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Complete encapsulation of nucleic acids by lipid-based nanoparticles (LNPs) is often thought to be one of the main prerequisites for successful nucleic acid delivery, as the lipid environment protects mRNA from degradation by external nucleases and assists in initiating delivery processes. However, delivery of mRNA via a preformed vesicle approach (PFV-LNPs) defies this precondition. Unlike traditional LNPs, PFV-LNPs are formed via a solvent-free mixing process, leading to a superficial mRNA localization. While demonstrating low encapsulation efficiency in the RiboGreen assay, PFV-LNPs improved delivery of mRNA to the retina by up to 50% compared to the LNP analogs across several benchmark formulations, suggesting the utility of this approach regardless of the lipid composition. Successful mRNA and gene editors' delivery is observed in the retinal pigment epithelium and photoreceptors and validated in mice, non-human primates, and human retinal organoids. Deploying PFV-LNPs in gene editing experiments result in a similar extent of gene editing compared to analogous LNP (up to 3% on genomic level) in the Ai9 reporter mouse model; but, remarkably, retinal tolerability is significantly improved for PFV-LNP treatment. The study findings indicate that the LNP formulation process can greatly influence mRNA transfection and gene editing outcomes, improving LNP treatment safety without sacrificing efficacy., (© 2024 Wiley‐VCH GmbH.)

Lipid nanoparticles (LNPs) largely rely on ionizable lipids to yield successful nucleic acid delivery via electrostatic disruption of the endosomal membrane. Here, we report the identification and evaluation of ionizable lipids containing a thiophene moiety (Thio-lipids). The Thio-lipids can be readily synthesized via the Gewald reaction, allowing for modular lipid design with functional constituents at various positions of the thiophene ring. Through the rational design of ionizable lipid structure, we prepared 47 Thio-lipids and identified some structural criteria required in Thio-lipids for efficient mRNA (messenger RNA) encapsulation and delivery in vitro and in vivo. Notably, none of the tested lipids have a pH-response profile like traditional ionizable lipids, potentially due to the electron delocalization in the thiophene core. Placement of the tails and localization of the ionizable headgroup in the thiophene core can endow the nanoparticles with the capability to reach various tissues. Using high-throughput formulation and barcoding techniques, we optimized the formulations to select two top lipids- 20b and 29d -and investigated their biodistribution in mice. Lipid 20b enabled LNPs to transfect the liver and spleen, and 29d LNP transfected the lung and spleen. Unexpectedly, LNP with lipid 20b was especially potent in mRNA delivery to the retina with no acute toxicity, leading to the successful delivery to the photoreceptors and retinal pigment epithelium in non-human primates., Competing Interests: Competing interests statement:Y.E., M. Gupta, J.K., M. Gautam, J.R., and G.S. are inventors on a patent application pertinent to this work filed by the Oregon State University. Y.E., D.N., and A.T. have stock options and advisory roles with EnterX Bio. G.S. is a cofounder of EnterX Bio. EnterX Bio has a scientific research agreement with Oregon State University. G.S. has a conflict management plan at Oregon State University. The other authors declare no competing interests.

Uropathogenic Escherichia coli (UPEC) is the most common bacterial agent associated with urinary tract infections, threatening public health systems with elevated medical costs and high morbidity rates. The successful establishment of the infection is associated with virulence factors encoded in its genome, in addition to antibacterial resistance genes, which could limit the treatment and resolution of the infection. In this sense, plant extracts from the genus Echeveria have traditionally been used to treat diverse infectious diseases. However, little is known about the effects of these extracts on bacteria and their potential mechanisms of action. This study aims to sequence a multidrug-resistant UPEC isolate (UTI-U7) and assess the multilocus sequence typing (MLST), virulence factors, antimicrobial resistance profile, genes, serotype, and plasmid content. Antimicrobial susceptibility profiling was performed using the Kirby-Bauer disk diffusion. The antibacterial and anti-adherent effects of the methanol extracts (ME) of Echeveria ( E. craigiana , E. kimnachii , and E. subrigida ) against UTI-U7 were determined. The isolate was characterized as an O25:H4-B2-ST2279-CH40 subclone and had resistant determinants to aminoglycosides, β-lactams, fluoroquinolones/quinolones, amphenicols, and tetracyclines, which matched with the antimicrobial resistance profile. The virulence genes identified encode adherence factors, iron uptake, protectins/serum resistance, and toxins. Identified plasmids belonged to the IncF group (IncFIA, IncFIB, and IncFII), alongside several prophage-like elements. After an extensive genome analysis that confirmed the pathogenic status of UTI-U7 isolate, Echeveria extracts were tested to determine their antibacterial effects; as an extract, E. subrigida (MIC, 5 mg/mL) displayed the best inhibitory effect. However, the adherence between UTI-U7 and HeLa cells was unaffected by the ME of the E. subrigida extract., Competing Interests: Conflict of interest: All authors declare no conflicts of interest in this paper., (© 2024 the Author(s), licensee AIMS Press.)

