Selective TLR4 Antagonism Prevents and Reverses Morphine-Induced Persistent Postoperative Cognitive Dysfunction, Dysregulation of Synaptic Elements, and Impaired BDNF Signaling in Aged Male Rats.

Perioperative neurocognitive disorders (PNDs) are characterized by confusion, difficulty with executive function, and episodic memory impairment in the hours to months following a surgical procedure. Postoperative cognitive dysfunction (POCD) represents such impairments that last beyond 30 d postsurgery and is associated with increased risk of comorbidities, progression to dementia, and higher mortality. While it is clear that neuroinflammation plays a key role in PND development, what factors underlie shorter self-resolving versus persistent PNDs remains unclear. We have previously shown that postoperative morphine treatment extends POCD from 4 d (without morphine) to at least 8 weeks (with morphine) in aged male rats, and that this effect is likely dependent on the proinflammatory capabilities of morphine via activation of toll-like receptor 4 (TLR4). Here, we extend these findings to show that TLR4 blockade, using the selective TLR4 antagonist lipopolysaccharide from the bacterium Rhodobacter sphaeroides (LPS-RS Ultrapure), ameliorates morphine-induced POCD in aged male rats. Using either a single central preoperative treatment or a 1 week postoperative central treatment regimen, we demonstrate that TLR4 antagonism (1) prevents and reverses the long-term memory impairment associated with surgery and morphine treatment, (2) ameliorates morphine-induced dysregulation of the postsynaptic proteins postsynaptic density 95 and synaptopodin, (3) mitigates reductions in mature BDNF, and (4) prevents decreased activation of the BDNF receptor TrkB (tropomyosin-related kinase B), all at 4 weeks postsurgery. We also reveal that LPS-RS Ultrapure likely exerts its beneficial effects by preventing endogenous danger signal HMGB1 (high-mobility group box 1) from activating TLR4, rather than by blocking continuous activation by morphine or its metabolites. These findings suggest TLR4 as a promising therapeutic target to prevent or treat PNDs.SIGNIFICANCE STATEMENT With humans living longer than ever, it is crucial that we identify mechanisms that contribute to aging-related vulnerability to cognitive impairment. Here, we show that the innate immune receptor toll-like receptor 4 (TLR4) is a key mediator of cognitive dysfunction in aged rodents following surgery and postoperative morphine treatment. Inhibition of TLR4 both prevented and reversed surgery plus morphine-associated memory impairment, dysregulation of synaptic elements, and reduced BDNF signaling. Together, these findings implicate TLR4 in the development of postoperative cognitive dysfunction, providing mechanistic insight and novel therapeutic targets for the treatment of cognitive impairments following immune challenges such as surgery in older individuals.

N-glycomic profiling of capsid proteins from Adeno-Associated Virus serotypes.

Adeno-associated virus (AAV) vector has become the leading platform for gene delivery. Each serotype exhibits a different tissue tropism, immunogenicity, and in vivo transduction performance. Therefore, selecting the most suitable AAV serotype is critical for efficient gene delivery to target cells or tissues. Genome divergence among different serotypes is due mainly to the hypervariable regions of the AAV capsid proteins. However, the heterogeneity of capsid glycosylation is largely unexplored. In the present study, the N-glycosylation profiles of capsid proteins of AAV serotypes 1 to 9 have been systemically characterized and compared using a previously developed high-throughput and high-sensitivity N-glycan profiling platform. The results showed that all 9 investigated AAV serotypes were glycosylated, with comparable profiles. The most conspicuous feature was the high abundance mannosylated N-glycans, including FM3, M5, M6, M7, M8, and M9, that dominated the chromatograms within a range of 74 to 83%. Another feature was the relatively lower abundance of fucosylated and sialylated N-glycan structures, in the range of 23%-40% and 10%-17%, respectively. However, the exact N-glycan composition differed. These differences may be utilized to identify potential structural relationships between the 9 AAV serotypes. The current research lays the foundation for gaining better understanding of the importance of N-glycans on the AAV capsid surface that may play a significant role in tissue tropism, interaction with cell surface receptors, cellular uptake, and intracellular processing.

Short-term high fat diet impairs memory, exacerbates the neuroimmune response, and evokes synaptic degradation via a complement-dependent mechanism in a mouse model of Alzheimer's disease.

