Her2 overexpression is associated with an aggressive form of breast cancer and malignant transformation. We demonstrate in this work that nanolipoprotein particles (NLPs) synthesized in a cell-free manner can be used to transfer Her2 protein into the membrane of nonmalignant cells in 3D culture in a nontoxic and facile manner. With NLP-mediated Her2 protein delivery, we observed an increased probability of nonmalignant cells forming apolar nongrowth-arrested tumor-like structures. The NLP delivery system alone or Her2-NLPs plus the Her2 inhibitor trastuzumab showed no effect on the acinar organization rate, indicating that Her2 signaling is key to this process. Transcriptomics revealed essentially no effect of empty NLPs compared to untreated cells, whereas Her2-NLPs versus either untreated or empty-NLP-treated cells revealed upregulation of several factors associated with breast cancer. Pathway analysis also suggested that known nodes downstream of Her2 were activated in response to Her2-NLP treatment. This demonstrates that Her2 protein delivery with NLPs is sufficient for the malignant transformation of nonmalignant cells. Thus, this system offers a new model for studying cell surface receptor signaling without genomic modification or transformation techniques.

Nanolipoprotein particles (NLPs), composed of membrane scaffold proteins and lipids, have been used to support membrane proteins in a native-like bilayer environment for biochemical and structural studies. Traditionally, these NLPs have been prepared by the controlled removal of detergent from a detergent-solubilized protein-lipid mixture. Recently, an alternative method has been developed using direct cell-free expression of the membrane scaffold protein in the presence of preformed lipid vesicles, which spontaneously produces NLPs without the need for detergent at any stage. Using SANS/SAXS, we show here that NLPs produced by this cell-free expression method are structurally indistinguishable from those produced using detergent removal methodologies. This further supports the utility of single step cell-free methods for the production of lipid binding proteins. In addition, detailed structural information describing these NLPs can be obtained by fitting a capped core-shell cylinder type model to all SANS/SAXS data simultaneously.

In vivo delivery of large RNA molecules has significant implications for novel gene therapy, biologics delivery, and vaccine applications. We have developed cationic nanolipoprotein particles (NLPs) to enhance the complexation and delivery of large self-amplifying mRNAs (replicons) in vivo. NLPs are high-density lipoprotein (HDL) mimetics, comprised of a discoidal lipid bilayer stabilized by apolipoproteins that are readily functionalized to provide a versatile delivery platform. Herein, we systematically screened NLP assembly with a wide range of lipidic and apolipoprotein constituents, using biophysical metrics to identify lead candidates for in vivo RNA delivery. NLPs formulated with cationic lipids successfully complexed with RNA replicons encoding luciferase, provided measurable protection from RNase degradation, and promoted replicon in vivo expression. The NLP complexation of the replicon and in vivo transfection efficiency were further enhanced by modulating the type and percentage of cationic lipid, the ratio of cationic NLP to replicon, and by incorporating additive molecules.

Objective

Commonly-used youth anxiety measures may not comprehensively capture fears, worries, and experiences related to the pervasive impact of the COVID-19 pandemic. This study described the development of the Fear of Illness and Virus Evaluation (FIVE) scales and validated the caregiver-report version.

Method

After initial development, feedback was obtained from clinicians and researchers, who provided suggestions on item content/wording, reviewed edits, and provided support for the updated FIVE's content and face validity. Factor structure, measurement invariance, and psychometric properties were analyzed using data from a multi-site, longitudinal study of COVID-19-related effects on family functioning with 1599 caregivers from the United States and Canada.

Results

Confirmatory factor analyses indicated a hierarchical five-factor structure best fit the data, resulting in a 31-item measure with four lower-order subscales: (1) Fears about Contamination and Illness; (2) Fears about Social Distancing, (3) Avoidance Behaviors, and (4) Mitigation Behaviors, and a higher-order factor, (5) Total Fears, indicated by the two fear-related lower-order subscales. Measurement invariance by country of residence, child age, and child sex was found. All subscales demonstrated strong internal consistency, appropriate item-scale discrimination, and no floor or ceiling effects. The Total Fears subscale demonstrated appropriate test-retest reliability. Concurrent validity supported by strong correlation with a youth anxiety measure.

Discussion

The FIVE provides a psychometrically-sound measure of COVID-19-related fears and behaviors in youth in a caregiver-report format. Future research is necessary to evaluate correlates and longitudinal symptom patterns captured by the FIVE caregiver-report, as well as the validity and reliability of a youth self-report version of the FIVE.

