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This experiment aimed to investigate the in vitro antimicrobial activity of danofloxacin against Escherichia coli (E. coli) isolated from pigeons, as well as the pharmacokinetics of danofloxacin in pigeons following oral (PO), intramuscular (IM), and intravenous (IV) administration. The minimum inhibitory concentration (MIC) of danofloxacin was first determined for 38 clinical E. coli strains using the micro broth dilution method. Subsequently, 30 healthy pigeons were weighed and randomly divided into 3 groups: IM, IV, and PO, with 10 pigeons in each group. Danofloxacin was given at 5 mg/kg body weight (BW) through 3 different routes. Blood was collected, and plasma was separated at various time points from 0 to 48 h. Plasma samples were analyzed for danofloxacin concentrations using a validated HPLC method. Pharmacokinetic analysis was performed using Phoenix software and a noncompartmental analytical (NCA) method. The results indicated that danofloxacin had a strong antibacterial effect on E. coli, with a MIC50 of 0.5 µg/mL. The noncompartmental analysis showed that after PO and IM administration at 5 mg/kg in pigeons, peak plasma concentrations (Cmax) of 0.61 and 1.62 µg/mL were reached at 4.5 and 0.53 h, respectively. The oral and intramuscular bioavailability (F) were 68.08% ± 24.82% and 87.82% ± 25.36%, respectively. Following IV administration, danofloxacin was widely distributed in pigeons, with volume of distribution (VZ) and volume of distribution at steady state (VSS) values of 6.11 ± 2.01 and 4.65 ± 1.62 L/kg, respectively, and was eliminated slowly, with an elimination half-life (t1/2λz) of 6.41 ± 2.15 h. Based on the calculated ratio values of AUC/MIC, the current IV, IM, and PO doses of 5 mg/kg of danofloxacin would be expected to effectively treat pigeons infected with E. coli strains with MIC values equal to or less than 0.5 µg/mL.
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With the increasing attention paid to macrocyclic scaffolds in peptide drug development, genetically encoded peptide macrocycle libraries have become invaluable sources for the discovery of high-affinity peptide ligands targeting disease-associated proteins. The traditional phage display technique of constructing disulfide-tethered macrocycles by cysteine oxidation has the inherent drawback of reduction instability of the disulfide bond. Chemical macrocyclization solves the problem of disulfide bond instability, but the involved highly electrophilic reagents are usually toxic to phages and may bring undesirable side reactions. Here, we report a unique Sortase-mediated Peptide Ligation and One-pot Cyclization strategy (SPLOC) to generate peptide macrocycle libraries, avoiding the undesired reactions of electrophiles with phages. The key to this platform is to mine the unnatural promiscuity of sortase on the X residue of the pentapeptide recognition sequence (LPXTG). Low reactive electrophiles are incorporated into the X-residue side chain, enabling intramolecular cyclization with the cysteine residue of the phage-displayed peptide library. Utilizing the genetically encoded peptide macrocycle library constructed by the SPLOC platform, we found a high-affinity bicyclic peptide binding TEAD4 with a nanomolar KD value (63.9 nM). Importantly, the binding affinity of the bicyclic peptide ligand is 102-fold lower than that of the acyclic analogue. To our knowledge, this is the first time to mine the unnatural promiscuity of ligases to generate peptide macrocycles, providing a new avenue for the construction of genetically encoded cyclic peptide libraries.
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We describe a simple and robust oxidation strategy for preparing N-terminal thiazolidine-containing peptide thioesters from peptide hydrazides. We find for the first time that l-thioproline can be used as a protective agent to prevent the nitrosation of N-terminal thiazolidine during peptide hydrazide oxidation. The thioproline-based oxidation strategy has been successfully applied to the chemical synthesis of CC chemokine ligand-2 (69aa) and omniligase-C (113aa), thereby demonstrating its utility in hydrazide-based native chemical ligation.
