Long-term antigen-specific immune response by an oncolytic adenovirus encoding SP-SA-E7-4-1BBL in HPV-16 cancer model.

BACKGROUND: To describe an oncolytic adenovirus (OAd) encoding SP-SA-E7-4-1BBL that is capable of inducing tumor regression in therapeutic assays. Herein, we tested whether the antitumor effect is given by the induction of a tumor-specific immune response, as well as the minimum dose needed to elicit antitumor protection and monitor the OAd biodistribution over time. METHODS AND RESULTS: C57BL/6 mice (n = 5) per group were immunized twice with OAds encoding SP-SA-E7-4-1BBL, SA-E7-4-1BBL, or SP-SA-4-1BBL and challenged with TC-1 cancer cells. The DNA construct SP-SA-E7-4-1BBL was employed as a control via biolistic or PBS injection. Groups without tumor development at 47 days were rechallenged with TC-1 cells, and follow-up lasted until day 90. The minimum dose of OAd to induce the antitumor effect was established by immunization using serial dilution doses. The cytometry bead assay and the ELISpot assay were used to evaluate cytokine release in response to ex vivo antigenic stimulation. The distribution profile of the OAd vaccine was evaluated in the different organs by histological, immunohistochemical and qPCR analyses. The OAd SP-SA-E7-4-1BBL-immunized mice did not develop tumors even in a rechallenge. A protective antitumor effect was observed from a dose that is one hundredth of most reports of adenoviral vaccines. Immunization with OAd increases Interferon-gamma-producing cells in response to antigen stimulation. OAd was detected in tumors over time, with significant morphological changes, contrary to nontumor tissues. CONCLUSIONS: The OAd SP-SA-E7-4-1BBL vaccine confers a prophylactic, safe, long-lasting, and antigen-dependent antitumor effect mediated by a Th1 antitumor immune response.

Cooperative Binding of SRSF3 to Structured 3'ss-α Exon RNA during α Exon Inclusion in the ZO-1 mRNA.

ZO-1α+ and ZO-1α- proteins are expressed in hermetic and leaky tight junctions, respectively. Two cis-acting distant exonic elements partly activate the 240 nucleotide-long α exon producing the ZO-1α+ isoform. However, the elements within and around the α exon and their respective factors involved in its splicing are unknown. To study the dynamic interaction between SRSF3 and its bioinformatically predicted target sites around the 3'ss upstream of the α exon during its activation, we performed EMSA, crosslinking, and in vivo splicing assays by ZO-1 minigene expression and siRNA-mediated silencing in transfected cells. Using V1 RNase, we probed the possible formation of a hairpin RNA structure between the intronic and proximal exonic SRSF3 binding sites. The hairpin sufficed for complex formations in the EMSA. The interaction of SRSF3 with the intronic site promoted the cooperative binding of SRSF3 to the exonic site. Finally, SRSF3 restored α exon activation in SRSF3 knockdown transfectants. Altogether, our results show that SRSF3-hairpin RNA interaction is crucial in the early recognition of 3'ss for α exon activation. It remains to be explored whether SRSF3 recruits or stabilizes the binding of other factors or brings separate splice sites into proximity.

Sustainable, technological, and innovative challenges post Covid-19 in health, economy, and education sectors.

The impact of the Covid-19 pandemic has caused an evolution in the business use of Big Data, Artificial Intelligence and New Technologies in general. The general objective of the article is to assess how this process developed during the pandemic in the use and standardization of Big Data, digitalization, the use of data in the private sector and in the public administration and to assess whether it has been used to modernize and digitalize the post-pandemic society. The specific objectives of the article are: 1) the impact of new technologies on society during confinement; 2) to understand the use of Big Data for the creation of new products and businesses and 3) to assess which businesses and companies and from which economic sectors have emerged, which have been transformed and which have disappeared.

Ceramide synthase 2 knockdown suppresses trophozoite growth, migration, in vitro encystment and excystment of Entamoeba invadens.

