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Clinical and Translational Science Jul 2024The AIDA randomized clinical trial found no significant difference in clinical failure or survival between colistin monotherapy and colistin-meropenem combination... (Randomized Controlled Trial)
Randomized Controlled Trial
The AIDA randomized clinical trial found no significant difference in clinical failure or survival between colistin monotherapy and colistin-meropenem combination therapy in carbapenem-resistant Gram-negative infections. The aim of this reverse translational study was to integrate all individual preclinical and clinical pharmacokinetic-pharmacodynamic (PKPD) data from the AIDA trial in a pharmacometric framework to explore whether individualized predictions of bacterial burden were associated with the trial outcomes. The compiled dataset included for each of the 207 patients was (i) information on the infecting Acinetobacter baumannii isolate (minimum inhibitory concentration, checkerboard assay data, and fitness in a murine model), (ii) colistin plasma concentrations and colistin and meropenem dosing history, and (iii) disease scores and demographics. The individual information was integrated into PKPD models, and the predicted change in bacterial count at 24 h for each patient, as well as patient characteristics, was correlated with clinical outcomes using logistic regression. The in vivo fitness was the most important factor for change in bacterial count. A model-predicted growth at 24 h of ≥2-log (164/207) correlated positively with clinical failure (adjusted odds ratio, aOR = 2.01). The aOR for one unit increase of other significant predictors were 1.24 for SOFA score, 1.19 for Charlson comorbidity index, and 1.01 for age. This study exemplifies how preclinical and clinical anti-infective PKPD data can be integrated through pharmacodynamic modeling and identify patient- and pathogen-specific factors related to clinical outcomes - an approach that may improve understanding of study outcomes.
Topics: Humans; Acinetobacter baumannii; Meropenem; Middle Aged; Microbial Sensitivity Tests; Female; Male; Anti-Bacterial Agents; Colistin; Adult; Aged; Animals; Treatment Outcome; Mice; Acinetobacter Infections; Translational Research, Biomedical; Drug Therapy, Combination; Models, Biological
PubMed: 38952168
DOI: 10.1111/cts.13870 -
The New Phytologist Jul 2024Plant homeodomain leucine zipper IV (HD-Zip IV) transcription factors (TFs) contain an evolutionarily conserved steroidogenic acute regulatory protein (StAR)-related...
Plant homeodomain leucine zipper IV (HD-Zip IV) transcription factors (TFs) contain an evolutionarily conserved steroidogenic acute regulatory protein (StAR)-related lipid transfer (START) domain. While the START domain is required for TF activity, its presumed role as a lipid sensor is not clear. Here we used tandem affinity purification from Arabidopsis cell cultures to demonstrate that PROTODERMAL FACTOR2 (PDF2), a representative member that controls epidermal differentiation, recruits lysophosphatidylcholines (LysoPCs) in a START-dependent manner. Microscale thermophoresis assays confirmed that a missense mutation in a predicted ligand contact site reduces lysophospholipid binding. We additionally found that PDF2 acts as a transcriptional regulator of phospholipid- and phosphate (Pi) starvation-related genes and binds to a palindromic octamer with consensus to a Pi response element. Phospholipid homeostasis and elongation growth were altered in pdf2 mutants according to Pi availability. Cycloheximide chase experiments revealed a role for START in maintaining protein levels, and Pi starvation resulted in enhanced protein destabilization, suggesting a mechanism by which lipid binding controls TF activity. We propose that the START domain serves as a molecular sensor for membrane phospholipid status in the epidermis. Our data provide insights toward understanding how the lipid metabolome integrates Pi availability with gene expression.
PubMed: 38952028
DOI: 10.1111/nph.19917 -
Biotechnology and Bioengineering Jul 2024In recent decades, biocatalysis has emerged as an important alternative to chemical catalysis in pharmaceutical manufacturing. Biocatalysis is attractive because...
