Preclinical evaluation of immunogenicity, efficacy and safety of a recombinant plant-based SARS-CoV-2 RBD vaccine formulated with 3M-052-Alum adjuvant.

Cost-effective, and accessible vaccines are needed for mass immunization to control the ongoing coronavirus disease 2019 (COVID-19), especially in low- and middle-income countries (LMIC).A plant-based vaccine is an attractive technology platform since the recombinant proteins can be easily produced at large scale and low cost. For the recombinant subunit-based vaccines, effective adjuvants are crucial to enhance the magnitude and breadth of immune responses elicited by the vaccine. In this study, we report a preclinical evaluation of the immunogenicity, efficacy and safety of a recombinant plant-based SARS-CoV-2 RBD vaccine formulated with 3M-052 (TLR7/8 agonist)-Alum adjuvant. This vaccine formulation, named Baiya SARS-CoV-2 Vax 2, induced significant levels of RBD-specific IgG and neutralizing antibody responses in mice. A viral challenge study using humanized K18-hACE2 mice has shown that animals vaccinated with two doses of Baiya SARS-CoV-2 Vax 2 established immune protection against SARS-CoV-2. A study in nonhuman primates (cynomolgus monkeys) indicated that immunization with two doses of Baiya SARS-CoV-2 Vax 2 was safe, well tolerated, and induced neutralizing antibodies against the prototype virus and other viral variants (Alpha, Beta, Gamma, Delta, and Omicron subvariants). The toxicity of Baiya SARS-CoV-2 Vax 2 was further investigated in Jcl:SD rats, which demonstrated that a single dose and repeated doses of Baiya SARS-CoV-2 Vax 2 were well tolerated and no mortality or unanticipated findings were observed. Overall, these preclinical findings support further clinical development of Baiya SARS-CoV-2 Vax 2.

Effects of Cultured Cordycep militaris on Sexual Performance and Erectile Function in Streptozotocin-Induced Diabetic Male Rats.

Cordyceps militaris (CM), a valuable edible and medicinal fungus, has been used as traditional medicine to treat health conditions, as well as hyposexuality in Asian societies for over a century. Due to the high demand, several artificial cultivation methods have been developed for their biological activities. In this study, CM was cultured on medium that contained white rice and silkworm pupae, and the levels of cordycepin and adenosine, as well as its aphrodisiac effects in diabetes-induced erectile dysfunction (DIED), were evaluated. Diabetic rats were induced by streptozotocin (STZ) injection and administered orally with CM (0.1, 0.5, and 1.0 g/kg BW/day) for 3 weeks. Diabetic rats in negative and positive control groups received vehicle and sildenafil citrate (5 mg/kg), respectively. Results showed the changes in mating behaviour in which mount latency and intromission latency were significantly increased in diabetic rats, compared with the normal control group. Diabetic rats also showed a significant reduction in intracavernosal pressure (ICP) response to cavernous nerve stimulation, sperm count, testosterone level, penile nitric oxide synthase (NOS), and testicular superoxide dismutase (SOD) activities, when compared to the normal control group. Administration of CM (0.1, 0.5, and 1.0 g/kg BW/day) reversed the effects of diabetes on the mating behaviour, and the ICP responses to electrical stimulation. Moreover, the levels of penile NOS, testicular SOD activities, testosterone, and sperm count were significantly increased, and testicular malondialdehyde (MDA) levels were significantly decreased in these treated diabetic rats. Diabetic rats treated with sildenafil showed a significant induction in intromission frequency and NOS and SOD activities, as well as a marked increase in ICP responses. These results suggest that CCM exerts its aphrodisiac effect, possibly through activating testosterone production and suppressing oxidative stress to enhance erectile function in diabetic rats.

Aphrodisiac Activity of Eulophia macrobulbon Extract on Erectile Dysfunction in Male Aged Rats.

This study investigated the effect of Eulophia macrobulbon (EM) extract on sexual performance in aged-related erectile dysfunction (ED) rats. The ethanol EM extract at the doses of 15, 150, and 450 and sildenafil citrate at the dose of 5 mg/kg body weight (BW) were administered orally to the aged male rats once daily for 21 days. Mating parameters and intracavernosal pressure (ICP) were measured to evaluate their sexual and erection functions. Numbers of sperm and sperm motility as well as the diameter of seminiferous tubules were observed. The serum testosterone and 3',5'-cyclic guanosine monophosphate (cGMP) concentration in the rat penile tissue were analyzed. The results showed the significant increased sexual motivation, copulatory performance, and ICP of aged rats treated with sildenafil citrate and all doses of EM extract as compared to control aged rats. Moreover, their serum testosterone levels were slightly increased and significant increase in penile cGMP concentration was observed in these aged rats treated with sildenafil citrate and EM extract. The results suggest that treatment with EM could inhibit activity of PDE5 in penile tissue resulting in the increased cGMP level and bring to the improvement of erectile function and sexual performance.