Entamoeba invadens is the protozoan which causes multiple damages in reptiles and is considered a prototype for the study of the Entamoeba encystment/excystment in vitro. Here we report that EinCerS2 knockdown promoted decrease in sphingomyelin (SM) subspecies with long-chain fatty acids (24:0) down to 50% but increase sphingolipids with short-chain fatty acids (16:0) up to three times in both trophozoites and cysts of E. invadens. EinCerS2 silencing also resulted in decreased trophozoites' movement, proliferation, cysts formation, and trophozoites hatched after excystment. By immunofluorescence assays, a polyclonal antibody against EinCerS2 detected the enzyme in the cytoplasm of E. invadens trophozoites, colocalizing with Endoplasmic Reticulum-resident cognate EiSERCA. Interestingly, EinCerS2 was redistributed close to the plasma membrane during encystation, suggesting that the generation of diacylglycerol (DAG) via synthesis of sphingolipids and the activation protein kinase C might participate in the encystment process of E. invadens.

The metabolic pathway of sphingolipids biosynthesis and signaling in Entamoeba histolytica.

Sphingolipids (SLs) synthesis involves a complex metabolic pathway occurring between the endoplasmic reticulum (ER) and Golgi apparatus, generating ceramide synthesis and complex lipids, respectively. Here we show that E. histolytica, apparently lacking cellular organelles (ER and Golgi apparatus), synthesizes a wide variety of sphingolipid subspecies, being particularly abundant those of long-chain fatty acids. In silico analysis showed five putative genes coding for ceramide synthases (CerS), all of them coding for proteins containing the TLC domain, a region conserved in CerS of multiple organisms. These genes are abundantly expressed in different growth phases. Silencing and overexpression of CerS C4M4U4 (the closest homolog of human CerS 2 and 3) demonstrated its involvement in the synthesis of ceramide. Additionally, we identify C4M4U4, SMS2 and PKC (α, ßII) proteins and their subcellular localization of E. histolytica, suggesting that these subcellular compartments might be involved in the biosynthesis and signaling pathway of sphingolipids, and evidencing different sphingolipid synthesis pathways in Entamoeba.

In silico analysis of putative metal response elements (MREs) in the zinc-responsive genes from Trichomonas vaginalis and the identification of novel palindromic MRE-like motif.

Zinc is an essential micronutrient that plays an important role as a co-factor to several proteins, including zinc-responsive transcription factors. Trichomonas vaginalis is able to survive in the presence of high zinc concentrations in the male urogenital tract. Several genes in T. vaginalis have been shown to respond to changes in zinc concentrations, however, the zinc-dependent mechanism remains undetermined. Recently, we identified in T. vaginalis the zinc finger protein, TvZNF1, which is an ortholog of the mammal metal transcription factor (MTF1). We searched for several of the zinc-responsive genes in T. vaginalis to determine whether if they contain metal response elements (MRE), cis-acting DNA elements that specifically bind MTF1. Six highly conserved over-represented sequence motifs (TvMREs), which share similarity with other eukaryotic MREs, were identified in the zinc-responsive genes in T. vaginalis. We also demonstrated that some of the TvMREs assemble as divalent complexes either as two closely spaced TvMREs or as two overlapping TvMREs forming a palindromic-like sequence: TGCC(N3)GGCA. Electrophoretic mobility shift assays were used to detect the zinc-dependent binding of TvZNF1 and nuclear proteins from T. vaginalis to this specific palindromic motif. Our results support a novel mechanism used by T. vaginalis for the transcriptional regulation of associated zinc-responsive genes through a MTF1/MRE-like system.

Clinical characteristics and severity of COVID-19 among Mexican adults.

