Serum index rules prevent risk of analysing uncentrifuged tubes on automated biochemistry analysers.

Around 1.5% of the total clinical biochemistry tests performed in laboratories are affected by preanalytical errors. Large, automated chemistry analysers prevent errors and interference by using control systems such as spectrophotometric measurements to evaluate serum indices, i.e. haemolysis (H), icterus (I), and lipemia/turbidity (L). However, still preanalytical errors can remain undetected. Our laboratory experienced an incident caused by laboratory-induced preanalytical errors, where approximately 100 sedimented lithium heparin samples bypassed centrifugation and entered our automated analyser. Based on index results, we investigated the possibility of using turbidimetry measurement, as a mean to prevent analysis on uncentrifuged sedimented whole blood. 14078 L-indices from 8 days in August 2019 were extracted from the middleware and used to develop and evaluate stop rules. Similarly, a one-day validation dataset was identified in December 2020 and used for an independent validation. Three different types of stop rules were evaluated: (1) A single L-index result above a cut-off; (2) A sequence of an L-index results above a cut-off; (3) A simple moving average of n results above a cut-off. A stop rule using 3 consecutive L-indices of 40-60 was found to be superior. However, practical implementation in the instrument middleware on a Roche Cobas 8000 only allowed a simple moving average of 110 (n = 5). This rule was found to be able to identify and stop sedimented whole blood analysis. Additionally, the rule has minimal impact on daily routine production in the laboratory.

Isobutanol production freed from biological limits using synthetic biochemistry.

Cost competitive conversion of biomass-derived sugars into biofuel will require high yields, high volumetric productivities and high titers. Suitable production parameters are hard to achieve in cell-based systems because of the need to maintain life processes. As a result, next-generation biofuel production in engineered microbes has yet to match the stringent cost targets set by petroleum fuels. Removing the constraints imposed by having to maintain cell viability might facilitate improved production metrics. Here, we report a cell-free system in a bioreactor with continuous product removal that produces isobutanol from glucose at a maximum productivity of 4 g L-1 h-1, a titer of 275 g L-1 and 95% yield over the course of nearly 5 days. These production metrics exceed even the highly developed ethanol fermentation process. Our results suggest that moving beyond cells has the potential to expand what is possible for bio-based chemical production.

Facilitating chemical and biochemical experiments with electronic microcontrollers and single-board computers.

Since the advent of modern science, researchers have had to rely on their technical skills or the support of specialized workshops to construct analytical instruments. The notion of the 'fourth industrial revolution' promotes construction of customized systems by individuals using widely available, inexpensive electronic modules. This protocol shows how chemists and biochemists can utilize a broad range of microcontroller boards (MCBs) and single-board computers (SBCs) to improve experimental designs and address scientific questions. We provide seven example procedures for laboratory routines that can be expedited by implementing this technology: (i) injection of microliter-volume liquid plugs into microscale capillaries for low-volume assays; (ii) transfer of liquid extract to a mass spectrometer; (iii) liquid-gas extraction of volatile organic compounds (called 'fizzy extraction'), followed by mass spectrometric detection; (iv) monitoring of experimental conditions over the Internet cloud in real time; (v) transfer of analytes to a mass spectrometer via a liquid microjunction interface, data acquisition, and data deposition into the Internet cloud; (vi) feedback control of a biochemical reaction; and (vii) optimization of sample flow rate in direct-infusion mass spectrometry. The protocol constitutes a primer for chemists and biochemists who would like to take advantage of MCBs and SBCs in daily experimentation. It is assumed that the readers have not attended any courses related to electronics or programming. Using the instructions provided in this protocol and the cited material, readers should be able to assemble simple systems to facilitate various procedures performed in chemical and biochemical laboratories in 1-2 d.

A modified gas-trapping method for high-throughput metabolic experiments in Drosophila melanogaster.

Metabolism is often studied in animal models, with the Drosophila melanogaster fruit fly model offering ease of genetic manipulation and high-throughput studies. Fly metabolism is typically studied using end-point assays that are simple but destructive, and do not provide information on the utilization of specific nutrients. To address these limitations, we adapted existing gas-trapping protocols to measure the oxidation of radiolabeled substrates (such as glucose) in multi-well plates. This protocol is cost effective, simple, and offers precise control over experimental diet and measurement time, thus being amenable to high-throughput studies. Furthermore, it is nondestructive, enabling time-course experiments and multiplexing with other parameters. Overall, this protocol is useful for merging fly genetics with metabolic studies to understand whole organism responses to different macronutrients.

