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1.
Front Bioeng Biotechnol ; 10: 896751, 2022.
Artículo en Inglés | MEDLINE | ID: mdl-35519622

RESUMEN

The ongoing pandemic caused by the novel coronavirus (SARS-CoV-2) has led to more than 445 million infections and the underlying disease, COVID-19, resulted in more than 6 million deaths worldwide. The scientific world is already predicting future zoonotic diseases. Hence, rapid response systems are needed to tackle future epidemics and pandemics. Here, we present the use of eukaryotic cell-free systems for the rapid response to novel zoonotic diseases represented by SARS-CoV-2. Non-structural, structural and accessory proteins encoded by SARS-CoV-2 were synthesized by cell-free protein synthesis in a fast and efficient manner. The inhibitory effect of the non-structural protein 1 on protein synthesis could be shown in vitro. Structural proteins were quantitatively detected by commercial antibodies, therefore facilitating cell-free systems for the validation of available antibodies. The cytotoxic envelope protein was characterized in electrophysiological planar lipid bilayer measurements. Hence, our study demonstrates the potential of eukaryotic cell-free systems as a rapid response mechanism for the synthesis, functional characterization and antibody validation against a viral pathogen.

2.
FASEB J ; 36 Suppl 12022 May.
Artículo en Inglés | MEDLINE | ID: mdl-35552902

RESUMEN

SARS-CoV-2 - induced COVID-19 causes acute respiratory distress syndrome (ARDS) and multiorgan failure. COVID-19 can also cause psycho-social problems, including increased alcohol consumption and consequent complications. Alcohol abuse is recognized as an independent factor that increases by three- to four-fold the incidence of ARDS, a severe form of acute lung injury with a mortality rate of 40 to 50 percent. This translates to tens of thousands of excess deaths in the United States each year from alcohol-associated lung injury. We developed a combined ARDS and alcohol abuse mouse model by intratracheally instilling the S1 subunit of SARS-CoV-2 Spike protein in K18-hACE2 transgenic mice that express the human angiotensin-converting enzyme 2 (ACE2) receptor for SARS-CoV-2 and which are kept on an ethanol diet. 72 hours after S1SP instillation, mice kept on the ethanol diet exhibited a strong decline in body weight, a dramatic increase in white blood cell content of bronchoalveolar lavage fluid (BALF), and an augmented "cytokine storm", compared to S1SP treated mice on control diet. Histologic examination of lung tissue demonstrated abnormal recruitment of immune cells in the alveolar space, destructive effects on parenchymal architecture, and an overall worsening of the lung injury score (LIS) of S1SP- and alcohol-treated animals. Along with the activation of pro-inflammatory biomarkers (NF-κB, STAT3, NLRP3 inflammasome), lung tissue homogenates from mice on alcohol diet, demonstrated overexpression of ACE2 compared to mice on control diet. In summary, K18-hACE2 transgenic mice on an alcohol diet exhibit a more severe S1SP-induced ARDS than corresponding mice on a control diet and may thus represent a useful model for the development of therapeutic interventions against alcohol-exacerbated COVID-19.

3.
Front Surg ; 9: 858117, 2022.
Artículo en Inglés | MEDLINE | ID: mdl-35388366

RESUMEN

The procedure for installation of a percutaneous bone-conducting device has undergone significant improvements since its introduction 40 years ago. Today, the linear incision technique with tissue preservation (LITT-P) and the minimally invasive procedure (MIPS) are the most commonly used approaches. In both these techniques, a gradual increase of the osteotomy using a three-step drilling sequence is utilized, as this approach can allow a stepwise deepening and widening of the osteotomy in the mastoid and can prevent bone overheating. A new minimally invasive procedure (MONO) has been developed that allows an osteotomy to be performed and enables complete removal of the bone volume in one single drill step for a 4 mm implant using a novel parabolic twist drill. Here, the feasibility of the MONO procedure was qualitatively and quantitatively evaluated in terms of the dura response to drill trauma in comparison with the outcomes achieved with guide drills used for the LITT-P and MIPS techniques. Fresh frozen temporal bone from a human cadaver was subjected to penetration by three drills beyond the base of the mastoid bone to different depths. The sites were evaluated, and the damage to and possible penetration of the dura were determined. The results showed that for a drill depth exceeding mastoid bone thickness by not more than 1 mm, damage to the dura was limited or nonexistent, whereas for a drill depth exceeding bone thickness by 2 mm, damage increased, or the dura was penetrated. There was a trend toward more damage and penetration for both the round burr and MIPS guide drill compared with the MONO drill bit. From this experimental ex vivo study, it can be concluded that if the dura is encountered, the MONO system is not more inclined to penetrate the dura than the conventional LITT-P and MIPS systems.