INTRODUCTION: Obesity, diabetes, hypertension and age have been pointed at as factors that influence on the progression of COVID-19; however, evidence for other conditions is inconclusive. OBJECTIVE: To identify which clinical characteristics are related to COVID-19 severity and to determine whether age acts a modifier of the relationship between cardio-metabolic comorbidities (CMC) and COVID-19 progression. METHOD: Data on ≥ 20-year-old confirmed cases (n = 159,017) were analyzed. Hospitalization, development of pneumonia, intubation requirement, intensive care unit admission and death were the dependent variables in Poisson regression models estimation, whereas the interaction between age and different CMCs were the independent variables. RESULTS: Having CMCs, as well as other comorbidities, was directly related to COVID-19 progression, whereas chronic obstructive pulmonary disease was only related to an increase in the risk of dying. The risk for COVID-19 severity was lower as age was more advanced. Asthma and smoking were not risk factors for the progression of COVID-19. CONCLUSION: In the Mexican population, the risk of COVID-19 progression associated with comorbidities was higher in young adults.

INTRODUCCIÓN: Se ha señalado que factores como obesidad, diabetes, hipertensión y edad influyen en la progresión de COVID-19; sin embargo, la evidencia para otras condiciones no es concluyente. OBJETIVO: Identificar qué antecedentes clínicos están relacionados con la gravedad de COVID-19 y si la edad funge como un modificador de efecto de la relación entre comorbilidades cardiometabólicas (CCM) y progresión de COVID-19. MÉTODO: Se analizaron los datos de casos confirmados ≥ 20 años (n = 155 017). La hospitalización, el desarrollo de neumonía, el requerimiento de intubación, el ingreso a la unidad de cuidados intensivos y la muerte constituyeron las variables dependientes en la estimación de modelos de regresión de Poisson y la interacción entre edad y CCM, las independientes. RESULTADOS: Tener CCM, así como otras comorbilidades, se relacionó directamente con la progresión de COVID-19. El riesgo de gravedad de COVID-19 asociado a las CCM fue menor conforme la edad era mayor. El asma y el tabaquismo no fueron factores de riesgo para la progresión de COVID-19. CONCLUSIÓN: En la población mexicana, el riesgo de progresión de COVID-19 asociada a comorbilidades fue mayor en los adultos jóvenes.

Ouabain Modulates the Adherens Junction in Renal Epithelial Cells.

BACKGROUND/AIMS: Ouabain, a well-known plant-derived toxin, is also a hormone found in mammals at nanomolar levels that binds to a site located in the a-subunit of Na⁺,K⁺-ATPase. Our main goal was to understand the physiological roles of ouabain. Previously, we found that ouabain increases the degree of tight junction sealing, GAP junction-mediated communication and ciliogenesis. Considering our previous results, we investigated the effect of ouabain on adherens junctions. METHODS: We used immunofluorescence and immunoblot methods to measure the effect of 10 nM ouabain on the cellular and nuclear content of E-cadherin, ß-catenin and γ-catenin in cultured monolayers of Marin Darby canine renal cells (MDCK). We also studied the effect of ouabain on adherens junction biogenesis through sequential Ca²âº removal and replenishment. Then, we investigated whether c-Src and ERK1/2 kinases are involved in these responses. RESULTS: Ouabain enhanced the cellular content of the adherens junction proteins E-cadherin, ß-catenin and γ-catenin and displaced ß-catenin and γ-catenin from the plasma membrane into the nucleus. Ouabain also increased the expression levels of E-cadherin and ß-catenin in the plasma membrane after Ca²âº replenishment. These effects on adherens junctions were sensitive to PP2 and PD98059, suggesting that they depend on c-Src and ERK1/2 signaling. The translocation of ß-catenin and γ-catenin into the nucleus was specific because ouabain did not change the localization of the tight junction proteins ZO-1 and ZO-2. Moreover, in ouabain-resistant MDCK cells, which express a Na⁺,K⁺-ATPase α1-subunit with low affinity for ouabain, this hormone was unable to regulate adherens junctions, indicating that the ouabain receptor that regulates adherens junctions is Na⁺,K⁺-ATPase. CONCLUSION: Ouabain (10 nM) upregulated adherens junctions. This novel result supports the proposition that one of the physiological roles of this hormone is the modulation of cell contacts.

