Expanding Extender Substrate Selection for Unnatural Polyketide Biosynthesis by Acyltransferase Domain Exchange within a Modular Polyketide Synthase.

Modular polyketide synthases (PKSs) are polymerases that employ α-carboxyacyl-CoAs as extender substrates. This enzyme family contains several catalytic modules, where each module is responsible for a single round of polyketide chain extension. Although PKS modules typically use malonyl-CoA or methylmalonyl-CoA for chain elongation, many other malonyl-CoA analogues are used to diversify polyketide structures in nature. Previously, we developed a method to alter an extension substrate of a given module by exchanging an acyltransferase (AT) domain while maintaining protein folding. Here, we report in vitro polyketide biosynthesis by 13 PKSs (the wild-type PKS and 12 AT-exchanged PKSs with unusual ATs) and 14 extender substrates. Our ∼200 in vitro reactions resulted in 13 structurally different polyketides, including several polyketides that have not been reported. In some cases, AT-exchanged PKSs produced target polyketides by >100-fold compared to the wild-type PKS. These data also indicate that most unusual AT domains do not incorporate malonyl-CoA and methylmalonyl-CoA but incorporate various rare extender substrates that are equal to in size or slightly larger than natural substrates. We developed a computational workflow to predict the approximate AT substrate range based on active site volumes to support the selection of ATs. These results greatly enhance our understanding of rare AT domains and demonstrate the benefit of using the proposed PKS engineering strategy to produce novel chemicals in vitro.

Core outcomes for pressure ulcer prevention trials: results of an international consensus study: Classification: Outcomes and qualitative research.

BACKGROUND: There is substantial heterogeneity between trial outcomes in pressure ulcer prevention research. The development of core outcome sets is one strategy to improve comparability between trial results and thus increase the quality of evidence. OBJECTIVES: To identify core outcomes for pressure ulcer prevention trials. METHODS: A workshop was held with service users to discuss their views and understanding of the outcomes identified by a scoping review and to identify any missing outcomes. In a next step, a Delphi survey comprising three rounds was conducted to evaluate a compiled list of outcomes by their importance. Afterwards the preselection from the Delphi survey was discussed in a virtual consensus meeting with the aim of agreeing on a final set of core outcomes. Individuals who had completed all three rounds of the Delphi survey were eligible to participate in this meeting. Participants included practitioners, service users, researchers and industry representatives. The OUTPUTs project is registered in the COMET database and is part of the Cochrane Skin Core Outcome Set Initiative. RESULTS: The workshop did not reveal any missing outcomes, but highlighted the need for further efforts to make lay people understand what an outcome is in a study setting. The Delphi survey took place between December 2020 and June 2021. After the three rounds, 18 out of 37 presented outcomes were rated to be critically important. In the following consensus meeting, six outcomes were prioritized to be included in the core outcome set for pressure ulcer prevention trials: (i) pressure ulcer occurrence; (ii) pressure ulcer precursor signs and symptoms; (iii) mobility; (iv) acceptability and comfort of intervention; (v) adherence/compliance; and (vi) adverse events/safety. CONCLUSIONS: Based on a comprehensive list of outcomes in pressure ulcer prevention research, there was clear agreement on the six identified core outcomes in three international Delphi rounds and in the consensus meeting. Although outcome measurement instruments need to be identified next, the six identified core outcomes should already be considered in future trials, as service users, practitioners, researchers and industry representatives have agreed that they are critically important. What is already known about this topic? There are numerous trials on pressure ulcer prevention, but evidence on the effectiveness of preventive measures is limited due to heterogeneity between trial outcomes. The development of a core outcome set is one strategy to improve comparability between trial results. What does this study add? A service user workshop, a three-round Delphi survey and an online consensus meeting with practitioners, service users, researchers and industry representatives were conducted to identify core outcomes for pressure ulcer prevention trials. Six core outcomes were defined: (i) pressure ulcer occurrence, (ii) pressure ulcer precursor signs and symptoms, (iii) mobility, (iv) acceptability and comfort of intervention, (v) adherence/compliance and (vi) adverse events/safety. What are the clinical implications of this work? Better evidence of interventions for pressure ulcer prevention will help health professionals and service users to decide which interventions are most appropriate and effective. Better evidence may contribute to better pressure ulcer prevention.