4.
Cancers (Basel) ; 14(7)2022 Mar 24.
Artículo en Inglés | MEDLINE | ID: mdl-35406416

RESUMEN

Incontinence after robot-assisted radical prostatectomy (RARP) is feared by most patients with prostate cancer. Many risk factors for incontinence after RARP are known, but a paucity of data integrates them. Prospectively acquired data from 680 men who underwent RARP January 2008-December 2015 and met inclusion/exclusion criteria were queried retrospectively and then divided into model development (80%) and validation (20%) cohorts. The UCLA-PCI-Short Form-v2 Urinary Function questionnaire was used to categorize perfect continence (0 pads), social continence (1-2 pads), or incontinence (≥3 pads). The observed incontinence rates were 26% at 6 months, 7% at 12 months, and 3% at 24 months. Logistic regression was used for model development, with variables identified using a backward selection process. Variables found predictive included age, race, body mass index, and preoperative erectile function. Internal validation and calibration were performed using standard bootstrap methodology. Calibration plots and receiver operating curves were used to evaluate model performance. The initial model had 6-, 12-, and 24-month areas under the curves (AUCs) of 0.64, 0.66, and 0.80, respectively. The recalibrated model had 6-, 12-, and 24-month AUCs of 0.52, 0.52, and 0.76, respectively. The final model was superior to any single clinical variable for predicting the risk of incontinence after RARP.

5.
Front Physiol ; 13: 827280, 2022.
Artículo en Inglés | MEDLINE | ID: mdl-35399274

RESUMEN

Intravital multiphoton microscopy has empowered investigators to study dynamic cell and subcellular processes in vivo within normal and disease organs. Advances in hardware, software, optics, transgenics and fluorescent probe design and development have enabled new quantitative approaches to create a disruptive technology pioneering advances in understanding of normal biology, disease pathophysiology and therapies. Offering superior spatial and temporal resolution with high sensitivity, investigators can follow multiple processes simultaneously and observe complex interactions between different cell types, intracellular organelles, proteins and track molecules for cellular uptake, intracellular trafficking, and metabolism in a cell specific fashion. The technique has been utilized in the kidney to quantify multiple dynamic processes including capillary flow, permeability, glomerular function, proximal tubule processes and determine the effects of diseases and therapeutic mechanisms. Limitations include the depth of tissue penetration with loss of sensitivity and resolution due to scattered emitted light. Tissue clearing technology has virtually eliminated penetration issues for fixed tissue studies. Use of multiphoton microscopy in preclinical animal models offers distinct advantages resulting in new insights into physiologic processes and the pathophysiology and treatment of diseases.

6.
Protein Sci ; 31(5): e4314, 2022 05.
Artículo en Inglés | MEDLINE | ID: mdl-35481629

RESUMEN

IMP dehydrogenase(IMPDH) is an essential enzyme that catalyzes the rate-limiting step in the guanine nucleotide pathway. In eukaryotic cells, GTP binding to the regulatory domain allosterically controls the activity of IMPDH by a mechanism that is fine-tuned by post-translational modifications and enzyme polymerization. Nonetheless, the mechanisms of regulation of IMPDH in bacterial cells remain unclear. Using biochemical, structural, and evolutionary analyses, we demonstrate that, in most bacterial phyla, (p)ppGpp compete with ATP to allosterically modulate IMPDH activity by binding to a, previously unrecognized, conserved high affinity pocket within the regulatory domain. This pocket was lost during the evolution of Proteobacteria, making their IMPDHs insensitive to these alarmones. Instead, most proteobacterial IMPDHs evolved to be directly modulated by the balance between ATP and GTP that compete for the same allosteric binding site. Altogether, we demonstrate that the activity of bacterial IMPDHs is allosterically modulated by a universally conserved nucleotide-controlled conformational switch that has divergently evolved to adapt to the specific particularities of each organism. These results reconcile the reported data on the crosstalk between (p)ppGpp signaling and the guanine nucleotide biosynthetic pathway and reinforce the essential role of IMPDH allosteric regulation on bacterial GTP homeostasis.