De novo synthesis of sphingolipids plays an important role during in vitro encystment of Entamoeba invadens.

Entamoeba invadens is a protozoan, which causes multiple damages in reptiles and is considered a prototype for the study of the Entamoeba encystment in vitro. Here we report for the first time the role of the de novo synthesis pathway of sphingolipids during the encystment of E. invadens. In silico analysis showed that this parasite has six putative genes coding for ceramide synthases (CerS), all of them coding for proteins containing the Lag1p motif, a region conserved in the ceramide synthases of multiple organisms, suggesting that they might be bona fide CerS. The six genes of E. invadens are differentially expressed at different time intervals in both stages trophozoite and cyst, based on the results obtained through qRT-PCR assays, the genes involved in the synthesis of sphingolipids with long-chain fatty acids CerS 2,3,4 (EIN_046610, EIN_097030, EIN_130350) have maximum points of relative expression in both stages of the E. invadens life cycle, which strongly suggest that the signaling exerted from the synthesis pathway of sphingolipids is essential for the encystment of E. invadens, since the generation of the more abundant sphingomyelin (SM) subspecies with long-chain fatty acids are fundamental for the parasite to reach its conversion from trophozoite to cyst. When myriocin was used as an inhibitor of serine palmitoyl CoA transferase (SPT), first enzyme in the de novo biosynthesis of sphingolipids, the trophozoites of E. invadens were unable to reach the encystment. Since the effect of myriocin was reversed with exogenous d-erythrosphingosine (DHS), it was demonstrated that the inhibition was specific and it was confirmed that the synthesis of sphingolipids play an essential role during the encystment process of E. invadens.

The mRVG-9R peptide as a potential therapeutic vector to the central nervous system cells.

Our research group has developed a cell-penetrating peptide-based delivery system that includes the Asn194Lys mutation in the rabies virus glycoprotein-9R peptide (mRVG-9R). This system has the capacity to deliver DNA in astrocytes and SH-SY5Y cells. The aim of this study was to evaluate the ability of the mRVG-9R peptide to deliver DNA molecules to murine brain cells. The mRVG-9R peptide, a karyophilic peptide (KP) and a plasmid encoding green fluorescent protein (GFP) were bound by electrostatic charges to form the mRVG-9R complex. mRVG-9R complex was injected into the cerebral cortex, striatum and hippocampus of C57BL/6 mice by stereotactic surgery. After 2, 4, and 20 days, the animals were sacrificed and their brains were prepared for quantitative reverse-transcription polymerase chain reaction and histological analysis. We detected the GFP expression in neurons and glial cells in the cerebral cortex, striatum, and hippocampus of the murine brain. The results suggest that the mRVG-9R peptide has the ability to deliver DNA molecules to murine brain cells. Also, the expression of the reporter gene is maintained at least up to 20 days after injection in neurons, astrocytes, oligodendrocytes, and microglia cells. Thus, the in vivo transfection ability of the mRVG-9R peptide, makes it a promising candidate as a therapeutic gene delivery vector to the central nervous system cells.

Kidney-specific WNK1 isoform (KS-WNK1) is a potent activator of WNK4 and NCC.

Familial hyperkalemic hypertension (FHHt) can be mainly attributed to increased activity of the renal Na+:Cl- cotransporter (NCC), which is caused by altered expression and regulation of the with-no-lysine (K) 1 (WNK1) or WNK4 kinases. The WNK1 gene gives rise to a kidney-specific isoform that lacks the kinase domain (KS-WNK1), the expression of which occurs primarily in the distal convoluted tubule. The role played by KS-WNK1 in the modulation of the WNK/STE20-proline-alanine rich kinase (SPAK)/NCC pathway remains elusive. In the present study, we assessed the effect of human KS-WNK1 on NCC activity and on the WNK4-SPAK pathway. Microinjection of oocytes with human KS-WNK1 cRNA induces remarkable activation and phosphorylation of SPAK and NCC. The effect of KS-WNK1 was abrogated by eliminating a WNK-WNK-interacting domain and by a specific WNK inhibitor, WNK463, indicating that the activation of SPAK/NCC by KS-WNK1 is due to interaction with another WNK kinase. Under control conditions in oocytes, the activating serine 335 of the WNK4 T loop is not phosphorylated. In contrast, this serine becomes phosphorylated when the intracellular chloride concentration ([Cl-]i) is reduced or when KS-WNK1 is coexpressed with WNK4. KS-WNK1-mediated activation of WNK4 is not due to a decrease of the [Cl-]i. Coimmunoprecipitation analysis revealed that KS-WNK1 and WNK4 interact with each other and that WNK4 becomes autophosphorylated at serine 335 when it is associated with KS-WNK1. Together, these observations suggest that WNK4 becomes active in the presence of KS-WNK1, despite a constant [Cl-]i.

