Broadening the Scope of Sapofection: Cationic Peptide-Saponin Conjugates Improve Gene Delivery In Vitro and In Vivo.

Gene therapies represent promising new therapeutic options for a variety of indications. However, despite several approved drugs, its potential remains untapped. For polymeric gene delivery, endosomal escape represents a bottleneck. SO1861, a naturally occurring triterpene saponin with endosomal escape properties isolated from Saponaria officinalis L., has been described as additive agent to enhance transfection efficiency (sapofection). However, the challenge to synchronize the saponin and gene delivery system in vivo imposes limitations. Herein, we address this issue by conjugating SO1861 to a peptide-based gene vector using a pH-sensitive hydrazone linker programmed to release SO1861 at the acidic pH of the endosome. Nanoplexes formulated with SO1861-equipped peptides were investigated for transfection efficiency and tolerability in vitro and in vivo. In all investigated cell lines, SO1861-conjugated nanoplexes have shown superior transfection efficiency and cell viability over supplementation of transfection medium with free SO1861. Targeted SO1861-equipped nanoplexes incorporating a targeting peptide were tested in vitro and in vivo in an aggressively growing neuroblastoma allograft model in mice. Using a suicide gene vector encoding the cytotoxic protein saporin, a slowed tumor growth and improved survival rate were observed for targeted SO1861-equipped nanoplexes compared to vehicle control.

Mitigating the Functional Deficit after Neurotoxic Motoneuronal Loss by an Inhibitor of Mitochondrial Fission.

Amyotrophic lateral sclerosis (ALS) is an extremely complex neurodegenerative disease involving different cell types, but motoneuronal loss represents its main pathological feature. Moreover, compensatory plastic changes taking place in parallel to neurodegeneration are likely to affect the timing of ALS onset and progression and, interestingly, they might represent a promising target for disease-modifying treatments. Therefore, a simplified animal model mimicking motoneuronal loss without the other pathological aspects of ALS has been established by means of intramuscular injection of cholera toxin-B saporin (CTB-Sap), which is a targeted neurotoxin able to kill motoneurons by retrograde suicide transport. Previous studies employing the mouse CTB-Sap model have proven that spontaneous motor recovery is possible after a subtotal removal of a spinal motoneuronal pool. Although these kinds of plastic changes are not enough to counteract the functional effects of the progressive motoneuron degeneration, it would nevertheless represent a promising target for treatments aiming to postpone ALS onset and/or delay disease progression. Herein, the mouse CTB-Sap model has been used to test the efficacy of mitochondrial division inhibitor 1 (Mdivi-1) as a tool to counteract the CTB-Sap toxicity and/or to promote neuroplasticity. The homeostasis of mitochondrial fission/fusion dynamics is indeed important for cell integrity, and it could be affected during neurodegeneration. Lesioned mice were treated with Mdivi-1 and then examined by a series of behavioral test and histological analyses. The results have shown that the drug may be capable of reducing functional deficits after the lesion and promoting synaptic plasticity and neuroprotection, thus representing a putative translational approach for motoneuron disorders.

The role of the Ventral Nucleus of the Trapezoid Body in the auditory prepulse inhibition of the acoustic startle reflex.

