Novel sensor-integrated proteome on chip (SPOC) platform with thousands of folded proteins on a 1.5 sq-cm biosensor chip to enable high-throughput real-time label-free screening for kinetic analysis.

An automated proteomic platform for producing and screening an array of functional proteins on biosensor surfaces was developed to address the challenges of measuring proteomic interaction kinetics in high throughput (HTP). This technology is termed Sensor-Integrated Proteome On Chip (SPOC®) which involves in-situ cell-free protein expression in nano-liter volume wells (nanowells) directly from rapidly customizable arrays of plasmid DNA, facilitating simultaneous capture-purification of up to 2400 unique full-length folded proteins onto a 1.5 sq-cm surface of a single gold biosensor chip. Arrayed SPOC sensors can then be screened by real-time label-free analysis, including surface plasmon resonance (SPR) to generate kinetic affinity, avidity data. Fluorescent and SPR assays were used to demonstrate zero crosstalk between protein spots. The functionality of the SPOC protein array was validated by antibody binding assay, post-translational modification, mutation-mediated differential binding kinetics, and catalytic activity screening on model SPOC protein arrays containing p53, Src, Jun, Fos, HIST1H3A, and SARS-CoV-2 receptor binding domain (RBD) protein variants of interest, among others. Monoclonal antibodies were found to selectively bind their target proteins on the SPOC array. A commercial anti-RBD antibody was used to demonstrate discriminatory binding to numerous SARS-CoV-2 RBD variants of concern with comprehensive kinetic information. With advantages of HTP, flexibility, low-cost, quick turnaround time, and real-time kinetic affinity profiling, the SPOC proteomic platform addresses the challenges of interrogating protein interactions at scale and can be deployed in various research and clinical applications.

Association between Obesity and Omega-3 Status in Healthy Young Women.

Omega-3 polyunsaturated fatty acids (n-3 PUFAs) are essential for healthy development and protect against metabolic disease. However, individuals with obesity may be pre-disposed to experiencing lower n-3 PUFA status than normal-weight individuals. This cross-sectional study examined the relationship between the omega-3 index (O3I), body mass index (BMI) and dietary intake in healthy young women (n = 300; age = 18-35 y), a group not previously focused on. Intake was adjusted for energy using the residuals method, and associations were explored using independent t-tests and Pearson's correlations. Participants with obesity were found to have significantly lower O3I than normal-weight participants (p < 0.0001); however, no significant differences were observed in mean n-3 PUFA intakes. Even so, energy-adjusted intakes of n-3 PUFAs, with the exception of alpha-linolenic acid, were significantly correlated with O3I. This study demonstrates that O3I is influenced by both BMI and diet in young women; however the relationship between these two variables may be complex. Current intakes of n-3 PUFA observed in young women may not be effective in achieving target O3I levels in those with obesity, and further research is needed to find effective ways of improving n-3 PUFA status in a group already at increased risk of metabolic disease.

Targeting Small Molecule Delivery to the Brain and Spinal Cord via Intranasal Administration of Rabies Virus Glycoprotein (RVG29)-Modified PLGA Nanoparticles.

Alternative routes of administration are one approach that could be used to bypass the blood-brain barrier (BBB) for effective drug delivery to the central nervous system (CNS). Here, we focused on intranasal delivery of polymer nanoparticles. We hypothesized that surface modification of poly(lactic-co-glycolic acid) (PLGA) nanoparticles with rabies virus glycoprotein (RVG29) would increase residence time and exposure of encapsulated payload to the CNS compared to non-targeted nanoparticles. Delivery kinetics and biodistribution were analyzed by administering nanoparticles loaded with the carbocyanine dye 1,1'-Dioctadecyl-3,3,3',3'-Tetramethylindotricarbocyanine Iodide (DiR) to healthy mice. Intranasal administration yielded minimal exposure of nanoparticle payload to most peripheral organs and rapid, effective delivery to whole brain. Regional analysis of payload delivery within the CNS revealed higher delivery to tissues closest to the trigeminal nerve, including the olfactory bulb, striatum, midbrain, brainstem, and cervical spinal cord. RVG29 surface modifications presented modest targeting benefits to the striatum, midbrain, and brainstem 2 h after administration, although targeting was not observed 30 min or 6 h after administration. Payload delivery to the trigeminal nerve was 3.5× higher for targeted nanoparticles compared to control nanoparticles 2 h after nanoparticle administration. These data support a nose-to-brain mechanism of drug delivery that closely implicates the trigeminal nerve for payload delivery from nanoparticles via transport of intact nanoparticles and eventual diffusion of payload. Olfactory and CSF routes are also observed to play a role. These data advance the utility of targeted nanoparticles for nose-to-brain drug delivery of lipophilic payloads and provide mechanistic insight to engineer effective delivery vectors to treat disease in the CNS.