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Oxidación-Reducción , Péptidos , Tiazolidinas , Tiazolidinas/química , Tiazolidinas/síntesis química , Estructura Molecular , Péptidos/química , Péptidos/síntesis química , Hidrazinas/química , Prolina/química , Ésteres/química , Compuestos de Sulfhidrilo/químicaRESUMEN
The current study aimed to investigate the pharmacokinetics of bromhexine hydrochloride in broilers after single intravenous (IV) and oral (PO) administration at 2.5 mg/kg body weight (BW). The trial adopted a randomized, parallel-controlled design, where 20 twelve-wk-old broilers were randomly assigned to either the PO or IV group. Blood samples were collected at predetermined time points, and plasma was further separated for analysis. The bromhexine hydrochloride concentrations in plasma samples were determined using an ultra-performance liquid chromatography-tandem quadrupole mass spectrometry (UPLC-MS/MS) method. Noncompartmental analysis (NCA) using Phoenix software was conducted to analyze the concentration versus time data of bromhexine hydrochloride in every chicken. Subsequently, the main pharmacokinetic parameters between the 2 groups were statistically analyzed using SPSS software. Results from NCA revealed that after oral administration at 2.5 mg/kg BW, bromhexine hydrochloride exhibited slow absorption, reaching an average peak concentration of 32.72 ng/mL at 1.78 h. However, incomplete absorption was observed, with an absolute bioavailability of only 20.06% ± 10.84%. Additionally, bromhexine hydrochloride displayed wide distribution, with a steady-state distribution volume (VSS) of 22.55 ± 13.45 L/kg, and slow elimination, with a clearance (Cl) of 1.52 ± 0.38 L/h/kg. Furthermore, gender effects were assessed on the pharmacokinetics of bromhexine hydrochloride in broilers, revealing better absorption in male broilers compared to females. This disparity may be attributed to the faster blood flow and richer blood volume typically found in male broilers.
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Bromhexina , Pollos , Animales , Masculino , Administración Oral , Femenino , Bromhexina/farmacocinética , Bromhexina/administración & dosificación , Inyecciones Intravenosas/veterinaria , Espectrometría de Masas en Tándem/veterinaria , Distribución Aleatoria , Disponibilidad Biológica , Administración Intravenosa/veterinariaRESUMEN
To study thermal behaviour during spontaneous combustion of an open-pit coal mine, mixed slag (coal, oil shale, and coal gangue) was taken as the research object. Laser thermal conductivity analyser and differential scanning calorimetry were used to test thermophysical parameters and heat release characteristics of the minerals. The parameters can be employed to calculate the apparent activation energy using the Arrhenius equation and evaluate the thermal behaviour of open-pit mixed slag. The results indicate that thermophysical parameters have stage characteristics. Thermal diffusivity and thermal conductivity of minerals, especially mixed slag, have a strong correlation with temperature. Heat flow of minerals exhibits five characteristic stages, and heat flow of the samples is consistent with the change in heating rate. During the heating process, thermal diffusivity and heat flow of the mixed slag are between those of a single mineral. Except for the mixed slag at 15 and 20 °C/min, the initial exothermic temperature of the other samples is mainly concentrated at 50-80 °C. Thermal energy release of the sample is mainly concentrated in the accelerated exothermic stage and rapid exothermic stage. Thermal energy release of mixed slag in rapid exothermic stage is always greater than that in accelerated exothermic stage, and the proportion of thermal energy release in these two stages exceeds 98 %. The apparent activation energy during the accelerated exothermic stage is lower, making it easier to release heat, and rapid exothermic stage is relatively high, which can readily lead to heat accumulation. Thermal analysis reveals that the thermal behaviour of mixed slag is significantly different from that of a single mineral. Its unique exothermic characteristics can provide a more accurate theoretical basis for the prevention and control of environmental pollution caused by slag spontaneous combustion.
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This study aimed to compare the residue depletion of gamithromycin in yellow-feather and white-feather broilers, using Sanhuang and Arbor Acres chickens as typical examples, respectively. Each breed (54 chickens) received a single subcutaneous dose of gamithromycin at 7.5 mg/kg bodyweight (BW). Tissues, including muscle, skin + fat, liver, kidney, and injection site, were collected at 6 h, 3, 5, 7, 10, 14, 21, 28, and 35 d postdrug administration. Gamithromycin concentrations in these tissues were determined using ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). The kinetics of gamithromycin were analyzed in different tissues using a noncompartmental method in the Phoenix software. Differences were observed in gamithromycin concentrations and kinetic characteristics in both breeds of chickens, with higher residue concentrations and longer residue times found in yellow-feathered broilers. In Sanhuang broilers, the elimination rates of gamithromycin followed this order: injection site > muscle > liver > kidney > skin + fat. The corresponding elimination half-lives (t1/2λzs) in these samples were 1.22, 1.30, 1.71, 2.04, and 2.52 d, respectively. In contrast, in Arbor Acres broilers, a different order was noted: muscle > injection site > kidney > liver > skin + fat, with corresponding t1/2λzs of 1, 1.23, 1.88, 1.93, and 2.21 d, respectively. These differences may be related to variations in pigments in various tissues of chickens of the 2 breeds. However, further investigations are warranted to discern the underlying reasons.
