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Methods in Molecular Biology (Clifton,... 2018Quantitative protein extraction and high-yield generation of peptides from biological samples are the prerequisite for successful bottom-up type proteomic analysis....
Quantitative protein extraction and high-yield generation of peptides from biological samples are the prerequisite for successful bottom-up type proteomic analysis. Filter aided sample preparation (FASP) is a method for processing of SDS-solubilized cells in a proteomic reactor format. In FASP, disposable centrifugal ultrafiltration units allow for detergent depletion, protein digestion, and isolation of peptides released by proteases from undigested material. Consecutive protein digestion with two or three proteases enables generation of peptide fractions with minimal overlap and considerably increases the number of identifications and protein sequence coverage. FASP is useful for analysis of samples varying in size from a few micrograms to several milligrams of total protein.
Topics: Centrifugation; Filtration; Proteolysis; Proteomics; Workflow
PubMed: 30259475
DOI: 10.1007/978-1-4939-8695-8_1 -
The Journal of Allergy and Clinical... Mar 2023An unmet clinical need exists in the management of treatment-refractory allergic bronchopulmonary aspergillosis (ABPA). Omalizumab has shown promising effects in case... (Meta-Analysis)
Meta-Analysis
BACKGROUND
An unmet clinical need exists in the management of treatment-refractory allergic bronchopulmonary aspergillosis (ABPA). Omalizumab has shown promising effects in case series and cohort studies; however, evidence to support its routine clinical use is lacking.
OBJECTIVE
The aim of this systematic review and meta-analysis was to evaluate the clinical effectiveness and safety of omalizumab in patients with ABPA.
METHODS
We conducted a systematic search across standard databases using specific key words until May 13, 2021. We performed a meta-analysis to compare the effectiveness (exacerbations, oral corticosteroid [OCS] use, lung function, and patient-reported asthma control) and safety of pre- and post-omalizumab treatment. Subgroup analyses were performed for treatment duration and underlying disease.
RESULTS
In total, 49 studies (n = 267) were included in the qualitative synthesis and 14 case series (n = 186) in the quantitative meta-analysis. Omalizumab treatment significantly reduced the annualized exacerbation rate compared with pretreatment (mean difference, -2.09 [95% CI, -3.07 to -1.11]; P < .01). There was a reduction in OCS use (risk difference, 0.65 [95% CI, 0.46-0.84]; P < .01), an increase in termination of OCS use (risk difference, 0.53 [95% CI, 0.24-0.82]; P < .01), and a reduction in OCS dose (milligrams per day) (mean difference, -14.62 [95% CI, -19.86 to -9.39]; P < .01) in ABPA patients receiving omalizumab. Omalizumab improved FEV % predicted by 11.9% (95% CI, 8.2-15.6; P < .01) and asthma control, and was well-tolerated.
CONCLUSIONS
Omalizumab treatment reduced exacerbations and OCS use, improved lung function and asthma control in patients with ABPA, and was well-tolerated. The results highlight the potential role of omalizumab in the treatment of ABPA.
Topics: Humans; Omalizumab; Aspergillosis, Allergic Bronchopulmonary; Cystic Fibrosis; Asthma; Adrenal Cortex Hormones
PubMed: 36581073
DOI: 10.1016/j.jaip.2022.12.012 -
Methods in Enzymology 2020Siderophores have important functions for bacteria in iron acquisition and as virulence factors. In this chapter we will discuss the engineering of cyclic hydroxamate...
Siderophores have important functions for bacteria in iron acquisition and as virulence factors. In this chapter we will discuss the engineering of cyclic hydroxamate siderophores by various biochemical approaches based on the example of Shewanella algae. The marine gamma-proteobacterium S. algae produces three different cyclic hydroxamate siderophores as metabolites via a single biosynthetic gene cluster and one of them is an important key player in interspecies competition blocking swarming of Vibrio alginolyticus. AvbD is the key metabolic enzyme assembling the precursors into three different core structures and hence an interesting target for metabolic and biochemical engineering. Synthetic natural and unnatural precursors can be converted in vitro with purified AvbD to generate siderophores with various ring sizes ranging from analytical to milligram scale. These engineered siderophores can be applied, for example, as swarming inhibitors against V. alginolyticus. Here, we describe the synthesis of the natural and unnatural siderophore precursors HS[X]A and provide our detailed protocols for protein expression of AvbD, conversion of HS[X]A with the enzyme to produce ring-size engineered siderophores and secondly for a biosynthetic feeding strategy that allows to extract engineered siderophores in the milligram scale.