Cholinergic signaling is essential to mediate the auditory prepulse inhibition (PPI), an operational measure of sensorimotor gating, that refers to the reduction of the acoustic startle reflex (ASR) when a low-intensity, non-startling acoustic stimulus (the prepulse) is presented just before the onset of the acoustic startle stimulus. The cochlear root neurons (CRNs) are the first cells of the ASR circuit to receive cholinergic inputs from non-olivocochlear neurons of the ventral nucleus of the trapezoid body (VNTB) and subsequently decrease their neuronal activity in response to auditory prepulses. Yet, the contribution of the VNTB-CRNs pathway to the mediation of PPI has not been fully elucidated. In this study, we used the immunotoxin anti-choline acetyltransferase (ChAT)-saporin as well as electrolytic lesions of the medial olivocochlear bundle to selectively eliminate cholinergic VNTB neurons, and then assessed the ASR and PPI paradigms. Retrograde track-tracing experiments were conducted to precisely determine the site of lesioning VNTB neurons projecting to the CRNs. Additionally, the effects of VNTB lesions and the integrity of the auditory pathway were evaluated via auditory brain responses tests, ChAT- and FOS-immunohistochemistry. Consequently, we established three experimental groups: 1) intact control rats (non-lesioned), 2) rats with bilateral lesions of the olivocochlear bundle (OCB-lesioned), and 3) rats with bilateral immunolesions affecting both the olivocochlear bundle and the VNTB (OCB/VNTB-lesioned). All experimental groups underwent ASR and PPI tests at several interstimulus intervals before the lesion and 7, 14, and 21 days after it. Our results show that the ASR amplitude remained unaffected both before and after the lesion across all experimental groups, suggesting that the VNTB does not contribute to the ASR. The%PPI increased across the time points of evaluation in the control and OCB-lesioned groups but not in the OCB/VNTB-lesioned group. At the ISI of 50 ms, the OCB-lesioned group exhibited a significant increase in%PPI (p < 0.01), which did not occur in the OCB/VNTB-lesioned group. Therefore, the ablation of cholinergic non-olivocochlear neurons in the OCB/VNTB-lesioned group suggests that these neurons contribute to the mediation of auditory PPI at the 50 ms ISI through their cholinergic projections to CRNs. Our study strongly reinforces the notion that auditory PPI encompasses a complex mechanism of top-down cholinergic modulation, effectively attenuating the ASR across different interstimulus intervals within multiple pathways.

Biocontrol Potential of Sodin 5, Type 1 Ribosome-Inactivating Protein from Salsola soda L. Seeds.

Sodin 5 is a type 1 ribosome-inactivating protein isolated from the seeds of Salsola soda L., an edible halophytic plant that is widespread in southern Europe, close to the coast. This plant, known as 'agretti', is under consideration as a new potential crop on saline soils. Considering a possible defence role of sodin 5 in the plant, we report here its antifungal activity against different halophilic and halotolerant fungi. Our results show that sodin 5 at a concentration of 40 µg/mL (1.4 µM) was able to inhibit the growth of the fungi Trimmatostromma salinum (35.3%), Candida parapsilosis (24.4%), Rhodotorula mucilaginosa (18.2%), Aspergillus flavus (12.2%), and Aureobasidium melanogenum (9.1%). The inhibition observed after 72 h was concentration-dependent. On the other hand, very slight growth inhibition was observed in the fungus Hortaea werneckii (4.2%), which commonly inhabits salterns. In addition, sodin 5 showed a cytotoxic effect on the Sf9 insect cell line, decreasing the survival of these cells to 63% at 1.0 µg/mL (34.5 nM). Structural analysis of sodin 5 revealed that its N-terminal amino acid residue is blocked. Using mass spectrometry, sodin 5 was identified as a homologous to type 1 polynucleotide:adenosine glycosylases, commonly known as ribosome-inactivating proteins from the Amaranthaceae family. Twenty-three percent of its primary structure was determined, including the catalytic site.

Genotoxicity Assessment of Quinoin, a Ribosome Inactivating Protein from Quinoa Seeds, in the Teleost Danio rerio.

BACKGROUND: Ribosome inactivating proteins (RIPs) are N-glycosylases found in various plants that are able to specifically and irreversibly inhibit protein translation, thereby leading to cell death. Their cytotoxic properties have attracted attention in the medical field in the context of developing new anticancer therapies. Quinoin is a novel toxic enzyme obtained from quinoa seeds and classified as a type 1 RIP (Chenopodium quinoa Willd.). Recently, quinoin was found to be cytotoxic to normal fibroblasts and keratinocytes in vitro, as well as to several tumor cell lines. METHODS: The aim of this study was to evaluate the in vitro and in vivo genotoxicity of quinoin in a zebrafish model. We evaluated its ability to induce DNA fragmentation, genomic instability, and reactive oxygen species (ROS) generation by means of terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) reaction, randomly amplified polymorphic DNA (RAPD) Polymerase Chain Reaction (PCR) technique, and dichlorofluorescine (DCF) assay, respectively. RESULTS: Quinoin was found to cause genomic damage in zebrafish, as shown by DNA fragmentation, polymorphic variations leading to genomic instability, and oxidative stress. Interestingly, longer quinoin treatment caused less damage than shorter treatments. CONCLUSIONS: This study demonstrated ROS-mediated genotoxicity of quinoin toward the zebrafish genome. The reduced damage observed after longer quinoin treatment could indicate the activation of detoxification mechanisms, activation of repair mechanisms, or the loss of protein activity due to enzymatic digestion. In order to clarify the genotoxic actions of quinoin, further investigations of the response pathways to DNA damage are needed. Overall, the ability of quinoin to cause breaks and instability in DNA, together with its clear cytotoxicity, make it an interesting candidate for the development of new drugs for cancer treatment.

