Immunogenic fusion proteins induce neutralizing SARS-CoV-2 antibodies in the serum and milk of sheep.

Antigen-specific polyclonal immunoglobulins derived from the serum, colostrum, or milk of immunized ruminant animals have potential as scalable therapeutics for the control of viral diseases including COVID-19. Here we show that the immunization of sheep with fusions of the SARS-CoV-2 receptor binding domain (RBD) to ovine IgG2a Fc domains promotes significantly higher levels of antigen-specific antibodies compared to native RBD or full-length spike antigens. This antibody population contained elevated levels of neutralizing antibodies that suppressed binding between the RBD and hACE2 receptors in vitro. A second immune-stimulating fusion candidate, Granulocyte-macrophage colony-stimulating factor (GM-CSF), induced high neutralizing responses in select animals but narrowly missed achieving significance. We further demonstrated that the antibodies induced by these fusion antigens were transferred into colostrum/milk and possessed cross-neutralizing activity against diverse SARS-CoV-2 variants. Our findings highlight a new pathway for recombinant antigen design in ruminant animals with applications in immune milk production and animal health.

Patterns of hydrophobicity found in the first and second transmembrane domains of solute transporters suggest a possible role in nascent protein anchoring and organization.

Solute transporters (STs) are an important subgroup of integral membrane proteins that facilitate the translocation of a diverse range of solutes such as sugars, amino acids, and neurotransmitters across cell membranes. Sequence analysis indicates that STs possess multiple stretches of hydrophobic-rich amino acids that are organized into the transmembrane domains (TMDs) of the functional protein, but exactly how the correct spatial arrangement of these domains is achieved remains a challenging problem. We hypothesized that perhaps differences in interdomain hydrophobicity might play some role in this process. To test this hypothesis, we generated a heptadic model of the alpha helix and mapped the average hydrophobicities (coaxial) and hydrophobic moments (radial) of 108 TMDs found in 9 different human ST proteins. Our results, taken together with earlier work from other groups, suggest that spatial patterns of hydrophobicity found in TMDs 1 and 2 are consistent with a role for these domains in the initial anchoring of the nascent ST protein to the endoplasmic reticulum (ER), as it emerges from the ribosome complex and perhaps in the subsequent spatial organisation of STs.

Maternal supplementation with a complex milk lipid mixture during pregnancy and lactation alters neonatal brain lipid composition but lacks effect on cognitive function in rats.

Complex milk lipids (CMLs) provide a critical nutritional source for generating both energy and essential nutrients for the growth of the newborn. The present study investigated nutritional supplementation with a CML containing gangliosides and phospholipids in pregnant and lactating rats on learning behavior and postnatal growth in male offspring. Wistar female rats were supplemented during pregnancy and lactation with either control or CML to provide gangliosides at a dose of 0.01% (low) and 0.05% (high) based on total food intake. The CML-supplemented dams showed no differences in comparison to controls regarding growth, food intake, and litter characteristics. There were significant differences in brain composition in male offspring at postnatal day 2 (P2) with higher concentrations of gangliosides (high dose, P < .05) and lower concentrations of phospholipids (low and high dose, P < .05) in the CML-supplemented groups. The distribution of individual ganglioside species was not significantly different between treatment groups. Brain weight at P2 was also significantly higher in the CML groups. Differences in the brain composition and weight were not significant by weaning (P21). As adults (P80), adiposity was reduced in the low CML-supplemented group compared to controls. No significant differences were detected between any of the treatment groups in any of the behavioral tasks (water maze, object recognition, and operant learning). These data suggest that maternal supplementation with a CML during pregnancy and lactation is safe and has a significant early impact on brain weight and ganglioside and phospholipid content in offspring but did not alter long-term behavioral function using standard behavioral techniques.

NNZ-2591, a novel diketopiperazine, prevented scopolamine-induced acute memory impairment in the adult rat.