Stability evaluation of morphine, hydromorphone, metamizole and esketamine containing analgesic mixtures applied for patient-controlled analgesia in hospice and palliative care.

In this study, different injection solutions containing opioid and nonopioid compounds used for patient-controlled analgesia in hospice and palliative care were evaluated in terms of analyte stability. Investigated injection solutions contained different combinations of morphine, hydromorphone, metamizole and esketamine. For the practical implementation, samples from infusion pumps were daily drawn over a period of 7 days at 22 and 37°C. Quantitative measurements were performed on a high-performance liquid chromatography system with ultraviolet detection applying a validated analytical method. All compounds apart from morphine showed no evident changes in concentration. However, a significant loss of morphine was observed for injection mixtures containing both morphine and metamizole at 37°C. After 7 days, only 72% of the initially measured morphine concentration was measured in the binary and 77% in the ternary mixture. Furthermore, an additional compound was detected that could represent the morphine-metamizole-adduct, "metamorphine". Based on these results, a significantly reduced morphine concentration must be expected after only 3 days if an injection solution mixture containing both morphine and metamizole is administered to a patient at 37°C. Since the analgesic effects of morphine-metamizole adducts have not yet been thoroughly investigated, further clinical studies are necessary before accurate conclusions can be drawn in this regard.

Molecular characterization of xerosis cutis: A systematic review.

BACKGROUND: Xerosis cutis or dry skin is a highly prevalent dermatological disorder especially in the elderly and in patients with underlying health conditions. In the past decades, numerous molecular markers have been investigated for their association with the occurrence or severity of skin dryness. The aim of this review was to summarize the molecular markers used in xerosis cutis research and to describe possible associations with different dry skin etiologies. METHODS: We conducted a systematic review of molecular markers of xerosis cutis caused by internal or systemic changes. References published between 1990 and September 2020 were searched using 'MEDLINE', 'EMBASE' and 'Biological abstracts' databases. Study results were summarized and analyzed descriptively. The review protocol was registered in PROSPERO database (CRD42020214173). RESULTS: A total of 21 study reports describing 72 molecules were identified including lipids, natural moisturizing factors (NMFs), proteins including cytokines and metabolites or metabolic products. Most frequently reported markers were ceramides, total free fatty acids, triglycerides and selected components of NMFs. Thirty-one markers were reported only once. Although, associations of these molecular markers with skin dryness were described, reports of unclear and/or no association were also frequent for nearly every marker. CONCLUSION: An unexpectedly high number of various molecules to quantify xerosis cutis was found. There is substantial heterogeneity regarding molecular marker selection, tissue sampling and laboratory analyses. Empirical evidence is also heterogeneous regarding possible associations with dry skin. Total free fatty acids, total ceramide, ceramide (NP), ceramide (NS), triglyceride, total free amino acids and serine seem to be relevant, but the association with dry skin is inconsistent. Although the quantification of molecular markers plays an important role in characterizing biological processes, pathogenic processes or pharmacologic responses, it is currently unclear which molecules work best in xerosis cutis.

Comparing the effects of three different multilayer dressings for pressure ulcer prevention on sacral skin after prolonged loading: An exploratory crossover trial.