In this study, we investigate a gene augmentation therapy candidate for the treatment of retinitis pigmentosa (RP) due to cyclic nucleotide-gated channel beta 1 (CNGB1) mutations. We use an adeno-associated virus serotype 5 with transgene under control of a novel short human rhodopsin promoter. The promoter/capsid combination drives efficient expression of a reporter gene (AAV5-RHO-eGFP) exclusively in rod photoreceptors in primate, dog, and mouse following subretinal delivery. The therapeutic vector (AAV5-RHO-CNGB1) delivered to the subretinal space of CNGB1 mutant dogs restores rod-mediated retinal function (electroretinographic responses and vision) for at least 12 months post treatment. Immunohistochemistry shows human CNGB1 is expressed in rod photoreceptors in the treated regions as well as restoration of expression and trafficking of the endogenous alpha subunit of the rod CNG channel required for normal channel formation. The treatment reverses abnormal accumulation of the second messenger, cyclic guanosine monophosphate, which occurs in rod photoreceptors of CNGB1 mutant dogs, confirming formation of a functional CNG channel. In vivo imaging shows long-term preservation of retinal structure. In conclusion, this study establishes the long-term efficacy of subretinal delivery of AAV5-RHO-CNGB1 to rescue the disease phenotype in a canine model of CNGB1-RP, confirming its suitability for future clinical development., Competing Interests: Declaration of interests C.R.O. and A.F. are employees of Sanofi. S.M. is listed as inventor on the patent application WO2018172961A1 ‘‘Gene therapy for the treatment of cngb1-linked retinitis pigmentosa’’ and is co-founder of the gene therapy company ViGeneron GmbH., (Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.)

Giardia is a protozoan that affects humans as well as a wide range of domestic species. It is distributed worldwide, and the highest frequency is seen in developing countries. Due to the potential for domestic cats to be carriers of this parasite and subsequently transmit the infection to humans, it is important to know the risk of transmission. For this reason, the objective of this study was to determine the frequency of this parasite in the cat population of the city of Santiago de Queretaro, Mexico, and identify the assemblages present to determine the role this host plays in public health, this being the first study of its type to be performed in the country. This was a cross-sectional study during which 200 fecal samples were collected from cats of both sexes and varying ages and strata of origin. The samples were analyzed by microscopy following the flotation technique, having obtained a general frequency of 25%. Giardia cysts were found at higher frequency in pasty stools. The assemblages found were zoonotic, specifically assemblage A, which suggests that the cat poses an important risk for the dissemination of the parasite to humans, making it an important public health problem.

Lipid nanoparticle (LNP)-based mRNA delivery holds promise for the treatment of inherited retinal degenerations. Currently, LNP-mediated mRNA delivery is restricted to the retinal pigment epithelium (RPE) and Müller glia. LNPs must overcome ocular barriers to transfect neuronal cells critical for visual phototransduction, the photoreceptors (PRs). We used a combinatorial M13 bacteriophage-based heptameric peptide phage display library for the mining of peptide ligands that target PRs. We identified the most promising peptide candidates resulting from in vivo biopanning. Dye-conjugated peptides showed rapid localization to the PRs. LNPs decorated with the top-performing peptide ligands delivered mRNA to the PRs, RPE, and Müller glia in mice. This distribution translated to the nonhuman primate eye, wherein robust protein expression was observed in the PRs, Müller glia, and RPE. Overall, we have developed peptide-conjugated LNPs that can enable mRNA delivery to the neural retina, expanding the utility of LNP-mRNA therapies for inherited blindness.