In rats, cyclo-L-glycyl-L-2-allylproline (NNZ-2591), a diketopiperazine, is neuroprotective after ischemic brain injury and also improves motor function in a rat model of Parkinson's disease. Given nootropic actions of diketopiperazines, we investigated the effects of and potential role for acetylcholine neurotransmission in NNZ-2591 on spatial memory after scopolamine-induced amnesia in rats. Adult male Wistar rats were assigned to four groups: saline/water; saline/NNZ-2591; scopolamine/water and scopolamine/NNZ-2591. Morris Water Maze (MWM) tasks were used to determine spatial learning and memory. Thirty minutes prior to each of four daily acquisition trials, rats were intraperitoneally injected with either scopolamine (0.5 mg/kg) or saline. Either NNZ-2591 (30 mg/kg) or water was administered orally (gavages) 10 min after the injection. Immediately after completion of the day 4 acquisition trial a spatial probe trial was performed. The brains were then collected for immunohistochemical analysis. Scopolamine impaired spatial learning and memory compared to saline treated group, particularly in the day 1 acquisition trial. NNZ-2591 did not reverse this deficit, however it significantly improved memory retention by showing more time spent in the correct quadrant. NNZ-2591 also counteracted the scopolamine-induced up-regulation of choline-acetyltransferase positive neurons in the striatum and similarly counteracted the increased synaptophysin density in the hippocampus. Furthermore, a scopolamine-independent antagonistic effect on muscarinic M2 acetylcholine receptors was found after NNZ-2591 treatment, supporting its modulation of acetylcholine neurotransmission. The data suggest that NNZ-2591 prevents scopolamine-induced acute impairment in memory and modulation of acetylcholine neurotransmission may be the mode of action underlying the memory improvement.

Supplementation with a mixture of complex lipids derived from milk to growing rats results in improvements in parameters related to growth and cognition.

Alterations in nutritional factors during early development can exert long-term effects on growth, neural function, and associated behaviors. The lipid component of milk provides a critical nutritional source for generating both energy and essential nutrients for the growth of the newborn. The present study, therefore, investigated the hypothesis that nutritional supplementation with a complex milk lipid (CML) preparation, derived from the milk fat globule membrane rich in phospholipids and gangliosides from young rats, has beneficial effects on learning behavior and postnatal growth and development. Male Wistar rat offspring from normal pregnancies were treated from neonatal day 10 until postnatal day 80 with either vehicle or CML at a dose of 0.2% (low) and 1.0% (high) based on total food intake (n = 16 per group). Neonatal dosing was via daily oral gavage, while postweaning dosing was via gel supplementation to a standard chow diet. Animals underwent behavioral tasks related to spatial memory, learning, and cognitive function. Complex milk lipid supplementation significantly increased linear growth rate (P < .05), and the improved growth trajectory was not related to changes in body composition as quantified by dual-energy x-ray absorptiometry scanning or altered plasma lipid profiles. Moreover, this effect was not dose dependent and not attributable to the contribution to total energy intake of the CML composition. Supplementation of the CML to growing rats resulted in statistically significant improvements in parameters related to novelty recognition (P < .02) and spatial memory (P < .05) using standard behavioral techniques, but operant testing showed no significant differences between treatment groups. Supplementation with a CML containing gangliosides had positive growth and learning behavioral effects in young normal growing rats.

Genetic linkage analysis of the X chromosome in autism, with emphasis on the fragile X region.

The higher prevalence of autism in males than in females suggests the possible involvement of the X chromosome. To test the hypothesis that there are mutations increasing susceptibility to autism on the X chromosome, and in particular the distal portion of the long arm that encompasses the FMRI and MECP2 loci, a genetic linkage study was performed. Twenty-two fragile X-negative families multiplex for autism and related disorders were used for the study. Linkage analysis, for markers in the Xq27-q28 region, using model-free likelihood-based analysis, produced a maximum MLOD of 1.7 for the narrowest diagnostic category of the typical autism/severe autism spectrum, and nonparametric analysis produced a maximum non-parametric lod (NPL) score of 2.1 for a broad phenotype diagnostic model. Thus, this study offers modest support for a susceptibility locus for autism within the Xq27-q28 region. Further genetic investigations of this region are warranted.