Evidence suggests that preventive dressings applied on sacral skin help to prevent pressure ulcers. However, possible performance differences of different dressing types are unclear. An exploratory randomized crossover trial with intra-individual comparisons was conducted to compare the effects of three different multi-layer foam dressings (Mepilex Border Sacrum, ALLEVYN Life Sacrum and Optifoam Gentle Sacrum) compared to no dressing on the sacral skin. Healthy female volunteers (n = 12, mean age 72 years) wore three different dressings on their sacral skin for 3.5 hours while lying supine on a standard hospital mattress. At regular intervals, subjects performed standardized movements to enhance shear loads. Skin surface temperature, stratum corneum hydration, erythema, skin roughness and the interleukin 1 alpha (IL-1α) concentration per total protein were measured at baseline and after the lying periods. After 3.5 hours, the median skin temperature increased in all four groups between 3.0°C and 3.8°C with only minor differences between the no dressing and the dressing groups. Median stratum corneum hydration increased during the lying period in all groups with highest increases in the Optifoam Gentle Sacrum (7.3 arbitrary units) and no dressing group (7.0 arbitrary units). There was a median decrease of the mean roughness (Rz) in the Optifoam Gentle Sacrum group of -6.3 µm but no relevant changes in the other groups. After loading, the erythema index was highest in the ALLEVYN Life Sacrum and no dressing groups. Highest releases of IL-1α were observed in the ALLEVYN Life Sacrum and Optifoam Gentle Sacrum groups, in the Mepilex Border Sacrum group changes were minor. Study results indicate, that the application of preventive dressings on sacral skin during loading do not cause additional occlusion compared to loading without dressings when lying supine. Different dressings cause different cutaneous responses during loading.

Author Correction: Complete biosynthesis of cannabinoids and their unnatural analogues in yeast.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Anisotropic Thermal Transport in Spray-Coated Single-Phase Two-Dimensional Materials: Synthetic Clay Versus Graphene Oxide.

Directional control on material properties such as mechanical moduli or thermal conductivity are of paramount importance for the development of nanostructured next-generation devices. Two-dimensional materials are particularly interesting in this context owing to their inherent structural anisotropy. Here, we compare graphene oxide (GO) and synthetic clay sodium fluorohectorite (Hec) with respect to their thermal transport properties. The unique sheet structure of both allows preparation of highly ordered Bragg stacks of these pure materials. The thermal conductivity parallel to the platelets strongly exceeds that perpendicular to them. We find a significant difference in the performance between GO and synthetic clay. Our analysis of the textured structure, size of the platelets, and chemical composition shows that Hec is a superior two-dimensional component to GO. Consequently, synthetic clay is a promising material for thermal management applications in electronic devices where electrically insulating materials are prerequisites.

Tunable Thermoelastic Anisotropy in Hybrid Bragg Stacks with Extreme Polymer Confinement.

Controlling thermomechanical anisotropy is important for emerging heat management applications such as thermal interface and electronic packaging materials. Whereas many studies report on thermal transport in anisotropic nanocomposite materials, a fundamental understanding of the interplay between mechanical and thermal properties is missing, due to the lack of measurements of direction-dependent mechanical properties. In this work, exceptionally coherent and transparent hybrid Bragg stacks made of strictly alternating mica-type nanosheets (synthetic hectorite) and polymer layers (polyvinylpyrrolidone) were fabricated at large scale. Distinct from ordinary nanocomposites, these stacks display long-range periodicity, which is tunable down to angstrom precision. A large thermal transport anisotropy (up to 38) is consequently observed, with the high in-plane thermal conductivity (up to 5.7 W m-1 K-1 ) exhibiting an effective medium behavior. The unique hybrid material combined with advanced characterization techniques allows correlating the full elastic tensors to the direction-dependent thermal conductivities. We, therefore, provide a first analysis on how the direction-dependent Young's and shear moduli influence the flow of heat.

Comparing skin characteristics and molecular markers of xerotic foot skin between diabetic and non-diabetic subjects: An exploratory study.

BACKGROUND: Xerosis cutis of the feet is one of the most common skin conditions among type 2 diabetics. Whether skin dryness among diabetic patients is different from 'general' skin dryness is unclear. The overall aim was to compare the structure, function and molecular markers of dry and cracked foot skin between diabetics and non-diabetics. METHODS: The foot skin of 40 diabetics and 20 non-diabetics was evaluated. A clinical assessment of skin dryness was performed and transepidermal water loss, stratum corneum hydration, skin surface pH, epidermal thickness, skin roughness, elasticity and structural stiffness were measured. Ceramides, natural moisturizing factors, histamines, proteins and molecular markers of oxidative stress were analyzed based on a non-invasive sampling method for collection of surface biomarkers. RESULTS: The mean number of superficial fissures in the diabetic group was nearly three times higher than in the non-diabetic group (11.0 (SD 6.2) vs. 3.9 (SD 4.2)). The skin stiffness was higher in the diabetic group and the values of almost all molecular markers showed considerably higher values compared to non-diabetics. Malondialdehyde and glutathione were lower in the diabetic sample. CONCLUSIONS: The high number of superficial fissures may be based on an increased stiffness of dry diabetic foot skin combined with different concentrations of molecular markers in the stratum corneum compared to dry foot skin of non-diabetics.