Solvent-assisted preparation of supported lipid bilayers.

The supported lipid bilayer (SLB) platform is a popular cell membrane mimic that is utilized in the chemical, biological, materials science, and medical fields. To date, SLB preparation has proven challenging because of the need for specialized fabrication equipment, domain-specific knowledge about topics relevant to lipid self-assembly, and extensive training in the interfacial science field. Existing methods, such as vesicle fusion, also work with only a narrow range of lipid compositions and material supports. Here, we describe a recently developed simple and versatile protocol to form SLBs. The protocol is simple because it requires minimal sample preparation and only basic microfluidics, making it technically accessible to researchers across different scientific disciplines. The protocol is versatile because it works on a wide range of material supports, such as silicon oxide, gold, and graphene, and is compatible with diverse lipid compositions, including sterols and signaling lipids. The main stages of the procedure involve dissolving a lipid sample in an organic solvent, depositing the lipid solution on a solid support, and replacing the organic solvent with aqueous buffer. In addition, we provide procedures for characterizing the quality of the prepared SLBs and present examples of biofunctionalization procedures. The protocol takes 1-2 h and is broadly useful in various application contexts, including clinical diagnostics, biosensing, and cellular interfaces.

Application and methodology of dissolution dynamic nuclear polarization in physical, chemical and biological contexts.

Dissolution dynamic nuclear polarization (d-DNP) is a versatile method to enhance nuclear magnetic resonance (NMR) spectroscopy. It boosts signal intensities by four to five orders of magnitude thereby providing the potential to improve and enable a plethora of applications ranging from the real-time monitoring of chemical or biological processes to metabolomics and in-cell investigations. This perspectives article highlights possible avenues for developments and applications of d-DNP in biochemical and physicochemical studies. It outlines how chemists, biologists and physicists with various fields of interest can transform and employ d-DNP as a powerful characterization method for their research.

Full in vivo characterization of carbonate chemistry at the site of calcification in corals.

Reef-building corals form their calcium carbonate skeletons within an extracellular calcifying medium (ECM). Despite the critical role of the ECM in coral calcification, ECM carbonate chemistry is poorly constrained in vivo, and full ECM carbonate chemistry has never been characterized based solely on direct in vivo measurements. Here, we measure pHECM in the growing edge of Stylophora pistillata by simultaneously using microsensors and the fluorescent dye SNARF-1, showing that, when measured at the same time and place, the results agree. We then conduct microscope-guided microsensor measurements of pH, [Ca2+], and [CO3 2-] in the ECM and, from this, determine [DIC]ECM and aragonite saturation state (Ωarag), showing that all parameters are elevated with respect to the surrounding seawater. Our study provides the most complete in vivo characterization of ECM carbonate chemistry parameters in a coral species to date, pointing to the key role of calcium- and carbon-concentrating mechanisms in coral calcification.

Review on Sperm Sorting Technologies and Sperm Properties toward New Separation Methods via the Interface of Biochemistry and Material Science.

Successful fertilization in mammals requires spermatozoa to efficiently traverse the female reproductive tract to meet the egg. This process naturally selects high quality sperm cells for fertilization, but when artificial reproductive technologies are used such as in vitro fertilization, intracytoplasmic sperm injection, or intrauterine insemination, other methods of sperm selection are required. Currently, technology enables sperm sorting based on motility, maturity as defined by zeta potential or hyaluronic acid binding site expression, absence of apoptotic factors, appropriate morphology, and even sex. This review summarizes current knowledge on all known methods of sperm cell sorting, compares their efficiency, and discusses the advantages and limitations of each technique. Scope for further refinement and improvement of current methods are discussed as is the potential to utilize a variety of materials to innovate new methods of sperm separation.

Breath water-based doubly labelled water method for the noninvasive determination of CO2 production and energy expenditure in mice.