Curcumin, a natural polyphenol derived from the spice turmeric (Curcuma longa), contains antioxidant, anti-inflammatory, and anti-cancer properties. However, curcumin bioavailability is inherently low due to poor water solubility and rapid metabolism. Here, we further refined for use curcumin incorporated into "biomimetic" nanolipoprotein particles (cNLPs) consisting of a phospholipid bilayer surrounded by apolipoprotein A1 and amphipathic polymer scaffolding moieties. Our cNLP formulation improves the water solubility of curcumin over 30-fold and produces nanoparticles with ~350 µg/mL total loading capacity for downstream in vitro and in vivo applications. We found that cNLPs were well tolerated in AG05965/MRC-5 human primary lung fibroblasts compared to cultures treated with curcumin solubilized in DMSO (curDMSO). Pre-treatment with cNLPs of quiescent G0/G1-phase MRC-5 cultures improved cell survival following ¹³⁷Cs gamma ray irradiations, although this finding was reversed in asynchronously cycling log-phase cell cultures. These findings may be useful for establishing cNLPs as a method to improve curcumin bioavailability for administration as a radioprotective and/or radiomitigative agent against ionizing radiation (IR) exposures in non-cycling cells or as a radiosensitizing agent for actively dividing cell populations, such as tumors.

131I-metaiodobenzylguanidine (131I-mIBG) is a targeted radiation therapy developed for the treatment of advanced neuroblastoma. We have previously shown that this patient cohort can be used to predict absorbed dose associated with early 131I exposure, 72 h after treatment. We now expand these studies to identify gene expression differences associated with 131I-mIBG exposure 15 days after treatment. Total RNA from peripheral blood lymphocytes was isolated from 288 whole blood samples representing 59 relapsed or refractory neuroblastoma patients before and after 131I-mIBG treatment. We found that several transcripts predictive of early exposure returned to baseline levels by day 15, however, selected transcripts did not return to baseline. At 72 h, all 17 selected pathway-specific transcripts were differentially expressed. Transcripts CDKN1A (P < 0.000001), FDXR (P < 0.000001), DDB2 (P < 0.000001), and BBC3 (P < 0.000001) showed the highest up-regulation at 72 h after 131I-mIBG exposure, with mean log2 fold changes of 2.55, 2.93, 1.86 and 1.85, respectively. At day 15 after 131I-mIBG, 11 of the 17 selected transcripts were differentially expressed, with XPC, STAT5B, PRKDC, MDM2, POLH, IGF1R, and SGK1 displaying significant up-regulation at 72 h and significant down-regulation at day 15. Interestingly, transcripts FDXR (P = 0.01), DDB2 (P = 0.03), BCL2 (P = 0.003), and SESN1 (P < 0.0003) maintained differential expression 15 days after 131I-mIBG treatment. These results suggest that transcript levels for DNA repair, apoptosis, and ionizing radiation-induced cellular stress are still changing by 15 days after 131I-mIBG treatment. Our studies showcase the use of biodosimetry gene expression panels as predictive biomarkers following early (72 h) and late (15 days) internal 131I exposure. Our findings also demonstrate the utility of our transcript panel to differentiate exposed from non-exposed individuals up to 15 days after exposure from internal 131I.

INTRODUCTION: Missions beyond low Earth orbit (LEO) will expose astronauts to ionizing radiation (IR) in the form of solar energetic particles (SEP) and galactic cosmic rays (GCR) including high atomic number and energy (HZE) nuclei. The gastrointestinal (GI) system is documented to be highly radiosensitive with even relatively low dose IR exposures capable of inducing mucosal lesions and disrupting epithelial barrier function. IR is also an established risk factor for colorectal cancer (CRC) with several studies examining long-term GI effects of SEP/GCR exposure using tumor-prone APC mouse models. Studies of acute short-term effects of modeled space radiation exposures in wildtype mouse models are more limited and necessary to better define charged particle-induced GI pathologies and test novel medical countermeasures (MCMs) to promote astronaut safety. METHODS: In this study, we performed ground-based studies where male and female C57BL/6J mice were exposed to γ-rays, 50 MeV protons, or 1 GeV/n Fe-56 ions at the NASA Space Radiation Laboratory (NSRL) with histology and immunohistochemistry endpoints measured in the first 24 h post-irradiation to define immediate SEP/GCR-induced GI alterations. RESULTS: Our data show that unlike matched γ-ray controls, acute exposures to protons and iron ions disrupts intestinal function and induces mucosal lesions, vascular congestion, epithelial barrier breakdown, and marked enlargement of mucosa-associated lymphoid tissue. We also measured kinetics of DNA double-strand break (DSB) repair using gamma-H2AX- specific antibodies and apoptosis via TUNEL labeling, noting the induction and disappearance of extranuclear cytoplasmic DNA marked by gamma-H2AX only in the charged particle-irradiated samples. We show that 18 h pre-treatment with curcumin-loaded nanolipoprotein particles (cNLPs) delivered via IV injection reduces DSB-associated foci levels and apoptosis and restore crypt villi lengths. DISCUSSION: These data improve our understanding of physiological alterations in the GI tract immediately following exposures to modeled space radiations and demonstrates effectiveness of a promising space radiation MCM.