Outcomes for Pressure Ulcer Trials (OUTPUTs): protocol for the development of a core domain set for trials evaluating the clinical efficacy or effectiveness of pressure ulcer prevention interventions.

BACKGROUND: Core outcome sets (COS) are being developed in many clinical areas to increase the quality and comparability of clinical trial results as well as to ensure their relevance for patients. A COS represents an agreed standardized set of outcomes that describes the minimum that should be consistently reported in all clinical trials of a defined area. It comprises a core domain set (defining what core outcomes should be measured) and a core measurement set (defining measurement/assessment instruments for each core domain). For pressure ulcer prevention trials a COS is lacking. The great heterogeneity of reported outcomes in this field indicates the need for a COS. METHODS/DESIGN: The first part of this project aims to develop a core domain set by following established methods, which incorporates four steps: (1) definition of the scope, (2) conducting a scoping review, (3) organizing facilitated workshops with service users, (4) performing Delphi surveys and establishing consensus in a face-to-face meeting with different stakeholders. DISCUSSION: After achieving consensus on the core domain set, further work will be undertaken to determine a corresponding core measurement set. This will lead to better pressure ulcer prevention research in the future. There are a number of methodological challenges in the field of COS development. To meet these challenges and to ensure a high-quality COS, the OUTPUTS project affiliates to current standards and works in close collaboration with international experts and with existing international service user groups. TRIAL REGISTRATION: The OUTPUTs project is registered in the COMET database: ( http://www.comet-initiative.org/studies/details/283 ). Registered on 2015.

Direct Measurement of the In-Plane Thermal Diffusivity of Semitransparent Thin Films by Lock-In Thermography: An Extension of the Slopes Method.

We present an extension of the well-known slopes method for characterization of the in-plane thermal diffusivity of semitransparent polymer films. We introduce a theoretical model which considers heat losses due to convection and radiation mechanisms, as well as semitransparency of the material to the exciting laser heat source (visible range) and multiple reflections at the film surfaces. Most importantly, a potential semitransparency of the material in the IR detection range is also considered. We prove by numerical simulations and by an asymptotic expansion of the surface temperature that the slopes method is also valid for any semitransparent film in the thermally thin regime. Measurements of the in-plane thermal diffusivity performed on semitransparent polymer films covering a wide range of absorption coefficients (to the exciting wavelength and in the IR detection range of our IR camera) validate our theoretical findings.

Complete biosynthesis of cannabinoids and their unnatural analogues in yeast.

Cannabis sativa L. has been cultivated and used around the globe for its medicinal properties for millennia1. Some cannabinoids, the hallmark constituents of Cannabis, and their analogues have been investigated extensively for their potential medical applications2. Certain cannabinoid formulations have been approved as prescription drugs in several countries for the treatment of a range of human ailments3. However, the study and medicinal use of cannabinoids has been hampered by the legal scheduling of Cannabis, the low in planta abundances of nearly all of the dozens of known cannabinoids4, and their structural complexity, which limits bulk chemical synthesis. Here we report the complete biosynthesis of the major cannabinoids cannabigerolic acid, Δ9-tetrahydrocannabinolic acid, cannabidiolic acid, Δ9-tetrahydrocannabivarinic acid and cannabidivarinic acid in Saccharomyces cerevisiae, from the simple sugar galactose. To accomplish this, we engineered the native mevalonate pathway to provide a high flux of geranyl pyrophosphate and introduced a heterologous, multi-organism-derived hexanoyl-CoA biosynthetic pathway5. We also introduced the Cannabis genes that encode the enzymes involved in the biosynthesis of olivetolic acid6, as well as the gene for a previously undiscovered enzyme with geranylpyrophosphate:olivetolate geranyltransferase activity and the genes for corresponding cannabinoid synthases7,8. Furthermore, we established a biosynthetic approach that harnessed the promiscuity of several pathway genes to produce cannabinoid analogues. Feeding different fatty acids to our engineered strains yielded cannabinoid analogues with modifications in the part of the molecule that is known to alter receptor binding affinity and potency9. We also demonstrated that our biological system could be complemented by simple synthetic chemistry to further expand the accessible chemical space. Our work presents a platform for the production of natural and unnatural cannabinoids that will allow for more rigorous study of these compounds and could be used in the development of treatments for a variety of human health problems.