Lesion of NPY Receptor-expressing Neurons in Perifornical Lateral Hypothalamus Attenuates Glucoprivic Feeding.

Glucoprivic feeding is one of several counterregulatory responses (CRRs) that facilitates restoration of euglycemia following acute glucose deficit (glucoprivation). Our previous work established that glucoprivic feeding requires ventrolateral medullary (VLM) catecholamine (CA) neurons that coexpress neuropeptide Y (NPY). However, the connections by which VLM CA/NPY neurons trigger increased feeding are uncertain. We have previously shown that glucoprivation, induced by an anti-glycolygic agent 2-deoxy-D-glucose (2DG), activates perifornical lateral hypothalamus (PeFLH) neurons and that expression of NPY in the VLM CA/NPY neurons is required for glucoprivic feeding. We therefore hypothesized that glucoprivic feeding and possibly other CRRs require NPY-sensitive PeFLH neurons. To test this, we used the ribosomal toxin conjugate NPY-saporin (NPY-SAP) to selectively lesion NPY receptor-expressing neurons in the PeFLH of male rats. We found that NPY-SAP destroyed a significant number of PeFLH neurons, including those expressing orexin, but not those expressing melanin-concentrating hormone. The PeFLH NPY-SAP lesions attenuated 2DG-induced feeding but did not affect 2DG-induced increase in locomotor activity, sympathoadrenal hyperglycemia, or corticosterone release. The 2DG-induced feeding response was also significantly attenuated in NPY-SAP-treated female rats. Interestingly, PeFLH NPY-SAP lesioned male rats had reduced body weights and decreased dark cycle feeding, but this effect was not seen in female rats. We conclude that a NPY projection to the PeFLH is necessary for glucoprivic feeding, but not locomotor activity, hyperglycemia, or corticosterone release, in both male and female rats.

The effect of NK3-Saporin injection within the arcuate nucleus on puberty, the LH surge, and the response to Senktide in female sheep†.

The timing of puberty onset is reliant on increased gonadotropin-releasing hormone (GnRH). This elicits a corresponding increase in luteinizing hormone (LH) due to a lessening of sensitivity to the inhibitory actions of estradiol (E2). The mechanisms underlying the increase in GnRH release likely involve a subset of neurons within the arcuate (ARC) nucleus of the hypothalamus that contain kisspeptin, neurokinin B (NKB), and dynorphin (KNDy neurons). We aimed to determine if KNDy neurons in female sheep are critical for: timely puberty onset; the LH surge; and the response to an intravenous injection of the neurokinin-3 receptor (NK3R) agonist, senktide. Prepubertal ewes received injections aimed at the ARC containing blank-saporin (control, n = 5) or NK3-saporin (NK3-SAP, n = 6) to ablate neurons expressing NK3R. Blood samples taken 3/week for 65 days following surgery were assessed for progesterone to determine onset of puberty. Control ewes exhibited onset of puberty at 33.2 ± 3.9 days post sampling initiation, whereas 5/6 NK3-SAP treated ewes didn't display an increase in progesterone. After an artificial LH surge protocol, surge amplitude was lower in NK3-SAP ewes. Finally, ewes were treated with senktide to determine if an LH response was elicited. LH pulses were evident in both groups in the absence of injections, but the response to senktide vs saline was similar between groups. These results show that KNDy cells are necessary for timely puberty onset and for full expresson of the LH surge. The occurrence of LH pulses in NK3-SAP treated ewes may indicate a recovery from an apulsatile state.

Regulation of nociception threshold by norepinephrine through adrenergic α2 receptor in rat models of Parkinson's disease.