TvZNF1 is a C2H2 zinc finger protein of Trichomonas vaginalis.

The zinc fingers proteins (ZNF) are the largest family of DNA binding proteins and can act as transcriptional factors in eukaryotes. ZNF are implicated in activation in response to environmental stimulus by biometals such as Zn2+. Many of these proteins have the classical C2H2 zinc finger motifs (C2H2-ZNFm) of approximately 30 amino acids, where a Zn2+ ion is coordinated by two cysteine and two histidine residues. Trichomonas vaginalis is a protozoan parasite than responds to environmental changes including Zn2+. Until now has not been described any ZNF that could be involved in the regulation of genic expression of T. vaginalis. Here, we characterized in silico and experimentally an annoted ZNF (TvZNF1) from T. vaginalis and isolated the gene, tvznf1 encoding it. TvZNF1 have eight C2H2-ZNFm with residues that maybe involved in the structural stability of DNA binding motifs. In this work we confirmed the Zn2+ upregulation expression of tvznf1 gene. Recombinant TvZNF1 was able to bind to specific DNA sequences according to EMSA assay. Additionally, we demonstrated that recombinant TvZNF1 bind to MRE signature in vitro, which strongly suggests its role in transcriptional regulation, similar to the one observed for mammalian MTF-1. This result suggested a conserved mechanism of genic regulation mediated by ZNFs in T. vaginalis.

The Effect of WNK4 on the Na+-Cl- Cotransporter Is Modulated by Intracellular Chloride.

It is widely recognized that the phenotype of familial hyperkalemic hypertension is mainly a consequence of increased activity of the renal Na(+)-Cl(-) cotransporter (NCC) because of altered regulation by with no-lysine-kinase 1 (WNK1) or WNK4. The effect of WNK4 on NCC, however, has been controversial because both inhibition and activation have been reported. It has been recently shown that the long isoform of WNK1 (L-WNK1) is a chloride-sensitive kinase activated by a low Cl(-) concentration. Therefore, we hypothesized that WNK4 effects on NCC could be modulated by intracellular chloride concentration ([Cl(-)]i), and we tested this hypothesis in oocytes injected with NCC cRNA with or without WNK4 cRNA. At baseline in oocytes, [Cl(-)]i was near 50 mM, autophosphorylation of WNK4 was undetectable, and NCC activity was either decreased or unaffected by WNK4. A reduction of [Cl(-)]i, either by low chloride hypotonic stress or coinjection of oocytes with the solute carrier family 26 (anion exchanger)-member 9 (SLC26A9) cRNA, promoted WNK4 autophosphorylation and increased NCC-dependent Na(+) transport in a WNK4-dependent manner. Substitution of the leucine with phenylalanine at residue 322 of WNK4, homologous to the chloride-binding pocket in L-WNK1, converted WNK4 into a constitutively autophosphorylated kinase that activated NCC, even without chloride depletion. Elimination of the catalytic activity (D321A or D321K-K186D) or the autophosphorylation site (S335A) in mutant WNK4-L322F abrogated the positive effect on NCC. These observations suggest that WNK4 can exert differential effects on NCC, depending on the intracellular chloride concentration.