Phosphodiesterase 5 Inhibitors from Derris scandens.

Phosphodiesterase 5 inhibitors have been used as a first-line medicine for the treatment of erectile dysfunction. In the search for new phosphodiesterase 5 inhibitors from natural sources, we found that the 95% ethanol extract of Derris scandens stem showed phosphodiesterase 5 inhibitory activity with an IC50 value of about 7 µg/mL. Seven isoflavones and a coumarin constituent isolated from this plant were investigated for phosphodiesterase 5 inhibitory activity. The results showed that osajin (8: ), 4',5,7-trihydroxybiprenylisoflavone (4: ), and derrisisoflavone A (2: ) had the ability to inhibit phosphodiesterase 5 with IC50 values of 4, 8, and 9 µM, respectively. These compounds exhibited selectivity on phosphodiesterase 5 over phosphodiesterase 1, however, the selectivity on phosphodiesterase 5 over phosphodiesterase 6 was low. In order to quantitatively determine these bioactive constituents in D. scandens extract, LC-QTOF-MS method has been developed and validated. The limit of quantitation values in the range of 0.1 - 5 µg/mL were obtained. The assay showed satisfactory precision and accuracy. The results from our method showed that the 95% ethanol extract of D. scandens stem was comprised of all eight compounds, with derrisisoflavone A (2: ) and lupalbigenin (3: ) presenting as the major constituents.

Acute toxicity and teratogenicity of α-mangostin in zebrafish embryos.

IMPACT STATEMENT: α-Mangostin has been reported to have anticancer properties both in vitro and in vivo models. Although there are several studies that evaluated the toxicity of the compound in rodent models, we are the first to evaluate the teratogenicity of α-mangostin. In the present work, we found that α-mangostin induced mortality and malformations in zebrafish embryos. In addition, we exhibited that the compound also disrupted the reactive oxygen species and hemoglobin levels. These findings suggest that α-mangostin may possibly cause the same adverse effects on human health. The mechanisms of these toxicological effects of the compound will be further elucidated and the effects found in zebrafish embryos need to be verified in other animal models.

Dopamine and glutamate interaction mediates reinstatement of drug-seeking behavior by stimulation of the ventral subiculum.

BACKGROUND: Drug addiction is a chronic brain disease characterized by recurrent episodes of relapse to drug-seeking/-taking behaviors. The ventral subiculum, the primary output of the hippocampus, plays a critical role in mediating drug-seeking behavior. METHODS: A d-amphetamine intravenous self-administration rat model was employed along with focal electrical stimulation of the ventral subiculum (20 Hz/200 pulses) to examine its role in reinstatement of drug-seeking behavior. Dopamine efflux in the nucleus accumbens was measured by in vivo microdialysis and subsequent HPLC-ED analyses. Pharmacological antagonism of dopamine and ionotropic glutamate receptors locally within the nucleus accumbens was employed to assess the role of glutamate and dopamine in reinstatement of drug-seeking behavior induced by stimulation of the ventral subiculum. RESULTS: Here, we demonstrate that reinstatement of drug-seeking behavior following extinction of d-amphetamine self-administration by rats was induced by electrical stimulation in the ventral subiculum but not the cortex. This reinstatement was accompanied by a significant increase in dopamine efflux in the nucleus accumbens and was disrupted by microinfusion of a dopamine D1 or D2 antagonist into the nucleus accumbens. Inhibition of N-methyl-D-aspartate or non- N-methyl-D-aspartate receptors had no effect on the reinstatement induced by ventral subiculum stimulation, whereas co-infusion of D1 and N-methyl-D-aspartate antagonists at formerly ineffective doses prevented drug-seeking behavior. CONCLUSIONS: These data support the hypothesis that dopamine/glutamate interactions within the ventral striatum related to memory processes are involved in relapse to addictive behavior.

The effects of Tabernaemontana divaricata root extract on amyloid beta-peptide 25-35 peptides induced cognitive deficits in mice.

