RESUMEN
Need for the Study: The mental branch of the trigeminal nerve, which supplies sensation to the lower lip, originates in the mandibular canal, making it an essential anatomical structure for dentists and oral surgeons to access. It is not well known that there is a spectrum of normal that includes variants in which there may be more than one nerve entry site, which means that if the mental foramen is not protected, normal feeling in the lower lip may be lost permanently. The diagnostic value of global radiographic landmarks like the mandibular trench and the dental foramen in identifying skeletal problems has been investigated. Materials and Methods: Four hundred patients over 40 who fit the inclusion/exclusion criteria were chosen. Patients were recruited from Vananchal Dental College and Hospital, Garhwa's Out Patient section for Oral Medicine, Diagnosis, and Radiology. The following patients with their consent are subjected for digital orthopantomography (Cephalometric Device for Rotograph EVOD Ref 930790001, SN 14112930, Villa Sistem Medical) and the captured images are then interpreted for the variations in mandibular canal. Result: The results of our study are as follows: In this study, the mean age of the subjects was 47.27 years, with a range of 40-75 yearsMajority of cases were females (53.75%) and 46.25% were males.The top of the residual ridge, located between the mental foramen and the mandibular canal, is clearly visible to all observers (grade I).Only 1.75% of the people surveyed had a bifid canal, despite the fact that the majority of the people surveyed had a single mandibular canal on both sides (98.25%).Right side mean was 18,682.017, whereas left side mean was 16,331.851; nevertheless, this difference was not statistically significant (P = 0.0860 NS). Conclusion: The dental foramen was located close to where the mandible and the area housing the next premolar met. These findings may be utilized to improve the safety of peri-apical surgical procedures. Therefore, it is therapeutically relevant to get insight into the structural alterations of the mental foramen and locate its location in preoperative radiological scans.
RESUMEN
We described a mass spectrometry-based assay to rapidly quantify the production of primary alcohols directly from cell cultures. This novel assay used the combination of TEMPO-based oxidation chemistry and oxime ligation, followed by product analysis based on Nanostructure-Initiator Mass Spectrometry. This assay enables quantitative monitor both C5 to C18 alcohols as well as glucose and gluconate in the growth medium to support strain characterization and optimization. We find that this assay yields similar results to gas chromatography for isoprenol production but required much less acquisition time per sample. We applied this assay to gain new insights into P. Putida's utilization of alcohols and find that this strain largely could not grow on heptanol and octanol.
Asunto(s)
Nanoestructuras , Cromatografía de Gases y Espectrometría de Masas , Espectrometría de Masas/métodos , Nanoestructuras/química , Glucosa , EtanolRESUMEN
Introduction Treatment with dexamethasone reduces mortality in patients with coronavirus disease 2019 (COVID-19) pneumonia requiring supplemental oxygen, but the optimal dose has not been determined. Objective To determine whether weight-based dexamethasone of 0.2 mg/kg is superior to 6 mg daily in reducing 28-day mortality in patients with COVID-19 and hypoxemia. Materials and methods A multicenter, open-label, randomized clinical trial was conducted between March 2021 and December 2021 at seven hospitals within Northwell Health. A total of 142 patients with confirmed COVID-19 and hypoxemia were included. Participants were randomized in a 1:1 ratio to dexamethasone 0.2 mg/kg intravenously daily (n = 70) or 6 mg daily (n = 72) for up to 10 days. Results There was no statistically significant difference in the primary outcome of 28-day all-cause mortality with deaths in 12 of 70 patients (17.14%) in the intervention group and 15 of 72 patients (20.83%) in the control group (p = 0.58). There were no statistically significant differences among the secondary outcomes. Conclusion In patients with COVID-19 and hypoxemia, the use of weight-based dexamethasone dosing was not superior to dexamethasone 6 mg in reducing all-cause mortality at 28 days. Clinical trial registration This study was registered under ClinicalTrials.gov (identifier: NCT04834375).