We present an optimized protocol for guided differentiation of retinal pigment epithelium (RPE) cells from human-induced pluripotent stem cells (iPSC). De novo-generated RPE cells are mature, polarized, and mimic the cellular and molecular profile of primary RPE; they are also suitable for in vivo cell transplantation studies. The protocol includes an enrichment step, making it useful for large-scale GMP manufacturing. RPE cells produced following this protocol are appropriate for cell replacement therapy for macular degeneration and disease modeling. For complete details on the use and execution of this protocol, please refer to Surendran et al. (2021)., Competing Interests: The authors declare no competing interests., (© 2022 The Author(s).)

Introduction: Diarrheagenic Escherichia coli pathotypes are important aetiological agents of diarrhoeal illness among children from less developed areas, worldwide. Diarrheagenic E. coli pathotypes strains are increasingly becoming drug resistant, thus effective and accessible therapeutic alternatives are required for their treatment; herbal extracts may be a potential alternative., Aims: to evaluate Echeveria craigiana, E. kimnachii, and E. subrigida methanol extracts antibacterial effect on six diarrheagenic E. coli reference strains and on human colorectal adenocarcinoma cells viability and cytokine production., Methodology: Diarrheagenic E. coli pathotypes reference strains: typical enteropathogenic E2348/69, enterotoxigenic H10407, enterohaemorrhagic O157:H7/EDL933, enteroinvasive E11, diffusely adherent C18451-A, and enteroaggregative 042 E. coli. E craigiana, E. kimnachii, and E. subrigida leaves, collected at Sinaloa, Mexico, were freeze-dried and macerated in methanol solvent. Antibacterial activity was determined by a novel method developed in our laboratory, bacterial oxygen consumption by polarographic oxygen electrode technique and membrane integrity by two methods (live/dead and protein leakage assays). Colorectal adenocarcinoma cells viability by MTT assay and cytokine production using a Cytometric Bead Array kit., Results: Extracts concentrations of 100 μg/mL and 5-hour incubation, reduced more than 93% the growth of all diarrheagenic E. coli pathotypes tested strains and significantly decreased bacterial oxygen consumption, like bacteriostatic antibiotics. After 24-hour incubation methanol extracts had a differential antibacterial effect on each diarrheagenic E. coli pathotypes strain. Echeveria extracts did not have any effect on viability and cytokine production of colorectal adenocarcinoma cells., Conclusions: Echeveria methanol extracts have a bacteriostatic effect on all diarrheagenic E. coli pathotypes strains, thus potentially they could be used as antibacterial agents on diarrheagenic E. coli pathotypes-contaminated products and on patients with diarrheagenic E. coli pathotypes infections., Competing Interests: No Conflict of Interest is declared, (Copyright (c) 2022 Sandra Olivas-Quintero, Rodolfo Bernal-Reynaga, Catalina Lopez-Saucedo, Samantha Maldonado-Puga, Sylvia Paz Diaz-Camacho, Salvador Uribe-Carvajal, Francisco Delgado-Vargas, Teresa Estrada-Garcia.)

Integrase strand-transfer inhibitors (INSTI) are the latest class of antiretrovirals registered in Mexico. They include raltegravir (RAL), elvitegravir/cobicistat (EVG/c), dolutegravir (DTG) and bictegravir (BIC). Along with international guidelines, Mexico adopted the use of INSTI about two years ago as initial antiretroviral therapy (ART). This is partially due to the increase in the pre-treatment resistance to non-nucleoside reverse transcriptase inhibitors (NNRTI), mainly efavirenz (EFV). Furthermore, INSTI depict greater efficacy, safety and less drug-drug interactions than NNRTI and protease inhibitors (PI). DTG is a second generation INSTI with a high barrier to resistance. It is recommended in international and national guidelines in a wide variety of clinical scenarios for persons living with human immunodeficiency virus (HIV) (PLWHIV), including treatment-naïve, first-line NNRTI treatment failure, simplification switch in suppressed patients, pregnancy, women with childbearing potential, adolescents and children over 6 years of age. DTG is mostly metabolized by the liver UDP-glucuronosyltransferase, and exhibits low drug-drug interactions overall; on the other hand, it has an extremely low renal elimination, therefore may be used in PLWHIV with advanced kidney disease without dose modification. Tuberculosis is a common coinfection in Mexico that requires rifampin-based anti-tuberculosis therapy, which requires increasing DTG to double dosing (50 mg BID). In Mexico, DTG-based regimens are likely to be cost-effective in many scenarios, given its acquisition costs and the particularities of the HIV population and associated clinical conditions, including a relatively high proportion of the following: i) new HIV diagnoses presenting at acquired immunodeficiency syndrome (AIDS) stage; ii) high rate of tuberculosis coinfection; iii) frequent first-line NNRTI treatment failures; and iv) relatively high proportion of infected children and adolescents.

