STUDIES OF THE EFFECTS OF DOPAMINE NEURON STIMULATING PEPTIDES IN RODENT MODELS OF NORMAL AND DYSFUNCTIONAL DOPAMINERGIC SYSTEMS

A theoretical post-translational processing model of the proprotein form of glial cell line-derived neurotrophic factor (GDNF) likely produces three biologically active peptides. The three prospective peptides formed are 5, 11, and 17 amino acid peptides, entitled dopamine neuron stimulating peptide -5 (DNSP-5), -11 (DNSP-11), and -17 (DNSP-17), respectively. The DNSPs were hypothesized to increase dopaminergic neuron function because of their relationship to GDNF: a molecule with established neurotrophic actions on dopaminergic neurons. The DNSPs have the potential to provide a therapeutic molecule similar to GDNF, but with increased ease of delivery and improved bioavailability. Neurochemical effects of DNSPs were examined in the nigrostriatal pathway of normal Fischer 344 rats, and DNSP-11 was found to be the most effective in increasing dopamine neurochemical function. Striatal microdialysis, four weeks after a single intranigral administration of DNSP-11, showed significant increases in the baseline concentrations of dopamine, DOPAC, and HVA. In addition, both, potassium and d-amphetamine-evoked dopamine overflow were significantly increased. DNSP-11 was delivered intranigrally to aged Fischer 344 rats to examine DNSP-11’s ability to improve dopaminergic function in aged dopamine neurons. DNSP-11 affected striatal dopaminergic function 28 days after treatment by decreasing baseline concentrations of dopamine and evoked dopamine release. Investigation of DNSP-11 continued, using two models of neurotoxin-induced dopamine neuron loss that model cell loss associated with Parkinson’s disease. The neuroprotective properties of DNSP-11 were evaluated by delivering DNSP-11 prior to the neurotoxic insult. DNSP-11 treatment was unable to protect dopaminergic neurons, but significantly increased dopamine metabolism. In a model of severe dopamine neuron loss, DNSP-11 treatment significantly improved apomorphine-induced rotation behavior, indicative of alterations in the function of nigrostriatal dopaminergic neurons. Subsequent examination of dopamine content within the SN revealed significant increases in dopamine and DOPAC levels by DNSP-11. Taken together, DNSP-11 treatments modified dopamine neurochemistry in all investigated rodent models. The observed long-term alterations of dopamine neurochemistry by DNSP-11 and subsequent behavioral changes support a potential use for DNSP-11 as a therapeutic for dopaminergic cell loss. Increased dopaminergic function by DNSP-11 is evidence for the novel concept that peptides contained within the prodomain of trophic factors can have neurotrophic actions

Breadth of Vaccinated Cancer Patient Humoral Response to SARS-CoV-2 Spike Protein and RBD Variants

SARS-CoV-2, the virus responsible for the COVID-19 of which several variants have emerged, such as the B.1.351 SARS-CoV-2 variant. The Receptor Binding Domain (RBD), located within the Spike protein is an immunogenic epitope for potent neutralizing antibodies. Current mRNA vaccines encode for the Spike protein, allowing the body to build antigen-specific antibodies. Assays measuring protective antibodies are essential to manage the COVID-19 pandemic and can be used as a platform for variant screening. RBD-foldon 2.2 is a novel antigen produced by fusing RBD with the trimerization domain Fibritin from Bacteriophage T4. Its amino acid sequence is based on the original Wuhan strain. (Breckenridge, 2021). B.1.351 RBD-foldon 2.2 antigen is identical to RBD-foldon 2.2, except it uses the B.1.351 variant RBD sequence. Using cancer patient sera samples, the breadth and robustness of response was examined in comparison to patients that indicated “no chronic conditions”. We hypothesized there would be a difference in humoral response to RBD-variant antigens in COVID-19 vaccinated cancer patients undergoing treatment vs patients with no chronic conditions. For sample selection, cancer patients were age/sex matched to individuals with no underlying health conditions, that received the same mRNA vaccine within 2 weeks of each other. To quantify antibody levels, ELISA end-point titers were performed. ELISAs detected levels of IgG and IgA antibodies against Spike, RBD-foldon, RBD-foldon 2.2, and RBD-foldon B.1.351. (Bushau, 2021). The statistical analysis used was a two-tailed student’s t-test to compare mean value of end-point titers between experimental and control groups. No significant difference between experimental and control groups for any antibody-antigen combination. B.1.351 RBD-foldon appears to elicit a lower response than RBD-foldon 2.2. Lower response may be explained by the mRNA sequence used in current vaccines encodes for original Wuhan SARS-CoV-2 spike protein. The platform is predictive of the level of antibody protection for variant screening

The rapid assessment of aggregated wastewater samples for genomic surveillance of sars-cov-2 on a city-wide scale