Alzheimer's Disease (AD) is a neurodegenerative disease characterized by profound memory impairments, synaptic loss, neuroinflammation, and hallmark pathological markers. High-fat diet (HFD) consumption increases the risk of developing AD even after controlling for metabolic syndrome, pointing to a role of the diet itself in increasing risk. In AD, the complement system, an arm of the immune system which normally tags redundant or damaged synapses for pruning, becomes pathologically overactivated leading to tagging of healthy synapses. While the unhealthy diet to AD link is strong, the underlying mechanisms are not well understood in part due to confounding variables associated with long-term HFD which can independently influence the brain. Therefore, we experimented with a short-term diet regimen to isolate the diet's impact on brain function without causing obesity. This project investigated the effect of short-term HFD on 1) memory, 2) neuroinflammation including complement, 3) AD pathology markers, 4) synaptic markers, and 5) in vitro microglial synaptic phagocytosis in the 3xTg-AD mouse model. Following the consumption of either standard chow or HFD, 3xTg-AD and non-Tg mice were tested for memory impairments. In a separate cohort of mice, levels of hippocampal inflammatory markers, complement proteins, AD pathology markers, and synaptic markers were measured. For the last set of experiments, BV2 microglial phagocytosis of synapses was evaluated. Synaptoneurosomes isolated from the hippocampus of 3xTg-AD mice fed chow or HFD were incubated with equal numbers of BV2 microglia. The number of BV2 microglia that phagocytosed synaptoneurosomes was tracked over time with a live-cell imaging assay. Finally, we incubated BV2 microglia with a complement receptor inhibitor (NIF) and repeated the assay. Behavioral analysis showed 3xTg-AD mice had significantly impaired long-term contextual and cued fear memory compared to non-Tg mice that was further impaired by HFD. HFD significantly increased inflammatory markers and complement expression while decreasing synaptic marker expression only in 3xTg-AD mice, without altering AD pathology markers. Synaptoneurosomes from HFD-fed 3xTg-AD mice were phagocytosed at a significantly higher rate than those from chow-fed mice, suggesting the synapses were altered by HFD. The complement receptor inhibitor blocked this effect in a dose-dependent manner, demonstrating the HFD-mediated increase in phagocytosis was complement dependent. This study indicates HFD consumption increases neuroinflammation and over-activates the complement cascade in 3xTg-AD mice, resulting in poorer memory. The in vitro data point to complement as a potential mechanistic culprit and therapeutic target underlying HFD's influence in increasing cognitive vulnerability to AD.

Post-operative cognitive dysfunction is exacerbated by high-fat diet via TLR4 and prevented by dietary DHA supplementation.

Post-operative cognitive dysfunction (POCD) is an abrupt decline in neurocognitive function arising shortly after surgery and persisting for weeks to months, increasing the risk of dementia diagnosis. Advanced age, obesity, and comorbidities linked to high-fat diet (HFD) consumption such as diabetes and hypertension have been identified as risk factors for POCD, although underlying mechanisms remain unclear. We have previously shown that surgery alone, or 3-days of HFD can each evoke sufficient neuroinflammation to cause memory deficits in aged, but not young rats. The aim of the present study was to determine if HFD consumption before surgery would potentiate and prolong the subsequent neuroinflammatory response and memory deficits, and if so, to determine the extent to which these effects depend on activation of the innate immune receptor TLR4, which both insults are known to stimulate. Young-adult (3mo) & aged (24mo) male F344xBN F1 rats were fed standard chow or HFD for 3-days immediately before sham surgery or laparotomy. In aged rats, the combination of HFD and surgery caused persistent deficits in contextual memory and cued-fear memory, though it was determined that HFD alone was sufficient to cause the long-lasting cued-fear memory deficits. In young adult rats, HFD + surgery caused only cued-fear memory deficits. Elevated proinflammatory gene expression in the hippocampus of both young and aged rats that received HFD + surgery persisted for at least 3-weeks after surgery. In a separate experiment, rats were administered the TLR4-specific antagonist, LPS-RS, immediately before HFD onset, which ameliorated the HFD + surgery-associated neuroinflammation and memory deficits. Similarly, dietary DHA supplementation for 4 weeks prior to HFD onset blunted the neuroinflammatory response to surgery and prevented development of persistent memory deficits. These results suggest that HFD 1) increases risk of persistent POCD-associated memory impairments following surgery in male rats in 2) a TLR4-dependent manner, which 3) can be targeted by DHA supplementation to mitigate development of persistent POCD.

Understanding spatiotemporal effects of food supplementation on host-parasite interactions using community-based science.