Giardia intestinalis is a protozoan of worldwide distribution capable of infecting a large number of species, including humans and domestic animals. Dogs represent a risk to public health due to cross-infections by the zoonotic assemblages. However, there is little information concerning the prevalence and frequency of this parasite and its assemblages in dogs of the central region of Mexico, thus this study aimed to contribute to this matter. A total of 402 feces samples from dogs of different settings (shelter, breeding establishments, domestic and stray) were obtained and direct coproparasitoscopic examination by flotation revealed a prevalence of 25%. PCR was performed for amplification of the β-Giardin gene, to which 24 samples were positive. Assemblages were obtained through RFLP analysis, using enzymes Hae III to obtain the main genotypes (A-G), and Hha I to subtype assemblage A. All 24 samples were genotyped as assemblage A, with 83% as AI and 17% as AII. Thus, these findings confirm that dogs in the central region of Mexico are a risk for zoonotic transmission of this parasite, emphasizing the importance of a much needed control of the disease in this species.

Individuals with X-HIGM syndrome fail to express functional CD40 ligand; consequently they cannot mount effective protective antibody responses against pathogenic bacteria. We evaluated, compared, and characterized the humoral immune response of wild type (WT) and C57-CD40L deficient (C57-CD40L(-/-)) mice infected with Citrobacter rodentium. Basal serum isotype levels were similar for IgM and IgG3 among mice, while total IgG and IgG2b concentrations were significantly lower in C57-CD40L(-/-) mice compared with WT. Essentially IgG1 and IgG2c levels were detectable only in WT mice. C57-CD40L(-/-) animals, orally inoculated with 2 × 10(9) CFU, presented several clinical manifestations since the second week of infection and eventually died. In contrast at this time point no clinical manifestations were observed among C57-CD40L(-/-) mice infected with 1 × 10(7) CFU. Infection was subclinical in WT mice inoculated with either bacterial dose. The serum samples from infected mice (1 × 10(7) CFU), collected at day 14 after infection, had similar C. rodentium-specific IgM titres. Although C57-CD40L(-/-) animals had lower IgG and IgG2b titres than WT mice, C57-CD40L(-/-) mice sera displayed complement-mediated bactericidal activity against C. rodentium. C. rodentium-infected C57-CD40L(-/-) mice are capable of producing antibodies that are protective. C57-CD40L(-/-) mouse is a useful surrogate model of X-HIGM syndrome for studying immune responses elicited against pathogens.

Enteroaggregative Escherichia coli (EAEC) is a heterogeneous emerging enteric pathogen. Identified during the 1980's when EAEC strains where isolated from cases of acute and persistent diarrhea among infants from developing countries and of traveler's diarrhea. Subsequently, EAEC strains were linked with foodborne outbreaks and diarrhea illness in adults and children from industrialized countries, HIV-infected subjects and stunting of malnourished poor children. Nowadays, EAEC is increasingly recognized as a major cause of acute diarrhea in children recurring hospitalization and of traveler's diarrhea worldwide. EAEC strains defining phenotype is the aggregative adherence (AA) pattern on epithelial cells. AggR a transcriptional regulator of several EAEC virulence genes has been a key factor in both understanding EAEC pathogenesis and defining typical EAEC (tEAEC) strains. EAEC virulence genes distribution among these strains is highly variable. Present challenges are the identification of key virulence genes and how they coordinately function in the setting of enteric disease.