Dry skin and the use of leave-on products in nursing care: A prevalence study in nursing homes and hospitals.

AIMS: To describe the prevalence of dry skin in nursing homes and hospitals and to describe relationships between topical skincare interventions and dry skin. DESIGN: Two multicentre descriptive cross-sectional prevalence studies. METHODS: The studies were performed in German nursing homes and hospitals in 2015 and 2016. Data were collected by trained nurses based on a standardized data collection form. The severity of dry skin was measured using the Overall Dry Skin Score. RESULTS: In total, 1,662 nursing home residents and 1,486 hospital patients participated. The prevalence of dry skin was 41.2% in nursing homes and 55.2% in hospitals. In case of skincare dependency, the proportions of participants with dry skin were higher, particularly in hospitals (70.2%). In both institutions, the application of leave-on products increased when dry skin was present but remained lower in hospitals. Considering the high amount of skin dryness in skincare-dependent participants, interventions seem not to be successful. Results indicate a need for skincare improvement in future.

Engineered Production of Short-Chain Acyl-Coenzyme A Esters in Saccharomyces cerevisiae.

Short-chain acyl-coenzyme A esters serve as intermediate compounds in fatty acid biosynthesis, and the production of polyketides, biopolymers and other value-added chemicals. S. cerevisiae is a model organism that has been utilized for the biosynthesis of such biologically and economically valuable compounds. However, its limited repertoire of short-chain acyl-CoAs effectively prevents its application as a production host for a plethora of natural products. Therefore, we introduced biosynthetic metabolic pathways to five different acyl-CoA esters into S. cerevisiae. Our engineered strains provide the following acyl-CoAs: propionyl-CoA, methylmalonyl-CoA, n-butyryl-CoA, isovaleryl-CoA and n-hexanoyl-CoA. We established a yeast-specific metabolite extraction protocol to determine the intracellular acyl-CoA concentrations in the engineered strains. Propionyl-CoA was produced at 4-9 µM; methylmalonyl-CoA at 0.5 µM; and isovaleryl-CoA, n-butyryl-CoA, and n-hexanoyl-CoA at 6 µM each. The acyl-CoAs produced in this study are common building blocks of secondary metabolites and will enable the engineered production of a variety of natural products in S. cerevisiae. By providing this toolbox of acyl-CoA producing strains, we have laid the foundation to explore S. cerevisiae as a heterologous production host for novel secondary metabolites.

Dry skin and pressure ulcer risk: A multi-center cross-sectional prevalence study in German hospitals and nursing homes.

BACKGROUND: Pressure ulcers are a serious health problem in medical and nursing care. Therefore, effective prevention is crucial. Major pressure ulcer risk factors have been identified but the particular role of dry skin (xerosis cutis) is unclear. OBJECTIVES: To investigate possible associations between dry skin and pressure ulcers focusing on the sacrum/trochanter and at heel/ankle skin areas. DESIGN: Two multicenter cross-sectional studies. SETTINGS/PARTICIPANTS: In 2014 and 2015 thirty nursing homes and thirteen hospitals in Germany participated. In total 3837 participants were included. Mean age was 76.1 (SD 15.5) years. METHODS: Skin assessments and data collection were performed by trained nurses based on a standardized data collection form. Descriptive comparisons and multilevel logistic regressions predicting pressure ulcers at sacrum/trochanter and ankle/heel were conducted. RESULTS: The prevalence of skin dryness at the trunk was significantly higher for subjects with pressure ulcers category 2+ at the sacral area compared to without (39.0% vs. 24.4%, p=0.010). Adjusted to demographic variables, mobility and type of institution dry skin at the trunk was no longer associated with pressure ulceration (OR 1.11 (95% CI 0.62-2.00)). 71.9% of patients with heel/ankle pressure ulcers category 2+ were affected by dry skin at legs or feet, compared to 42.8% of subjects without pressure ulcers (p<0.001). In the adjusted analysis the OR was 1.85 (95% CI 0.83-4.14). CONCLUSIONS: Study results indicate that dry skin at the feet may be considered as a risk factor for heel pressure ulcer development. Skin dryness may be less important for sacral pressure ulcers. Therefore, the variable skin status should be better defined in future studies and pressure ulcer risk models. Results further support differences in pressure ulcer aetiologies between anatomical locations.