Topics: Antibiosis; Bacterial Proteins; Diamines; Escherichia coli; Hydroxamic Acids; Metabolic Engineering; Movement; Peptides, Cyclic; Putrescine; Recombinant Proteins; Shewanella; Siderophores; Succinates; Vibrio alginolyticus
PubMed: 32046852
DOI: 10.1016/bs.mie.2019.10.030 -
Chemistry (Weinheim An Der Bergstrasse,... Mar 2020High-throughput experimentation (HTE) is a growing, enabling technology that allows the execution of large, parallel sets of experiments. Often, automation is required... (Review)
Review
High-throughput experimentation (HTE) is a growing, enabling technology that allows the execution of large, parallel sets of experiments. Often, automation is required to dose compounds on milligram to sub-milligram scale, to run many parallel reactions, and to analyse large datasets. Unique approaches to screen design, implementation, and analysis are required, distinct from traditional synthetic organic chemistry. The discipline also presents a profitable opportunity for individual scientists to learn about and explore fields adjacent to chemistry, including data science, robotics and equipment engineering, and computer programming. This perspective presents the author's viewpoints on the field of HTE, its implementation within a chemistry career, and the automated future of organic chemistry technology.
PubMed: 32125726
DOI: 10.1002/chem.202000656 -
Science Translational Medicine May 2023This first-in-human study evaluated RO7122290, a bispecific fusion protein carrying a split trimeric 4-1BB (CD137) ligand and a fibroblast activation protein α (FAP)...
This first-in-human study evaluated RO7122290, a bispecific fusion protein carrying a split trimeric 4-1BB (CD137) ligand and a fibroblast activation protein α (FAP) binding site that costimulates T cells for improved tumor cell killing in FAP-expressing tumors. Patients with advanced or metastatic solid tumors received escalating weekly intravenous doses of RO7122290 as a single agent ( = 65) or in combination with a 1200-milligram fixed dose of the anti-programmed death-ligand 1 (anti-PD-L1) antibody atezolizumab given every 3 weeks ( = 50), across a tested RO7122290 dose range of 5 to 2000 milligrams and 45 to 2000 milligrams, respectively. Three dose-limiting toxicities were reported, two at different RO7122290 single-agent doses (grade 3 febrile neutropenia and grade 3 cytokine release syndrome) and one for the combination (grade 3 pneumonitis). No maximum tolerated dose was identified. The pharmacokinetic profile of RO7122290 suggested nonlinearity in elimination. The observed changes in peripheral and tissue pharmacodynamic (PD) biomarkers were consistent with the postulated mechanism of action. Treatment-induced PD changes included an increase in proliferating and activated T cells in peripheral blood both in the single-agent and combination arms. Increased infiltration of intratumoral CD8 and Ki67CD8 T cells was observed for both treatment regimens, accompanied by the up-regulation of T cell activation genes and gene signatures. Eleven patients experienced a complete or partial response, six of whom were confirmed to be immune checkpoint inhibitor naive. These results support further evaluation of RO7122290 in combination with atezolizumab or other immune-oncology agents for the treatment of solid tumors.
Topics: Humans; CD8-Positive T-Lymphocytes; Neoplasms; Fibroblasts
PubMed: 37163618
DOI: 10.1126/scitranslmed.abp9229 -
Protein Expression and Purification Jun 2017Chinese Hamster Ovary (CHO) cells are the principal mammalian host used for stable cell line generation and biotherapeutic protein production. Until recently, production... (Comparative Study)
Comparative Study
Chinese Hamster Ovary (CHO) cells are the principal mammalian host used for stable cell line generation and biotherapeutic protein production. Until recently, production of milligrams to grams of protein in CHO transient systems was challenging. As such, Human Embryonic Kidney (HEK293) cells are the most common mammalian cell type used for transient transfection. The post-translational modifications (PTMs) of a protein are dictated in part by the cell line used for expression, and changes in PTMs have been shown to affect both the activity and biophysical properties of proteins. Therefore, it is potentially advantageous to keep the host cell type consistent throughout drug discovery and development. To this end, we compared the ExpiCHO system, a high density CHO-S transient transfection system, to the Expi293 and FreeStyle MAX CHO transient systems. Fourteen proteins were expressed in both the Expi293 and ExpiCHO systems. For a majority of proteins tested, the protein titers observed with the ExpiCHO system were higher than those seen with both the FreeStyle MAX CHO and Expi293 systems. Antibodies expressed using the ExpiCHO system had glycosylation patterns more similar to antibodies produced in stable CHO cell lines than Expi293-derived antibodies. However, culture duration and temperature were found to affect protein titer, monodispersity, enzyme activity, and PTMs and should be carefully selected when using the ExpiCHO system. The ExpiCHO transient transfection systems allows for facile production of milligrams to grams of protein in CHO cells and de-risks the transition from transient to stable material during drug development.