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Antibacterianos , Pollos , Residuos de Medicamentos , Animales , Antibacterianos/administración & dosificación , Antibacterianos/farmacocinética , Antibacterianos/análisis , Residuos de Medicamentos/análisis , Inyecciones Subcutáneas/veterinaria , Plumas/química , Macrólidos/administración & dosificación , Macrólidos/farmacocinética , Macrólidos/análisis , Espectrometría de Masas en Tándem/veterinaria , MasculinoRESUMEN
We report here an enzymatic strategy for asparaginyl endopeptidase-mediated peptide cyclization. Incorporation of chloroacetyl groups into the recognition sequence of OaAEP1 enabled intramolecular cyclization with Cys residues. Combining this strategy and phage display, we identified nanomolar macrocyclic peptide ligands targeting TEAD4. One of the bicyclic peptides binds to TEAD4 with a KD value of 139 nM, 16 times lower than its linear analogue, demonstrating the utility of this platform in discovering high-affinity macrocyclic peptide ligands.
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Bacteriófagos , Péptidos , Ciclización , Péptidos/química , Cisteína Endopeptidasas , Ligandos , Bacteriófagos/metabolismo , Biblioteca de Péptidos , Péptidos Cíclicos/químicaRESUMEN
This study aimed to investigate the in vitro antibacterial activity of danofloxacin against Escherichia coli isolated from Gushi chickens, as well as the tissue distribution and residue depletion of danofloxacin in Gushi chickens following multiple oral administration. A total of 42 clinical E. coli strains were isolated from the cloaca of locally farmed Gushi chickens between August and October 2023. Then the minimum inhibitory concentration (MIC) of danofloxacin against these isolates was determined by broth microdilution method. Additionally, 42 healthy Gushi chickens were randomly divided into 6 groups, and danofloxacin was orally administered at a dose of 5 mg/kg body weight (BW) for 3 consecutive days. Plasma, intestinal content, and tissue samples, including muscle, skin + fat, liver, kidney, lung, and intestine, were collected at 4, 12, 24, 48, 72, and 120 h after the last administration. Danofloxacin concentrations in all samples were determined using a high-performance liquid chromatography (HPLC) method. The average concentration vs. time data were then subjected to noncompartmental analysis using Phoenix software, and withdrawal periods for danofloxacin in Gushi chickens were further determined with WT1.4 software, setting a 95% confidence interval. Results indicated a notable inhibitory effect of danofloxacin on E. coli, with an MIC50 of 0.5 µg/mL. Additionally, danofloxacin exhibited widespread distribution in Gushi chickens, detectable in all collected samples. Among all tissues, the liver exhibited the highest concentration, followed by the intestine. Even on the fifth day postadministration, danofloxacin persisted in skin + fat, liver, and lung. The elimination half-lives (t1/2λzs) of danofloxacin varied across samples: skin + fat (47.87 h), lung (30.61 h), liver (22.07 h), plasma (16.05 h), muscle (12.53 h), intestine (9.83 h), and kidney (6.34 h). Considering residue depletion and the maximum residue limit (MRL) of danofloxacin in poultry set by Chinese regulatory authorities, withdrawal periods for the kidney, muscle, liver, and skin + fat were determined as 1.03, 1.38, 3.34, and 5.85 d, respectively, rounded to a final withdrawal time of 6 d.