BACKGROUND: The mechanism of pain symptoms in Parkinson's disease (PD) is unclear. Norepinephrine (NE) regulates neuropathic pain through ascending and descending pathways. However, the loss of NE neurons in the brain of patients with PD is obvious, it is speculated that NE is involved in the occurrence of PD pain symptoms. AIMS: To investigate the effect of NE on the activation of brain cells through adrenergic α2 receptor, so as to regulate the nociception threshold in a 6-OHDA-induced animal model of PD. METHODS: PD rat model was established by 6-OHDA injection (6-OHDA group). DSP-4 (or anti-DBH-saporin) was used to reduce the NE level of the PD rat brain. The heat sensitivity threshold (HST) and pressure withdrawal threshold (PWT) were measured. Tyrosine hydroxylase and NE in rat brains were detected by Elisa. The percentage of GFAP-positive cells in the prefrontal cortex, cingulate gyrus and striatum of rats was detected by immunohistochemistry and immunofluorescence. GFAP protein was semiquantified by method of western blot. Then yohimbine and guanfacine were used to increase the NE level in PD rats, and the above experimental changes were observed after drug application. RESULTS: The contents of NE in the brain of 6-OHDA-induced PD rats were lower than that of control group. After DSP-4 (or anti-DBH-saporin) injection, PD rats showed the lowest NE level (compared with 6-OHDA group, p ≤ 0.05), and after yohimbine and guanfacine were applied to 6-OHDA group, the contents of NE increased in the prefrontal cortex of rats. The HST and PWT of 6-OHDA group were significantly lower than those of control group, and after DSP-4 (or anti-DBH-saporin) injection, the HST and PWT of rats were lower than those of 6-OHDA group, and after the administration of yohimbine and guanfacine, both HST and PWT were significantly increased. GFAP-positive cells increased in prefrontal cortex and anterior cingulate gyrus of 6-OHDA group rats, and more significantly increased after DSP-4 (or anti-DBH-saporin) injection, and significantly reduced after yohimbine and guanfacine were used. CONCLUSIONS: The change of norepinephrine content can affect the activation of prefrontal and cingulate gyrus glial cells and participate in the regulation of nociception threshold in PD rats. Adrenergic α2 receptor agonist and central presynaptic membrane α2 receptor blocker both affect cell activation and improve hyperalgesia.

Lesions of KNDy and Kiss1R Neurons in the Arcuate Nucleus Produce Different Effects on LH Pulse Patterns in Female Sheep.

The current model for the synchronization of GnRH neural activity driving GnRH and LH pulses proposes that a set of arcuate (ARC) neurons that contain kisspeptin, neurokinin B, and dynorphin (KNDy neurons) is the GnRH pulse generator. This study tested the functional role of ovine KNDy neurons in pulse generation and explored the roles of nearby Kiss1 receptor (Kiss1R)-containing cells using lesions produced with saporin (SAP) conjugates. Injection of NK3-SAP ablated over 90% of the KNDy cells, while Kiss-SAP (saporin conjugated to kisspeptin-54) lesioned about two-thirds of the Kiss1R population without affecting KNDy or GnRH cell number. Both lesions produced a dramatic decrease in LH pulse amplitude but had different effects on LH pulse patterns. NK3-SAP increased interpulse interval, but Kiss-SAP did not. In contrast, Kiss-SAP disrupted the regular hourly occurrence of LH pulses, but NK3-SAP did not. Because Kiss1R is not expressed in KNDy cells, HiPlex RNAScope was used to assess the colocalization of 8 neurotransmitters and 3 receptors in ARC Kiss1R-containing cells. Kiss1R cells primarily contained transcript markers for GABA (68%), glutamate (28%), ESR1 (estrogen receptor-α) mRNA, and OPRK1 (kappa opioid receptor) mRNA. These data support the conclusion that KNDy neurons are essential for GnRH pulses in ewes, whereas ARC Kiss1R cells are not but do maintain the amplitude and regularity of GnRH pulses. We thus propose that in sheep, ARC Kiss1R neurons form part of a positive feedback circuit that reinforces the activity of the KNDy neural network, with GABA or glutamate likely being involved.

Differential role of GABAergic and cholinergic ventral pallidal neurons in behavioral despair, conditioned fear memory and active coping.