Omega-3 polyunsaturated fatty acids status and cognitive function in young women.

BACKGROUND: Research indicates that low omega-3 polyunsaturated fatty acid (n-3 PUFA) may be associated with decreased cognitive function. This study examined the association between n-3 PUFA status and cognitive function in young Australian women. METHODS: This was a secondary outcome analysis of a cross-sectional study that recruited 300 healthy women (18-35 y) of normal weight (NW: BMI 18.5-24.9 kg/m2) or obese weight (OB: BMI ≥30.0 kg/m2). Participants completed a computer-based cognition testing battery (IntegNeuro™) evaluating the domains of impulsivity, attention, information processing, memory and executive function. The Omega-3 Index (O3I) was used to determine n-3 PUFA status (percentage of EPA (20:5n-3) plus DHA (22:6n3) in the red cell membrane) and the participants were divided into O3I tertile groups: T1 < 5.47%, T2 = 5.47-6.75%, T3 > 6.75%. Potential confounding factors of BMI, inflammatory status (C-reactive Protein), physical activity (total MET-min/wk), alpha1-acid glycoprotein, serum ferritin and hemoglobin, were assessed. Data reported as z-scores (mean ± SD), analyses via ANOVA and ANCOVA. RESULTS: Two hundred ninety-nine women (26.9 ± 5.4 y) completed the study (O3I data, n = 288). The ANOVA showed no overall group differences but a significant group × cognition domain interaction (p < 0.01). Post hoc tests showed that participants in the low O3I tertile group scored significantly lower on attention than the middle group (p = 0.01; ES = 0.45 [0.15-0.74]), while the difference with the high group was borderline significant (p = 0.052; ES = 0.38 [0.09-0.68]). After confounder adjustments, the low group had lower attention scores than both the middle (p = 0.01) and high (p = 0.048) groups. These findings were supported by univariate analyses which found significant group differences for the attention domain only (p = 0.004). CONCLUSIONS: Cognitive function in the attention domain was lower in women with lower O3I, but still within normal range. This reduced but normal level of cognition potentially provides a lower baseline from which cognition would decline with age. Further investigation of individuals with low n-3 PUFA status is warranted.

Association between Haem and Non-Haem Iron Intake and Serum Ferritin in Healthy Young Women.

Iron is an essential micronutrient for human health and inadequate intake may result in iron deficiency (ID) or iron deficiency anaemia (IDA). Unlike other recent studies investigating iron status in young women, this cross-sectional study analysed dietary intake and biochemical data from healthy young (18-35 years) women (n = 299) to determine the association between both haem iron (HI) and non-haem iron (NHI) intakes and serum ferritin (SF). Dietary restraint and possible inflammation secondary to obesity were also measured and accounted for, and energy intake was adjusted for using the residuals method. Independent samples t-tests and chi-squared tests were performed, and factors found to be significantly different between iron replete (IR) and ID/IDA participants were analysed using general linear modelling. ID/IDA participants consumed significantly lower total energy than iron replete (IR) (p = 0.003). Lower energy intake was also associated with higher levels of dietary restraint (p = 0.001). Both HI and NHI were positively associated with SF with HI was found to be a stronger predictor (ß = 0.128, p = 0.009) than NHI (ß = 0.037, p = 0.028). The study demonstrates that intake of both HI and NHI, as well as adequate dietary energy, are associated with normal iron status levels in young women, and that restrained eaters may be at greater risk of low iron status.

Iron Deficiency Anemia, Not Iron Deficiency, Is Associated with Reduced Attention in Healthy Young Women.