We explored a novel doubly labelled water (DLW) method based on breath water (BW-DLW) in mice to determine whole body CO2 production and energy expenditure noninvasively. The BW-DLW method was compared to the DLW based on blood plasma. Mice (n = 11, 43.5 ± 4.6 g body mass (BM)) were administered orally a single bolus of doubly labelled water (1.2 g H218O kg BM-1 and 0.4 g 2H2O kg BM-1, 99 atom% (AP) 18O or 2H). To sample breath water, the mice were placed into a respiration vessel. The exhaled water vapour was condensed in a cold-trap. The isotope enrichments of breath water were compared with plasma samples. The 2H/1H and 18O/16O isotope ratios were measured by means of isotope ratio mass spectrometry. The CO2 production (RCO2) was calculated from the 2H and 18O enrichments in breath water and plasma over 5 days. The isotope enrichments of breath water vs. plasma were correlated (R2 = 0.89 for 2H and 0.95 for 18O) linearly. The RCO2 determined based on breath water and plasma was not different (113.2 ± 12.7 vs. 111.4 ± 11.0 mmol d-1), respectively. In conclusion, the novel BW-DLW method is appropriate to obtain reliable estimates of RCO2 avoiding blood sampling.

Use of biochemical miniaturized galleries, rRNA based lateral flow assay and Real Time PCR for Cronobacter spp. confirmation.

Identification of Cronobacter represent a major challenge for laboratories testing powdered infant formula (PIF). In the present study, two biochemical galleries and three molecular methods have been applied to confirm 276 Cronobacter spp. and non-Cronobacter isolates from different sources. Using the latest database of API 20 E and ID 32 E biochemical miniaturized kits, 53% and 78% of the isolates were identified respectively. From the available results, total accuracy for Cronobacter detection was in 97% (API 20 E) and 99% (ID 32 E). The three molecular methods were based on rRNA based lateral flow, Real Time PCR combined with either a hybridization or hydrolysis probe. For all three methods total accuracy was more than 99%. A pilot trial using Next Generation Sequencing (NGS) correctly identified 58 out of 66 isolates (88%) in DNA mixtures. The results indicate that the commercially available approaches such as ID 32 E, rRNA based lateral flow and Real Time PCR are all suitable for Cronobacter identification at the genus level. The NGS method may become a suitable alternative in the future, provided that the sequence database is improved.

Human Milk Lipidomics: Current Techniques and Methodologies.

Human milk contains a complex combination of lipids, proteins, carbohydrates, and minerals, which are essential for infant growth and development. While the lipid portion constitutes only 5% of the total human milk composition, it accounts for over 50% of the infant's daily energy intake. Human milk lipids vary throughout a feed, day, and through different stages of lactation, resulting in difficulties in sampling standardization and, like blood, human milk is bioactive containing endogenous lipases, therefore appropriate storage is critical in order to prevent lipolysis. Suitable sample preparation, often not described in studies, must also be chosen to achieve the aims of the study. Gas chromatography methods have classically been carried out to investigate the fatty acid composition of human milk lipids, but with the advancement of other chromatographic techniques, such as liquid and supercritical fluid chromatography, as well as mass spectrometry, intact lipids can also be characterized. Despite the known importance, concise and comprehensive analysis of the human milk lipidome is limited, with gaps existing in all areas of human milk lipidomics, discussed in this review. With appropriate methodology and instrumentation, further understanding of the human milk lipidome and the influence it has on infant outcomes can be achieved.

Cobalamin-Dependent C-Methyltransferases From Marine Microbes: Accessibility via Rhizobia Expression.

Cobalamin-dependent radical S-adenosylmethionine (rSAM) methyltransferases catalyze chemically challenging methylation reactions on diverse natural products at unactivated carbon centers. In vivo reconstitution and biosynthetic studies of natural product gene clusters encoding these enzymes are often severely limited by ineffective heterologous expression hosts, including the otherwise versatile Escherichia coli. In this chapter, we describe the use of rhizobia bacteria as effective expression hosts for cobalamin-dependent rSAM C-methyltransferases. We chose the natural product pathway encoding the heavily modified cytotoxic peptides, the polytheonamides, as our model pathway due to the presence of two methyltransferases responsible for a total of 17 C-methylations. Detailed protocols are given for vector construction, transformation, and heterologous expression in Rhizobium leguminosarum bv. viciae 3841. Additional methods pertaining to analytical separation and mass spectrometric analysis of modified peptides are also entailed. As genomics continues to uncover new enzymes and pathways from unknown and uncultivated microbes, use of metabolically distinct heterologous expression hosts like rhizobia will be a necessary tool to unravel the catalytic and metabolic diversity of marine microbial life.