RESUMEN
We present a droplet-based microfluidic system that enables CRISPR-based gene editing and high-throughput screening on a chip. The microfluidic device contains a 10 × 10 element array, and each element contains sets of electrodes for two electric field-actuated operations: electrowetting for merging droplets to mix reagents and electroporation for transformation. This device can perform up to 100 genetic modification reactions in parallel, providing a scalable platform for generating the large number of engineered strains required for the combinatorial optimization of genetic pathways and predictable bioengineering. We demonstrate the system's capabilities through the CRISPR-based engineering of two test cases: (1) disruption of the function of the enzyme galactokinase (galK) in E. coli and (2) targeted engineering of the glutamine synthetase gene (glnA) and the blue-pigment synthetase gene (bpsA) to improve indigoidine production in E. coli.
RESUMEN
BACKGROUND: SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) continues to be a global challenge due to the lack of definitive treatment strategies. We sought to determine the efficacy of early administration of anti-interleukin 6 therapy in reducing hospital mortality and progression to mechanical ventilation. METHODS: This was a retrospective chart review of 11,512 patients infected with SARS-CoV-2 who were admitted to a New York health system from March to May 2020. Tocilizumab was administered to subjects at the nasal cannula level of oxygen support to maintain an oxygen saturation of >88%. The Charlson comorbidity index was used as an objective assessment of the burden of comorbidities to predict 10-year mortality. The primary outcome of interest was hospital mortality. Secondary outcomes were progression to mechanical ventilation; the prevalence of venous thromboembolism and renal failure; and the change in C-reactive protein, D-dimer, and ferritin levels after tocilizumab administration. Propensity score matching by using a 1:2 protocol was used to match the tocilizumab and non-tocilizumab groups to minimize selection bias. The groups were matched on baseline demographic characteristics, including age, sex, and body mass index; Charlson comorbidity index score; laboratory markers, including ferritin, D-dimer, lactate dehydrogenase, and C-reactive protein values; and the maximum oxygen requirement at the time of tocilizumab administration. Mortality outcomes were evaluated based on the level of oxygen requirement and the day of hospitalization at the time of tocilizumab administration. RESULTS: The overall hospital mortality was significantly reduced in the tocilizumab group when tocilizumab was administered at the nasal cannula level (10.4% vs 22.0%; P = .002). In subjects who received tocilizumab at the nasal cannula level, the progression to mechanical ventilation was reduced versus subjects who were initially on higher levels of oxygen support (6.3% vs 18.7%; P < .001). There was no improvement in mortality when tocilizumab was given at the time of requiring non-rebreather, high-flow nasal cannula, noninvasive ventilator, or invasive ventilator. CONCLUSIONS: Early use of anti-interleukin 6 therapy may be associated with improved hospital mortality and reduction in progression to more severe coronavirus disease 2019.
Asunto(s)
Tratamiento Farmacológico de COVID-19 , SARS-CoV-2 , Anticuerpos Monoclonales Humanizados , Humanos , Respiración Artificial , Estudios Retrospectivos , Resultado del TratamientoRESUMEN
Progress in sequencing, microfluidics, and analysis strategies has revolutionized the granularity at which multicellular organisms can be studied. In particular, single-cell transcriptomics has led to fundamental new insights into animal biology, such as the discovery of new cell types and cell type-specific disease processes. However, the application of single-cell approaches to plants, fungi, algae, or bacteria (environmental organisms) has been far more limited, largely due to the challenges posed by polysaccharide walls surrounding these species' cells. In this perspective, we discuss opportunities afforded by single-cell technologies for energy and environmental science and grand challenges that must be tackled to apply these approaches to plants, fungi and algae. We highlight the need to develop better and more comprehensive single-cell technologies, analysis and visualization tools, and tissue preparation methods. We advocate for the creation of a centralized, open-access database to house plant single-cell data. Finally, we consider how such efforts should balance the need for deep characterization of select model species while still capturing the diversity in the plant kingdom. Investments into the development of methods, their application to relevant species, and the creation of resources to support data dissemination will enable groundbreaking insights to propel energy and environmental science forward.