Women of reproductive age are at increased risk for iron deficiency (ID) and iron deficiency anemia (IDA), with both implicated in decreased cognitive function (CF). Obesity may complicate this association via inflammatory-mediated ferritin elevation. This cross-sectional study examined the association between hematological iron status (iron replete (IR), ID or IDA) and CF in healthy, young (18-35 years) women of normal-weight (NW: BMI 18.5-24.9 kg/m²) or obese-weight (OB: BMI >30 kg/m²). Participants completed a validated, computer-based cognition assessment evaluating impulsivity, attention, information processing, memory and executive function; CF reported as z-scores (mean ± SD). Iron status and CF were compared between groups via ANOVA, with adjustment for potential confounders (BMI, physical activity, C-reactive protein) via ANCOVA. A total of 157 NW and 142 OB women (25.8 ± 5.1 years) participated. Prevalence of ID and IDA were 14% and 6% respectively, with no significant difference between NW and OB groups. Women with IDA scored significantly lower on attention (although within normal range; ±1 z-score), compared to ID (IDA: -0.75 ± 1.89; ID: 0.53 ± 1.37; p = 0.004) but not IR (0.03 ± 1.33, p = 0.21) groups; there were no significant differences between ID and IR groups (p = 0.34). Adjustment for confounders did not significantly alter these results. In conclusion, women with IDA showed significantly reduced attention compared to women with ID.

Relationship between Obesity and Cognitive Function in Young Women: The Food, Mood and Mind Study.

Limited research addresses links between obesity and cognitive function in young adults. Objective. To investigate the relationship between obesity and cognitive function in young women. Methods. This cross-sectional study recruited healthy, young (18-35 y) women of normal (NW: BMI = 18.5-24.9 kg·m-2) or obese (OB: BMI ≥ 30.0 kg·m-2) weight. Participants completed a validated, computer-based cognitive testing battery evaluating impulsivity, attention, information processing, memory, and executive function. Questionnaires on depression and physical activity and a fasting blood sample for C-reactive protein and the Omega-3 Index were also collected. Cognition data are presented as z-scores (mean ± SD), and group comparisons were assessed via ANOVA. Potential confounding from questionnaire and blood variables were evaluated using ANCOVA. Results. 299 women (NW: n = 157; OB: n = 142) aged 25.8 ± 5.1 y were enrolled. Cognition scores were within normal range (±1 z-score), but OB had lower attention (NW: 0.31 ± 1.38; OB: -0.25 ± 1.39; ES: 0.41, CI: 0.17-0.64; p < 0.001) and higher impulsivity (NW: 0.36 ± 1.14; OB: -0.07 ± 1.07; ES: 0.39, CI: 0.15-0.62; p=0.033). Confounder adjustment had minimal impact on results. Conclusion. The OB group had normal but significantly lower performance on attention and were more impulsive compared to NW participants. This may indicate early cognitive decline, but longitudinal research confirming these findings is warranted.

A critical evaluation of drug delivery from ligand modified nanoparticles: Confounding small molecule distribution and efficacy in the central nervous system.

In this work, we sought to test how surface modification of poly(lactic-co-glycolic acid) (PLGA) nanoparticles with peptide ligand alters the brain specific delivery of encapsulated molecules. For biodistribution studies, nanoparticles modified with rabies virus glycoprotein (RVG29) were loaded with small molecule drug surrogates and administered to healthy mice by lateral tail vein injection. Mice were perfused 2h after injection and major anatomical regions of the CNS were dissected (striatum, midbrain, cerebellum, hippocampus, cortex, olfactory bulb, brainstem, and cervical, thoracic, lumbar and sacral spinal cord). For functional studies, surface modified nanoparticles were loaded with the chemotherapeutic camptothecin (CPT) and administered to mice bearing intracranial GL261-Luc2 gliomas. Outcome measures included tumor growth, as measured by bioluminescent imaging, and median survival time. We observed that small molecule delivery from PLGA nanoparticles varied by as much as 150% for different tissue regions within the CNS. These differences were directly correlated to regional differences in cerebral blood volume. Although the presence of RVG29 enhanced apparent brain delivery for multiple small molecule payloads, we observed minimal evidence for targeting to muscle or spinal cord, which are the known sites for rabies virus entry into the CNS, and enhancements in brain delivery were not prolonged due to an apparent aqueous instability of the RVG29 ligand. Furthermore, we have identified concerning differences in apparent delivery kinetics as measured by different payloads: nanoparticle encapsulated DiR was observed to accumulate in the brain, whereas encapsulated Nile red was rapidly cleared. Although systemically administered CPT loaded nanoparticles slowed the growth of orthotopic brain tumors to prolong survival, the presence of RVG29 did not enhance therapeutic efficacy compared to control nanoparticles. These data are consistent with a model of delivery of hydrophobic small molecules to the brain that does not rely on internalization of polymer nanoparticles in target tissue. We discuss an important risk for discordance between biodistribution, as typically measured by drug surrogate, and therapeutic outcome, as determined by clinically relevant measurement of drug function in a disease model. These results pose critical considerations for the methods used to design and evaluate targeted drug delivery systems in vivo.