In situ deposition of nanobodies by an engineered commensal microbe promotes survival in a mouse model of enterohemorrhagic E. coli.

Engineered smart microbes that deliver therapeutic payloads are emerging as treatment modalities, particularly for diseases with links to the gastrointestinal tract. Enterohemorrhagic E coli (EHEC) is a causative agent of potentially lethal hemolytic uremic syndrome. Given concerns that antibiotic treatment increases EHEC production of Shiga toxin (Stx), which is responsible for systemic disease, novel remedies are needed. EHEC encodes a type III secretion system (T3SS) that injects Tir into enterocytes. Tir inserts into the host cell membrane, exposing an extracellular domain that subsequently binds intimin, one of its outer membrane proteins, triggering the formation of attaching and effacing (A/E) lesions that promote EHEC mucosal colonization. Citrobacter rodentium (Cr), a natural A/E mouse pathogen, similarly requires Tir and intimin for its pathogenesis. Mice infected with Cr(ΦStx2dact), a variant lysogenized with an EHEC-derived phage that produces Stx2dact, develop intestinal A/E lesions and toxin-dependent disease. Stx2a is more closely associated with human disease. By developing an efficient approach to seamlessly modify the C. rodentium genome, we generated Cr_Tir-MEHEC(ΦStx2a), a variant that expresses Stx2a and the EHEC extracellular Tir domain. We found that mouse pre-colonization with HS-PROT3EcT-TD4, a human commensal E. coli strain (E. coli HS) engineered to efficiently secrete- an anti-EHEC Tir nanobody, delayed bacterial colonization and improved survival after challenge with Cr_Tir-MEHEC(ΦStx2a). This study provides the first evidence to support the efficacy of engineered commensal E. coli to intestinally deliver therapeutic payloads that block essential enteric pathogen virulence determinants, a strategy that may serve as an antibiotic-independent antibacterial therapeutic modality.

Modeling Choroideremia Disease with Isogenic Induced Pluripotent Stem Cells.

Choroideremia (CHM) is a rare X-linked chorioretinal dystrophy causing progressive vision loss due to mutations in the CHM gene, leading to Rab escort protein 1 loss of function. CHM disease is characterized by a progressive degeneration of the choroid, the retinal pigment epithelium (RPE), and the retina. The RPE is a monolayer of polarized cells that supports photoreceptors, providing nutrients, growth factors, and ions, and removes retinal metabolism waste products, having a central role in CHM pathogenesis. Commonly used models such as ARPE-19 cells do not reproduce accurately the nature of RPE cells. Human induced pluripotent stem cells (hiPSCs) can be differentiated into RPE cells (hiPSC-RPE), which mimic key features of native RPE, being more suited to study retinal diseases. Therefore, we took advantage of hiPSCs to generate new human-based CHM models. Two isogenic hiPSC lines were generated through CRISPR/Cas9: a CHM knock-out line from a healthy donor and a corrected CHM patient line using a knock-in approach. The differentiated hiPSC-RPE lines exhibited critical morphological and physiological characteristics of native RPE, including the presence of the tight junction markers Claudin-19 and Zonula Occludens-1, phagocytosis of photoreceptor outer segments, pigmentation, a postmitotic state, and the characteristic polygonal shape. In addition, all the studied cells were able to form retinal organoids. This work resulted in the establishment of isogenic hiPSC lines, representing a new and important CHM cellular model. To our knowledge, this is the first time that isogenic cell lines have been developed to model CHM disease, providing a valuable tool for studying the mechanisms at the onset of RPE degeneration.

Complications and Patient-Centered Outcomes of an Implant- Supported Single Crown after Immediate and Delayed Loading: A Systematic Review and Meta-analysis.

Single implants are a predictable treatment, and immediate loading can be an option with acceptable survival rates. Clinical and patient-centered outcomes comparing immediate and delayed protocol of single implants are unclear. The purpose of this study was to assess complications, satisfaction, and quality of life of patients rehabilitated with delayed and immediate loading single crowns. An electronic search was conducted in PubMed/MEDLINE, Scopus, Web of Science, Cochrane Library, and Embase databases up to February 2023. Only prospective studies, randomized and non-randomized clinical trials comparing immediate and delayed loading were included. For the quantitative analysis, dichotomous and continuous variables were evaluated with a 95% confidence interval. A total of 20 studies were evaluated. No statistically significant difference between protocols was observed: satisfaction (I2: 0%; P = 0.42), quality of life (I2: 0 %; P = 0.05), biological complications (I2: 9%; P = 0.17) mechanical complications (I2: 58%; P = 0.84), and survival rate (I2: 0%; P = 0.38). Subgroup analysis showed significant differences only for marginal bone loss when immediate implants were placed in the mandible (IÇ: 15%; P = 0.01) and posterior zone (I2: 0%; P = 0.001). Complications and patient-centered outcomes for immediate single-implant crowns were comparable to delayed loading. Scientific evidence showed no significant difference between loading protocols for survival rates. Although several factors could interfere with the complication events, implant failures, and marginal bone loss, the subgroup analysis evidenced that only immediate implants placed in the posterior mandible zone had higher statistically significant mean marginal bone loss.

