αB-Crystallin Peptide Fused with Elastin-like Polypeptide: Intracellular Activity in Retinal Pigment Epithelial Cells Challenged with Oxidative Stress.

BACKGROUND: Oxidative stress-induced retinal degeneration is among the main contributing factors of serious ocular pathologies that can lead to irreversible blindness. αB-crystallin (cry) is an abundant component of the visual pathway in the vitreous humor, which modulates protein and cellular homeostasis. Within this protein exists a 20 amino acid fragment (mini-cry) with both chaperone and antiapoptotic activity. This study fuses this mini-cry peptide to two temperature-sensitive elastin-like polypeptides (ELP) with the goal of prolonging its activity in the retina. METHODS: The biophysical properties and chaperone activity of cry-ELPs were confirmed by mass spectrometry, cloud-point determination, and dynamic light scattering 'DLS'. For the first time, this work compares a simpler ELP architecture, cry-V96, with a previously reported ELP diblock copolymer, cry-SI. Their relative mechanisms of cellular uptake and antiapoptotic potential were tested using retinal pigment epithelial cells (ARPE-19). Oxidative stress was induced with H2O2 and comparative internalization of both cry-ELPs was made using 2D and 3D culture models. We also explored the role of lysosomal membrane permeabilization by confocal microscopy. RESULTS: The results indicated successful ELP fusion, cellular association with both 2D and 3D cultures, which were enhanced by oxidative stress. Both constructs suppressed apoptotic signaling (cleaved caspase-3); however, cry-V96 exhibited greater lysosomal escape. CONCLUSIONS: ELP architecture is a critical factor to optimize delivery of therapeutic peptides, such as the anti-apoptotic mini-cry peptide; furthermore, the protection of mini-cry via ELPs is enhanced by lysosomal membrane permeabilization.

Biomimetic SARS-CoV-2 Spike Protein Nanoparticles.

COVID-19 is an infectious respiratory disease caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). This virus contains a crucial coat protein that engages with target cells via a receptor binding domain (RBD) on its spike protein. To better study the RBD and its therapeutic opportunities, we genetically engineered a simple fusion with a thermo-responsive elastin-like polypeptide (ELP). These fusions express in Escherichia coli at a high yield in the soluble fraction and were easily purified using ELP-mediated phase separation (79 mg/L culture). Interestingly, they assembled peptide-based nanoparticles (Rh = 71.4 nm), which was attributed to oligomerization of RBDs (25.3 kDa) counterbalanced by steric stabilization by a soluble ELP (73.4 kDa). To investigate their biophysical properties, we explored the size, shape, and binding affinity for the human angiotensin-converting enzyme 2 (hACE2) and cellular uptake. Biomimetic nanoparticles such as these may enable future strategies to target the same cells, tissues, and cell-surface receptors as those harnessed by SARS-CoV-2.

The Promising Therapeutic Potential of Oligonucleotides for Pulmonary Fibrotic Diseases.

INTRODUCTION: Fibrotic lung diseases represent a large subset of diseases with an unmet clinical need. Oligonucleotide therapies (ONT) are a promising therapeutic approach for the treatment of pulmonary disease as they can inhibit pathways that are otherwise difficult to target. Additionally, targeting the lung specifically with ONT is advantageous because it reduces the possibilities of systemic side effects and tolerability concerns. AREAS COVERED: This review presents the chemical basis of designing various ONTs currently known to treat fibrotic lung diseases. Further, the authors have also discussed the delivery vehicle, routes of administration, physiological barriers of the lung, and toxicity concerns with ONTs. EXPERT OPINION: ONTs provide a promising therapeutic approach for the treatment of fibrotic diseases of the lung, particularly because ONTs directly delivered to the lung show little systemic side effects compared to current therapeutic strategies. Dry powder aerosolized inhalers may be a good strategy for getting ONTs into the lung in humans. However, as of now, no dry powder ONTs have been approved for use in the clinical setting, and this challenge must be overcome for future therapies. Various delivery methods that can aid in direct targeting may also improve the use of ONTs for lung fibrotic diseases.

Hydra-Elastin-like Polypeptides Increase Rapamycin Potency When Targeting Cell Surface GRP78.