UNLABELLED: ETHNOPHAMACOLOGICAL RELEVANCE: Tabernaemontana divaricata (TD), a Thai medicinal herb, has been widely used as an analgesic, sedative, or a cough syrup. Moreover, it has been used in traditional rejuvenation remedies as for preventing forgetfulness and improving the memory. AIM OF STUDY: The present study aimed to determine the effect of TD on Abeta25-35 peptides induced cognitive deficits and acetylcholinesterase activity in mice. MATERIALS AND METHODS: Mice were pretreated with TDE (250, 500 and 1,000 mg/kg body weight) for 28 days and then received i.c.v. injection of Abeta25-35 peptides. Cognitive performance was evaluated using the Morris water maze (MWM) and step-down avoidance test. The Ellman's colorimetric method was used to investigate the levels of cortical and hippocampal AChE activity. RESULTS: Abeta25-35 peptides induced the memory impairment and the increased levels of cortical and hippocampal AChE activity. The consumption of TDE significantly improved the memory impairment and attenuated the brain levels of AChE activity induced by Abeta25-35 peptides. CONCLUSIONS: These findings suggest that subchronic administration of TDE might prevent the Abeta25-35 peptides induced memory deficits by decreasing the AChE activity level. Therefore TDE could potentially be one of nootropic supplements for those elderly people suffering from dementia such as the AD patients.

Reductions of acetylcholine release and nerve growth factor expression are correlated with memory impairment induced by interleukin-1beta administrations: effects of omega-3 fatty acid EPA treatment.

Interleukin (IL)-1beta may play an important role in Alzheimer's disease. However, the relationships between glucocorticoids and acetylcholine (ACh), and between neurotrophins and ACh in IL-1-induced memory deficits are unknown. While ethyl-eicosapentaenoate (E-EPA) has recently been reported to reduce inflammation and improve memory, cholinergic and neurotrophic mechanisms by which E-EPA improves memory is unclear. This study evaluated: (i) the correlation between ACh release and memory impairment; (ii) the effect of glucocorticoids on ACh release; (iii) the relationship between nerve growth factor (NGF) and inflammation; and (iv) the effects of E-EPA treatment on IL-1beta-induced changes. Intracerebroventricular IL-1beta administrations produced a significant reduction in hippocampal ACh release in rats fed control diet, which was partially attenuated by mifepristone (RU 486) and completely blocked by IL-1 receptor antagonist. In eight-arm radial maze, significantly less ACh release was correlated with the memory deficits after IL-1beta administrations. mRNA expression of hippocampal NGF was lower, whereas IL-1beta was higher when compared with controls. E-EPA treatment significantly improved the memory, which was correlated with normalizing ACh release, and expressions of NGF and IL-1beta. This study revealed important mechanisms by which IL-1beta impairs, while E-EPA improves memory through IL-1-glucocorticoid-ACh release and IL-1-NGF-ACh release pathways.

Neural circuits engaged in ventral hippocampal modulation of dopamine function in medial prefrontal cortex and ventral striatum.

Dopamine (DA) transmission in the medial prefrontal cortex (mPFC) and nucleus accumbens (NAc) is crucial for various cognitive processes. However, our understanding of the regulation of DA efflux by glutamatergic afferents to these areas is incomplete. Using microdialysis in freely moving rats, we provide evidence in the present study that brief stimulation (20 Hz, 10 s) of the ventral hippocampus potently increases DA efflux in the mPFC, NAc, and ventral tegmental area for 30-40 min. Subsequent experiments show that the stimulation-evoked increase in DA efflux in the mPFC depends on local activation of alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA)/kainate, but not N-methyl-D-aspartate, receptors in the mPFC. Additionally, neural activity and ionotropic glutamate receptor activation in the ventral tegmental area are necessary for ventral hippocampal stimulation to increase mPFC DA efflux. Blocking neural activity or ionotropic glutamate receptors in the ventral tegmental area also attenuated the stimulation-evoked increase in DA efflux in the NAc. Evidence in support of a role for the mPFC in the stimulation-evoked increase in NAc DA was not obtained. Taken together, these observations highlight the important role of the ventral hippocampus in modulating forebrain DA efflux via separate neural circuits.

Electrical stimulation of the hippocampus disrupts prepulse inhibition in rats: frequency- and site-dependent effects.