Topics: Animals; CHO Cells; Cricetinae; Cricetulus; Gene Expression; HEK293 Cells; Humans; Protein Processing, Post-Translational; Recombinant Proteins; Transfection
PubMed: 28342833
DOI: 10.1016/j.pep.2017.03.018 -
Endocrinology, Diabetes & Metabolism... 2016Estrogen is used to induce puberty in peripubertal girls with hypogonadism. Although both synthetic and natural forms are available, along with different routes of...
UNLABELLED
Estrogen is used to induce puberty in peripubertal girls with hypogonadism. Although both synthetic and natural forms are available, along with different routes of administration, in the UK oral ethinyl estradiol and the low-dose oral contraceptive pill are commonly used as hormone replacement therapy for practical reasons. We present five peripubertal girls (aged 12.5-14.9 years) with hypogonadism (two with primary hypogonadism due to Turner syndrome and three with central (secondary) hypogonadism as part of multiple pituitary hormone deficiency) who for a variety of reasons have received milligram doses of estradiol (E2) in error for between 6 weeks and 6 months, instead of the expected microgram doses of ethinyl estradiol. Although there are no direct comparisons in peripubertal girls between synthetic and natural estrogens, all girls had vaginal bleeding whilst receiving the milligram doses and have ended up with reduced final heights, below the 9th centile in 1 and below the 2nd centile in 4. Whilst reduction in final height may be part of the underlying condition (especially in Turner syndrome) the two girls with height predictions performed prior to receiving the estrogen overdose have not achieved their predicted height. Estrogen is one of the few drugs which is available in both milligram and microgram formulations. Clinicians need to be alert to the possibility of patients receiving the wrong formulation and dosage in error.
LEARNING POINTS
Girls with primary and secondary gonadal failure require assistance with pubertal induction.Although several different formulations and route of administration are available, for practical reasons, the majority of girls in the UK receive oral ethinyl estradiol.Estrogen preparations are available in both milligram and microgram formulations, with potential for receiving the wrong dose.Girls receiving milligram rather than microgram preparations all had vaginal bleeding and a short final height.
PubMed: 26843960
DOI: 10.1530/EDM-15-0096 -
Nature Biotechnology Dec 2017Linear, medium-chain (C8-C12) hydrocarbons are important components of fuels as well as commodity and specialty chemicals. As industrial microbes do not contain pathways... (Review)
Review
Linear, medium-chain (C8-C12) hydrocarbons are important components of fuels as well as commodity and specialty chemicals. As industrial microbes do not contain pathways to produce medium-chain chemicals, approaches such as overexpression of endogenous enzymes or deletion of competing pathways are not available to the metabolic engineer; instead, fatty acid synthesis and reversed β-oxidation are manipulated to synthesize medium-chain chemical precursors. Even so, chain lengths remain difficult to control, which means that purification must be used to obtain the desired products, titers of which are typically low and rarely exceed milligrams per liter. By engineering the substrate specificity and activity of the pathway enzymes that generate the fatty acyl intermediates and chain-tailoring enzymes, researchers can boost the type and yield of medium-chain chemicals. Development of technologies to both manipulate chain-tailoring enzymes and to assay for products promises to enable the generation of g/L yields of medium-chain chemicals.
Topics: Biofuels; Biomass; Escherichia coli; Fatty Acids; Hydrocarbons; Lignin; Metabolic Engineering; Saccharomyces cerevisiae
PubMed: 29220020
DOI: 10.1038/nbt.4022 -
Expert Opinion on Drug Safety Jul 2016The benefits of opioid therapy must be balanced by any adverse effects. In recent years, prescription opioids have been increasingly prescribed, but have also been... (Review)
Review
INTRODUCTION
The benefits of opioid therapy must be balanced by any adverse effects. In recent years, prescription opioids have been increasingly prescribed, but have also been associated with increased abuse, overdose and death.