The ß1-subunit of the MaxiK channel associates with the thromboxane A2 receptor and reduces thromboxane A2 functional effects.

The large conductance voltage- and Ca(2+)-activated K(+) channel (MaxiK, BK(Ca), BK) is composed of four pore-forming α-subunits and can be associated with regulatory ß-subunits. One of the functional roles of MaxiK is to regulate vascular tone. We recently found that the MaxiK channel from coronary smooth muscle is trans-inhibited by activation of the vasoconstricting thromboxane A(2) prostanoid receptor (TP), a mechanism supported by MaxiK α-subunit (MaxiKα)-TP physical interaction. Here, we examined the role of the MaxiK ß1-subunit in TP-MaxiK association. We found that the ß1-subunit can by itself interact with TP and that this association can occur independently of MaxiKα. Subcellular localization analysis revealed that ß1 and TP are closely associated at the cell periphery. The molecular mechanism of ß1-TP interaction involves predominantly the ß1 extracellular loop. As reported previously, TP activation by the thromboxane A(2) analog U46619 caused inhibition of MaxiKα macroscopic conductance or fractional open probability (FP(o)) as a function of voltage. However, the positive shift of the FP(o) versus voltage curve by U46619 relative to the control was less prominent when ß1 was coexpressed with TP and MaxiKα proteins (20 ± 6 mV, n = 7) than in cells expressing TP and MaxiKα alone (51 ± 7 mV, n = 7). Finally, ß1 gene ablation reduced the EC(50) of the U46619 agonist in mediating aortic contraction from 18 ± 1 nm (n = 12) to 9 ± 1 nm (n = 12). The results indicate that the ß1-subunit can form a tripartite complex with TP and MaxiKα, has the ability to associate with each protein independently, and diminishes U46619-induced MaxiK channel trans-inhibition as well as vasoconstriction.

Genome-Wide Classification of Myb Domain-Containing Protein Families in Entamoeba invadens.

Entamoeba histolytica, the causative agent of amebiasis, is the third leading cause of death among parasitic diseases globally. Its life cycle includes encystation, which has been mostly studied in Entamoeba invadens, responsible for reptilian amebiasis. However, the molecular mechanisms underlying this process are not fully understood. Therefore, we focused on the identification and characterization of Myb proteins, which regulate the expression of encystation-related genes in various protozoan parasites. Through bioinformatic analysis, we identified 48 genes in E. invadens encoding MYB-domain-containing proteins. These were classified into single-repeat 1R (20), 2R-MYB proteins (27), and one 4R-MYB protein. The in-silico analysis suggests that these proteins are multifunctional, participating in transcriptional regulation, chromatin remodeling, telomere maintenance, and splicing. Transcriptomic data analysis revealed expression signatures of eimyb genes, suggesting a potential orchestration in the regulation of early and late encystation-excystation genes. Furthermore, we identified probable target genes associated with reproduction, the meiotic cell cycle, ubiquitin-dependent protein catabolism, and endosomal transport. In conclusion, our findings suggest that E. invadens Myb proteins regulate stage-specific proteins and a wide array of cellular processes. This study provides a foundation for further exploration of the molecular mechanisms governing encystation and unveils potential targets for therapeutic intervention in amebiasis.

New tricks of an old pattern: structural versatility of scorpion toxins with common cysteine spacing.

Scorpion venoms are a rich source of K(+) channel-blocking peptides. For the most part, they are structurally related small disulfide-rich proteins containing a conserved pattern of six cysteines that is assumed to dictate their common three-dimensional folding. In the conventional pattern, two disulfide bridges connect an α-helical segment to the C-terminal strand of a double- or triple-stranded ß-sheet, conforming a cystine-stabilized α/ß scaffold (CSα/ß). Here we show that two K(+) channel-blocking peptides from Tityus scorpions conserve the cysteine spacing of common scorpion venom peptides but display an unconventional disulfide pattern, accompanied by a complete rearrangement of the secondary structure topology into a CS helix-loop-helix fold. Sequence and structural comparisons of the peptides adopting this novel fold suggest that it would be a new elaboration of the widespread CSα/ß scaffold, thus revealing an unexpected structural versatility of these small disulfide-rich proteins. Acknowledgment of such versatility is important to understand how venom structural complexity emerged on a limited number of molecular scaffolds.