Asunto(s)
Conservación de los Recursos Energéticos/métodos , Bases de Datos como Asunto , Ciencia Ambiental/métodos , Plantas , Análisis de la Célula Individual/métodos , Tecnología/instrumentaciónRESUMEN
Fabricated ecosystems (EcoFABs) offer an innovative approach to in situ examination of microbial establishment patterns around plant roots using nondestructive, high-resolution microscopy. Previously high-resolution imaging was challenging because the roots were not constrained to a fixed distance from the objective. Here, we describe a new 'Imaging EcoFAB' and the use of this device to image the entire root system of growing Brachypodium distachyon at high resolutions (20×, 40×) over a 3-week period. The device is capable of investigating root-microbe interactions of multimember communities. We examined nine strains of Pseudomonas simiae with different fluorescent constructs to B. distachyon and individual cells on root hairs were visible. Succession in the rhizosphere using two different strains of P. simiae was examined, where the second addition was shown to be able to establish in the root tissue. The device was suitable for imaging with different solid media at high magnification, allowing for the imaging of fungal establishment in the rhizosphere. Overall, the Imaging EcoFAB could improve our ability to investigate the spatiotemporal dynamics of the rhizosphere, including studies of fluorescently-tagged, multimember, synthetic communities.
Asunto(s)
Brachypodium/microbiología , Microtecnología/instrumentación , Imagen Molecular/métodos , Raíces de Plantas/microbiología , Pseudomonas/fisiología , Rizosfera , Brachypodium/metabolismo , Raíces de Plantas/metabolismo , Microbiología del SueloRESUMEN
Lignocellulosic biomass is composed of three major biopolymers: cellulose, hemicellulose and lignin. Analytical tools capable of quickly detecting both glycan and lignin deconstruction are needed to support the development and characterization of efficient enzymes/enzyme cocktails. Previously we have described nanostructure-initiator mass spectrometry-based assays for the analysis of glycosyl hydrolase and most recently an assay for lignin modifying enzymes. Here we integrate these two assays into a single multiplexed assay against both classes of enzymes and use it to characterize crude commercial enzyme mixtures. Application of our multiplexed platform based on nanostructure-initiator mass spectrometry enabled us to characterize crude mixtures of laccase enzymes from fungi Agaricus bisporus (Ab) and Myceliopthora thermophila (Mt) revealing activity on both carbohydrate and aromatic substrates. Using time-series analysis we determined that crude laccase from Ab has the higher GH activity and that laccase from Mt has the higher activity against our lignin model compound. Inhibitor studies showed a significant reduction in Mt GH activity under low oxygen conditions and increased activities in the presence of vanillin (common GH inhibitor). Ultimately, this assay can help to discover mixtures of enzymes that could be incorporated into biomass pretreatments to deconstruct diverse components of lignocellulosic biomass.
Asunto(s)
Enzimas/química , Lignina/química , Espectrometría de Masas/métodos , N-Glicosil Hidrolasas/química , Activación Enzimática , Pruebas de Enzimas , Estructura MolecularRESUMEN
Chemokine receptor CXCR4 overexpression in solid tumors has been strongly associated with poor prognosis and adverse clinical outcome. However, blockade of CXCL12-CXCR4 signaling axis by inhibitors like Nox-A12, FDA approved CXCR4 inhibitor drug AMD3100 have shown limited clinical success in cancer treatment. Therefore, exclusive contribution of CXCR4-CXCL12 signaling in pro-tumorigenic function is questionable. In our pursuit to understand the impact of chemokine signaling in carcinogenesis, we reveal that instead of CXCR4-CXCL12 signaling, presence of CXCR4 intracellular protein augments paclitaxel resistance and pro-tumorigenic functions. In search of pro-apoptotic mechanisms for CXCR4 mediated drug resistance; we discover that DR5 is a new selective target of CXCR4 in breast and colon cancer. Further, we detect that CXCR4 directs the differential recruitment of transcription factors p53 and YY1 to the promoter of DR5 in course of its transcriptional repression. Remarkably, inhibiting CXCR4-ligand-mediated signals completely fails to block the above phenotype. Overexpression of different mutant versions of CXCR4 lacking signal transduction capabilities also result in marked downregulation of DR5 expression in colon cancer indeed confirms the reverse relationship between DR5 and intracellular CXCR4 protein expression. Irrespective of CXCR4 surface expression, by utilizing stable gain and loss of function approaches, we observe that intracellular CXCR4 protein selectively resists and sensitizes colon cancer cells against paclitaxel therapy in vitro and in vivo. Finally, performing TCGA data mining and using human breast cancer patient samples, we demonstrate that expression of CXCR4 and DR5 are inversely regulated. Together, our data suggest that targeting CXCR4 intracellular protein may be critical to dampen the pro-tumorigenic functions of CXCR4.