AREAS COVERED
This review will categorize the common risks of opioid administration. Recognized adverse effects of opioid therapy include constipation, tolerance, endocrinopathies, sleep disorders, cognitive effects, respiratory depression, overdose and addiction. Studies have shown that there is increased risk of overdose and death with higher daily opioid doses, particularly above a morphine equivalent oral daily dose of 100 milligrams. Extended-release/long acting (ER/LA) opioid formulations may be beneficial for the compliant patient, yet may expose a higher risk for abuse if used inappropriately since each tablet carries a larger dose of medication.
EXPERT OPINION
Prospective, controlled one-year trials are needed to establish the efficacy and safety profile of chronic opioid therapy. In addition to the well known side effects of chronic opioid therapy, the influence and serious effect of opioids on sleep and central sleep apnea is only recently being investigated. The lowest possible daily opioid must be used to manage chronic pain, and all clinicians should be cautious in the use of daily morphine equivalent doses above 50-100 milligrams.
Topics: Analgesics, Opioid; Chronic Pain; Delayed-Action Preparations; Dose-Response Relationship, Drug; Drug Overdose; Drug Tolerance; Humans; Opioid-Related Disorders; Time Factors
PubMed: 27070052
DOI: 10.1080/14740338.2016.1177509 -
American Journal of Obstetrics and... Jul 2018Women commonly receive opioid prescriptions following hospitalization. The rise of the opioid epidemic in the United States underscores the importance of a better...
BACKGROUND
Women commonly receive opioid prescriptions following hospitalization. The rise of the opioid epidemic in the United States underscores the importance of a better understanding of prescribing patterns. Although delivery is the most frequent reason for hospitalization in the United States, there is inadequate knowledge regarding opioid prescribing at postpartum hospital discharge.
OBJECTIVE
We sought to describe opioid prescribing patterns at the time of discharge following delivery in a large, diverse cohort, and to describe the relationship of these patterns with objective and subjective measures of pain prior to discharge.
STUDY DESIGN
This is a retrospective cohort study of all deliveries at a single, high-volume tertiary care center over a 1-year period. Women were excluded from analysis if they had evidence of recent opioid use, or their labor, delivery, or postpartum course was notable for rare, nonroutine events anticipated to increase pain. Medical records were queried for demographic and clinical data, including whether an opioid prescription was provided at discharge, and if so, details of that prescription. The primary outcome was amount of opioid morphine milligram equivalents prescribed at discharge, described separately for women after vaginal and cesarean deliveries. Among women who received a prescription, we additionally assessed associations between prescription quantity and subjective (patient-reported pain score) and objective (inpatient opioid requirement during the final 24 hours of hospitalization) assessments of pain. Descriptive and bivariable analyses were performed.
RESULTS
Of the total 12,611 women, 12,326 were eligible for inclusion. Of 9038 women postvaginal delivery and 3288 women postcesarean delivery, 30.4% and 86.7% received an opioid prescription at discharge, respectively. Of women receiving discharge opioid prescriptions, median morphine milligram equivalents received was 200 (interquartile range: 120-300) following vaginal and 300 (interquartile range: 200-300) following cesarean delivery. Nearly half (45.7%) of women postvaginal delivery and 18.5% of women postcesarean delivery who received an opioid prescription used 0 morphine milligram equivalent during the final hospital day. Similarly, 26.5% and 18.5% of women after vaginal and cesarean delivery, respectively, reported a pain score of 0 of 10 prior to discharge. Regardless of delivery mode, the amount of opioids prescribed did not differ between those who reported a pain score of 0 of 10 and those who reported a pain score of >0 of 10 immediately prior to discharge. Similarly, for women who underwent cesarean delivery, the morphine milligram equivalents prescribed did not differ between those who used 0 morphine milligram equivalents and those who used >0 in the 24 hours prior to hospital discharge.
CONCLUSION
Postpartum women are commonly prescribed opioids at the time of postpartum hospital discharge. There is a wide range of morphine milligram equivalents prescribed at hospital discharge following delivery, highlighting a lack of standardization. Furthermore, regardless of objective and subjective measures of pain prior to discharge, women received similar amounts of prescription morphine milligram equivalents following either vaginal or cesarean deliveries.
Topics: Adult; Analgesics, Opioid; Anti-Inflammatory Agents, Non-Steroidal; Cesarean Section; Cohort Studies; Delivery, Obstetric; Female; Humans; Pain Measurement; Pain, Postoperative; Patient Discharge; Postnatal Care; Practice Patterns, Physicians'; Pregnancy; Retrospective Studies
PubMed: 29630887
DOI: 10.1016/j.ajog.2018.04.003