Asunto(s)
Nucleótidos de Guanina , IMP Deshidrogenasa , Adenina , Adenosina Trifosfato , Guanosina Pentafosfato , Guanosina Trifosfato/metabolismo , Homeostasis , IMP Deshidrogenasa/genética , IMP Deshidrogenasa/metabolismo , Modelos Moleculares
7.
Physiol Rev ; 2022 Apr 04.
Artículo en Inglés | MEDLINE | ID: mdl-35378997

RESUMEN

For nearly fifty years the proximal tubule has been known to reabsorb, process, and either catabolize or transcytose albumin from the glomerular filtrate. Innovative techniques and approaches have provided insights into these processes, and several genetic diseases, nonselective proximal tubule cell (PTC) defects, chronic kidney disease and acute PTC injury lead to significant albuminuria, reaching nephrotic range. Albumin is also known to stimulate PTC injury cascades. Thus, the mechanisms of albumin reabsorption, catabolism and transcytosis are being reexamined utilizing techniques that allow for novel molecular and cellular discoveries. Megalin, a scavenger receptor, cubilin, amnionless, and Dab2 form a nonselective multi-receptor complex that mediates albumin binding, uptake and directs proteins for lysosomal degradation following endocytosis. The neonatal Fc receptor mediates albumin transcytosis by its pH-dependent binding affinity in endosomal compartments. This transcytotic, reclamation, pathway minimizes urinary losses and cellular catabolism of albumin thus prolonging its serum half-life. It also serves as a PTC molecular sorting mechanism to preserve and reclaim physiologic albumin while allowing "altered" albumin that does not bind to FcRn to enter the lysosomal pathway. The clinical importance of PTC albumin metabolism has also increased as albumin is now being used to bind therapeutic agents to extend their half-life and minimize filtration and kidney injury. The purpose of this review is to update and integrate evolving information regarding the reabsorption and processing of albumin by proximal tubule cells including discussing genetic disorders and therapeutic considerations.

8.
Am J Pathol ; 2022 Mar 26.
Artículo en Inglés | MEDLINE | ID: mdl-35351468

RESUMEN

Idiopathic pulmonary fibrosis (IPF) is a dramatic disease without cure. The US Food and Drug Administration-approved drugs, pirfenidone and nintedanib, only slow disease progression. The clinical investigation of novel therapeutic approaches for IPF is an unmet clinical need. Nucleotide-binding oligomerization domain-like receptor or NOD-like receptors are pattern recognition receptors capable of binding a large variety of stress factors. NLR family pyrin domain-containing protein 3 (NLRP3), once activated, promotes IL-1ß, IL-18 production, and innate immune responses. Multiple reports indicate that the inflammasome NLRP3 is overactivated in IPF patients, leading to increased production of class I IL and collagens. Similarly, data from animal models of pulmonary fibrosis confirm the role of NLRP3 in the development of chronic lung injury and pulmonary fibrosis. In this report, we review evidence of NLRP3 activation in IPF and of NLRP3 inhibition in different animal models of fibrosis, and highlight the recent advances in direct and indirect NLRP3 inhibitors.