Lysine Methyltransferase EhPKMT2 Is Involved in the In Vitro Virulence of Entamoeba histolytica.

Lysine methylation, a posttranslational modification catalyzed by protein lysine methyltransferases (PKMTs), is involved in epigenetics and several signaling pathways, including cell growth, cell migration and stress response, which in turn may participate in virulence of protozoa parasites. Entamoeba histolytica, the etiologic agent of human amebiasis, has four PKMTs (EhPKMT1 to EhPKMT4), but their role in parasite biology is unknown. Here, to obtain insight into the role of EhPKMT2, we analyzed its expression level and localization in trophozoites subjected to heat shock and during phagocytosis, two events that are related to amoeba virulence. Moreover, the effect of EhPKMT2 knockdown on those activities and on cell growth, migration and cytopathic effect was investigated. The results indicate that this enzyme participates in all these cellular events, suggesting that it could be a potential target for development of novel therapeutic strategies against amebiasis.

CDNF overexpression prevents motor-cognitive dysfunction by intrastriatal CPP-based delivery system in a Parkinson's disease animal model.

Parkinson's disease (PD) is characterized by the loss of dopaminergic neurons in the substantia nigra pars compact (SNpc), and no effective treatment has yet been established to prevent PD. Neurotrophic factors, such as cerebral dopamine neurotrophic factor (CDNF), have shown a neuroprotective effect on dopaminergic neurons. Previously, we developed a cell-penetrating-peptide-based delivery system that includes Asn194Lys mutation in the rabies virus glycoprotein-9R peptide (mRVG9R), which demonstrated a higher delivery rate than the wild-type. In this study, using a mouse PD-like model, we evaluated the intrastriatal mRVG9R-KP-CDNF gene therapy through motor and cognitive tests and brain cell analysis. The mRVG9R-KP-CDNF complex was injected into the striatum on days 0 and 20. To induce the PD-like model, mice were intraperitoneally administered Paraquat (PQ) twice a week for 6 weeks. Our findings demonstrate that mRVG9R-KP-CDNF gene therapy effectively protects brain cells from PQ toxicity and prevents motor and cognitive dysfunction in mice. We propose that the mRVG9R-KP-CDNF complex inhibits astrogliosis and microglia activation, safeguarding dopaminergic neurons and oligodendrocytes from PQ-induced damage. This study presents an efficient CDNF delivery system, protecting neurons and glia in the nigrostriatal pathway from PQ-induced damage, which is known to lead to motor and cognitive dysfunction in neurodegenerative diseases such as PD.

Enhanced antitumor activity induced by a DNA vaccine encoding E7 antigen fused to an ERAD-targeting sequence.

The endoplasmic reticulum (ER) is a key organelle in cell homeostasis and cell health through antigen presentation to immune cells. Thus, the ER has become a therapeutic target to induce cellular immune responses. We previously reported the antitumor effect of a DNA vaccine that expresses the E7 antigen fused to the cyclooxygenase-2 (COX-2) protein. This inflammation-related enzyme contains a degradation cassette associated with the endoplasmic reticulum-associated degradation (ERAD) pathway. To avoid the use of full-length COX-2 and any risk of adverse effects due to the activity of its catalytic site, we designed new versions of the fusion protein. These new constructs encode the E7 antigen fused to the signal peptide and the ERAD sequence of COX-2 with or without the membrane-binding domain (MBD) as well as deletion of the catalytic site. We evaluated the antigen-specific antitumor effect of these DNA constructs in murine prophylactic and therapeutic cancer models. These assays showed that the ERAD cassette is the minimum sequence in the COX-2 protein that induces an antitumor effect when fused to the E7 antigen with the advantage of eliminating any potential adverse effects from the use of full-length COX-2.