Asunto(s)
Neoplasias de la Mama/genética , Resistencia a Antineoplásicos/efectos de los fármacos , Receptores CXCR4/metabolismo , Receptores del Ligando Inductor de Apoptosis Relacionado con TNF/metabolismo , Línea Celular Tumoral , Femenino , HumanosRESUMEN
The rhizosphere is a dynamic ecosystem shaped by complex interactions between plant roots, soil, microbial communities and other micro- and macro-fauna. Although studied for decades, critical gaps exist in the study of plant roots, the rhizosphere microbiome and the soil system surrounding roots, partly due to the challenges associated with measuring and parsing these spatiotemporal interactions in complex heterogeneous systems such as soil. To overcome the challenges associated with in situ study of rhizosphere interactions, specialized plant growth chamber systems have been developed that mimic the natural growth environment. This review discusses the currently available lab-based systems ranging from widely known rhizotrons to other emerging devices designed to allow continuous monitoring and non-destructive sampling of the rhizosphere ecosystems in real-time throughout the developmental stages of a plant. We categorize them based on the major rhizosphere processes it addresses and identify their unique challenges as well as advantages. We find that while some design elements are shared among different systems (e.g., size exclusion membranes), most of the systems are bespoke and speaks to the intricacies and specialization involved in unraveling the details of rhizosphere processes. We also discuss what we describe as the next generation of growth chamber employing the latest technology as well as the current barriers they face. We conclude with a perspective on the current knowledge gaps in the rhizosphere which can be filled by innovative chamber designs.
RESUMEN
The mutagenic APOBEC3B (A3B) cytosine deaminase is frequently over-expressed in cancer and promotes tumour heterogeneity and therapy resistance. Hence, understanding the mechanisms that underlie A3B over-expression is important, especially for developing therapeutic approaches to reducing A3B levels, and consequently limiting cancer mutagenesis. We previously demonstrated that A3B is repressed by p53 and p53 mutation increases A3B expression. Here, we investigate A3B expression upon treatment with chemotherapeutic drugs that activate p53, including 5-fluorouracil, etoposide and cisplatin. Contrary to expectation, these drugs induced A3B expression and concomitant cellular cytosine deaminase activity. A3B induction was p53-independent, as chemotherapy drugs stimulated A3B expression in p53 mutant cells. These drugs commonly activate ATM, ATR and DNA-PKcs. Using specific inhibitors and gene knockdowns, we show that activation of DNA-PKcs and ATM by chemotherapeutic drugs promotes NF-κB activity, with consequent recruitment of NF-κB to the A3B gene promoter to drive A3B expression. Further, we find that A3B knockdown re-sensitises resistant cells to cisplatin, and A3B knockout enhances sensitivity to chemotherapy drugs. Our data highlight a role for A3B in resistance to chemotherapy and indicate that stimulation of A3B expression by activation of DNA repair and NF-κB pathways could promote cancer mutations and expedite chemoresistance.
Asunto(s)
Citidina Desaminasa/genética , Antígenos de Histocompatibilidad Menor/genética , Neoplasias/genética , Factor de Transcripción ReIA/genética , Proteína p53 Supresora de Tumor/genética , Proteínas de la Ataxia Telangiectasia Mutada/genética , Sistemas CRISPR-Cas/genética , Cisplatino/farmacología , Reparación del ADN/efectos de los fármacos , Etopósido/farmacología , Fluorouracilo/farmacología , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Heterogeneidad Genética , Células HCT116 , Humanos , Células MCF-7 , Mutación/genética , FN-kappa B/genética , Neoplasias/patologíaRESUMEN
The ADP-ribosylhydrolase ARH3 plays a key role in DNA damage repair, digesting poly(ADP-ribose) and removing ADP-ribose from serine residues of the substrates. Specific inhibitors that selectively target ARH3 would be a useful tool to examine DNA damage repair, as well as a possible strategy for tumor suppression. However, efforts to date have not identified any suitable compounds. Here, we used in silico and biochemistry screening to search for ARH3 inhibitors. We discovered a small molecule compound named ARH3 inhibitor 26 (AI26) as, to our knowledge, the first ARH3 inhibitor. AI26 binds to the catalytic pocket of ARH3 and inhibits the enzymatic activity of ARH3 with an estimated IC50 of â¼2.41 µm in vitro Moreover, hydrolysis of DNA damage-induced ADP-ribosylation was clearly inhibited when cells were pretreated with AI26, leading to defects in DNA damage repair. In addition, tumor cells with DNA damage repair defects were hypersensitive to AI26 treatment, as well as combinations of AI26 and other DNA-damaging agents such as camptothecin and doxorubicin. Collectively, these results reveal not only a chemical probe to study ARH3-mediated DNA damage repair but also a chemotherapeutic strategy for tumor suppression.