9.
J Vis Exp ; (181)2022 03 04.
Artículo en Inglés | MEDLINE | ID: mdl-35311826

RESUMEN

Applying novel microscopy methods to suitable animal disease models to explore the dynamic physiology of the kidney remains a challenge. Rats with surface glomeruli provide a unique opportunity to investigate physiological and pathophysiological processes using intravital 2-photon microscopy. Quantification of glomerular capillary blood flow and vasoconstriction and dilatation in response to drugs, permeability, and inflammation are just some of the processes that can be studied. In addition, transgenic rats, i.e., podocytes labeled with fluorescent dyes and other molecular biomarker approaches, provide increased resolution to directly monitor and quantify protein-protein interactions and the effects of specific molecular alterations. In mice, which lack surface glomeruli after four weeks of age, unilateral ureteral obstruction (UUO) for several weeks has been used to induce surface glomeruli. As this induction model does not allow for baseline studies, we quantified the effects of UUO on glomerular processes in the UUO model in Munich Wistar Frömter (MWF) rats, which have surface glomeruli under physiologic conditions. The UUO model for five weeks or more induced significant alterations to gross renal morphology, the peritubular and glomerular microvasculature, as well as the structure and function of tubular epithelia. Glomerular and peritubular red blood cell (RBC) flow decreased significantly (p < 0.01), probably due to the significant increase in the adherence of white blood cells (WBCs) within glomerular and peritubular capillaries. The glomerular sieving coefficient of albumin increased from 0.015 ± 0.002 in untreated MWFs to 0.045 ± 0.05 in 5-week-old UUO MWF rats. Twelve weeks of UUO resulted in further increases in surface glomerular density and glomerular sieving coefficient (GSC) for albumin. Fluorescent albumin filtered across the glomeruli was not reabsorbed by the proximal tubules. These data suggest that using UUO to induce surface glomeruli limits the ability to study and interpret normal glomerular processes and disease alterations.


Asunto(s)
Obstrucción Ureteral , Animales , Tasa de Filtración Glomerular , Riñón/metabolismo , Glomérulos Renales/metabolismo , Ratones , Microscopía , Ratas , Ratas Wistar
10.
Cells ; 11(6)2022 03 19.
Artículo en Inglés | MEDLINE | ID: mdl-35326496

RESUMEN

Hydrochloric acid (HCl) exposure causes asthma-like conditions, reactive airways dysfunction syndrome, and pulmonary fibrosis. Heat Shock Protein 90 (HSP90) is a molecular chaperone that regulates multiple cellular processes. HSP90 inhibitors are undergoing clinical trials for cancer and are also being studied in various pre-clinical settings for their anti-inflammatory and anti-fibrotic effects. Here we investigated the ability of the heat shock protein 90 (HSP90) inhibitor AT13387 to prevent chronic lung injury induced by exposure to HCl in vivo and its protective role in the endothelial barrier in vitro. We instilled C57Bl/6J mice with 0.1N HCl (2 µL/g body weight, intratracheally) and after 24 h began treatment with vehicle or AT13387 (10 or 15 mg/kg, SC), administered 3×/week; we analyzed histological, functional, and molecular markers 30 days after HCl. In addition, we monitored transendothelial electrical resistance (TER) and protein expression in a monolayer of human lung microvascular endothelial cells (HLMVEC) exposed to HCl (0.02 N) and treated with vehicle or AT13387 (2 µM). HCl provoked persistent alveolar inflammation; activation of profibrotic pathways (MAPK/ERK, HSP90); increased deposition of collagen, fibronectin and elastin; histological evidence of fibrosis; and a decline in lung function reflected in a downward shift in pressure-volume curves, increased respiratory system resistance (Rrs), elastance (Ers), tissue damping (G), and hyperresponsiveness to methacholine. Treatment with 15 mg/kg AT13387reduced alveolar inflammation, fibrosis, and NLRP3 staining; blocked activation of ERK and HSP90; and attenuated the deposition of collagen and the development of chronic lung injury and airway hyperreactivity. In vitro, AT13387 prevented HCl-induced loss of barrier function and AKT, ERK, and ROCK1 activation, and restored HSP70 and cofilin expression. The HSP90 inhibitor, AT13387, represents a promising drug candidate for chronic lung injury that can be administered subcutaneously in the field, and at low, non-toxic doses.