In recent decades, biocatalysis has emerged as an important alternative to chemical catalysis in pharmaceutical manufacturing. Biocatalysis is attractive because enzymatic cascades can synthesize complex molecules with incredible selectivity, yield, and in an environmentally benign manner. Enzymes for pharmaceutical biocatalysis are typically used in their unpurified state, since it is time-consuming and cost-prohibitive to purify enzymes using conventional chromatographic processes at scale. However, impurities present in crude enzyme preparations can consume substrate, generate unwanted byproducts, as well as make the isolation of desired products more cumbersome. Hence, a facile, nonchromatographic purification method would greatly benefit pharmaceutical biocatalysis. To address this issue, here we have captured enzymes into membraneless compartments by fusing enzymes with an intrinsically disordered protein region, the RGG domain from LAF-1. The RGG domain can undergo liquid-liquid phase separation, forming liquid condensates triggered by changes in temperature or salt concentration. By centrifuging these liquid condensates, we have successfully purified enzyme-RGG fusions, resulting in significantly enhanced purity compared to cell lysate. Furthermore, we performed enzymatic reactions utilizing purified fusion proteins to assay enzyme activity. Results from the enzyme assays indicate that enzyme-RGG fusions purified by the centrifugation method retain enzymatic activity, with greatly reduced background activity compared to crude enzyme preparations. Our work focused on three different enzymes-a kinase, a phosphorylase, and an ATP-dependent ligase. The kinase and phosphorylase are components of the biocatalytic cascade for manufacturing molnupiravir, and we demonstrated facile co-purification of these two enzymes by co-phase separation. To conclude, enzyme capture by RGG tagging promises to overcome difficulties in bioseparations and biocatalysis for pharmaceutical synthesis.
PubMed: 38951956
DOI: 10.1002/bit.28787 -
Parasites & Vectors Jul 2024Abiotic factors play a significant role in the evolution of Leishmania infantum infection due to its vectorial nature. This study aims to assess the evolution in the...
BACKGROUND
Abiotic factors play a significant role in the evolution of Leishmania infantum infection due to its vectorial nature. This study aims to assess the evolution in the detection of new L. infantum infection cases in Valdeorras (Ourense, Northwestern Spain) over a 20-year period and how different climatic variables and preventive measures may have affected it.
METHODS
Indirect immunofluorescence antibody tests (IFAT) were performed on serum samples collected from dogs attending the 'Servicios Veterinarios de Sil' veterinary clinic (Valdeorras, Northwestern Spain) between May 2003 and April 2023 to detect L. infantum exposure. The percentage of new cases of L. infantum infection was calculated from May of one year to April of the following year. Climatic conditions in the region, global sales of ectoparasiticides and the number of vaccines against L. infantum delivered in the veterinary clinic from 2003 to 2022 were recorded. Statistical analyses were conducted to determine the associations between these factors and the percentage of new cases of L. infantum infection.
RESULTS
A total of 2909 dogs were assessed, and 3785 IFAT tests were performed between May 2003 and April 2023. The mean percentage of new seropositive cases over the 20-year period studied was 21.65 ± 10.8%, with a decline from the beginning to the end of the period studied. The percentage was significantly higher between May 2003 and April 2008 compared with the other periods (May 2008 to April 2013, May 2013 to April 2018 and May 2018 to April 2023). There was a positive correlation between the percentage of new cases of L. infantum infection and the maximum relative humidity in winter. Conversely, there was a negative correlation between the percentage of new cases and sales of ectoparasiticides and vaccination against L. infantum.
CONCLUSIONS
This study is one of the longest evaluations of the evolution of L. infantum infection in a fixed location and its association with external factors including climatic conditions and preventive measures. The results confirm that Valdeorras is a high-risk area for L. infantum infection. The use of ectoparasiticides and vaccines against L. infantum has been shown to play a significant role in preventing L. infantum infection, highlighting the crucial role of veterinarians in the fight against this disease.