The ventral pallidum (VP), a major component of the reward circuit, is well-associated with appetitive behaviors. Recent evidence suggests that this basal forebrain nucleus may have an overarching role in affective processing, including behavioral responses to aversive stimuli. We investigated this by utilizing selective immunotoxin lesions and a series of behavioral tests in adult male Wistar rats. We made bilateral GAT1-Saporin, 192-IgG-Saporin or PBS (vehicle) injections into the VP to respectively eliminate GABAergic and cholinergic neurons, and tested the animals in the forced swim test (FST), open field test (OFT), elevated plus maze (EPM), Morris water maze (MWM) and cued fear conditioning. Both GAT1-Saporin and 192-IgG-Saporin injections reduced behavioral despair without altering general locomotor activity. During the acquisition phase of cued fear conditioning, this antidepressant effect was accompanied by reduced freezing and increased darting in the 192-IgG-Saporin group, and increased jumping in the GAT1-Saporin group. In the extinction phase, cholinergic lesions impaired fear memory irrespective of the context, while GABAergic lesions reduced memory durability only during the early phases of extinction in a novel context. In line with this, selective cholinergic, but not GABAergic, lesions impaired spatial memory in the MWM. We observed no consistent effect in anxiety-like behavior assessed in the OFT and EPM. These findings indicate that both the GABAergic and cholinergic neuronal groups of the VP may contribute to emotion regulation through modulation of behavioral despair and acquired fear by suppressing active coping and promoting species-specific passive behaviors.

Structural and functional characterization of the cytotoxic protein ledodin, an atypical ribosome-inactivating protein from shiitake mushroom (Lentinula edodes).

We have purified ledodin, a cytotoxic 22-kDa protein from shiitake mushroom (Lentinula edodes) consisting of a 197 amino acid chain. Ledodin possessed N-glycosylase activity on the sarcin-ricin loop of mammalian 28S rRNA and inhibited protein synthesis. However, it was not active against insect, fungal, and bacterial ribosomes. In vitro and in silico studies suggested that ledodin exhibits a catalytic mechanism like that of DNA glycosylases and plant ribosome-inactivating proteins. Moreover, the sequence and structure of ledodin was not related to any protein of known function, although ledodin-homologous sequences were found in the genome of several species of fungi, some edible, belonging to different orders of the class Agaricomycetes. Therefore, ledodin could be the first of a new family of enzymes widely distributed among this class of basidiomycetes. The interest of these proteins lies both, in the fact that they can be a toxic agent of some edible mushrooms and in their application in medicine and biotechnology.

Streptavidin-Saporin: Converting Biotinylated Materials into Targeted Toxins.

Streptavidin-Saporin can be considered a type of 'secondary' targeted toxin. The scientific community has taken advantage of this conjugate in clever and fruitful ways using many kinds of biotinylated targeting agents to send saporin into a cell selected for elimination. Saporin is a ribosome-inactivating protein that causes inhibition of protein synthesis and cell death when delivered inside a cell. Streptavidin-Saporin, mixed with biotinylated molecules to cell surface markers, results in powerful conjugates that are used both in vitro and in vivo for behavior and disease research. Streptavidin-Saporin harnesses the 'Molecular Surgery' capability of saporin, creating a modular arsenal of targeted toxins used in applications ranging from the screening of potential therapeutics to behavioral studies and animal models. The reagent has become a well-published and validated resource in academia and industry. The ease of use and diverse functionality of Streptavidin-Saporin continues to have a significant impact on the life science industry.

Recombinant anti-HIV MAP30, a ribosome inactivating protein: against plant virus and bacteriophage.

The ribosome inactivating proteins (RIPs) efficiently decrease the microbial infections in plants. Momordica charantia MAP30 is a type I RIP that has not been investigated against plant viruses or bacteriophages. To evaluate of these activities, the recombinant MAP30 (rMAP30) was produced in the hairy roots of Nicotiana tabacum. Inoculation of 3 µg of transgenic total protein or 0.6 µg of rMAP30 against 0.1 µg of TMV reduced the leaf necrotic spots to 78.23% and 82.72%, respectively. The treatment of 0.1 µg of CMV with rMAP30 (0.6 µg) showed the reduction in the leaf necrotic spots to 85.8%. While the infection was increased after rMAP30 dilution. In the time interval assays, the leaves were first inoculated with 1 µg of rMAP30 or 0.1 µg of purified TMV or CMV agent for 6 h, then virus or protein was applied in order. This led the spot reduction to 35.22% and 67% for TMV, and 38.61% and 55.31% for CMV, respectively. In both the pre- and co-treatments of 1:10 or 1:20 diluted bacteriophage with 15 µg of transgenic total protein, the number and diameter of the plaques were reduced. The results showed that the highest inhibitory effect was observed in the pre-treatment assay of bacteriophage with transgenic total protein for 24 h. The decrease in the growth of bacteriophage caused more growth pattern of Escherichia coli. The results confirm that rMAP30 shows antibacterial activity against Streptococcus aureus and E. coli, antifungal activity against Candida albicans, and antiviral activity against CMV and TMV. Moreover, rMAP30 exhibits anti-phage activity for the first time. According to our findings, rMAP30 might be a valuable preservative agent in foods and beverages in the food industry as well as an antiviral and antimicrobial mixture in agriculture.