Rapalogues are powerful therapeutic modalities for breast cancer; however, they suffer from low solubility and dose-limiting side effects. To overcome these challenges, we developed a long-circulating multiheaded drug carrier called 5FA, which contains rapamycin-binding domains linked with elastin-like polypeptides (ELPs). To target these "Hydra-ELPs" toward breast cancer, we here linked 5FA with four distinct peptides which are reported to engage the cell surface form of the 78 kDa glucose-regulated protein (csGRP78). To determine if these peptides affected the carrier solubility, this library was characterized by light scattering and mass spectrometry. To guide in vitro selection of the most potent functional carrier for rapamycin, its uptake and inhibition of mTORC1 were monitored in a ductal breast cancer model (BT474). Using flow cytometry to track cellular association, it was found that only the targeted carriers enhanced cellular uptake and were susceptible to proteolysis by SubA, which specifically targets csGRP78. The functional inhibition of mTOR was monitored by Western blot for pS6K, whereby the best carrier L-5FA reduced mTOR activity by 3-fold compared to 5FA or free rapamycin. L-5FA was further visualized using super-resolution confocal laser scanning microscopy, which revealed that targeting increased exposure to the carrier by ∼8-fold. This study demonstrates how peptide ligands for GRP78, such as the L peptide (RLLDTNRPLLPY), may be incorporated into protein-based drug carriers to enhance targeting.

Intracellular Dynamin Elastin-like Polypeptides Assemble into Rodlike, Spherical, and Reticular Dynasomes.

Dynamin (DNM) is a family of large GTPases possessing a unique mechanical ability to "pinch" off vesicles entering cells. DNM2 is the most ubiquitously expressed member of the DNM family. We developed a novel tool based on elastin-like polypeptide (ELP) technology to quickly, precisely, and reversibly modulate the structure of DNM2. ELPs are temperature-sensitive biopolymers that self-assemble into microdomains above sharp transition temperatures. When linked together, DNM2 and a temperature-sensitive ELP fusion organize into a range of distinct temperature-dependent structures above a sharp transition temperature, which were not observed with wild-type DNM2 or a temperature-insensitive ELP fusion control. The structures comprised three different morphologies, which were prevalent at different temperature ranges. The size of these structures was influenced by an inhibitor of the DNM2 GTPase activity, dynasore; furthermore, they appear to entrap co-expressed cytosolic ELPs. Having demonstrated an unexpected diversity of morphologically distinct structures, DNM2-ELP fusions may have applications in the exploration of dynamin-dependent biology.

High Genomic Identity between Clinical and Environmental Strains of Herbaspirillum frisingense Suggests Pre-Adaptation to Different Hosts and Intrinsic Resistance to Multiple Drugs.

The genus Herbaspirillum is widely studied for its ability to associate with grasses and to perform biological nitrogen fixation. However, the bacteria of the Herbaspirillum genus have frequently been isolated from clinical samples. Understanding the genomic characteristics that allow these bacteria to switch environments and become able to colonize human hosts is essential for monitoring emerging pathogens and predicting outbreaks. In this work, we describe the sequencing, assembly, and annotation of the genome of H. frisingense AU14559 isolated from the sputum of patients with cystic fibrosis, and its comparison with the genomes of the uropathogenic strain VT-16-41 and the environmental strains GSF30, BH-1, IAC152, and SG826. The genes responsible for biological nitrogen fixation were absent from all strains except for GSF30. On the other hand, genes encoding virulence and host interaction factors were mostly shared with environmental strains. We also identified a large set of intrinsic antibiotic resistance genes that were shared across all strains. Unlike other strains, in addition to unique genomic islands, AU14559 has a mutation that renders the biosynthesis of rhamnose and its incorporation into the exopolysaccharide unfeasible. These data suggest that H. frisingense has characteristics that provide it with the metabolic diversity needed to infect and colonize human hosts.

Single-Cell Quantification of the Transition Temperature of Intracellular Elastin-like Polypeptides.