Assaying Oxidative Coupling Activity of CYP450 Enzymes.

Cytochrome P450 (CYP450) enzymes are ubiquitous catalysts in natural product biosynthetic schemes where they catalyze numerous different transformations using radical intermediates. In this protocol, we describe procedures to assay the activity of a marine bacterial CYP450 enzyme Bmp7 which catalyzes the oxidative radical coupling of polyhalogenated aromatic substrates. The broad substrate tolerance of Bmp7, together with rearrangements of the aryl radical intermediates leads to a large number of products to be generated by the enzymatic action of Bmp7. The complexity of the product pool generated by Bmp7 thus presents an analytical challenge for structural elucidation. To address this challenge, we describe mass spectrometry-based procedures to provide structural insights into aryl crosslinked products generated by Bmp7, which can complement subsequent spectroscopic experiments. Using the procedures described here, for the first time, we show that Bmp7 can efficiently accept polychlorinated aryl substrates, in addition to the physiological polybrominated substrates for the biosynthesis of polyhalogenated marine natural products.

Preanalytical influence of pneumatic tube delivery system on results of routine biochemistry and haematology analysis.

Pneumatic tube delivery system (PTS) enables to reduce considerably turnaround times. The aim of the study was to assess the influence of the PTS on the quality of routine biochemical and hematological tests in our laboratory. Blood samples from 6 hospitalized patients and 8 healthy volunteers were analyzed. Blood samples were delivered to the laboratory by a PTS and by a human courier. We performed the following analysis: ionized calcium, sodium, potassium, lactate deshydrogenase (LDH), aspartate aminotransferase (ASAT), arterial blood gas, complete blood count and coagulation test as prothrombin time, activated partial thromboplastin time, factors V and VIII. Results were compared between the both method of transport according to the recommendation of the Société française de biologie clinique and the French committee for accreditation (SH-GTA01, norme NF ISO 5275-6). The hemolysis index of plasma was similar between the groups and no morphological differences were found on blood cells. For three samples, when delivered by PTS, LDH levels (two samples) and neutrophil polynuclear count (one sample) were above the recommended guidelines compared to those delivered by courier. Conversely, LDH levels and FVIII were below in two samples delivered by PTS. LDH levels, PNN count or factor VIII can be affected by PTS without the clinical interpretation being modified. We concluded that the PTS can be used to transport blood samples for routine biochemical and hematological analysis in our hospital.

Saposin-Lipoprotein Scaffolds for Structure Determination of Membrane Transporters.

Membrane proteins depend on their natural lipid environment for function, which makes them more difficult to study in isolation. A number of approaches that mimic the lipid bilayer of biological membranes have been described (nanodiscs, SMALPs), enabling novel ways to assay activity and elucidate structures of this important class of proteins. More recently, the use of saposin A, a protein that is involved in lipid transport, to form Salipro (saposin-lipid-protein) complexes was demonstrated for a range of membrane protein targets (Frauenfeld et al., 2016). The method is fast and requires few resources. The saposin-lipid-scaffold adapts to various sizes of transmembrane regions during self-assembly, forming a minimal lipid nanoparticle. This results in the formation of a well-defined membrane protein-lipid complex, which is desirable for structural characterization. Here, we describe a protocol to reconstitute the sarco-endoplasmic reticulum calcium ATPase (SERCA) into Salipro nanoparticles. The complex formation is analyzed using negative stain electron microscopy (EM), allowing to quickly determine an initial structure of the membrane protein and to evaluate sample conditions for structural studies using single-particle cryo-EM in a detergent-free environment.

Controlled modification of biomolecules by ultrashort laser pulses in polar liquids.