Supplemental feeding can increase the overall health of animals but also can have variable effects on how animals defend themselves against parasites. However, the spatiotemporal effects of food supplementation on host-parasite interactions remain poorly understood, likely because large-scale, coordinated efforts to investigate them are difficult. Here, we introduce the Nest Parasite Community Science Project, which is a community-based science project that coordinates studies with bird nest box 'stewards' from the public and scientific community. This project was established to understand broad ecological patterns between hosts and their parasites. The goal of this study was to determine the effect of food supplementation on eastern bluebirds (Sialia sialis) and their nest parasite community across the geographic range of the bluebirds from 2018 to 2021. We received 674 nests from 69 stewards in 26 states in the eastern United States. Nest box stewards reported whether or not they provided mealworms or suet near nesting bluebirds, then they followed the nesting success of the birds (number of eggs laid and hatched, proportion that hatched, number and proportion of nestlings that successfully fledged). We then identified and quantified parasites in the nests. Overall, we found that food supplementation increased fledging success. The most common nest parasite taxon was the parasitic blow fly (Protocalliphora sialia), but a few nests contained fleas (Ceratophyllus idius, C. gallinae and Orchopeas leucopus) and mites (Dermanyssus spp. and Ornithonyssus spp.). Blow flies were primarily found at northern latitudes, where food supplementation affected blow fly prevalence. However, the direction of this effect varied substantially in direction and magnitude across years. More stewards fed bluebirds at southern latitudes than at northern latitudes, which contradicts the findings of other community-based science projects. Overall, food supplementation of birds was associated with increased host fitness but did not appear to play a consistent role in defence against these parasites across all years. Our study demonstrates the importance of coordinated studies across years and locations to understand the effects of environmental heterogeneity, including human-based food supplementation, on host-parasite dynamics.

Quantitative profiling of N-glycosylation of SARS-CoV-2 spike protein variants.

With the global spread of the corona virus disease-2019 pandemic, new spike variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continuously emerge due to increased possibility of virus adaptive amino acid mutations. However, the N-glycosylation profiles of different spike variants are yet to be explored extensively, although the spike protein is heavily glycosylated and surface glycans are well-established to play key roles in viral infection and immune response. Here, we investigated quantitatively the N-glycosylation profiles of seven major emerging spike variants including Original, Alpha (B.1.1.7), Beta (B.1.351), Gamma (P.1), Kappa (B.1.671.1), Delta (B.1.671.2), and Omicron (B.1.1.529). The aim was to understand the changing pattern of N-glycan profiles in SARS-CoV-2 evolution in addition to the widely studied amino acid mutations. Different spike variants exhibit substantial variations in the relative abundance of different glycan peaks and subclasses, although no specific glycan species are exclusively present in or absent from any specific variant. Cluster analysis shows that the N-glycosylation profiles may hold the potential for SARS-CoV-2 spike variants classification. Alpha and Beta variants exhibit the closest similarity to the Original, and the Delta variant displays substantial similarity to Gamma and Kappa variants, while the Omicron variant is significantly different from its counterparts. We demonstrated that there is a quantifiable difference in N-glycosylation profiles among different spike variants. The current study and observations herein provide a valuable framework for quantitative N-glycosylation profiling of new emerging viral variants and give us a more comprehensive picture of COVID-19 evolution.

Multi-attribute analysis of adeno-associated virus by size exclusion chromatography with fluorescence and triple-wavelength UV detection.

Adeno-associated virus (AAV) is the leading platform for in vivo gene therapy to treat numerous genetic diseases. Comprehensive analysis of the AAV particles is essential to ensure desired safety and efficacy. An array of techniques is required to evaluate their critical quality attributes. However, many of these techniques are expensive, time-consuming, labour-intensive, and varying in accuracy. Size exclusion chromatography coupled with fluorescence and triple-wavelength ultraviolet detection (SEC-FLD-TWUV) and incorporating an aromatic amino acid of tryptophan as an internal standard offers a simple, rapid, and reliable approach for simultaneous multi-attribute analysis of AAVs. In the current study, we demonstrate its capability for AAV characterization and quantification, that includes capsid concentration, empty to full capsid ratio, vector genome concentration, and the presence of aggregates or fragments. All were performed in 20-min chromatographic runs with minimal sample handling. Data analysis involves the assessment of intrinsic fluorescence and UV absorbance of samples at three wavelengths that can be utilised to determine the content of the capsid protein and genome copy number. The separation efficiency using SEC columns with different pore sizes, and elution buffers of varying compositions, ionic strength, and pH values was also evaluated. This SEC-FLD-TWUV method may serve as a powerful yet cost-effective tool for responsive quality evaluation of AAVs. This may enhance performance, robustness, and safety of bioprocessing for AAV vectors to be used in gene therapy.

CD8+ T cells contribute to diet-induced memory deficits in aged male rats.