Asunto(s)
Antineoplásicos , Lesión Pulmonar , Fibrosis Pulmonar , Animales , Antineoplásicos/farmacología , Benzamidas , Colágeno/metabolismo , Células Endoteliales/metabolismo , Proteínas HSP90 de Choque Térmico/metabolismo , Ácido Clorhídrico/efectos adversos , Inflamación/patología , Isoindoles , Lesión Pulmonar/inducido químicamente , Lesión Pulmonar/tratamiento farmacológico , Lesión Pulmonar/prevención & control , Ratones , Ratones Endogámicos C57BL , Fibrosis Pulmonar/inducido químicamente , Fibrosis Pulmonar/tratamiento farmacológico , Fibrosis Pulmonar/prevención & control
11.
Sci Data ; 9(1): 57, 2022 02 16.
Artículo en Inglés | MEDLINE | ID: mdl-35173163

RESUMEN

The common vampire bat (Desmodus rotundus) is a sanguivorous (i.e., blood-eating) bat species distributed in the Americas from northern Mexico southwards to central Chile and Argentina. Desmodus rotundus is one of only three mammal species known to feed exclusively on blood, mainly from domestic mammals, although large wildlife and occasionally humans can also serve as a food source. Blood feeding makes D. rotundus an effective transmissor of pathogens to its prey. Consequently, this species is a common target of culling efforts by various individuals and organizations. Nevertheless, little is known about the historical distribution of D. rotundus. Detailed occurrence data are critical for the accurate assessment of past and current distributions of D. rotundus as part of ecological, biogeographical, and epidemiological research. This article presents a dataset of D. rotundus historical occurrence reports, including >39,000 locality reports across the Americas to facilitate the development of spatiotemporal studies of the species. Data are available at https://doi.org/10.6084/m9.figshare.15025296 .


Asunto(s)
Quirópteros , Animales , Argentina , Humanos , México , Estados Unidos
12.
Microorganisms ; 10(2)2022 Jan 24.
Artículo en Inglés | MEDLINE | ID: mdl-35208721

RESUMEN

Bats are natural reservoirs of a variety of zoonotic viruses, many of which cause severe human diseases. Characterizing viruses of bats inhabiting different geographical regions is important for understanding their viral diversity and for detecting viral spillovers between animal species. Herein, the diversity of DNA viruses of five arthropodophagous bat species from Argentina was investigated using metagenomics. Fecal samples of 29 individuals from five species (Tadarida brasiliensis, Molossus molossus, Eumops bonariensis, Eumops patagonicus, and Eptesicus diminutus) living at two different geographical locations, were investigated. Enriched viral DNA was sequenced using Illumina MiSeq, and the reads were trimmed and filtered using several bioinformatic approaches. The resulting nucleotide sequences were subjected to viral taxonomic classification. In total, 4,520,370 read pairs were sequestered by sequencing, and 21.1% of them mapped to viral taxa. Circoviridae and Genomoviridae were the most prevalent among vertebrate viral families in all bat species included in this study. Samples from the T. brasiliensis colony exhibited lower viral diversity than samples from other species of New World bats. We characterized 35 complete genome sequences of novel viruses. These findings provide new insights into the global diversity of bat viruses in poorly studied species, contributing to prevention of emerging zoonotic diseases and to conservation policies for endangered species.

13.
Nat Struct Mol Biol ; 29(1): 47-58, 2022 01.
Artículo en Inglés | MEDLINE | ID: mdl-35013599

RESUMEN

Inosine-5'-monophosphate dehydrogenase (IMPDH), a key regulatory enzyme in purine nucleotide biosynthesis, dynamically assembles filaments in response to changes in metabolic demand. Humans have two isoforms: IMPDH2 filaments reduce sensitivity to feedback inhibition, while IMPDH1 assembly remains uncharacterized. IMPDH1 plays a unique role in retinal metabolism, and point mutants cause blindness. Here, in a series of cryogenic-electron microscopy structures we show that human IMPDH1 assembles polymorphic filaments with different assembly interfaces in extended and compressed states. Retina-specific splice variants introduce structural elements that reduce sensitivity to GTP inhibition, including stabilization of the extended filament form. Finally, we show that IMPDH1 disease mutations fall into two classes: one disrupts GTP regulation and the other has no effect on GTP regulation or filament assembly. These findings provide a foundation for understanding the role of IMPDH1 in retinal function and disease and demonstrate the diverse mechanisms by which metabolic enzyme filaments are allosterically regulated.