Topics: Dogs; Animals; Spain; Dog Diseases; Leishmania infantum; Leishmaniasis, Visceral; Climate; Antibodies, Protozoan; Male; Fluorescent Antibody Technique, Indirect; Female
PubMed: 38951858
DOI: 10.1186/s13071-024-06357-8 -
Scientific Reports Jul 2024To assess malnutrition contribution to the functional status and health related quality of life after hospitalization due to COVID-19 pneumonia, 66 selected adults...
To assess malnutrition contribution to the functional status and health related quality of life after hospitalization due to COVID-19 pneumonia, 66 selected adults referred for physical rehabilitation accepted to participate in the study; none of them required oxygen supply or had history of lung/musculoskeletal/neurological/immune/rheumatic disease or trauma, or contraindication for respiratory-function tests. At three evaluations, with 3 months in-between, assessments included: self-report of functional status, the St. George's Respiratory Questionnaire, spirometry, the 6-min-walk-test, the MRC-scale, the 30-s sit-to-stand-test, the timed-up-and-go-test, nutritional status, and ultrasound imaging (vastus medialis and diaphragm). At referral, patients had nutritional deficits with protein deficiency, which gradually improved; while muscle thickness (of both vastus medialis and diaphragm) increased, along with muscle strength and mobility (ANOVA, p < 0.05). Contrarywise, the distance covered during the 6-min-walk-test decreased (ANOVA, p < 0.05), with a negative influence from excess body mass. During rehabilitation, health-related quality of life and functional status improved, with negative influence from a history of tobacco use and referral delay, respectively. After hospitalization due to COVID-19, early diagnosis of both protein deficiency and decrease of skeletal muscle thickness could be relevant for rehabilitation, while pondering the negative impact of excess body mass on submaximal exercise performance.
Topics: Humans; COVID-19; Quality of Life; Male; Female; Malnutrition; Middle Aged; Aged; Follow-Up Studies; Functional Status; Nutritional Status; SARS-CoV-2; Adult; Hospitalization; Muscle Strength; Surveys and Questionnaires
PubMed: 38951534
DOI: 10.1038/s41598-024-65698-7 -
Methods in Molecular Biology (Clifton,... 2024Quantitative immunoassays, such as the traditional enzyme-linked immunosorbent assay (ELISA), are used to determine concentrations of an antigen in a matrix of unknown...
Quantitative immunoassays, such as the traditional enzyme-linked immunosorbent assay (ELISA), are used to determine concentrations of an antigen in a matrix of unknown antigen concentration. Magnetic immunoassays, such as the Luminex xMAP technology, allow for the simultaneous detection of multiple analytes and offer heightened sensitivity, specificity, low sample volume requirements, and high-throughput capabilities. Here, we describe a quantitative immunoassay using the Luminex MAGPIX® System to determine the antigen concentration from liquid samples with unknown concentrations. In detail, we describe a newly developed assay for determining production yields of Drosophila S2-produced Marburg virus (MARV) glycoprotein in insect-cell-culture-derived supernatant. The potential applications of this assay could extend to the quantification of viral antigens in fluids derived from both in vitro and in vivo models infected with live MARV, thereby providing additional applications for virological research.
Topics: Animals; Microspheres; Immunoassay; Antigens, Viral; Marburgvirus; Drosophila; Cell Culture Techniques; Cell Line; Enzyme-Linked Immunosorbent Assay
PubMed: 38951344
DOI: 10.1007/978-1-0716-3961-0_22 -
Methods in Molecular Biology (Clifton,... 2024The Baculovirus Expression Vector System (BEVS) is used with cultured insect cells to produce a wide variety of heterologous proteins, which can be secreted into the...