Asunto(s)
Daño del ADN , Reparación del ADN/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Glicósido Hidrolasas/antagonistas & inhibidores , Glicósido Hidrolasas/metabolismo , Línea Celular Tumoral , Glicósido Hidrolasas/genética , HumanosRESUMEN
ZBTB24, encoding a protein of the ZBTB family of transcriptional regulators, is one of four known genes-the other three being DNMT3B, CDCA7 and HELLS-that are mutated in immunodeficiency, centromeric instability and facial anomalies (ICF) syndrome, a genetic disorder characterized by DNA hypomethylation and antibody deficiency. The molecular mechanisms by which ZBTB24 regulates gene expression and the biological functions of ZBTB24 are poorly understood. Here, we identified a 12-bp consensus sequence [CT(G/T)CCAGGACCT] occupied by ZBTB24 in the mouse genome. The sequence is present at multiple loci, including the Cdca7 promoter region, and ZBTB24 binding is mostly associated with gene activation. Crystallography and DNA-binding data revealed that the last four of the eight zinc fingers (ZFs) (i.e. ZF5-8) in ZBTB24 confer specificity of DNA binding. Two ICF missense mutations have been identified in the ZBTB24 ZF domain, which alter zinc-binding cysteine residues. We demonstrated that the corresponding C382Y and C407G mutations in mouse ZBTB24 abolish specific DNA binding and fail to induce Cdca7 expression. Our analyses indicate and suggest a structural basis for the sequence specific recognition by a transcription factor centrally important for the pathogenesis of ICF syndrome.
Asunto(s)
Proteínas de Ciclo Celular/genética , Cara/anomalías , Genoma , Mutación Missense , Proteínas Nucleares/genética , Enfermedades de Inmunodeficiencia Primaria/genética , Proteínas Represoras/química , Factores de Transcripción/química , Dedos de Zinc/genética , Animales , Sitios de Unión , Proteínas de Ciclo Celular/metabolismo , Clonación Molecular , Escherichia coli/genética , Escherichia coli/metabolismo , Cara/patología , Expresión Génica , Sitios Genéticos , Vectores Genéticos , Humanos , Ratones , Modelos Moleculares , Proteínas Nucleares/metabolismo , Motivos de Nucleótidos , Enfermedades de Inmunodeficiencia Primaria/metabolismo , Enfermedades de Inmunodeficiencia Primaria/patología , Regiones Promotoras Genéticas , Unión Proteica , Conformación Proteica en Hélice alfa , Conformación Proteica en Lámina beta , Dominios y Motivos de Interacción de Proteínas , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas Represoras/genética , Proteínas Represoras/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
BACKGROUND: Producing valuable fuels and chemicals from lignin is a key factor for making lignocellulosic biomass economically feasible; however, significant roadblocks exist due to our lack of detailed understanding of how lignin is enzymatically depolymerized and of the range of possible lignin fragments that can be produced. Development of suitable enzymatic assays for characterization of putative lignin active enzymes is an important step towards improving our understanding of the catalytic activities of relevant enzymes. Previously, we have successfully built an assay platform based on glycan substrates containing a charged perfluorinated tag and nanostructure-initiator mass spectrometry to study carbohydrate active enzymes, especially various glycosyl hydrolyses. Here, we extend this approach to develop a reliable and rapid assay to study lignin-modifying enzymes. RESULTS: Two ß-aryl ether bond containing model lignin dimer substrates, designed to be suitable for studying the activities of lignin-modifying enzymes (LMEs) by nanostructure-initiator mass spectrometry (NIMS), were successful synthesized. Small-angle neutron scattering experiments showed that these substrates form micelles in solution. Two LMEs, laccase from the polypore mushroom Trametes versicolor, and manganese peroxidase (MnP) from white rot fungus Nematoloma frowardii, were tested for catalytic activity against the two model substrates. We show that the reaction of laccase and MnP with phenolic substrate yields products that arise from the cleavage of the carbon-carbon single bond between the α-carbon and the adjacent aryl carbon, consistent with the mechanism for producing phenoxy radical as reaction intermediates. Reactions of the nonphenolic substrate with laccase, on the other hand, adopt a different pathway by producing an α-oxidation product; as well as the cleavage of the ß-aryl ether bond. No cleavage of the carbon-carbon