During spaceflight, astronauts are exposed to multiple unique environmental factors, particularly microgravity and ionizing radiation, that can cause a range of harmful health consequences. Over the past decades, increasing evidence demonstrates that the space environment can induce changes in gene expression and RNA processing. Long non-coding RNA (lncRNA) represent an emerging area of focus in molecular biology as they modulate chromatin structure and function, the transcription of neighboring genes, and affect RNA splicing, stability, and translation. They have been implicated in cancer development and associated with diverse cardiovascular conditions and associated risk factors. However, their role on astronauts' health after spaceflight remains poorly understood. In this perspective article, we provide new insights into the potential role of exosomal lncRNA after spaceflight. We analyzed the transcriptional profile of exosomes isolated from peripheral blood plasma of three astronauts who flew on various Shuttle missions between 1998-2001 by RNA-sequencing. Computational analysis of the transcriptome of these exosomes identified 27 differentially expressed lncRNAs with a Log₂ fold change, with molecular, cellular, and clinical implications.

Chlamydia trachomatis is a sexually transmitted bacterium that infects over 130 million individuals worldwide annually. To implement a vaccine, we developed a cell-free co-translational system to express the Chlamydia muridarum major outer membrane protein (MOMP). This approach uses a nanolipoprotein particles (tNLP) made from ApoA1 protein, amphiphilic telodendrimer and lipids that self-assemble to form 10-25 nm discs. These tNLP provide a protein-encapsulated lipid support to solubilize and fold membrane proteins. The cell-free system co-translated MOMP and ApoA1 in the presence of telodendrimer mixed with lipids. The MOMP-tNLP complex was amenable to CpG and FSL-1 adjuvant addition. To investigate the ability of MOMP-tNLP+CpG+FSL-1 to induce protection against an intranasal (i.n.) C. muridarum challenge, female mice were vaccinated intramuscularly (i.m.) or i.n. and i.m. simultaneously 4 weeks apart. Following vaccination with MOMP-tNLP+CpG+FSL-1, mice mounted significant humoral and cell-mediated immune responses. Following the i.n. challenge, mice vaccinated with MOMP-tNLP+CpG+FSL-1 i.n. + i.m. group were protected as determined by the percentage change in body weight and by the number of C. muridarum inclusion forming units (IFU) recovered from the lungs. To our knowledge, this is the first time a MOMP-based vaccine formulated in tNLP has been shown to protect against C. muridarum.

Subunit vaccines offer advantages over more traditional inactivated or attenuated whole-cell-derived vaccines in safety, stability, and standard manufacturing. To achieve an effective protein-based subunit vaccine, the protein antigen often needs to adopt a native-like conformation. This is particularly important for pathogen-surface antigens that are membrane-bound proteins. Cell-free methods have been successfully used to produce correctly folded functional membrane protein through the co-translation of nanolipoprotein particles (NLPs), commonly known as nanodiscs. This strategy can be used to produce subunit vaccines consisting of membrane proteins in a lipid-bound environment. However, cell-free protein production is often limited to small scale (<1 mL). The amount of protein produced in small-scale production runs is usually sufficient for biochemical and biophysical studies. However, the cell-free process needs to be scaled up, optimized, and carefully tested to obtain enough protein for vaccine studies in animal models. Other processes involved in vaccine production, such as purification, adjuvant addition, and lyophilization, need to be optimized in parallel. This paper reports the development of a scaled-up protocol to express, purify, and formulate a membrane-bound protein subunit vaccine. Scaled-up cell-free reactions require optimization of plasmid concentrations and ratios when using multiple plasmid expression vectors, lipid selection, and adjuvant addition for high-level production of formulated nanolipoprotein particles. The method is demonstrated here with the expression of a chlamydial major outer membrane protein (MOMP) but may be widely applied to other membrane protein antigens. Antigen effectiveness can be evaluated in vivo through immunization studies to measure antibody production, as demonstrated here.