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Pollos , Escherichia coli , Animales , Administración Oral , Antibacterianos , Fluoroquinolonas/farmacologíaRESUMEN
This study investigates the combined effects of chitooligosaccharide (COS) and florfenicol (FLO) on the inhibition of Escherichia coli in vitro, as well as the pharmacokinetic interactions between these compounds in healthy chickens. The minimum inhibitory concentration (MIC) of COS and FLO alone and the fractional inhibitory concentration index (FICI) after combined treatment were determined using the broth microdilution method and checkerboard method, respectively. Additionally, we evaluated the pharmacokinetic interactions between the 2 types of COS and FLO in healthy chickens. Thirty chickens were randomly divided into 3 groups: Florfenicol group (30 mg/kg), COS J85 group (COS J85 20 mg/kg + florfenicol 30 mg/kg), COS H85 group (COS H85 20 mg/kg + florfenicol 30 mg/kg). Either FLO or COS was orally administered by gavage. The concentrations of FLO in chicken plasma were measured at different time points after the drug withdrawal using high-performance liquid chromatography (HPLC), and pharmacokinetic parameters were calculated by a compartmental method. The results showed that COS J85 and COS H85, when combined with FLO, had FICI values of 0.1875 to 0.75 and 0.3125 to 1, respectively, indicating good synergistic or additive effects against Escherichia coli. The pharmacokinetics of FLO alone and in combination with COS followed a 1-compartment model with first-order absorption and elimination. Furthermore, the pharmacokinetic analysis revealed that the elimination half-life (t1/2ke) of florfenicol was significantly increased in the COS H85 group compared to oral administration of florfenicol alone (P < 0.05). Other pharmacokinetic parameters did not show significant changes (P > 0.05), except between the 2 combined treatment groups, where statistical differences were observed for various parameters, excluding the area under the concentration-time curve from the time of dosing to infinity (AUC) and peak concentration (Cmax).
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Pollos , Quitosano , Oligosacáridos , Tianfenicol , Tianfenicol/análogos & derivados , Animales , Escherichia coli , Tianfenicol/farmacología , Antibacterianos , SemividaRESUMEN
A sustainable pathway for the synthesis of tetracyclic purinium salts via ruthenium-catalyzed electro-oxidative annulation of C6-arylpurine nucleosides with alkynes without a stoichiometric metal oxidant has been developed. The protocol described herein exhibits high regioselectivity, broad scope, and wide functional group tolerance, allowing efficient coupling of various biologically important molecules including acyclic, ribosyl, arabinosyl, and deoxyribosyl purine nucleoside derivatives. A novel purinoisoquinolinium-coordinated ruthenium(0) sandwich intermediate has been isolated, crystallographically characterized, and electrochemically analyzed, offering direct mechanistic insight.
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Coal spontaneous combustion often wastes resources and causes environmental pollution. Rapid and accurate identification of high temperature areas in coal is essential to reducing such combustion and environmental pollution. The acoustic thermometry method has the benefits of large temperature measurement space, non-contact, and high interference resistance. Determining the attenuation characteristics of acoustic waves in loose coal is the basis and premise for realizing acoustic temperature measurement. Four types of bituminous coal were scanned by computer tomography equipment. A self-designed acoustic attenuation test device was used to test coal samples under different temperatures and particle sizes. The study result demonstrates that the distribution characteristics of loose coal voids are mainly related to the particle size. The smaller the particle size range, the more uniform the void distribution. As the size of the coal particles increases, the voids become larger. The acoustic attenuation coefficients of four coal samples showed an increasing trend as frequency increased. The influence of coal particle size distribution on the acoustic attenuation coefficient was greater than that of temperature and metamorphic degree. The peak values of coal sound attenuation for different particle sizes were around 400, 700, 1100, and 1600 Hz. This indicated that the distribution of voids was the main factor affecting the propagation of acoustic waves. By analysing the attenuation mechanism of the acoustic wave in loose coal, the attenuation of acoustic temperature measurement signal was caused by the combined effect of loose coal on acoustic wave absorption and scattering. The study results provide theoretical support for the realization of acoustic wave detection of high temperature point in loose coal spontaneous combustion.
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We report here a robust and practical strategy for chemical protein synthesis using an o-nitrobenzyl group as a temporary protective group for an N-terminal cysteine residue of intermediate hydrazide fragments. By reinvestigating the photoremoval of an o-nitrobenzyl group, we establish a robust and reliable strategy for its quantitative photodeprotection. The o-nitrobenzyl group is completely stable to oxidative NaNO2 treatment and has been applied to the convergent chemical synthesis of programmed death ligand 1 fragment, providing a practical avenue for hydrazide-based native chemical ligation.