Gamma-delta T cells suppress microbial metabolites that activate striatal neurons and induce repetitive/compulsive behavior in mice.

Intestinal γδ T cells play an important role in shaping the gut microbiota, which is critical not only for maintaining intestinal homeostasis but also for controlling brain function and behavior. Here, we found that mice deficient for γδ T cells (γδ-/-) developed an abnormal pattern of repetitive/compulsive (R/C) behavior, which was dependent on the gut microbiota. Colonization of WT mice with γδ-/- microbiota induced R/C behavior whereas colonization of γδ-/- mice with WT microbiota abolished the R/C behavior. Moreover, γδ-/- mice had elevated levels of the microbial metabolite 3-phenylpropanoic acid in their cecum, which is a precursor to hippurate (HIP), a metabolite we found to be elevated in the CSF. HIP reaches the striatum and activates dopamine type 1 (D1R)-expressing neurons, leading to R/C behavior. Altogether, these data suggest that intestinal γδ T cells shape the gut microbiota and their metabolites and prevent dysfunctions of the striatum associated with behavior modulation.

Gene therapy for inherited retinal diseases: exploiting new tools in genome editing and nanotechnology.

Inherited retinal diseases (IRDs) encompass a diverse group of genetic disorders that lead to progressive visual impairment and blindness. Over the years, considerable strides have been made in understanding the underlying molecular mechanisms of IRDs, laying the foundation for novel therapeutic interventions. Gene therapy has emerged as a compelling approach for treating IRDs, with notable advancements achieved through targeted gene augmentation. However, several setbacks and limitations persist, hindering the widespread clinical success of gene therapy for IRDs. One promising avenue of research is the development of new genome editing tools. Cutting-edge technologies such as CRISPR-Cas9 nucleases, base editing and prime editing provide unprecedented precision and efficiency in targeted gene manipulation, offering the potential to overcome existing challenges in gene therapy for IRDs. Furthermore, traditional gene therapy encounters a significant challenge due to immune responses to viral vectors, which remain crucial obstacles in achieving long-lasting therapeutic effects. Nanotechnology has emerged as a valuable ally in the quest to optimize gene therapy outcomes for ocular diseases. Nanoparticles engineered with nanoscale precision offer improved gene delivery to specific retinal cells, allowing for enhanced targeting and reduced immunogenicity. In this review, we discuss recent advancements in gene therapy for IRDs and explore the setbacks that have been encountered in clinical trials. We highlight the technological advances in genome editing for the treatment of IRDs and how integrating nanotechnology into gene delivery strategies could enhance the safety and efficacy of gene therapy, ultimately offering hope for patients with IRDs and potentially paving the way for similar advancements in other ocular disorders.

Early life history of the Caribbean coral Orbicella faveolata (Scleractinia: Merulinidae) / Historia de vida temprana del coral caribeño Orbicella faveolata (Scleractinia: Merulinidae)

Introduction: Rehabilitation of hermatypic coral species that have declined in the Caribbean in recent decades is a priority. Production of sexual recruits is considered the best restoration method to aid affected populations. Objective: To gain knowledge of early life stages of Orbicella faveolata and to enhance production of new sexual recruits. Methods: Gamete bundles from the coral species O. faveolata were collected over two years (2018 and 2019) from Los Corales del Rosario y de San Bernardo Natural National Park, Cartagena, Colombia. Assisted fertilization, larval rearing, settlement (onto crustose coralline algae, CCA) and post settlement survival in laboratory conditions were monitored. Results: Embryonic and larval development were documented over 55 hours after the first cleavage, when larvae were fully developed and started pre-settlement behavior. Settlement began 7 days after first cleavage and after 37 days polyps had acquired zooxanthellae. Larval settlement was higher on Lythophyllum congestum and Titanoderma prototypum than in response to Porolithon pachydermum, Neogoniolithon sp., Hydrolithon sp., and Lythophyllum sp. Larvae did not settle on dead coral or on the negative control (sterilized seawater). After the first week post settlement survival was 59 % amongst O. faveolata recruits. During the second week, survival dropped to 42 %, and was further reduced to 0 % at the end of the third week. Conclusions: O. faveolata larvae require cues from certain CCA species to settle, they do not settle in absence of CCA. Increased larvae availability is possible through assisted fertilization in the laboratory, however, due to the high mortality in early post-settlement phases, additional research needs to be conducted in order to scale up larvae production and improve understanding of the cues that enhance settlement and the factors which cause post-settlement mortality.