Elastin-like polypeptides (ELPs) are modular, stimuli-responsive materials that self-assemble into protein-rich microdomains in response to heating. By cloning ELPs to effector proteins, expressed intracellular fusions can even modulate cellular pathways. A critical step in engineering these fusions is to determine and control their intracellular phase transition temperature (Tt). To do so, this Method paper describes a simple live-cell imaging technique to estimate the Tt of non-fluorescent ELP fusion proteins by co-transfection with a fluorescent ELP marker. Intracellular microdomain formation can then be visualized in live cells through the co-assembly of the non-fluorescent and fluorescent ELP fusion proteins. If the two ELP fusions have different Tt, the intracellular ELP mixture phase separates at the temperature corresponding to the fusion with the lower Tt. In addition, co-assembled ELP microdomains often exhibit pronounced differences in size or number, compared to single transfected treatments. These features enable live-cell imaging experiments and image analysis to determine the intracellular Tt of a library of related ELP fusions. As a case study, we employ the recently reported Caveolin1-ELP library (CAV1-ELPs). In addition to providing a detailed protocol, we also report the development of a useful FIJI plugin named SIAL (Simple Image Analysis Library), which contains programs for image randomization and blinding, phenotype scoring, and ROI selection. These tasks are important parts of the protocol detailed here and are also commonly employed in other image analysis workflows.

Tiempo de sondaje postquirúrgico recomendado para evitar complicaciones en cirugías endourológicas del tracto urinario inferior / Recommended postsurgical urinary catheterization time to avoid complications in endourological surgeries of the lower urinary tract

RESUMEN Objetivo: determinar el tiempo de sondaje postquirúrgico recomendado para evitar complicaciones en cirugías endourológicas del tracto urinario inferior. Materiales y métodos: se realizó una revisión bibliográfica actualizada sobre el tiempo de sondaje postquirúrgico recomendado para evitar complicaciones en cirugías endourológicas del tracto urinario inferior. Resultados: Se encontró asociación entre un mayor tiempo de sondaje y la ocurrencia de posteriores complicaciones relacionadas cirugías endourológicas del tracto urinario inferior, siendo el período menor a 72h el óptimo para la permanencia de la sonda ve-sical postquirúrgica en este tipo de intervenciones. No se identificaron factores de riesgo asociados a la prolongación del tiempo de sondaje postquirúrgico. Recomendaciones: El tipo de abordaje endoscópico se ha convertido en la actualidad en el tratamiento estándar de oro para la mayoría de las patologías del tracto urinario inferior; sin embargo, se requiere realizar más estudios sobre las complicaciones y factores de riesgo relacionados al tiempo de sondaje postquirúrgico para evitar complicaciones en cirugías endourológicas del tracto urinario inferior

ABSTRACT Objective: to determine the recommended post-surgical probing time to avoid complications in endourological surgeries of the lower urinary tract. Materials and methods: an updated bibliographic review was carried out on the recommended post-surgical catheterization time to avoid complications in endou-rological surgeries of the lower urinary tract. Results: An association was found between a longer catheterization time and the occurrence of subsequent complications related to endourological surgeries of the lower urinary tract, with the period of less than 72h being the optimal period for the permanence of the postsurgical urinary catheter in this type of intervention. No risk factors associated with prolonged postoperative probing time were identified. Recom-mendations: The type of endoscopic approach has now become the gold standard treatment for most lower urinary tract pathologies; However, more studies are required on the complications and risk factors related to the time of postoperative catheterization to avoid complications in endourological surgeries of the lower urinary tract

Malformación congénita de la vía aérea pulmonar. Reporte de un caso / Congenital malformation of the pulmonary airway. Report of a case

RESUMEN El objetivo de este estudio es realizar una revisión de la Malformación Congénita Pulmonar a propósito del caso de un feto de una paciente de 34 años que cursaba su segunda gesta, cuyo diagnóstico se realizó a la semana 30 de gestación. Además se realizó una revisión bibliográfica de la patología y de casos similares.

ABSTRACT The objective of this study is to perform a review of Congenital Pulmonary Malformation regarding a case of a fetus in a 34-year-old woman who was in her second pregnancy, whose diagnosis was made at week 30 of gestation. In addition, a bibliographic review of the pathology and similar cases was carried out

Metaplasia ósea del endometrio. Reporte de un caso y revisión de la bibliografía / Endometrial osseous metaplasia. Case report and literatura review

RESUMEN La metaplasia ósea endometrial es una patología poco común que se asocia a infertilidad secundaria. La mayoría de casos se presenta en mujeres con antecedente de gestación no evolutiva. La sospecha se hace mediante el hallazgo de un endometrio hiperecogénico que se asemeja a la imagen de un dispositivo intrauterino. El gold standard para el diagnóstico y tratamiento es la histeroscopia con el estudio histopatológico del material obtenido en el pre-cedimeinto. La importancia de su descripción es su presentación en una paciente nuligesta. Se describe el caso clínico y revisión de la literatura. Palabras claves: Metaplasia ósea endometrial, infertilidad secundaria, endometrio hiperecogénico

ABSTRACT Endometrial osseoum metaplasia is a rare condition associated with secondary infertility. Most cases occur in women with a histo-ry of non-evolutionary pregnancy. The suspicion is made by the finding of a hyperechoic endometrium that is similar of an intrauterine de-vice. The diagnostic and therapeutic gold standard is made by hysteroscopy with histopathological study of the material obtained in the precedure. The importance of this case is its presentation in a nulliparous patient. The clinical case and literature review are described

Caveolin elastin-like polypeptide fusions mediate temperature-dependent assembly of caveolar microdomains.