Throughout the course of the ongoing SARS-CoV-2 pandemic there has been a need for approaches that enable rapid monitoring of public health using an unbiased and minimally invasive means. A major way this has been accomplished is through the regular assessment of wastewater samples by qRT-PCR to detect the prevalence of viral nucleic acid with respect to time and location. Further expansion of SARS-CoV-2 wastewater monitoring efforts to include the detection of variants of interest/concern through next-generation sequencing has enhanced the understanding of the SARS-CoV-2 outbreak. In this report, we detail the results of a collaborative effort between public health and metropolitan wastewater management authorities and the University of Louisville to monitor the SARS-CoV-2 pandemic through the monitoring of aggregate wastewater samples over a period of 28 weeks. Through the use of next-generation sequencing approaches the polymorphism signatures of Variants of Concern / Interest were evaluated to determine the likelihood of their prevalence within the community on the basis of their relative dominance within sequence datasets. Our data indicate that wastewater monitoring of water quality treatment centers and smaller neighbor-hood-scale catchment areas is a viable means by which the prevalence and genetic variation of SARS-CoV-2 within a metropolitan community of approximately one million individuals may be monitored, as our efforts detected the introduction and emergence of variants of concern in the city of Louisville. Importantly, these efforts confirm that regional emergence and spread of variants of interest/concern may be detected as readily in aggregate wastewater samples as compared to the individual wastewater sheds. Furthermore, the information gained from these efforts enabled targeted public health efforts including increased outreach to at-risk communities and the deployment of mobile or community-focused vaccination campaigns

A Phase 1a/1b Clinical Trial Design to Assess Safety, Acceptability, Pharmacokinetics and Tolerability of Intranasal Q-Griffithsin for COVID-19 Prophylaxis

Background: The COVID-19 pandemic remains an ongoing threat to global public health. Q-Griffithsin (Q-GRFT) is a lectin that has demonstrated potent broad-spectrum inhibitory activity in preclinical studies in models of Nipah virus and the beta coronaviruses SARS-CoV, MERS-CoV, and SARS-CoV-2. Methods: Here, we propose a clinical trial design to test the safety, pharmacokinetics (PK), and tolerability of intranasally administered Q-GRFT for the prevention of SARS-CoV-2 infection as a prophylaxis strategy. The initial Phase 1a study will assess the safety and PK of a single dose of intranasally administered Q-GRFT. If found safe, the safety, PK, and tolerability of multiple doses of intranasal Q-GRFT will be assessed in a Phase 1b study. Group 1 participants will receive 3 mg of intranasal Q-GRFT (200 μL/nostril) once daily for 7 days. If this dose is tolerated, participants will be enrolled in Group 2 to receive 3 mg twice daily for 7 days. Secondary endpoints of the study will be user perceptions, acceptability, and the impact of product use on participants’ olfactory sensation and quality of life. Discussion: Results from this study will support further development of Q-GRFT as a prophylactic against respiratory viral infections in future clinical trials

Technoeconomic Modeling of Plant-Based Griffithsin Manufacturing

Griffithsin is a marine algal lectin that exhibits broad-spectrum antiviral activity by binding oligomannose glycans on viral envelope glycoproteins, including those found in HIV-1, HSV-2, SARS, HCV and other enveloped viruses. An efficient, scalable and cost-effective manufacturing process for Griffithsin is essential for the adoption of this drug in human antiviral prophylaxis and therapy, particularly in cost-sensitive indications such as topical microbicides for HIV-1 prevention. The production of certain classes of recombinant biologics in plants can offer scalability, cost and environmental impact advantages over traditional biomanufacturing platforms. Previously, we showed the technical viability of producing recombinant Griffithsin in plants. In this study, we conducted a technoeconomic analysis (TEA) of plant-produced Griffithsin manufactured at commercial launch volumes for use in HIV microbicides. Data derived from multiple non-sequential manufacturing batches conducted at pilot scale and existing facility designs were used to build a technoeconomic model using SuperPro Designer® modeling software. With an assumed commercial launch volume of 20 kg Griffithsin/year for 6.7 million doses of Griffithsin microbicide at 3 mg/dose, a transient vector expression yield of 0.52 g Griffithsin/kg leaf biomass, recovery efficiency of 70%, and purity of >99%, we calculated a manufacturing cost for the drug substance of $0.32/dose and estimated a bulk product cost of$0.38/dose assuming a 20% net fee for a contract manufacturing organization (CMO). This is the first report modeling the manufacturing economics of Griffithsin. The process analyzed is readily scalable and subject to efficiency improvements and could provide the needed market volumes of the lectin within an acceptable range of costs, even for cost-constrained products such as microbicides. The manufacturing process was also assessed for environmental, health and safety impact and found to have a highly favorable environmental output index with negligible risks to health and safety. The results of this study help validate the plant-based manufacturing platform and should assist in selecting preferred indications for Griffithsin as a novel drug

Surface Hardness Impairment of Quorum Sensing and Swarming for Pseudomonas aeruginosa