Introducción: La rehabilitación de las especies de corales hermatípicos del Caribe que han disminuido en las últimas décadas es una prioridad. La producción de reclutas sexuales se considera el mejor método de restauración para ayudar a las poblaciones afectadas. Objetivo: Obtener conocimiento de las primeras etapas de la vida de O. faveolata y mejorar la producción de nuevos reclutas sexuales. Métodos: Por dos años (2018 y 2019), seis días después de luna llena en septiembre, se recolectaron paquetes gaméticos en arrecifes del Parque Nacional Natural Los Corales del Rosario y de San Bernardo, Cartagena, Colombia. Se siguió la fertilización asistida, la cría de larvas, el asentamiento y la supervivencia posterior al asentamiento en algas coralinas costrosas (ACC) en condiciones de laboratorio. Resultados: El desarrollo de embriones y larvas se documenta a lo largo de 55 h después del primer clivaje, cuando la larva está desarrollada completamente y comenzó el comportamiento previo al asentamiento. El asentamiento comienza 7 días después del primer clivaje y 37 días después, la mayoría de los pólipos presentan zooxantelas. El asentamiento larval fue más alto en Lythophyllum congestum y Titanoderma prototypum que en respuesta a Porolithon pachydermum, Neogoniolithon sp., Hydrolithon sp., y Lythophyllum sp. No hubo asentamiento sobre coral muerto ni en el control negativo (agua de mar esterilizada). La supervivencia bajó de un 59 % en la primera semana después del asentamiento, a 42 % durante la segunda semana y 0 % para el final de la tercera semana. Conclusiones: Las larvas de O. faveolata requieren señales de ciertas especies de ACC para asentarse, ellas no se asientan en ausencia de ACC. La disponibilidad de larvas es posible mediante la fertilización asistida en laboratorio. Sin embargo, debido a la alta mortalidad en las primeras fases posteriores al asentamiento, queda mucho por hacer para aumentar la producción de larvas y mejorar nuestro conocimiento y comprensión de las señales que mejoran el asentamiento y las que previenen o inhiben la supervivencia del recluta.

Treinamento (Entrenamiento) muscular inspiratório para o controle (el control) autonômico de indivíduos saudáveis (sanos) / Inspiratory muscle training in autonomic control in healthy individuals

Verificar o efeito (el efecto) agudo de diferentes sobrecargas de treinamento (entrenamiento) muscular inspiratório (TMI) sobre a (la) modulação autonômica cardiovascular em indivíduos saudáveis.Métodos: Ensaio (Ensayo) clínico randomizado cruzado. Foram (Fueron) incluídos voluntários saudáveis (sanos) entre 18 e 35 anos. A variabilidade da (de la) frequência cardíaca (VFC) foi analisada antes e após o (y después del) TMI a os (a los) 10 minutos, 60 minutos e 24 horas (agudo, subagudo e tardio, respectivamente). A força (La fuerza) muscular inspiratória foi avaliada (fue evaluada) através da (de la)medida de pressão inspiratória máxima (PImáx) com a utilização do (del) manovacuômetro digital MVD300 (Microhard System®, Globalmed, Porto Alegre, Brasil). O TMI foi realizado à 30% e 60% da PImáx ajustados no (en el) dispositivo pressórico linear Power breathe®. Resultados: Foram avaliados dezenove (diecinueve) indivíduos (47% homens, 25 ± 5 anos). Na (En la) fase aguda apenas com 60% da PImá houve redução (hubo una reducción) significativa da variabilidade dos (de los) intervalos RR e no (y enel) componente de alta frequência (HFnu), en quanto que o (mientras que el) componente de baixa(baja) frequência (LFnu) e o balanço (y el equilibrio) autonômico (LF/HF) aumentaram significativamente.Na (En la) fase subaguda, o mesmo comportamento foi observado para HFnu, LFnu, LF/HF. Quando comparadas as (Cuando se compararon las) sobrecargas, houve (hubo) aumento significativo na (en la) magnitude do efeito a 60% PImáx para NN50, LF/HF, LFnu, HFnu na fase aguda, bem como, para (así como para) RR, NN50, LFnu e HFnu na fase subaguda (p < 0.05). Conclusão: Agudamente, o efeito doTMI à 60% da PImáx foi maior deslocando a (fue más grande, desplazando la) modulação autonômicado sistema cardiovascular em indivíduos saudáveis para um predomínio simpático...