Asunto(s)
IMP Deshidrogenasa/genética , Retina/enzimología , Adenosina Trifosfato/metabolismo , Regulación Alostérica , Sitios de Unión , Dominio Catalítico , Guanosina Trifosfato/metabolismo , Células HEK293 , Humanos , IMP Deshidrogenasa/química , IMP Deshidrogenasa/ultraestructura , Modelos Moleculares , NAD/metabolismo , Enfermedades de la Retina/genética
14.
Ultrason Sonochem ; 82: 105909, 2022 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-35051841

RESUMEN

Ultrasound is a promising technology for the improvement of zeolite production, due to its beneficial effects on mass transfer and nucleation. However, a broad understanding of the sonication parameters that influence the growth of zeolites most is still lacking. In the present work, zeolite A was synthesized and the kinetic model of Gualtieri was used to obtain information about the crystallization parameters. The effect of the sonication power and duration on the relative crystallinity and particle size distribution were investigated using a Langevin-type transducer operating at 40 kHz. The experimental data shows that ultrasound has a significant effect on the nucleation and growth. With that, a reduction of up to 40 % of the initial synthesis time can be achieved. Additionally, a narrower particle size distribution is achieved when ultrasound is used during the zeolite A synthesis.

15.
Biomacromolecules ; 23(2): 467-477, 2022 02 14.
Artículo en Inglés | MEDLINE | ID: mdl-34097399

RESUMEN

From trauma wards to chemotherapy, red blood cells are essential in modern medicine. Current methods to bank red blood cells typically use glycerol (40 wt %) as a cryoprotective agent. Although highly effective, the deglycerolization process, post-thaw, is time-consuming and results in some loss of red blood cells during the washing procedures. Here, we demonstrate that a polyampholyte, a macromolecular cryoprotectant, synergistically enhances ovine red blood cell cryopreservation in a mixed cryoprotectant system. Screening of DMSO and trehalose mixtures identified optimized conditions, where cytotoxicity was minimized but cryoprotective benefit maximized. Supplementation with polyampholyte allowed 97% post-thaw recovery (3% hemolysis), even under extremely challenging slow-freezing and -thawing conditions. Post-thaw washing of the cryoprotectants was tolerated by the cells, which is crucial for any application, and the optimized mixture could be applied directly to cells, causing no hemolysis after 1 h of exposure. The procedure was also scaled to use blood bags, showing utility on a scale relevant for application. Flow cytometry and adenosine triphosphate assays confirmed the integrity of the blood cells post-thaw. Microscopy confirmed intact red blood cells were recovered but with some shrinkage, suggesting that optimization of post-thaw washing could further improve this method. These results show that macromolecular cryoprotectants can provide synergistic benefit, alongside small molecule cryoprotectants, for the storage of essential cell types, as well as potential practical benefits in terms of processing/handling.


Asunto(s)
Dimetilsulfóxido , Trehalosa , Animales , Criopreservación/métodos , Crioprotectores/farmacología , Dimetilsulfóxido/farmacología , Eritrocitos/metabolismo , Hemólisis , Sustancias Macromoleculares/metabolismo , Ovinos , Trehalosa/farmacología
16.
Eur J Anaesthesiol ; 2021 Dec 01.
Artículo en Inglés | MEDLINE | ID: mdl-34860715