We have previously shown that short-term (3-day) high fat diet (HFD) consumption induces a neuroinflammatory response and subsequent impairment of long-term memory in aged, but not young adult, male rats. However, the immune cell phenotypes driving this proinflammatory response are not well understood. Previously, we showed that microglia isolated from young and aged rats fed a HFD express similar levels of priming and proinflammatory transcripts, suggesting that additional factors may drive the exaggerated neuroinflammatory response selectively observed in aged HFD-fed rats. It is established that T cells infiltrate both the young and especially the aged central nervous system (CNS) and contribute to immune surveillance of the parenchyma. Thus, we investigated the modulating role of short-term HFD on T cell presence in the CNS in aged rats using bulk RNA sequencing and flow cytometry. RNA sequencing results indicate that aging and HFD altered the expression of genes and signaling pathways associated with T cell signaling, immune cell trafficking, and neuroinflammation. Moreover, flow cytometry data showed that aging alone increased CD4+ and CD8+ T cell presence in the brain and that CD8+, but not CD4+, T cells were further increased in aged rats fed a HFD. Based on these data, we selectively depleted circulating CD8+ T cells via an intravenous injection of an anti-CD8 antibody in aged rats prior to 3 days of HFD to infer the functional role these cells may be playing in long-term memory and neuroinflammation. Results indicate that peripheral depletion of CD8+ T cells lowered hippocampal cytokine levels and prevented the HFD-induced i) increase in brain CD8+ T cells, ii) memory impairment, and iii) alterations in pre- and post-synaptic structures in the hippocampus and amygdala. Together, these data indicate a substantial role for CD8+ T cells in mediating diet-induced memory impairments in aged male rats.

Total Ankle Replacement Versus Arthrodesis for End-Stage Ankle Osteoarthritis: A Randomized Controlled Trial.

BACKGROUND: End-stage ankle osteoarthritis causes severe pain and disability. There are no randomized trials comparing the 2 main surgical treatments: total ankle replacement (TAR) and ankle fusion (AF). OBJECTIVE: To determine which treatment is superior in terms of clinical scores and adverse events. DESIGN: A multicenter, parallel-group, open-label randomized trial. (ISRCTN registry number: 60672307). SETTING: 17 National Health Service trusts across the United Kingdom. PATIENTS: Patients with end-stage ankle osteoarthritis, aged 50 to 85 years, and suitable for either procedure. INTERVENTION: Patients were randomly assigned to TAR or AF surgical treatment. MEASUREMENTS: The primary outcome was change in Manchester-Oxford Foot Questionnaire walking/standing (MOXFQ-W/S) domain scores between baseline and 52 weeks after surgery. No blinding was possible. RESULTS: Between 6 March 2015 and 10 January 2019, a total of 303 patients were randomly assigned; mean age was 68 years, and 71% were men. Twenty-one patients withdrew before surgery, and 281 clinical scores were analyzed. At 52 weeks, the mean MOXFQ-W/S scores improved for both groups. The adjusted difference in the change in MOXFQ-W/S scores from baseline was -5.6 (95% CI, -12.5 to 1.4), showing that TAR improved more than AF, but the difference was not considered clinically or statistically significant. The number of adverse events was similar between groups (109 vs. 104), but there were more wound healing issues in the TAR group and more thromboembolic events and nonunion in the AF group. The symptomatic nonunion rate for AF was 7%. A post hoc analysis suggested superiority of fixed-bearing TAR over AF (-11.1 [CI, -19.3 to -2.9]). LIMITATION: Only 52-week data; pragmatic design creates heterogeneity of implants and surgical techniques. CONCLUSION: Both TAR and AF improve MOXFQ-W/S and had similar clinical scores and number of harms. Total ankle replacement had greater wound healing complications and nerve injuries, whereas AF had greater thromboembolism and nonunion, with a symptomatic nonunion rate of 7%. PRIMARY FUNDING SOURCE: National Institute for Health and Care Research Heath Technology Assessment Programme.

Construction of an InstantPC-derivatized glycan glucose unit database: A foundation work for high-throughput and high-sensitivity glycomic analysis.

The glycosylation profile of biotherapeutic glycoproteins is a critical quality attribute that is routinely monitored to ensure desired product quality, safety and efficacy. Additionally, as one of the most prominent and complex post-translational modifications, glycosylation plays a key role in disease manifestation. Changes in glycosylation may serve as a specific and sensitive biomarker for disease diagnostics and prognostics. However, the conventional 2-aminobenzamide-based N-glycosylation analysis procedure is time-consuming and insensitive with poor reproducibility. We have evaluated an innovative streamlined 96-well-plate-based platform utilizing InstantPC label for high-throughput, high-sensitivity glycan profiling, which is user-friendly, robust and ready for automation. However, the limited availability of InstantPC-labeled glycan standards has significantly hampered the applicability and transferability of this platform for expedited glycan structural profiling. To address this challenge, we have constructed a detailed InstantPC-labeled glycan glucose unit (GU) database through analysis of human serum and a variety of other glycoproteins from various sources. Following preliminary hydrophilic interaction liquid chromatography (HILIC) with fluorescence detection separation and analysis, glycoproteins with complex glycan profiles were subjected to further fractionation by weak anion exchange HILIC and exoglycosidase sequential digestion for cross-validation of the glycan assignment. Hydrophilic interaction ultra-performance liquid chromatography coupled with electrospray ionization mass spectrometry was subsequently utilized for glycan fragmentation and accurate glycan mass confirmation. The constructed InstantPC glycan GU database is accurate and robust. It is believed that this database will enhance the application of the developed platform for high-throughput, high-sensitivity glycan profiling and that it will eventually advance glycan-based biopharmaceutical production and disease biomarker discovery.