bond between the α-carbon and the aryl carbon was observed. To facilitate understanding of reaction kinetics, the reaction time course for laccase activity on the phenolic substrate (I) was generated by the simultaneous measurement of all products at different time points of the reaction. Withdrawal of only a small sample aliquot (0.2 µL at each time point) ensured minimum perturbation of the reaction. The time course can help us to understand the enzyme kinetics. CONCLUSIONS: A new assay procedure has been developed for studying lignin-modifying enzymes by nanostructure-initiator mass spectrometry. Enzyme assays of a laccase and a MnP on phenolic and nonphenolic ß-aryl ether substrates revealed different primary reaction pathways due to the availability of the phenoxy radical intermediates. Our assay provides a wealth of information on bond cleavage events not available using conventional colorimetric assays and can easily be carried out in microliter volumes and the quantitative analysis of product formation and kinetics is rapidly achieved by NIMS. This is the first time that NIMS technology was applied to study the activities of lignin-modifying enzymes. Unlike other previous works, our use of amphiphilic guaiacylglycerol ß-O-4 substrate (I) enables the formation of micelles. This approach helps avoid the re-polymerization of the resulting monomeric product. As a result, our assay can clearly demonstrate the degradation pathways of phenolic guaiacylglycerol ß-O-4 type of molecules with laccase and MnP.
RESUMEN
The challenges of diagnosing infectious disease, especially in the developing world, and the shortcomings of available instrumentation have exposed the need for portable, easy-to-use diagnostic tools capable of detecting the wide range of causative microbes while operating in low resource settings. We present a centrifugal microfluidic platform that combines ultrasensitive immunoassay and isothermal amplification-based screening for the orthogonal detection of both protein and nucleic acid targets at the point-of-care. A disposable disc with automatic aliquoting inlets is paired with a non-contact heating system and precise rotary control system to yield an easy-to-use, field-deployable platform with versatile screening capabilities. The detection of three enterotoxins (cholera toxin, Staphylococcal enterotoxin B, and Shiga-like toxin 1) and three enteric bacteria (C. jejuni, E. coli, and S. typhimurium) were performed independently and shown to be highly sensitive (limit of detection =â¯1.35-5.50â¯ng/mL for immunoassays and 1-30 cells for isothermal amplification), highly exclusive in the presence of non-specific targets, and capable of handling a complex sample matrix like stool. The full panel of toxins and bacteria were reliably detected simultaneously on a single disc at clinically relevant sample concentrations in less than an hour. The ability of our technology to detect multiple analyte types in parallel at the point-of-care can serve a variety of needs, from routine patient care to outbreak triage, in a variety of settings to reduce disease impact and expedite effective treatment.
Asunto(s)
Técnicas Biosensibles/métodos , Diarrea/diagnóstico , Enterobacteriaceae/fisiología , Enterotoxinas/análisis , Inmunoensayo , Técnicas de Amplificación de Ácido Nucleico , Técnicas Biosensibles/instrumentación , Enterobacteriaceae/genética , Heces/química , Heces/microbiología , Humanos , Límite de Detección , MicrofluídicaRESUMEN
Surface-assisted laser desorption ionization (SALDI) is an approach for gas-phase ion generation for mass spectrometry using laser excitation on typically conductive or semiconductive nanostructures. Here, we introduce insulator nanostructure desorption ionization mass spectrometry (INDI-MS), a nanostructured polymer substrate for SALDI-MS analysis of small molecules and peptides. INDI-MS surfaces are produced through the self-assembly of a perfluoroalkyl silsesquioxane nanostructures in a single chemical vapor deposition silanization-step. We find that surfaces formed from the perfluorooctyltrichlorosilane monomer assemble semielliptical features with a 10 nm height, diameters between 10 and 50 nm, and have attomole-femtomole sensitivities for selected analytes. Surfaces prepared with silanes that either lack the trichloro or perfluoro groups, lack sensitivity. Further, we demonstrate that hydrophobic INDI regions can be micropatterned onto hydrophilic surfaces to perform on-chip self-desalting in an array format.