RESUMEN

BACKGROUND: Training schoolchildren in resuscitation seems to improve rates of resuscitation by bystanders. Leading medical societies recommend comprehensive resuscitation education in schools. To date, no widespread implementation within the European Union has happened. OBJECTIVE: The study aim was to identify facilitators and barriers for the implementation of cardiopulmonary resuscitation training for schoolchildren within the European Union. DESIGN: Systematic review. DATA SOURCES: A literature search in PubMed was conducted between 1 January 1999 and 30 June 2020 in accordance with the PRISMA statement. The search terms 'resuscitation', 'children' and 'Europe' were combined with the Boolean Operator 'AND' and 'OR'. Medical subject heading terms were used in order to include relevant articles. ELIGIBILITY CRITERIA: Articles were included if cardiopulmonary resuscitation training specifically tailored for schoolchildren aged 12 to 18 years was considered in countries of the European Union. Articles that fulfilled the following criteria were excluded: duplicates, training methods only for specific patient groups, articles not accessible in the English language, and articles that did not include original data. Findings were structured by an evidence-based six-level approach to examine barriers and facilitators in healthcare. RESULTS: Thirty out of 2005 articles were identified. Large variations in cardiopulmonary resuscitation training approaches ranging from conventional to innovative training methods can be observed. Schoolteachers as resuscitation instructors act either as barrier or facilitator depending on their personal attitude and their exposure to training in resuscitation. Cardiopulmonary resuscitation training in schoolchildren is effective. The uncoordinated interplay between the generally motivated schools and the political orientation towards resuscitation training for schoolchildren serve as barrier. The lack of financial support, absent systematic organisation and standardisation of training create major barriers. CONCLUSION: Training schoolchildren in cardiopulmonary resuscitation is effective. More financial support and political guidance is needed. Until then, local initiatives, motivated teachers, and dedicated principles combined with innovative and low-cost training methods facilitate cardiopulmonary resuscitation training in schools.

17.
Nat Prod Res ; : 1-9, 2021 Dec 20.
Artículo en Inglés | MEDLINE | ID: mdl-34930096

RESUMEN

Pentacyclic triterpenes are found in a great variety of natural products and constitute an organic template for the development of new derivative compounds with therapeutic applications. In the present work, lupeol acetate isolated from Chrysophyllum cainito L. fruit was used as a template for the synthesis of novel N-alkyl-arylsulfonamide derivatives, and their synergistic effects with metronidazole against strains of Trichomonas vaginalis were tested. A library of 18 derivatives was synthesized. Ten compounds exhibited an IC50 < 100 µM against a metronidazole-sensitive strain of T. vaginalis. Only seven of these compounds (12, 15, 18-22) also showed activity against metronidazole-resistant strains. The compounds 20 (N-cyclohexyl-p-chlorobenzenesulfonamidolupeol acetate) and 22 (N-cyclohexyl-p-nitrobenzenesulfonamidolupeol acetate) exhibited a similar IC50 against both susceptible and resistant T. vaginalis strains and enhanced the efficacy of metronidazole in a partial and total synergistic way, respectively. These data provided evidence of the trichomonicidal effect of N-alkyl-arylsulfonamide derivatives of lupeol acetate, representing highly promising novel antiparasitic agents.

18.
Front Microbiol ; 12: 740914, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-34777288

RESUMEN

We have structurally and functionally characterized Skl and Pal endolysins, the latter being the first endolysin shown to kill effectively Streptococcus pneumoniae, a leading cause of deathly diseases. We have proved that Skl and Pal are cysteine-amidases whose catalytic domains, from CHAP and Amidase_5 families, respectively, share an α3ß6-fold with papain-like topology. Catalytic triads are identified (for the first time in Amidase_5 family), and residues relevant for substrate binding and catalysis inferred from in silico models, including a calcium-binding site accounting for Skl dependence on this cation for activity. Both endolysins contain a choline-binding domain (CBD) with a ß-solenoid fold (homology modeled) and six conserved choline-binding loci whose saturation induced dimerization. Remarkably, Pal and Skl dimers display a common overall architecture, preserved in choline-bound dimers of pneumococcal lysins with other catalytic domains and bond specificities, as disclosed using small angle X-ray scattering (SAXS). Additionally, Skl is proved to be an efficient anti-pneumococcal agent that kills multi-resistant strains and clinical emergent-serotype isolates. Interestingly, Skl and Pal time-courses of pneumococcal lysis were sigmoidal, which might denote a limited access of both endolysins to target bonds at first stages of lysis. Furthermore, their DTT-mediated activation, of relevance for other cysteine-peptidases, cannot be solely ascribed to reversal of catalytic-cysteine oxidation.