Serum N-glycomic profiling may provide potential signatures for surveillance of COVID-19.

Disease development and progression are often associated with aberrant glycosylation, indicating that changes in biological fluid glycome may potentially serve as disease signatures. The corona virus disease-2019 (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) represents a significant threat to global human health. However, the effect of SARS-CoV-2 infection on the overall serum N-glycomic profile has been largely unexplored. Here, we extended our 96-well-plate-based high-throughput, high-sensitivity N-glycan profiling platform further with the aim of elucidating potential COVID-19-associated serum N-glycomic alterations. Use of this platform revealed both similarities and differences between the serum N-glycomic fingerprints of COVID-19 positive and control cohorts. Although there were no specific glycan peaks exclusively present or absent in COVID-19 positive cohort, this cohort showed significantly higher levels of glycans and variability. On the contrary, the overall N-glycomic profiles for healthy controls were well-contained within a narrow range. From the serum glycomic analysis, we were able to deduce changes in different glycan subclasses sharing certain structural features. Of significance was the hyperbranched and hypersialylated glycans and their derived glycan subclass traits. T-distributed stochastic neighbor embedding and hierarchical heatmap clustering analysis were performed to identify 13 serum glycomic variables that potentially distinguished the COVID-19 positive from healthy controls. Such serum N-glycomic changes described herein may indicate or correlate to the changes in serum glycoproteins upon COVID-19 infection. Furthermore, mapping the serum N-glycome following SARS-CoV-2 infection may help us better understand the disease and enable "Long-COVID" surveillance to capture the full spectrum of persistent symptoms.

Preservation of endoplasmic reticulum (ER) Ca2+ stores by deletion of inositol-1,4,5-trisphosphate receptor type 1 promotes ER retrotranslocation, proteostasis, and protein outer segment localization in cyclic nucleotide-gated channel-deficient cone photoreceptors.

Endoplasmic reticulum (ER) Ca2+ homeostasis relies on an appropriate balance between efflux- and influx-channel activity responding to dynamic changes of intracellular Ca2+ levels. Dysregulation of this complex signaling network has been shown to contribute to neuronal and photoreceptor death in neuro- and retinal degenerative diseases, respectively. In mice with cone cyclic nucleotide-gated (CNG) channel deficiency, a model of achromatopsia/cone dystrophy, cones display early-onset ER stress-associated apoptosis and protein mislocalization. Cones in these mice also show reduced cytosolic Ca2+ level and subsequent elevation in the ER Ca2+ -efflux-channel activity, specifically the inositol-1,4,5-trisphosphate receptor type 1 (IP3 R1), and deletion of IP3 R1 results in preservation of cones. This work investigated how preservation of ER Ca2+ stores leads to cone protection. We examined the effects of cone specific deletion of IP3 R1 on ER stress responses/cone death, protein localization, and ER proteostasis/ER-associated degradation. We demonstrated that deletion of IP3 R1 improves trafficking of cone-specific proteins M-/S-opsin and phosphodiesterase 6C to cone outer segments and reduces localization to cone inner segments. Consistent with the improved protein localization, deletion of IP3 R1 results in increased ER retrotranslocation protein expression, reduced proteasome subunit expression, reduced ER stress/cone death, and reduced retinal remodeling. We also observed the enhanced ER retrotranslocation in mice that have been treated with a chemical chaperone, supporting the connection between improved ER retrotranslocation/proteostasis and alleviation of ER stress. Findings from this work demonstrate the importance of ER Ca2+ stores in ER proteostasis and protein trafficking/localization in photoreceptors, strengthen the link between dysregulation of ER Ca2+ homeostasis and ER stress/cone degeneration, and support an involvement of improved ER proteostasis in ER Ca2+ preservation-induced cone protection; thereby identifying IP3 R1 as a critical mediator of ER stress and protein mislocalization and as a potential target to preserve cones in CNG channel deficiency.

Evaluation of the Modified Barium Swallow Study Conducted With and Without Radiologist Supervision: A Pre- and Post-Observational Study.