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Cisteína , Proteínas , Cisteína/química , Ligadura , Nitrito de Sodio/químicaRESUMEN
Native chemical ligation is a widely used technique for peptide fragment condensation in aqueous solutions, which has broken through the length limitation of traditional solid-phase peptide synthesis. It can achieve high-efficient chemical synthesis of proteins containing more than 300 amino acid residues. Peptide hydrazide, as a valuable reagent equivalent to a thioester peptide, can be easily and efficiently prepared by the Fmoc-based SPPS method and has been widely used in native chemical ligation. Here we take the chemical synthesis of a SARS-CoV-2 miniprotein inhibitor LCB1 as an example to describe the detailed procedure of hydrazide-based native chemical ligation.
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COVID-19 , SARS-CoV-2 , Humanos , Hidrazinas , Péptidos/química , Técnicas de Síntesis en Fase SólidaRESUMEN
We present the finding of a dimeric ACE2 peptide mimetic designed through side chain cross-linking and covalent dimerization. It has a binding affinity of 16 nM for the SARS-CoV-2 spike RBD, and effectively inhibits the SARS-CoV-2 pseudovirus in Huh7-hACE2 cells with an IC50 of 190 nM and neutralizes the authentic SARS-CoV-2 in Caco2 cells with an IC50 of 2.4 µM. Our study should provide a new insight for the optimization of peptide-based anti-SARS-CoV-2 inhibitors.
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Antivirales/farmacología , Fragmentos de Péptidos/farmacología , Peptidomiméticos/farmacología , SARS-CoV-2/efectos de los fármacos , Secuencia de Aminoácidos , Enzima Convertidora de Angiotensina 2/química , Antivirales/síntesis química , Antivirales/metabolismo , Línea Celular Tumoral , Humanos , Pruebas de Sensibilidad Microbiana , Fragmentos de Péptidos/síntesis química , Fragmentos de Péptidos/metabolismo , Peptidomiméticos/síntesis química , Peptidomiméticos/metabolismo , Unión Proteica , Dominios Proteicos , Glicoproteína de la Espiga del Coronavirus/química , Glicoproteína de la Espiga del Coronavirus/metabolismoRESUMEN
We report a new pattern of a bicyclic helical peptide constructed through head-to-tail cross-linking. The described bicyclic helical peptide has a head-to-tail cross-linking arm and a C-terminal i, i + 4 cross-linking arm. This scaffold will provide a promising scaffold for designing a proteolytically resistant helix-constrained peptide.
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PéptidosRESUMEN
BACKGROUND: The prognosis of obstructive colorectal cancer (oCRC) is worse than that of nonobstructive colorectal cancer. However, no previous study has established an individualized prediction model for the prognosis of patients with oCRC. We aimed to screen the factors that affect the prognosis of oCRC and to use these findings to establish a nomogram model that predicts the individual prognosis of patients with oCRC. METHODS: This retrospective study collected data of 181 patients with oCRC from three medical hospitals between February 2012 and December 2017. Among them, 129 patients from one hospital were used as the training cohort. Univariate and multivariate analyses were used in this training cohort to select independent risk factors that affect the prognosis of oCRC, and a nomogram model was established. The other 52 patients from two additional hospitals were used as the validation cohort to verify the model. RESULTS: Multivariate analysis showed that carcinoembryonic antigen level (p = 0.037, hazard ratio [HR] = 2.872 [1.065-7.740]), N stage (N1 vs. N0, p = 0.028, HR = 3.187 [1.137-8.938]; N2 vs. N0, p = 0.010, HR = 4.098 [1.393-12.051]), and surgical procedures (p = 0.002, HR = 0.299 [0.139-0.643]) were independent prognostic factors of overall survival in patients with oCRC. These factors were used to construct the nomogram model, which showed good concordance and accuracy. CONCLUSION: Carcinoembryonic antigen, N stage, and surgical method are independent prognostic factors for overall survival in patients with oCRC, and the nomogram model can visually display these results.
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Neoplasias Colorrectales , Nomogramas , Biomarcadores de Tumor , Humanos , Pronóstico , Estudios RetrospectivosRESUMEN
Facile head-to-side chain cross-linking strategies are developed to generate helix-constrained peptides. In our strategies, a covalent cross-linker is incorporated at N, i+7 or N, i+1 positions to lock the peptide into a helical conformation. The described patterns of head-to-side chain cross-linking will provide new frameworks for constrained helical peptide.
