Lipid Nanoparticle Encapsulation Empowers Poly(I:C) to Activate Cytoplasmic RLRs and Thereby Increases Its Adjuvanticity.

Poly(I:C) is a synthetic analogue of dsRNA capable of activating both TLR3 and RLRs, such as MDA-5 and RIG-I, as pathogen recognition receptors. While poly(I:C) is known to provoke a robust type I IFN, type III IFN, and Th1 cytokine response, its therapeutic use as a vaccine adjuvant is limited due to its vulnerability to nucleases and poor uptake by immune cells. is encapsulated poly(I:C) into lipid nanoparticles (LNPs) containing an ionizable cationic lipid that can electrostatically interact with poly(I:C). LNP-formulated poly(I:C) triggered both lysosomal TLR3 and cytoplasmic RLRs, in vitro and in vivo, whereas poly(I:C) in an unformulated soluble form only triggered endosomal-localized TLR3. Administration of LNP-formulated poly(I:C) in mouse models led to efficient translocation to lymphoid tissue and concurrent innate immune activation following intramuscular (IM) administration, resulting in a significant increase in innate immune activation compared to unformulated soluble poly(I:C). When used as an adjuvant for recombinant full-length SARS-CoV-2 spike protein, LNP-formulated poly(I:C) elicited potent anti-spike antibody titers, surpassing those of unformulated soluble poly(I:C) by orders of magnitude and offered complete protection against a SARS-CoV-2 viral challenge in vivo, and serum from these mice are capable of significantly reducing viral infection in vitro.

Preparation and characterization of an aptamer-functionalized solid-phase microextraction fiber and its application in the selective monitoring of adenosine phosphates with liquid chromatography and tandem mass spectrometry.

An aptamer with adenosine triphosphate as a ligand was immobilized onto the surface of a porous-polymer-coated fiber to obtain an aptamer-functionalized porous-polymer-coated solid-phase microextraction fiber. The fiber was observed with a crosslinked and porous morphological surface structure. It shows specific selectivity to adenosine triphosphate with a selectivity coefficient of 22.1 compared to the scrambled oligonucleotide functionalized fiber, and the selectivity factors over other analogues and reference compounds were from 6.1 to 77.5. When the fiber-based solid-phase microextraction was coupled with liquid chromatography and tandem mass spectrometry, detection limits of 2.7, 29, and 34 µg/L were achieved for adenosine triphosphate, adenosine diphosphate, and adenosine monophosphate, respectively. The spiking recoveries of 77.6-91.9% were achieved for trace adenosine phosphates in human serum sample. Furthermore, the fibers showed high stability and good reusability and could be used over 50 times for the real serum sample pretreatment without remarkable performance reduction.

Use of pus metagenomic next-generation sequencing for efficient identification of pathogens in patients with sepsis.

The positive detection rate of blood metagenomic next-generation sequencing (mNGS) was still too low to meet clinical needs, while pus from the site of primary infection may be advantageous for identification of pathogens. To assess the value of mNGS using pus in patients with sepsis, thirty-five samples were collected. Pathogen identification and mixed infection diagnosis obtained by use of mNGS or cultivation methods were compared. Fifty-three aerobic or facultative anaerobes, 59 obligate anaerobes and 7 fungi were identified by the two methods. mNGS increased the accuracy rate of diagnosing aerobic or facultative anaerobic infections from 44.4% to 94.4%; mNGS also increased the sensitivity of diagnosing obligate anaerobic infections from 52.9% to 100.0%; however, mNGS did not show any advantage in terms of fungal infections. Culture and mNGS identified 1 and 24 patients with mixed infection, respectively. For obligate anaerobes, source of microorganisms was analyzed. The odontogenic bacteria all caused empyema (n = 7) or skin and soft tissue infections (n = 5), whereas the gut-derived microbes all caused intra-abdominal infections (n = 7). We also compared the clinical characteristics of non-obligate anaerobic and obligate anaerobic infection groups. The SOFA score [9.0 (7.5, 14.3) vs. 5.0 (3.0, 8.0), P = 0.005], procalcitonin value [4.7 (1.8, 39.9) vs. 2.50 (0.7, 8.0), P = 0.035], the proportion of septic shock (66.7% vs. 35.3%, P = 0.044) and acute liver injury (66.7% vs. 23.5%, P = 0.018) in the non-obligate anaerobic infection group were significantly higher than those in the obligate anaerobic infection group. In patients with sepsis caused by purulent infection, mNGS using pus from the primary lesion may yield more valuable microbiological information.

Lipid nanoparticle composition for adjuvant formulation modulates disease after influenza virus infection in quadrivalent influenza vaccine vaccinated mice.