Who seeks Teratology Information Service's advice? Assessing the risk of selection bias in observational cohort studies on drug risks in pregnancy.

All women of reproductive age should have access to Teratology Information Services (TIS). Observational cohort studies based on TIS data are one of the most important sources for risk characterization of drugs during pregnancy. A selection in TIS populations towards higher socioeconomic level would compromise the TIS' goal of health prevention and the quality of research. The aim of this study is to investigate in which respect Berlin TIS enquirers are different from the general female population of reproductive age in Germany. 5,239 women aged between 20 and 39 were compared with the general female population of reproductive age in Germany. Medium- and high-level educated women tend to be overrepresented among TIS enquirers. TIS should strive towards reaching subpopulations with poor access to health care. TIS-based observational studies require appropriate comparison cohorts from the same data pool with similar procedures of ascertainment to reduce the risk of selection bias.

Multi-omic data integration enables discovery of hidden biological regularities.

Rapid growth in size and complexity of biological data sets has led to the 'Big Data to Knowledge' challenge. We develop advanced data integration methods for multi-level analysis of genomic, transcriptomic, ribosomal profiling, proteomic and fluxomic data. First, we show that pairwise integration of primary omics data reveals regularities that tie cellular processes together in Escherichia coli: the number of protein molecules made per mRNA transcript and the number of ribosomes required per translated protein molecule. Second, we show that genome-scale models, based on genomic and bibliomic data, enable quantitative synchronization of disparate data types. Integrating omics data with models enabled the discovery of two novel regularities: condition invariant in vivo turnover rates of enzymes and the correlation of protein structural motifs and translational pausing. These regularities can be formally represented in a computable format allowing for coherent interpretation and prediction of fitness and selection that underlies cellular physiology.

The Need for Integrated Approaches in Metabolic Engineering.

This review highlights state-of-the-art procedures for heterologous small-molecule biosynthesis, the associated bottlenecks, and new strategies that have the potential to accelerate future accomplishments in metabolic engineering. We emphasize that a combination of different approaches over multiple time and size scales must be considered for successful pathway engineering in a heterologous host. We have classified these optimization procedures based on the "system" that is being manipulated: transcriptome, translatome, proteome, or reactome. By bridging multiple disciplines, including molecular biology, biochemistry, biophysics, and computational sciences, we can create an integral framework for the discovery and implementation of novel biosynthetic production routes.

A streamlined ribosome profiling protocol for the characterization of microorganisms.

Ribosome profiling is a powerful tool for characterizing in vivo protein translation at the genome scale, with multiple applications ranging from detailed molecular mechanisms to systems-level predictive modeling. Though highly effective, this intricate technique has yet to become widely used in the microbial research community. Here we present a streamlined ribosome profiling protocol with reduced barriers to entry for microbial characterization studies. Our approach provides simplified alternatives during harvest, lysis, and recovery of monosomes and also eliminates several time-consuming steps, in particular size-selection steps during library construction. Furthermore, the abundance of rRNAs and tRNAs in the final library is drastically reduced. Our streamlined workflow enables greater throughput, cuts the time from harvest to the final library in half (down to 3-4 days), and generates a high fraction of informative reads, all while retaining the high quality standards of the existing protocol.