A Novel EGFR Targeted Immunotoxin Based on Cetuximab and Type 1 RIP Quinoin Overcomes the Cetuximab Resistance in Colorectal Cancer Cells.

Cetuximab is a monoclonal antibody blocking the epidermal growth factor receptor (EGFR) in metastatic colorectal cancer (mCRC). However, cetuximab treatment has no clinical benefits in patients affected by mCRC with KRAS mutation or in the presence of constitutive activation of signalling pathways acting downstream of the EGFR. The aim of this study was to improve cetuximab's therapeutic action by conjugating cetuximab with the type 1 ribosome inactivating protein (RIP) quinoin isolated from quinoa seeds. A chemical conjugation strategy based on the use of heterobifunctional reagent succinimidyl 3-(2-pyridyldithio)propionate (SPDP) was applied to obtain the antibody-type 1 RIP chimeric immunoconjugate. The immunotoxin was then purified by chromatographic technique, and its enzymatic action was evaluated compared to quinoin alone. Functional assays were performed to test the cytotoxic action of the quinoin cetuximab immunoconjugate against the cetuximab-resistant GEO-CR cells. The novel quinoin cetuximab immunoconjugate showed a significant dose-dependent cytotoxicity towards GEO-CR cells, achieving IC50 values of 27.7 nM (~5.0 µg/mL) at 72 h compared to cetuximab (IC50 = 176.7 nM) or quinoin (IC50 = 149.3 nM) alone assayed in equimolar amounts. These results support the therapeutic potential of quinoin cetuximab immunoconjugate for the EGFR targeted therapy, providing a promising candidate for further development towards clinical use in the treatment of cetuximab-resistant metastatic colorectal cancer.

Reactive oxygen species-responsive branched poly (ß-amino ester) with robust efficiency for cytosolic protein delivery.

Protein therapy targeting the intracellular machinery holds great potentials for disease treatment, and therefore, effective cytosolic protein delivery technologies are highly demanded. Herein, we developed reactive oxygen species (ROS)-degradable, branched poly(ß-amino ester) (PBAE) with built-in phenylboronic acid (PBA) in the backbone and terminal-pendent arginine for the efficient cytosolic protein delivery. The PBAE could form stable and cell-ingestible nanocomplexes (NCs) with proteins via electrostatic interaction, nitrogen-boronate (N-B) coordination, and hydrogen bonding, while it can be degraded into small segments by the over-produced H2O2 in tumor cells to enable cytoplasmic protein release. As thus, PBAE exhibited high efficiency in delivering varieties of proteins with distinct molecular weights (12.4-430 kDa) and isoelectric points (4.7-10.5) into tumor cells, including enzymes, toxins, and antibodies. Moreover, PBAE mediated efficient delivery of saporin into tumor cells in vivo, provoking pronounced anti-tumor outcomes. This study provides a robust and versatile platform for cytosolic protein delivery, and the elaborately tailored PBAE may find promising applications for protein-based biological research and disease management. STATEMENT OF SIGNIFICANCE: Cytosolic delivery of native proteins holds great therapeutic potentials, which however, is limited by the lack of robust delivery carriers that can simultaneously feature strong protein encapsulation yet effective intracellular protein release. Herein, ROS-degradable, branched poly(ß-amino ester) (PBAE) with backbone-embedded phenylboronic acid (PBA) and terminal-pendent arginine was developed to synchronize these two processes. PBA and arginine moieties allowed PBAE to encapsulate proteins via N-B coordination, electrostatic interaction, hydrogen bonding, and salt bridging, while PBA could be oxidized by over-produced H2O2 inside cancer cells to trigger PBAE degradation and intracellular protein release. As thus, the top-performing PBAE mediated efficient cytosolic delivery of various proteins including enzymes, toxins, and antibodies. This study provides a powerful platform for cytosolic protein delivery, and may find promising utilities toward intracellular protein therapy against cancer and other diseases such as inflammation.