PURPOSE: Assess quality metrics of modified barium swallow studies (MBSS) with and without a radiologist present during the procedure. METHODS: Retrospective review of MBSS performed on adult inpatients at a tertiary care hospital 6-months pre- and post-institutional change from having to not having a radiologist present during the examination. FACTORS ASSESSED INCLUDED: fluoroscopy time; study duration; number of cine loops; number of images; efficiency of collimation (using a 5-point scoring system); time to final report; radiologist-speech language pathologist report disagreement; and recalls for inadequate studies. Statistical analysis was via Welch's t-test and a test of proportions for continuous and count data under the normal approximation. RESULTS: 106 and 119 MBSS were analyzed from the radiologist present and radiologist absent periods, respectively. No statistically significant differences were found for: average fluoroscopy time (116.1 s vs. 126.9 s; P = 0.161); study duration (400.4 s vs. 417.3 s; P = 0.453); number of cine loops (9.3 vs. 10.2; P = 0.075); number of images (620.5 vs. 581.1; P = 0.350); or report disagreement. There was improved performance without the radiologist present for collimation (1.92 vs. 1.43; P = 0.003) and fewer non-diagnostic images (6.5 vs. 4.5; P = 0.001). Time to final report was longer with the radiologist absent due to more reports with significant delays. There were no repeated studies because of inadequate technique in either group. CONCLUSION: MBSS performed by technologists without radiologist supervision is not inferior to those performed with radiologist supervision on multiple performance measures. This supports technologist operated MBSS without radiologist supervision, while acknowledging a need to further address radiologist report time delay.

High-throughput and high-sensitivity N-Glycan profiling: A platform for biopharmaceutical development and disease biomarker discovery.

Protein glycosylation contributes to critical biological function of glycoproteins. Glycan analysis is essential for the production of biopharmaceuticals as well as for the identification of disease biomarkers. However, glycans are highly heterogeneous, which has considerably hampered the progress of glycomics. Here, we present an improved 96-well plate format platform for streamlined glycan profiling that takes advantage of rapid glycoprotein denaturation, deglycosylation, fluorescent derivatization, and on-matrix glycan clean-up. This approach offers high sensitivity with consistent identification and quantification of diverse N-glycans across multiple samples on a high-throughput scale. We demonstrate its capability for N-glycan profiling of glycoproteins from various sources, including two recombinant monoclonal antibodies produced from Chinese Hamster Ovary cells, EG2-hFc and rituximab, polyclonal antibodies purified from human serum, and total glycoproteins from human serum. Combined with the complementary information obtained by sequential digestion from exoglycosidase arrays, this approach allows the detection and identification of multiple N-glycans in these complex biological samples. The reagents, workflow, and Hydrophilic interaction liquid chromatography with fluorescence detection (HILIC-FLD), are simple enough to be implemented into a straightforward user-friendly setup. This improved technology provides a powerful tool in support of rapid advancement of glycan analysis for biopharmaceutical development and biomarker discovery for clinical disease diagnosis.

Potential contribution of ryanodine receptor 2 upregulation to cGMP/PKG signaling-induced cone degeneration in cyclic nucleotide-gated channel deficiency.

Photoreceptor cyclic nucleotide-gated (CNG) channels regulate Ca2+ influx in rod and cone photoreceptors. Mutations in cone CNG channel subunits CNGA3 and CNGB3 are associated with achromatopsia and cone dystrophies. Mice lacking functional cone CNG channel show endoplasmic reticulum (ER) stress-associated cone degeneration. The elevated cyclic guanosine monophosphate (cGMP)/cGMP-dependent protein kinase (PKG) signaling and upregulation of the ER Ca2+ channel ryanodine receptor 2 (RyR2) have been implicated in cone degeneration. This work investigates the potential contribution of RyR2 to cGMP/PKG signaling-induced ER stress and cone degeneration. We demonstrated that the expression and activity of RyR2 were highly regulated by cGMP/PKG signaling. Depletion of cGMP by deleting retinal guanylate cyclase 1 or inhibition of PKG using chemical inhibitors suppressed the upregulation of RyR2 in CNG channel deficiency. Depletion of cGMP or deletion of Ryr2 equivalently inhibited unfolded protein response/ER stress, activation of the CCAAT-enhancer-binding protein homologous protein, and activation of the cyclic adenosine monophosphate response element-binding protein, leading to early-onset cone protection. In addition, treatment with cGMP significantly enhanced Ryr2 expression in cultured photoreceptor-derived Weri-Rb1 cells. Findings from this work demonstrate the regulation of cGMP/PKG signaling on RyR2 in the retina and support the role of RyR2 upregulation in cGMP/PKG signaling-induced ER stress and photoreceptor degeneration.

Dietary DHA prevents cognitive impairment and inflammatory gene expression in aged male rats fed a diet enriched with refined carbohydrates.