The importance of rhamnolipid to swarming of the bacterium Pseudomonas aeruginosa is well established. It is frequently, but not exclusively, observed that P. aeruginosa swarms in tendril patterns—formation of these tendrils requires rhamnolipid. We were interested to explain the impact of surface changes on P. aeruginosa swarm tendril development. Here we report that P. aeruginosa quorum sensing and rhamnolipid production is impaired when growing on harder semi-solid surfaces. P. aeruginosa wild-type swarms showed huge variation in tendril formation with small deviations to the “standard” swarm agar concentration of 0.5%. These macroscopic differences correlated with microscopic investigation of cells close to the advancing swarm edge using fluorescent gene reporters. Tendril swarms showed significant rhlA-gfp reporter expression right up to the advancing edge of swarming cells while swarms without tendrils (grown on harder agar) showed no rhlA-gfp reporter expression near the advancing edge. This difference in rhamnolipid gene expression can be explained by the necessity of quorum sensing for rhamnolipid production. We provide evidence that harder surfaces seem to limit induction of quorum sensing genes near the advancing swarm edge and these localized effects were sufficient to explain the lack of tendril formation on hard agar. We were unable to artificially stimulate rhamnolipid tendril formation with added acyl-homoserine lactone signals or increasing the carbon nutrients. This suggests that quorum sensing on surfaces is controlled in a manner that is not solely population dependent

Closing the loop in primate prefrontal cortex: inter-laminar processing

Prefrontal cortical (PFC) activity in the primate brain emerging from minicolumnar microcircuits plays a critical role in cognitive processes dealing with executive control of behavior. However, the specific operations of columnar laminar processing in prefrontal cortex (PFC) are not completely understood. Here we show via implementation of unique microanatomical recording and stimulating arrays, that minicolumns in PFC are involved in the executive control of behavior in rhesus macaque nonhuman primates (NHPs) performing a delayed-match-to-sample (DMS) task. PFC neurons demonstrate functional interactions between pairs of putative pyramidal cells within specified cortical layers via anatomically oriented minicolumns. Results reveal target-specific, spatially tuned firing between inter-laminar (layer 2/3 and layer 5) pairs of neurons participating in the gating of information during the decision making phase of the task with differential correlations between activity in layer 2/3 and layer 5 in the integration of spatial vs. object-specific information for correct task performance. Such inter-laminar processing was exploited by the interfacing of an online model which delivered stimulation to layer 5 locations in a pattern associated with successful performance thereby closing the columnar loop externally in a manner that mimicked normal processing in the same task. These unique technologies demonstrate that PFC neurons encode and process information via minicolumns which provides a closed loop form of “executive function,” hence disruption of such inter-laminar processing could form the bases for cognitive dysfunction in primate brain

Technoeconomic Modeling of Plant-Based Griffithsin Manufacturing.

Griffithsin is a marine algal lectin that exhibits broad-spectrum antiviral activity by binding oligomannose glycans on viral envelope glycoproteins, including those found in HIV-1, HSV-2, SARS, HCV and other enveloped viruses. An efficient, scalable and cost-effective manufacturing process for Griffithsin is essential for the adoption of this drug in human antiviral prophylaxis and therapy, particularly in cost-sensitive indications such as topical microbicides for HIV-1 prevention. The production of certain classes of recombinant biologics in plants can offer scalability, cost and environmental impact advantages over traditional biomanufacturing platforms. Previously, we showed the technical viability of producing recombinant Griffithsin in plants. In this study, we conducted a technoeconomic analysis (TEA) of plant-produced Griffithsin manufactured at commercial launch volumes for use in HIV microbicides. Data derived from multiple non-sequential manufacturing batches conducted at pilot scale and existing facility designs were used to build a technoeconomic model using SuperPro Designer® modeling software. With an assumed commercial launch volume of 20 kg Griffithsin/year for 6.7 million doses of Griffithsin microbicide at 3 mg/dose, a transient vector expression yield of 0.52 g Griffithsin/kg leaf biomass, recovery efficiency of 70%, and purity of >99%, we calculated a manufacturing cost for the drug substance of $0.32/dose and estimated a bulk product cost of$0.38/dose assuming a 20% net fee for a contract manufacturing organization (CMO). This is the first report modeling the manufacturing economics of Griffithsin. The process analyzed is readily scalable and subject to efficiency improvements and could provide the needed market volumes of the lectin within an acceptable range of costs, even for cost-constrained products such as microbicides. The manufacturing process was also assessed for environmental, health and safety impact and found to have a highly favorable environmental output index with negligible risks to health and safety. The results of this study help validate the plant-based manufacturing platform and should assist in selecting preferred indications for Griffithsin as a novel drug

Conformal ceramic electrodes that record glutamate release and corresponding neural activity in primate prefrontal cortex

Conformal ceramic electrodes utilized in prior recordings of nonhuman primate prefrontal cortical layer 2/3 and layer 5 neurons were used in this study to record tonic glutamate concentration and transient release in layer 2/3 PFC. Tonic glutamate concentration increased in the Match (decision) phase of a visual delayed-match-to-sample (DMS) task, while increased transient glutamate release occurred in the Sample (encoding) phase of the task. Further, spatial vs. object-oriented DMS trials evoked differential changes in glutamate concentration. Thus the same conformal recording electrodes were capable of electrophysiological and electrochemical recording, and revealed similar evidence of neural processing in layers 2/3 and layer 5 during cognitive processing in a behavioral task