Diffusely adherent Escherichia coli (DAEC) is thought to cause diarrhoea in children, and so too are other diarrhoeagenic E. coli (DEC); however, the evidence base is inconclusive. DEC pathotypes are differentiated on the basis of their pathogenic features, and thus cannot be quickly identified on selective culture media. Molecular techniques, not readily available in most clinical laboratories, are required to differentiate DEC strains from non-pathogenic E. coli in the stool flora. We report a case of persistent bloody diarrhoea, without fever, in a previously healthy 21-month infant from whom we isolated five DAEC strains. The child's stools movements were loose, with gross blood and mucus; fresh mount analysis revealed numerous faecal leukocytes and erythrocytes. Response to antimicrobial treatment with trimethoprim-sulfamethoxazole was poor despite susceptibility in vitro. Although the patient improved with azithromycin, blood was present in the patient's stools for over 30 days. The severe diarrhoea in this patient might be explained by the fact that these DAEC isolates harboured a siderophore receptor, which allows the bacteria to use iron derived from haem compounds that promote its multiplication. The isolates also induced in vitro secretion of several immunomodulatory cytokines that may account for the patient's loose stools and faecal leukocytes. DAEC may play a greater role than suspected in afebrile children with bloody diarrhoea.

Enterotoxigenic Escherichia coli (ETEC) are a major cause of diarrheal disease in humans, calves and pigs. In humans, these infections mainly occur in developing countries leading to a high diarrheal morbidity and infant mortality and to travellers' diarrhea. ETEC strains constitute a phenotypically and genetically diverse pathotype with as common characteristics the production of heat-labile (LT) and/or heat-stable enterotoxins (ST) as well as of one or more fimbrial colonization factors. Despite the global importance of these pathogens, a broadly ETEC protective vaccine is not yet available, partially due to the lack of a suitable animal model for human ETEC. Such model would allow to test more ETEC molecules as potential vaccine candidates. The C57-CD40 ligand deficient (C57-cd40l(-/-)) mouse has been successfully used to develop infection models of intestinal pathogens, but little is known about its humoral immune response. Therefore, the aims of this study were to characterize the humoral immune response of C57 and C57-cd40l(-/-) mice and to determine the persistence of ETEC H10407 and two of its variants after oral inoculation. The serum IgM, IgG and IgA and faecal IgG and IgA concentrations, of twelve mice per mouse strain (C57 and C57-cd40l(-/-)), were determined by ELISA. All serum immunoglobulins and the faecal IgG concentration were significantly lower in C57-cd40l(-/-) than in C57 mice. In contrast the faecal IgA concentration was significantly higher in the C57-cd40l(-/-) mice. This high intestinal IgA concentration might be a compensatory T cell-independent production of IgA production. Both mouse strains were orally inoculated with 5×10(8) ETEC H10407 (LT(+), ST-colonization factor antigen I (CFA/I)(+)) and ETEC in animal faeces was established by culture followed by st and lt loci identification by PCR until day 14 post infection. Most C57 mice eliminated the strain within 3 days whereas infection remained in C57-cd40l(-/-) mice until day 14. Subsequently both mouse strains were inoculated with ETEC H10407 variants and followed up until day 113. Likewise C57 mice eliminated both ETEC variants within 4 days. All C57-cd40l(-/-) mice had eliminated the LT(-) variant at day 31, whereas the ST-CFA/I(-) variant remained in mice stools until day 113. These observations suggest that C57-cd40l(-/-) mice are permissive for ETEC H10407 colonization., (Copyright © 2012 Elsevier B.V. All rights reserved.)

The vocal sac is a visually conspicuous attribute of most male frogs, but its role in visual communication has only been demonstrated recently in diurnally displaying frogs. Here we characterized the spectral properties of the inflated vocal sac of male túngara frogs (Physalaemus pustulosus), a nocturnal species, and túngara visual sensitivity to this cue across reproductive state and sex. We measured the spectral and total reflectance of different male body regions, including inflated and non-inflated vocal sacs, along with samples of the visual background against which males are perceived. Inflated vocal sacs were the most reflective of all body parts, being one log unit more reflective than background materials. We utilized an optomotor drum with black stripes and stripes that mimicked the spectral reflectance of the inflated vocal sacs with various nocturnal light intensities to measure the visual sensitivity thresholds of males, non-reproductive females and reproductive females. All three groups exhibited visual sensitivities corresponding to intensities below moonless conditions in open habitats or at the edge of secondary tropical forests. Reproductive females exhibited the greatest visual sensitivity of all groups, and were significantly more sensitive than non-reproductive females. Though the mechanism for this physiological difference between reproductive and non-reproductive females is unknown, it is consistent with previously observed patterns of light-dependent phonotaxic behavior in túngaras. We suggest that the visual ecology of the vocal sac, especially in nocturnal frogs, offers a rich source for investigations of visual ecology and physiological regulation of vision.