The consumption of a processed foods diet (PD) enriched with refined carbohydrates, saturated fats, and lack of fiber has increased in recent decades and likely contributed to increased incidence of chronic disease and weight gain in humans. These diets have also been shown to negatively impact brain health and cognitive function in rodents, non-human primates, and humans, potentially through neuroimmune-related mechanisms. However, mechanisms by which PD impacts the aged brain are unknown. This gap in knowledge is critical, considering the aged brain has a heightened state of baseline inflammation, making it more susceptible to secondary challenges. Here, we showed that consumption of a PD, enriched with refined carbohydrate sources, for 28 days impaired hippocampal- and amygdalar-dependent memory function in aged (24 months), but not young (3 months) F344 × BN rats. These memory deficits were accompanied by increased expression of inflammatory genes, such as IL-1ß, CD11b, MHC class II, CD86, NLRP3, and complement component 3, in the hippocampus and amygdala of aged rats. Importantly, we also showed that when the same PD is supplemented with the omega-3 polyunsaturated fatty acid DHA, these memory deficits and inflammatory gene expression changes were ameliorated in aged rats, thus providing the first evidence that DHA supplementation can protect against memory deficits and inflammatory gene expression in aged rats fed a processed foods diet. Lastly, we showed that while PD consumption increased weight gain in both young and aged rats, this effect was exaggerated in aged rats. Aging was also associated with significant alterations in hypothalamic gene expression, with no impact by DHA on weight gain or hypothalamic gene expression. Together, our data provide novel insights regarding diet-brain interactions by showing that PD consumption impairs cognitive function likely through a neuroimmune mechanism and that dietary DHA can ameliorate this phenomenon.

Oxidative damage increases with degree of simulated bacterial infection, but not ectoparasitism, in tree swallow nestlings.

The purpose of mounting an immune response is to destroy pathogens, but this response comes at a physiological cost, including the generation of oxidative damage. However, many studies on the effects of immune challenges employ a single high dose of a simulated infection, meaning that the consequences of more mild immune challenges are poorly understood. We tested whether the degree of immunological challenge in tree swallows (Tachycineta bicolor) affects oxidative physiology and body mass, and whether these metrics correlate with parasitic nest mite load. We injected 14 day old nestlings with 0, 0.01, 0.1 or 1 mg lipopolysaccharide (LPS) per kg body mass, then collected a blood sample 24 h later to quantify multiple physiological metrics, including oxidative damage (i.e. d-ROMs), circulating amounts of triglyceride and glycerol, and levels of the acute phase protein haptoglobin. After birds had fledged, we identified and counted parasitic nest mites (Dermanyssus spp. and Ornithonyssus spp.). We found that only nestlings injected with 1 mg LPS kg-1 body mass, which is a common dosage in ecoimmunological studies, lost more body mass than individuals from other treatment groups. However, every dose of LPS resulted in a commensurate increase in oxidative damage. Parasitic mite abundance had no effect on oxidative damage across treatments. The amount of oxidative damage correlated with haptoglobin levels, suggesting compensatory mechanisms to limit self-damage during an immune response. We conclude that while only the highest-intensity immune challenges resulted in costs related to body mass, even low-intensity immune challenges result in detectable increases in oxidative damage.

Correlates of bird collisions with buildings across three North American countries.

Collisions with buildings cause up to 1 billion bird fatalities annually in the United States and Canada. However, efforts to reduce collisions would benefit from studies conducted at large spatial scales across multiple study sites with standardized methods and consideration of species- and life-history-related variation and correlates of collisions. We addressed these research needs through coordinated collection of data on bird collisions with buildings at sites in the United States (35), Canada (3), and Mexico (2). We collected all carcasses and identified species. After removing records for unidentified carcasses, species lacking distribution-wide population estimates, and species with distributions overlapping fewer than 10 sites, we retained 269 carcasses of 64 species for analysis. We estimated collision vulnerability for 40 bird species with ≥2 fatalities based on their North American population abundance, distribution overlap in study sites, and sampling effort. Of 10 species we identified as most vulnerable to collisions, some have been identified previously (e.g., Black-throated Blue Warbler [Setophaga caerulescens]), whereas others emerged for the first time (e.g., White-breasted Nuthatch [Sitta carolinensis]), possibly because we used a more standardized sampling approach than past studies. Building size and glass area were positively associated with number of collisions for 5 of 8 species with enough observations to analyze independently. Vegetation around buildings influenced collisions for only 1 of those 8 species (Swainson's Thrush [Catharus ustulatus]). Life history predicted collisions; numbers of collisions were greatest for migratory, insectivorous, and woodland-inhabiting species. Our results provide new insight into the species most vulnerable to building collisions, making them potentially in greatest need of conservation attention to reduce collisions and into species- and life-history-related variation and correlates of building collisions, information that can help refine collision management.