The Baculovirus Expression Vector System (BEVS) is used with cultured insect cells to produce a wide variety of heterologous proteins, which can be secreted into the culture medium during the transient infection process (Smith et al. Mol Cell Biol 12:2156-2165, 1983). When the infection process is complete, centrifugation is often used to separate the desired protein from the spent insect cells. The desired product in the harvested supernatant is contaminated with baculovirus, amino acids, lipids, detergents, oils, lysed cells from the infection process, genomic DNA from the insect cells, and proteases due to the lytic nature of the baculovirus infection process and many other contaminants (Ikonomou et al. Appl Microbiol Biotechnol 62:1-20, 2003). All these contaminants that are present in the centrifuged supernatant with the desired secreted protein make the initial chromatographic capture step critical for effective purification of the desired protein. A purification scheme will be outlined for a slightly acidic secreted protein using cation exchange chromatography (Lundanes et al. Chromatography: basic principles, sample preparations and related methods, 1st edn. Wiley, 2013).
Topics: Animals; Baculoviridae; Chromatography, Ion Exchange; Recombinant Proteins; Insecta; Sf9 Cells; Genetic Vectors; Cell Line; Spodoptera
PubMed: 38951340
DOI: 10.1007/978-1-0716-3961-0_18 -
Methods in Molecular Biology (Clifton,... 2024Virus-like particles (VLPs) of the adeno-associated virus (AAV) can be produced using the baculovirus expression vector system. Insertion of small peptides on the...
Virus-like particles (VLPs) of the adeno-associated virus (AAV) can be produced using the baculovirus expression vector system. Insertion of small peptides on the surface of the AAV or AAV VLPs has been used to redirect the AAV to different target tissues and for vaccine development. Usually, the VLPs self-assemble intracellularly, and an extraction step must be performed before purification. Here, we describe the method we have used to extract AAV VLPs from insect cells successfully with peptide insertions on their surface.
Topics: Dependovirus; Animals; Peptides; Genetic Vectors; Virion; Baculoviridae; Sf9 Cells; Humans; Cell Line; Capsid Proteins
PubMed: 38951338
DOI: 10.1007/978-1-0716-3961-0_16 -
Methods in Molecular Biology (Clifton,... 2024Purification of rAAV is a crucial unit operation of the AAV production process. It enables the capture of AAV and removal of contaminants such as host cell proteins,...
Purification of rAAV is a crucial unit operation of the AAV production process. It enables the capture of AAV and removal of contaminants such as host cell proteins, host cell DNA, and other cell culture-related impurities. Here we describe the purification of rAAV produced in insect cells Sf9/rBEV by immuno-affinity capture chromatography. The method is fully scale-amenable unlike other traditional purification methods based on ultracentrifugation. The method reported herein has two main steps: (1) the clarification of cell lysate by depth filtration and (2) the selective capture and single-step purification of AAV via immune-affinity chromatography. This purification method has been successfully implemented to purify the majority of wild-type AAV serotypes.
Topics: Dependovirus; Animals; Chromatography, Affinity; Sf9 Cells; Genetic Vectors; Humans; Spodoptera
PubMed: 38951337
DOI: 10.1007/978-1-0716-3961-0_15 -
Archives of Virology Jun 2024Given the high prevalence of avian leukosis virus subgroup K (ALV-K) in chickens in China, the positive rate of ALV-K in local chickens in Henan province was...
Given the high prevalence of avian leukosis virus subgroup K (ALV-K) in chickens in China, the positive rate of ALV-K in local chickens in Henan province was investigated, and the genetic region encoding the glycoprotein gp85 of isolates from positive chickens was analyzed. The positive rate of ALV-K in local chickens in Henan was found to be 87.2% (41/47). Phylogenetic analysis of gp85 sequences revealed six clusters that differed in their host range regions (hr1 and hr2) and variable regions (vr1, vr2, and vr3). Evidence of recombination of hr1, hr2, vr1, vr2, and vr3 was observed between the different clusters. The isolate HN23LS02 appears to have obtained its hr1 and hr2 regions from separate lineages via recombination but without having a significant affect on the replication capacity of the virus.
Topics: Animals; Avian Leukosis Virus; Chickens; Phylogeny; Avian Leukosis; Viral Envelope Proteins; Poultry Diseases; Host Specificity; China; Recombination, Genetic
PubMed: 38951272
DOI: 10.1007/s00705-024-06083-7