Prepulse inhibition (PPI) is a normal reduction in the startle response produced when a brief, low intensity stimulus is presented prior to a startle-evoking stimulus. PPI is often disrupted in humans diagnosed with schizophrenia. As similar stimuli elicit PPI in rodents and humans, interventions in rodents that disrupt PPI may reveal aspects of neuronal dysfunction relevant to schizophrenia. Stimulation of the ventral hippocampus (vHip) with NMDA significantly increases dopamine (DA) efflux in the nucleus accumbens (NAc) and disrupts PPI, whereas NMDA infusion into the dorsal hippocampus (dHip) fails to alter PPI. Our previous research shows that brief periods of 20 Hz electrical vHip stimulation also significantly increase NAc DA efflux. The present experiments assessed the effects of stimulating the vHip or dHip on PPI and NAc DA efflux. As predicted, 20 Hz stimulation (10 s, 300 microA) of the vHip, but not the dHip, reversibly disrupted PPI. In contrast, 2 Hz stimulation (100 s, 300 microA) of the vHip failed to affect PPI. Microdialysis experiments revealed that 20 Hz stimulation of the vHip increased NAc DA efflux only in the hemisphere ipsilateral to the stimulating electrode, whereas 20 Hz stimulation of the dHip failed to affect NAc DA efflux. These data demonstrate the regional specificity and frequency-dependent effects of hippocampal activity on PPI. Additionally, it is intriguing that both chemical and electrical stimulation of the vHip disrupt PPI and increase NAc DA efflux, however, the relevance of these changes in NAc DA efflux to the disruption of PPI remains to be determined.

Neurochemical correlates of relapse to d-amphetamine self-administration by rats induced by stimulation of the ventral subiculum.

RATIONALE AND OBJECTIVES: Previous studies show that electrical stimulation of the ventral subiculum (vSub) can reinstate drug-seeking behavior in rats following extinction. This study examined whether vSub stimulation could also evoke reinitiation of d-amphetamine (d-AMPH) self-administration during voluntary abstinence following a prolonged bout of drug intake. Dynamic changes in extracellular levels of dopamine (DA) and metabolites in the nucleus accumbens (NAc) during 48-h unlimited access to d-AMPH by rats were monitored. Neurochemical correlates of relapse to d-AMPH administration induced by vSub stimulation or by experimenter administered d-AMPH infusions were also examined during voluntary abstinence in separate experiments. METHODS AND RESULTS: In vivo microdialysis using high-pressure liquid chromatography with electrochemical detector (HPLC-EC) was used to monitor changes in DA and metabolite efflux in the NAc during a continuous access-abstinence-relapse cycle of d-AMPH self-administration in a 48-h test. The initial pattern of drug intake was associated with significant increases in DA efflux in the NAc. During the abstinence phase, DA efflux was near pre-session baseline values. Electrical stimulation at the vSub after 2 h of abstinence immediately induced a significant increase in DA efflux and reinstatement of drug self-administration behavior. Evoked DA release and responses on drug-paired lever induced by vSub stimulation were significantly greater than those induced by experimenter-administered d-AMPH. CONCLUSIONS: Relapse to drug-taking behavior can be triggered by activation of the subicular glutamatergic pathway to the NAc. This study also confirmed that during abstinence the neurochemical response of the mesolimbic DA system to d-AMPH is attenuated and this can be reversed by vSub stimulation.

Glutamate receptor-dependent modulation of dopamine efflux in the nucleus accumbens by basolateral, but not central, nucleus of the amygdala in rats.

Dopaminergic neurotransmission in the nucleus accumbens (NAc) and neural processes in the basolateral (BLA) and central (CeN) amygdala nuclei are implicated in associative reward learning. Given their direct and indirect connections with the NAc and ventral tegmental area (VTA), both the BLA and CeN may regulate the mesoaccumbens dopamine (DA) system in rewarding situations. Electrical stimulation of the BLA (20 Hz, 10 sec, 300 microA) induced a long-lasting 25 +/- 4% increase in DA efflux in the NAc, measured by microdialysis in freely moving rats, whereas comparable stimulation of the CeN had no effect. Reverse dialysis of either the NMDA receptor antagonist APV (100 micrometer) or the AMPA-kainate receptor antagonist DNQX (100 micrometer), but not the metabotropic glutamate receptor antagonist (+/-)-amino-4-carboxy-methyl-phenylacetic acid (100 micrometer), into the NAc blocked the stimulation-evoked increase in DA efflux in the NAc. VTA infusion of lidocaine (lido; 4%) significantly reduced basal DA levels for approximately 30 min but failed to suppress the increase in NAc DA efflux resulting from BLA stimulation. Additionally, infusions of lido (4%) into the medial prefrontal cortex failed to block the stimulation-evoked increase in NAc DA efflux. These data support the hypothesis that the BLA can directly modulate DA efflux through local mechanisms in the NAc, independent of an action on DA cell bodies in the VTA. The finding that brief activation of the CeN had no long-lasting effects on DA efflux in the NAc suggests an important degree of functional independence between the CeN and BLA.