Correlaciones de las Colisiones de Aves contra Edificios en Tres Países de América del Norte Resumen Las colisiones contra los edificios causan hasta mil millones de fatalidades de aves al año en los Estados Unidos y en Canadá. Sin embargo, los esfuerzos por reducir estas colisiones se beneficiarían con estudios realizados a grandes escalas espaciales en varios sitios de estudio con métodos estandarizados y considerando las variaciones relacionadas a la historia de vida y a la especie y las correlaciones de las colisiones. Abordamos estas necesidades de investigación por medio de una recolección coordinada de datos sobre las colisiones de aves contra edificios en los Estados Unidos (35), Canadá (3) y México (2). Recolectamos todos los cadáveres y los identificamos hasta especie. Después de retirar los registros de cadáveres no identificados, las especies sin estimaciones poblacionales a nivel distribución y las especies con distribuciones traslapadas en menos de diez sitios, nos quedamos con 269 cadáveres de 64 especies para el análisis. Estimamos la vulnerabilidad a colisiones para 40 especies con ≥2 fatalidades con base en la abundancia poblacional para América del Norte, el traslape de su distribución entre los sitios de estudio y el esfuerzo de muestreo. De las diez especies que identificamos como las más vulnerables a las colisiones, algunas han sido identificadas previamente (Setophaga caerulescens), y otras aparecieron por primera vez (Sitta carolinensis), posiblemente debido a que usamos una estrategia de muestreo más estandarizada que en los estudios previos. El tamaño del edificio y el área del vidrio estuvieron asociados positivamente con el número de colisiones para cinco de ocho especies con suficientes observaciones para ser analizadas independientemente. La vegetación alrededor de los edificios influyó sobre las colisiones solamente para una de esas ocho especies Catharus ustulatus). Las historias de vida pronosticaron las colisiones; el número de colisiones fue mayor para las especies migratorias, insectívoras y aquellas que habitan en las zonas boscosas. Nuestros resultados proporcionan una nueva perspectiva hacia las especies más vulnerables a las colisiones contra edificios, lo que las pone en una necesidad potencialmente mayor de atención conservacionista para reducir estas colisiones y de estudio de las variaciones relacionadas con la especie y la historia de vida y las correlaciones de las colisiones contra edificios, información que puede ayudar a refinar el manejo de colisiones.

The role of protein hydrolysates in prolonging viability and enhancing antibody production of CHO cells.

Four independent mAb-producing CHO cell lines were grown in media supplemented with one of seven protein hydrolysates of animal and plant origin. This generated a 7x4 matrix of replicate cultures which was analysed for viable cell density and mAb productivity. In all cultures, a consistent growth rate was shown in batch culture up to 4 to 5 days. Differences between cultures appeared in the decline phase which was followed up to 7 days beyond the start of the cultures. There was a marginal but significant overall increase (x1.1) in the integral viable cell density (IVCD) in the presence of hydrolysate but a more substantial increase in the cell-specific mAb (qMab) productivity (x1.5). There were individual differences between hydrolysates in terms of enhancement of mAb productivity, the highest being a 166% increase of mAb titre (to 117 mg/L) in batch cultures of CHO-EG2 supplemented with UPcotton hydrolysate. The effect of one of the most active hydrolysates (HP7504) on antibody glycosylation was investigated. This showed no change in the predominant seven glycans produced but a significant increase in the galactosylation and sialylation of some but not all the antibodies. Overall, the animal hydrolysate, Primatone and two cotton-derived hydrolysates provided the most substantial benefit for enhanced productivity. The cotton-based hydrolysates can be viewed as valuable supplements for animal-derived component-free (ADCF) media and as a source for the investigation of chemically defined bioactive components. KEY POINTS: • Protein hydrolysates enhanced both IVCD & qMab; the effect on qMab being consistently greater. • Cotton-based hydrolysates showed high bioactivity and potential for use in serum-free media. • Enhanced galactosylation and sialylation was shown for some of the Mabs tested.

Parallel single-cell optical transit dielectrophoresis cytometer.

In this work, we present an optical transit DEP flow cytometer for parallel single-cell analysis. Each cell's dielectric property is inferred from velocity perturbations due to DEP actuation in a microfluidic channel. Dual LED sources facilitate velocity measurement by producing two transit shadows for each cell passing through the channel. These shadows are detected using a 256-pixel linear optical array detector. Massively parallel analysis is possible as each pixel of the detector can independently analyze the passing cells. A wide channel (∼18 mm) was employed to carry many particles simultaneously, and the system was capable of detecting the velocity of over 200 cells simultaneously. We have achieved analysis rates for 10 µm diameter polystyrene spheres response exceeding 250 per second. With appropriate calibration, this DEP cytometer can quantitatively measure the dielectric response. The dielectric response (Clausius-Mossotti factor) of viable CHO cells was measured over the frequency range of 100 kHz to 6 MHz, and the obtained response matches the previously measured values by our group. The DEP cytometer uses simple modular components to achieve high throughput label-free single-cell dielectric analysis and can begin analyzing particles within 10 s after starting to pump the sample into the channel.