosarcoma are typically refractory to treatment. Numerous lines of evidence suggest that cytotoxic T lymphocytes (CTLs) limit the development of metastatic osteosarcoma. We have investigated the role of PD-1, an inhibitory TNFR family protein expressed on CTLs, in limiting the efficacy of immune-mediated control of metastatic osteosarcoma. We show that human metastatic, but not primary, osteosarcoma tumors express a ligand for PD-1 (PD-L1) and that tumor-infiltrating CTLs express PD-1, suggesting this pathway may limit CTLs control of metastatic osteosarcoma in patients. PD-L1 is also expressed on the K7M2 osteosarcoma tumor cell line that establishes metastases in mice, and PD-1 is expressed on tumor-infiltrating CTLs during disease progression. Blockade of PD-1/PD-L1 interactions dramatically improves the function of osteosarcoma-reactive CTLs in vitro and in vivo, and results in decreased tumor burden and increased survival in the K7M2 mouse model of metastatic osteosarcoma. Our results suggest that blockade of PD-1/PD-L1 interactions in patients with metastatic osteosarcoma should be pursued as a therapeutic strategy.

Production of fuels and chemicals through a fermentation-based manufacturing process that uses renewable feedstock such as lignocellulosic biomass is a desirable alternative to petrochemicals. Although it is still in its infancy, synthetic biology offers great potential to overcome the challenges associated with lignocellulose conversion. In this review, we will summarize the identification and optimization of synthetic biological parts used to enhance the utilization of lignocellulose-derived sugars and to increase the biocatalyst tolerance for lignocellulose-derived fermentation inhibitors. We will also discuss the ongoing efforts and future applications of synthetic integrated biological systems used to improve lignocellulose conversion.