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Péptidos/síntesis química , Secuencia de Aminoácidos , Dicroismo Circular , Reactivos de Enlaces Cruzados , Estructura Molecular , Péptidos/química , Estructura Secundaria de ProteínaRESUMEN
Puerariae Radix (PR) serves as food and medicinal plant for thousands of years with explicit efficacy for heart diseases, while biological target specifically binding-oriented screening of the active components in PR remains a preliminary stage. Cell membrane chromatography (CMC) is newly developed approach where interactions between active components and certain biological targets can be effectively studied, Human umbilical vein endothelial cell (HUVEC) membrane, with its abundant receptors such as ß and AT1, is most eligible for constructing CMC. In this study, an HUVEC/CMC-LC-MS2 system was developed for screening active components in PR, 11 compounds were screened out and four of them were identified. Besides puerarin, the rest identified are daidzin, pueroside D and 3'-hydroxypuerarin. The study provides more reference for CMC applications and PR exploitation.
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Membrana Celular/metabolismo , Cromatografía de Afinidad/métodos , Medicamentos Herbarios Chinos/análisis , Isoflavonas/análisis , Espectrometría de Masas/métodos , Línea Celular , Medicamentos Herbarios Chinos/química , Medicamentos Herbarios Chinos/metabolismo , Células Endoteliales de la Vena Umbilical Humana , Humanos , Isoflavonas/química , Isoflavonas/aislamiento & purificación , Isoflavonas/metabolismo , Pueraria , Reproducibilidad de los ResultadosRESUMEN
BACKGROUND: Human adenoviruses (HAdV) are a common cause of viral conjunctivitis, making surveillance of them from sporadic cases of conjunctivitis important. METHODS: To acquire a better understanding of the epidemic genotypes of HAdV in outpatient children with adenoviral conjunctivitis in Jiangxi Province, China (2011-2012), 179 samples from cases with a high suspicion of HAdV were analyzed by PCR. Samples confirmed to be HAdV-positive by PCR were cultured in Hep-2 cells to isolate the viruses, which were then identified through hexon gene sequencing. RESULTS: The adenoviral conjunctivitis positivity rate was 74.86% (134/179), from which 71.64% (96/134) were infections in boys, and 92.54% (124/134) were infections in children under 5 years of age. Sixty-nine HAdV strains were isolated from the positive samples and 69 sequences were obtained. Phylogenetic analysis indicated that 33 strains (47.82%) clustered with HAdV-B7, 21 (30.43%) with HAdV-B3, 6 (8.70%) with HAdV-B55, 6 (8.70%) with HAdV-E4, 1 with HAdV-B21 (1.45%), 1 with HAdV-D37 (1.45%), and 1 with HAdV-D64 (1.45%). CONCLUSIONS: This is the first identification of HAdV-B55 relating to adenoviral conjunctivitis in China. These findings provide a firm basis for future surveillance of adenoviral conjunctivitis in China or other East Asian regions.
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Infecciones por Adenovirus Humanos/epidemiología , Adenovirus Humanos/genética , Conjuntivitis Viral/epidemiología , Niño , Preescolar , China/epidemiología , Conjuntiva/virología , ADN Viral/genética , Femenino , Genotipo , Humanos , Lactante , Masculino , Pacientes Ambulatorios , FilogeniaRESUMEN
Essential oils from the seed, pulp, and leaf of sea buckthorn were obtained with hydrodistillation, and their phytochemical composition was analyzed through gas chromatography-mass spectrometry. Furthermore, the antibacterial activity of the oils was tested on five food-borne bacteria by spectrometry and evaluated in terms of minimum inhibitory concentration. The results indicate that the composition of all essential oils is dominated by free fatty acids, esters, and alkanes. Minimum inhibitory concentration values on each bacterium were obtained for oils from different parts. The oils from different parts exhibited nearly equal inhibitory effect on Staphylococcus aureus. The pulp oil was found to be the most effective for the rest of bacteria tested except Escherichia coli, on which seed oil shows twice the inhibitory effect to that of leaf or pulp oil. Three natural inhibitory examples were found comparable with or even better than the positive control: pulp oil on Bacillus subtilis, and pulp oil and leaf oil on Bacillus coagulans.