There are considerable avenues through which currently licensed influenza vaccines could be optimized. We tested influenza vaccination in a mouse model with two adjuvants: Sendai virus-derived defective interfering (SDI) RNA, a RIG-I agonist; and an amphiphilic imidazoquinoline (IMDQ-PEG-Chol), a TLR7/8 agonist. The negatively charged SDI RNA was formulated into lipid nanoparticles (LNPs) facilitating direct delivery of SDI RNA to the cytosol, where RIG-I sensing induces inflammatory and type I interferon responses. We previously tested SDI RNA and IMDQ-PEG-Chol as standalone and combination adjuvants for influenza and SARS-CoV-2 vaccines. Here, we tested two different ionizable lipids, K-Ac7-Dsa and S-Ac7-Dog, for LNP formulations. The LNPs were incorporated with SDI RNA to determine its potential as a combination adjuvant with IMDQ-PEG-Chol by evaluating the host immune response to vaccination and infection in immunized BALB/c mice. Adjuvanticity of IMDQ-PEG-Chol with and without empty or SDI-loaded LNPs was validated with quadrivalent inactivated influenza vaccine (QIV), showing robust induction of antibody titers and T-cell responses. Depending on the adjuvant combination and LNP formulation, humoral and cellular vaccine responses could be tailored towards type 1 or type 2 host responses with specific cytokine profiles that correlated with the protective responses to viral infection. The extent of protection conferred by different vaccine/LNP/adjuvant combinations was tested by challenging mice with a vaccine-matched strain of influenza A virus A/Singapore/gp1908/2015 IVR-180 (H1N1). Groups that received either LNP formulated with SDI or IMDQ-PEG-Chol, or both, showed very low levels of viral replication in their lungs at 5 days post-infection (DPI). These studies provide evidence that the combination of vaccines with LNPs and/or adjuvants promote antigen-specific cellular responses that can contribute to protection upon infection. Interestingly, we observed differences in humoral and cellular responses to vaccination between different groups receiving K-Ac7-Dsa or S-Ac7-Dog lipids in LNP formulations. The differences were also reflected in inflammatory responses in lungs of vaccinated animals to infection, depending on LNP formulations. Therefore, this study suggests that the composition of the LNPs, particularly the ionizable lipid, plays an important role in inducing inflammatory responses in vivo, which is important for vaccine safety and to prevent adverse effects upon viral exposure.

Bioinspired Shear-Flow-Driven Layer-by-Layer in Situ Self-Assembly.

Layer-by-layer (LbL) assembly is widely applied as a coating technique for the nanoscale control of architecture and related properties. However, its translational applications are limited by the time-consuming and laborious nature of the process. Inspired by the blood-clotting process, herein, we develop a shear-flow-driven LbL (SF-LbL) self-assembly approach that accelerates the adsorption rate of macromolecules by mechanically configuring the polymer chain via a coil-stretch transition, which effectively simplifies and speeds the diffusion-controlled assembly process. The structural characteristics and surface homogeneity of the SF-LbL films are improved, and diverse three-dimensional structures can be achieved. Functional SF-LbL-assembled surfaces for corneal modification are successfully fabricated, and the surface of wounded rat corneas and skin can be directly decorated in situ with SF-LbL nanofilms due to the advantages of this approach. Furthermore, in situ SF-LbL self-assembly has promise as a simple approach for the wound dressing for interventional therapeutics in the clinic, as illustrated by the successful in situ fabrication of drug-free layers consisting of chitosan and heparin on the dorsal skin of diabetic mice to rescue defective wound healing. This bioinspired self-assembly approach is expected to provide a robust and versatile platform with which to explore the surface engineering of nanofilms in science, engineering, and medicine.

Novel liquid-liquid-solid microextraction method with molecularly imprinted polymer-coated stainless steel fiber for aqueous sample pretreatment.

A novel liquid-liquid-solid microextraction (LLSME) method was developed to overcome the well-known water-compatibility problem of molecularly imprinted polymers (MIPs). The enrichment factors with MIP-LLSME method were within 70-210 for trace chloroacetanilide herbicides under optimized extraction conditions. The method was characterized by simplicity, low solvent-consumption and high selectivity, and it was suitable for the one-step pretreatment of various aqueous samples such as river water and farm water.

Molecularly imprinted polymer coated on stainless steel fiber for solid-phase microextraction of chloroacetanilide herbicides in soybean and corn.

A molecularly imprinted polymer (MIP) with metolachlor as template was firstly coated on stainless steel fiber through chemical bonding strategy to solve the fragility problem of silica fiber substrate for solid-phase microextraction. The surface pretreatment of stainless steel fiber and the polymerization conditions were investigated systematically to enhance the preparation feasibility and MIP coating performance, and then a porous and highly cross-linked MIP coating with 14.8-microm thickness was obtained with over 200 times re-usability which was supported by non-fragile stainless steel fiber adoption. The MIP coating possessed specific selectivities to metolachlor, its metabolites and other chloroacetanilide herbicides with the factors of 1.1-4.6. Good extraction capacities of metolachlor, propisochlor and butachlor were found with MIP coating under quick adsorption and desorption kinetics, and the detection limits of 3.0, 9.6 and 38 microg L(-1) were achieved, respectively. Moreover, the MIP-coated stainless steel fiber was evaluated for trace metolachlor, propisochlor and butachlor extraction in the spiked soybean and corn samples, and the enrichment factors of 54-60, 27-31 and 15-20 were obtained, respectively.