Caveolae are membrane organelles formed by submicron invaginations in the plasma membrane, and are involved in mechanosensing, cell signaling, and endocytosis. Although implicated broadly in physiology and pathophysiology, better tools are required to elucidate the precise role of caveolar processes through selective activation and inactivation of their trafficking. Our group recently reported that thermally-responsive elastin-like polypeptides (ELPs) can trigger formation of 'genetically engineered protein microdomains (GEPMs)' functionalized with either Clathrin-light chain or the epidermal growth factor receptor. This manuscript is the first report of this strategy to modulate caveolin-1 (CAV1). By attaching different ELP sequences to CAV1, mild heating can be used to self-assemble CAV1-ELP microdomains inside of cells. The temperature of self-assembly can be controlled by tuning the ELP sequence. The formation of CAV1-ELP microdomains internalizes Cholera Toxin Subunit B, a commonly used marker of caveolae mediated endocytosis. CAV1-ELPs also colocalize with Cavin 1, an essential component of functional caveolae biogenesis. With the emerging significance of caveolae in health and disease and the lack of specific probes to rapidly and reversibly affect caveolar function, CAV1-ELP microdomains are a new tool to rapidly probe caveolae associated processes in endocytosis, cell signaling, and mechanosensing.

Historia de la investigación en pediatría / History of pediatric investigation

La investigación en niños es necesaria, ya que los niños no son adultos pequeños como se ha asumido durante las últimas tres décadas. Sin embargo, es necesario conciliar esta necesidad con la protección que la sociedad debe proporcionarles para evitar que se repitan abusos ocurridos en décadas previas. Tanto las agencias reguladoras como otras autoridades sanitarias y organizaciones e instituciones públicas internacionales, han elaborado una serie de recomendaciones para que estos estudios sean realizados respetando los principios y normas éticas básicas en investigación clínica, particularmente en las poblaciones consideradas vulnerables, como los niños. También, las agencias reguladoras de Estados Unidos, Canadá y Europa, han establecido una serie de medidas para fomentar la investigación en el campo de la población pediátrica a nivel mundial, y tratar de cambiar el concepto generalizado de que los niños son considerados "Huérfanos Terapéuticos...(AU)

Investigation in children is necessary, since children are not small adults as has been assumed during the last three decades. However, it is necessary to reconcile this need with the protection that society must provide them to avoid repeating abuses that occurred in previous decades. Regulatory agencies and other health authorities and international public institutions and organizations have prepared a series of recommendations for these studies to be carried out respecting the basic ethical principles and standards in clinical research, particularly in populations considered vulnerable, such as children. Also, the regulatory agencies of the United States, Canada and Europe, have established a series of measures to promote research in the field of pediatric population worldwide, and try to change the generalized concept that children are considered "Therapeutic Orphans"...(AU)

Structure-activity relationship of antibacterial chalcones.

The antibacterial activity of 31 chalcones was tested against bacterial strains, Bacillus cereus ATCC 11778, Escherichia coli ATCC 25922, Pseudomonas aeruginosa ATCC 27853, and Staphylococcus aureus ATCC 25923. Some of the tested chalcones showed fair to significant activity against Gram-positive bacteria. By comparison of the results obtained, the antibacterial activity can be related to features such as the presence of a C-4 hydroxyl group, a C-4' oxygenated substituent or a C-3' isoprenoid side chain, while the C-2' hydroxyl group might have importance for the stability of the molecule. The inhibitory effect of chalcones on human pathogenic microorganisms can be correlated with the substitution patterns of the aromatics rings.

Efficacy of ofloxacin otic solution once daily for 7 days in the treatment of otitis externa: a multicenter, open-label, phase III trial.