Long-term nucleus basalis cholinergic depletion induces attentional deficits and impacts cortical neurons and BDNF levels without affecting the NGF synthesis.

Basal forebrain cholinergic neurons (BFCNs) represent the main source of cholinergic innervation to the cortex and hippocampus and degenerate early in Alzheimer's disease (AD) progression. Phenotypic maintenance of BFCNs depends on levels of mature nerve growth factor (mNGF) and mature brain-derived neurotrophic factor (mBDNF), produced by target neurons and retrogradely transported to the cell body. Whether a reciprocal interaction where BFCN inputs impact neurotrophin availability and affect cortical neuronal markers remains unknown. To address our hypothesis, we immunolesioned the nucleus basalis (nb), a basal forebrain cholinergic nuclei projecting mainly to the cortex, by bilateral stereotaxic injection of 192-IgG-Saporin (the cytotoxin Saporin binds p75ntr receptors expressed exclusively by BFCNs) in 2.5-month-old Wistar rats. At 6 months post-lesion, Saporin-injected rats (SAP) showed an impairment in a modified version of the 5-Choice Serial Reaction Time Task (5-choice task). Postmortem analyses of the brain revealed a reduction of Choline Acetyltransferase-immunoreactive neurons compared to wild-type controls. A diminished number of cortical vesicular acetylcholine transporter-immunoreactive boutons was accompanied by a reduction in BDNF mRNA, mBDNF protein levels, markers of glutamatergic (vGluT1), and GABAergic (GAD65) neurons in the SAP-group compared to the controls. NGF mRNA, NGF precursor, and mNGF protein levels were not affected. Additionally, cholinergic markers correlated with the attentional deficit and BDNF levels. Our findings demonstrate that while cholinergic nb loss impairs cognition and reduces cortical neuron markers, it produces differential effects on neurotrophin availability, affecting BDNF but not NGF levels.

Neural Pathway for Gut Feelings: Vagal Interoceptive Feedback From the Gastrointestinal Tract Is a Critical Modulator of Anxiety-like Behavior.

BACKGROUND: Anxiety disorders are associated with an altered perception of the body's internal state. Therefore, understanding the neuronal basis of interoception can foster novel anxiety therapies. In rodents, the feeding status bidirectionally modulates anxiety-like behavior but how the sensing of gastrointestinal state affects anxiety remains unclear. METHODS: We combined chemogenetics, neuropharmacology, and behavioral approaches in male and female rats to test whether vagal afferents terminating in the gastrointestinal tract mediate feeding-induced tuning of anxiety. Using saporin-based lesions and transcriptomics, we investigated the chronic impact of this gut-brain circuit on anxiety-like behavior. RESULTS: Both feeding and selective chemogenetic activation of gut-innervating vagal afferents increased anxiety-like behavior. Conversely, chemogenetic inhibition blocked the increase in anxiety-like behavior induced by feeding. Using a selective saporin-based lesion, we demonstrate that the loss of gut-innervating vagal afferent signaling chronically reduces anxiety-like behavior in male rats but not in female rats. We next identify a vagal circuit that connects the gut to the central nucleus of the amygdala, using anterograde transsynaptic tracing from the nodose ganglia. Lesion of this gut-brain vagal circuit modulated the central amygdala transcriptome in both sexes but selectively affected a network of GABA (gamma-aminobutyric acid)-related genes only in males, suggesting a potentiation of inhibitory control. Blocking GABAergic signaling in the central amygdala re-established normal anxiety levels in male rats. CONCLUSIONS: Vagal sensory signals from the gastrointestinal tract are critical for baseline and feeding-induced tuning of anxiety via the central amygdala in rats. Our results suggest vagal gut-brain signaling as a target to normalize interoception in anxiety disorders.

Analysis of the Sequence Preference of Saporin by Deep Sequencing.