19.
Plant Physiol ; 186(3): 1487-1506, 2021 07 06.
Artículo en Inglés | MEDLINE | ID: mdl-34624108

RESUMEN

Because it is the precursor for various essential cellular components, the amino acid serine is indispensable for every living organism. In plants, serine is synthesized by two major pathways: photorespiration and the phosphorylated pathway of serine biosynthesis (PPSB). However, the importance of these pathways in providing serine for plant development is not fully understood. In this study, we examine the relative contributions of photorespiration and PPSB to providing serine for growth and metabolism in the C3 model plant Arabidopsis thaliana. Our analyses of cell proliferation and elongation reveal that PPSB-derived serine is indispensable for plant growth and its loss cannot be compensated by photorespiratory serine biosynthesis. Using isotope labeling, we show that PPSB-deficiency impairs the synthesis of proteins and purine nucleotides in plants. Furthermore, deficiency in PPSB-mediated serine biosynthesis leads to a strong accumulation of metabolites related to nitrogen metabolism. This result corroborates 15N-isotope labeling in which we observed an increased enrichment in labeled amino acids in PPSB-deficient plants. Expression studies indicate that elevated ammonium uptake and higher glutamine synthetase/glutamine oxoglutarate aminotransferase (GS/GOGAT) activity causes this phenotype. Metabolic analyses further show that elevated nitrogen assimilation and reduced amino acid turnover into proteins and nucleotides are the most likely driving forces for changes in respiratory metabolism and amino acid catabolism in PPSB-deficient plants. Accordingly, we conclude that even though photorespiration generates high amounts of serine in plants, PPSB-derived serine is more important for plant growth and its deficiency triggers the induction of nitrogen assimilation, most likely as an amino acid starvation response.


Asunto(s)
Arabidopsis/crecimiento & desarrollo , Proliferación Celular/efectos de los fármacos , Respiración de la Célula/efectos de los fármacos , Nitrógeno/metabolismo , Desarrollo de la Planta/fisiología , Reguladores del Crecimiento de las Plantas/metabolismo , Serina/biosíntesis , Vías Biosintéticas , Fosforilación
20.
PLoS Pathog ; 17(9): e1009488, 2021 09.
Artículo en Inglés | MEDLINE | ID: mdl-34492091

RESUMEN

Arenavirus entry into host cells occurs through a low pH-dependent fusion with late endosomes that is mediated by the viral glycoprotein complex (GPC). The mechanisms of GPC-mediated membrane fusion and of virus targeting to late endosomes are not well understood. To gain insights into arenavirus fusion, we examined cell-cell fusion induced by the Old World Lassa virus (LASV) GPC complex. LASV GPC-mediated cell fusion is more efficient and occurs at higher pH with target cells expressing human LAMP1 compared to cells lacking this cognate receptor. However, human LAMP1 is not absolutely required for cell-cell fusion or LASV entry. We found that GPC-induced fusion progresses through the same lipid intermediates as fusion mediated by other viral glycoproteins-a lipid curvature-sensitive intermediate upstream of hemifusion and a hemifusion intermediate downstream of acid-dependent steps that can be arrested in the cold. Importantly, GPC-mediated fusion and LASV pseudovirus entry are specifically augmented by an anionic lipid, bis(monoacylglycero)phosphate (BMP), which is highly enriched in late endosomes. This lipid also specifically promotes cell fusion mediated by Junin virus GPC, an unrelated New World arenavirus. We show that BMP promotes late steps of LASV fusion downstream of hemifusion-the formation and enlargement of fusion pores. The BMP-dependence of post-hemifusion stages of arenavirus fusion suggests that these viruses evolved to use this lipid as a cofactor to selectively fuse with late endosomes.


Asunto(s)
Endosomas/metabolismo , Fiebre de Lassa/metabolismo , Virus Lassa/fisiología , Lisofosfolípidos/metabolismo , Monoglicéridos/metabolismo , Internalización del Virus , Animales , Células COS , Chlorocebus aethiops , Células HEK293 , Humanos , Proteínas del Envoltorio Viral/metabolismo
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