BACKGROUND: Otitis externa (OE) is an infection of the external auditory canal that is typically treated with topically applied broad-spectrum antibiotics. Twice-daily topical treatment with ofloxacin otic 0.3% solution for 10 days has been reported to be as effective and well tolerated as the standard of care, neomycin sulfate/polymyxin B sulfate/hydrocortisone solution administered 4 times daily for 10 days. OBJECTIVE: This study evaluated the efficacy and safety profile of 7 days of a once-daily regimen of ofloxacin otic 0.3% solution in the treatment of OE. METHODS: This multicenter, open-label, Phase III study was conducted from June 12, 2002, to October 14, 2002. Eligible patients were aged > or = 6 months and had OE of <2 weeks' duration with moderate to severe edema and tenderness involving 1 or both ears and sufficient exudate for microbiologic culture. Ofloxacin otic solution was instilled once daily for 7 days (5 drops for children aged 6 months to <13 years, 10 drops for adolescents/adults aged > or = 13 years). Assessments were conducted at the end-of-treatment visit and 7 to 10 days later (the test-of-cure visit). Medication was supplied free of charge to study participants who incurred no costs for physician visits. RESULTS: Of 489 patients enrolled at 58 sites in 3 countries, 439 were clinically evaluable (173 children, 266 adolescents/adults; 52 % males, 48% females; 47% Hispanic, 45% white; 5% black, and 3% other). The cure rate among clinically evaluable patients was 91% (95% of children, 88% of adolescents/adults); 68% of patients were cured within 7 days. Forty-three potentially pathogenic strains were isolated from 253 microbiologically evaluable patients. Pseudomonas aeruginosa was isolated from 158 (62%) microbiologically evaluable patients and Staphylococcus aureus from 32 (13%). Eradication rates were 96% overall. No serious adverse events were observed. Minor adverse events were experienced by 15 (3%) of 489 patients included in the safety population. The most common adverse events were pruritus (5 patients), increased earache (4 patients), and application-site reactions (3 patients). Overall mean (SD) adherence to therapy was 98% (11.9). CONCLUSIONS: Ofloxacin otic 0.3% solution administered once daily for 7 days was well tolerated and effective in achieving clinical and microbiologic cure of OE. The compliance rates in this study suggests that this regimen may be better accepted by patients than longer, more repetitive regimens.

Resistencia bacteriana a antimicrobianos / Bacterial resistance to antimicrobials

Con la finalidad de conocer la resistencia bacteriana a los antimicrobianos, se estudiaron 266 cepas bacterianas de cultivos provenientes del Servicio de Pediatría del Hospital Universitario "Dr. Luis Razetti", de Barcelona, Venezuela, durante julio de 1991 a diciembre de 1992. Los resultados fueron: E.coli es resistente a ampicilina en 89,74 por ciento ; a amikacina: 10,42 por ciento y a gentamicina 18 por ciento . El S.aureus fue resistente en el 100 por ciento a penicilina; a ampicilina: 86,36 por ciento ; oxacilina: 25 por ciento ; vancomicina: 14,81 por ciento y siendo menos resistente a amikacina en el 8,33 por ciento . Enterobacter sp fue resistente a ampicilina, cefuroxima y cloranfenicol en el 100 por ciento de las cepas; amikacina: 15,32 por ciento y no hubo resistencia a gentamicina. La P.aeruginosa presentó resistencia a ceftazidime en el 25 por ciento ; cefoperazone: 31 por ciento y a amikacina en 8,33 por ciento . Se concluye que es importante conocer la resistencia bacteriana para establecer una conducta apropiada cuando no haya respuesta al tratamiento antimicrobiano habitual

El circuito abierto con Trilene en Anestesia Infantil

1.La narcosis con trilene, en el circuito abierto gota a gota, en anestesia pediatrica, puede emplearse en todo tipo de cirugia menor y en niños de cualquier edad, aún de días, 2.Según nuestra casuistica tuvimos 6 por ciento de complicaciones correspondientes a paro respiratorio, 3.Con este procedimiento se consigue un rapido despertar del paciente, por su facilidad de eliminación del anestesico. 4.La inducción es rápida suave y agradable para el paciente tomando un tiempo mínimo de 2 minutos, 5.Constituye una anestesia economicamente barata, no presisa de inhaladores especiales que demandan alto costo, 6.Brinda al médico recien egresado, que realiza su año de provincia, un método sencillo y de fácil manejo,en lugar donde existe carencia de material anestesico particulamente oxigeno