RESUMEN
Oligonucleotides are almost exclusively synthesized using the nucleoside phosphoramidite method, even though it is limited to the direct synthesis of â¼200 mers and produces hazardous waste. Here, we describe an oligonucleotide synthesis strategy that uses the template-independent polymerase terminal deoxynucleotidyl transferase (TdT). Each TdT molecule is conjugated to a single deoxyribonucleoside triphosphate (dNTP) molecule that it can incorporate into a primer. After incorporation of the tethered dNTP, the 3' end of the primer remains covalently bound to TdT and is inaccessible to other TdT-dNTP molecules. Cleaving the linkage between TdT and the incorporated nucleotide releases the primer and allows subsequent extension. We demonstrate that TdT-dNTP conjugates can quantitatively extend a primer by a single nucleotide in 10-20 s, and that the scheme can be iterated to write a defined sequence. This approach may form the basis of an enzymatic oligonucleotide synthesizer.
Asunto(s)
Replicación del ADN/genética , ADN Polimerasa Dirigida por ADN/genética , Nucleósidos/genética , Oligonucleótidos/genética , ADN Nucleotidilexotransferasa/química , ADN Nucleotidilexotransferasa/genética , ADN Polimerasa Dirigida por ADN/química , Nucleósidos/química , Oligonucleótidos/biosíntesis , Oligonucleótidos/química , Compuestos Organofosforados/químicaRESUMEN
The ability to observe lethal anthrax exotoxins translocating through size-constricting nanopores in vitro, combined with detailed sequence and structural data, has aided in elucidated mechanisms of exotoxin cell entry and toxicity. However, due to limited observations of anthrax exotoxins translocating through protective antigen nanopores in vitro and the instability of protective antigen-functionalized suspended lipid bilayers, questions remain regarding the native mechanisms of cell entry. Nanoporous hydrogel membranes offer a robust tool for studying protein translocation with ensemble measurements that complement conventional single-molecule translocation measurements. Here, we utilize nanoporous hydrogel membranes to assess the translocation of full-length anthrax lethal and edema factors through nanopores similar in diameter to protective antigen translocons. We find that, relative to globular serum and other proteins that do not translocate natively through nanopores, anthrax exotoxins demonstrate significantly reduced barriers to pore entry. Computed free-energy barriers to the unfolding of proteins and the dissociation of macromolecular complexes are generally found to coincide with translocation. Finally, a nanopore-blocking strategy is developed that utilizes nonspecific synthetic peptide constructs and effectively prevents LF translocation within the nanoporous hydrogel.
Asunto(s)
Nanoporos , Carbunco , Antígenos Bacterianos , Exotoxinas , Humanos , Hidrogeles , Membrana Dobles de LípidosRESUMEN
BACKGROUND: The presence of B lines on lung ultrasonography is a characteristic feature of both cardiogenic pulmonary edema (CPE) and noncardiogenic alveolar interstitial syndrome (NCAIS), so their presence does not allow the clinician to differentiate between the two entities. Our study used M-mode ultrasonography of the pleura to differentiate CPE from NCAIS. METHODS: A total of 43 subjects were enrolled in the study, and based on history, physical examination, and chart review, the patients were divided into three groups: an NCAIS group, a CPE group, and a control group. Three distinct pleural line morphologic categories were identified: a continuous pleural line, a fragmented pleural line, and a sinusoidal pleural line. In addition, two separate subpleural patterns were independently identified by the investigators: a horizontal pattern and a vertical pattern. These pleural and subpleural patterns were correlated with subject diagnoses. RESULTS: A fragmented pleural line and a vertical subpleural pattern on M-mode ultrasonography is associated with patients who have NCAIS. Most patients with CPE have a continuous pleural line and a vertical subpleural pattern on M-mode ultrasonography. A sinusoidal pleural line on M-mode ultrasonography is suggestive of the presence of a pleural effusion. CONCLUSIONS: Our results indicate that M-mode ultrasonography is useful to distinguish CPE from NCAIS based on the pleural and the subpleural morphologic features.