Ribosome-inactivating proteins (RIPs) are RNA:adenosine glycosidases that inactivate eukaryotic ribosomes by depurinating the sarcin-ricin loop (SRL) in 28S rRNA. The GAGA sequence at the top of the SRL or at the top of a hairpin loop is assumed to be their target motif. Saporin is a RIP widely used to develop immunotoxins for research and medical applications, but its sequence specificity has not been investigated. Here, we combine the conventional aniline cleavage assay for depurinated nucleic acids with high-throughput sequencing to study sequence-specific depurination of oligonucleotides caused by saporin. Our data reveal the sequence preference of saporin for different substrates and show that the GAGA motif is not efficiently targeted by this protein, neither in RNA nor in DNA. Instead, a preference of saporin for certain hairpin DNAs was observed. The observed sequence-specific activity of saporin may be relevant to antiviral or apoptosis-inducing effects of RIPs. The developed method could also be useful for studying the sequence specificity of depurination by other RIPs or enzymes.

Prenatal exposure to valproic acid causes allodynia associated with spinal microglial activation.

Autism spectrum disorder (ASD) is a neurodevelopmental disorder characterized by deficits in social communication and social interaction and the presence of restricted, repetitive behaviors. Additionally, difficulties in sensory processing commonly occur in ASD. Sensory abnormalities include heightened or reduced sensitivity to pain, but the mechanism underlying sensory phenotypes in ASD remain unknown. Emerging evidence suggests that microglia play an important role in forming and refining neuronal circuitry, and thus contribute to neuronal plasticity and nociceptive signaling. In the present study, we investigated the age-dependent tactile sensitivity in an animal model of ASD induced by prenatal exposure to valproic acid (VPA) and subsequently assessed the involvement of microglia in the spinal cord in pain processing. Pregnant ICR (CD1) mice were intraperitoneally injected with either saline or VPA (500 mg/kg) on embryonic day 12.5. Male offspring of VPA-treated mothers showed mechanical allodynia at both 4 and 8 weeks of age. In the spinal cord dorsal horn in prenatally VPA-treated mice, the numbers and staining intensities of ionized calcium-binding adapter molecule 1-positive cells were increased and the cell bodies became enlarged, indicating microglial activation. Treatment with PLX3397, a colony-stimulating factor 1 receptor inhibitor, for 10 days resulted in a decreased number of spinal microglia and attenuated mechanical allodynia in adult mice prenatally exposed to VPA. Additionally, intrathecal injection of Mac-1-saporin, a saporin-conjugated anti-CD11b antibody to deplete microglia, abolished mechanical allodynia. These findings suggest that prenatal VPA treatment causes allodynia and that spinal microglia contribute to the increased nociceptive responses.

Molecular cloning and characterization of an alpha-amylase inhibitor (TkAAI) gene from Trichosanthes kirilowii Maxim.

Trichosanthes kirilowii Maxim taxonomically belongs to the Cucurbitaceae family and Trichosanthes genus. Its whole fruit, fruit peel, seed and root are widely used in traditional Chinese medicines. A ribosome-inactivating protein with RNA N-glycosidase activity called Trichosanthrip was isolated and purified from the seeds of T. kirilowii in our recent previous research. To further explore the biological functions of Trichosanthrip, the cDNA of T. kirilowii alpha-amylase inhibitor (TkAAI) was cloned through rapid-amplification of cDNA ends and its sequence was analyzed. Also, the heterologous protein was expressed in Escherichia coli and its alpha-amylase activity was further measured under optimized conditions. The full-length cDNA of TkAAI was 613 bp. The speculated open reading frame sequence encoded 141 amino acids with a molecular weight of 16.14 kDa. Phylogenetic analysis demonstrated that the Alpha-Amylase Inhibitors Seed Storage domain sequence of TkAAI revealed significant evolutionary homology with the 2S albumin derived from the other plants in the Cucurbitaceae group. In addition, TkAAI was assembled into pET28a with eGFP to generate a prokaryotic expression vector and was induced to express in E. coli. The TkAAI-eGFP infusion protein was proven to exhibit alpha-amylase inhibitory activity against porcine pancreatic amylase in a suitable reaction system. Analysis of gene expression patterns proved that the relative expression level of TkAAI in seeds is highest. The results presented here forecasted that the TkAAI might play a crucial role during the development of T. kirilowii seeds and provided fundamental insights into the possibility of T. kirilowii derived medicine to treat diabetes related diseases.