Targeted chemical modification of peptides and proteins by laser pulses in a biologically relevant environment, i.e. aqueous solvent at room temperature, allows for accurate control of biological processes. However, the traditional laser methods of control of chemical reactions are applicable only to a small class of photosensitive biomolecules because of strong and ultrafast perturbations from biomolecule-solvent interactions. Here, we report excitation of harmonics of vibration modes of solvent molecules by femtosecond laser pulses to produce controlled chemical modifications of non-photosensitive peptides and proteins in polar liquids under room conditions. The principal modifications included lysine formylation and methionine sulfoxidation both of which occur with nearly 100% yield under atmospheric conditions. That modification occurred only if the laser irradiance exceeded certain threshold level. The threshold, type, and extent of the modifications were completely controlled by solvent composition, laser wavelength, and peak irradiance of ultrashort laser pulses. This approach is expected to assist in establishing rigorous control over a broad class of biological processes in cells and tissues at the molecular level.

Current Trends in Ligand Binding Real-Time Measurement Technologies.

Numerous advances in ligand binding assay (LBA) real-time measurement technologies have been made within the last several years, ranging from the development of novel platforms to drive technology expansion to the adaptation of existing platforms to optimize performance and throughput. In this review, we have chosen to focus on technologies that provide increased value to two distinct segments of the LBA community. First, experimentally, by measuring real-time binding events, these technologies provide data that can be used to interrogate receptor/ligand binding interactions. While overall the platforms are not new, they have made significant advances in throughput, multiplexing, and/or sensitivity. Second, clinically, these point-of-care (POC) technologies provide instantaneous information which facilitates rapid treatment decisions.

Analytical techniques for the study of polyphenol-protein interactions.

This mini review focuses on advances in biophysical techniques to study polyphenol interactions with proteins. Polyphenols have many beneficial pharmacological properties, as a result of which they have been the subject of intensive studies. The most conventional techniques described here can be divided into three groups: (i) methods used for screening (in-situ methods); (ii) methods used to gain insight into the mechanisms of polyphenol-protein interactions; and (iii) methods used to study protein aggregation and precipitation. All of these methods used to study polyphenol-protein interactions are based on modifications to the physicochemical properties of the polyphenols or proteins after binding/complex formation in solution. To date, numerous review articles have been published in the field of polyphenols. This review will give a brief insight in computational methods and biosensors and cell-based methods, spectroscopic methods including fluorescence emission, UV-vis adsorption, circular dichroism, Fourier transform infrared and mass spectrometry, nuclear magnetic resonance, X-ray diffraction, and light scattering techniques including small-angle X-ray scattering and small-angle neutron scattering, and calorimetric techniques (isothermal titration calorimetry and differential scanning calorimetry), microscopy, the techniques which have been successfully used for polyphenol-protein interactions. At the end the new methods based on single molecule detection with high potential to study polyphenol-protein interactions will be presented. The advantages and disadvantages of each technique will be discussed as well as the thermodynamic, kinetic or structural parameters, which can be obtained. The other relevant biophysical experimental techniques that have proven to be valuable, such electrochemical methods, hydrodynamic techniques and chromatographic techniques will not be described here.

Desafíos de la Bioquímica en la era hipertecnológica / Challenges of Biochemistry in the hyper technological era / Desafios da Bioquímica na era da hipertecnologia

En la era hipertecnológica, ¿somos asistidos por la Tecnología o asistentes de la Tecnología? La máquina inteligente, el robot ideal, ¿puede realizar todas las tareas del laboratorio, con igual o mayor eficacia que el hombre? Creemos que no, ya que la máquina puede tener solamente "pensamiento calculador". En cambio, el hombre posee "pensamiento reflexivo" y juicio ético, que le permiten un discernimiento crítico fundamental. La máquina debe asistirnos, pero es el profesional quien debe decidir lo atinente al pre y post análisis, al control del instrumental en la fase analítica, y a la atención al paciente. La simplificación de los métodos analíticos conlleva a la realización de los análisis deslocalizados, "al lado del paciente". Los progresos técnicos como la miniaturización, la automatización y la informática favorecen el proceso de deslocalización, que puede tener lugar en unidades críticas, guardias, quirófanos o, incluso, fuera del ámbito hospitalario. La rapidez de respuesta en casos críticos puede justificar los análisis deslocalizados, pero existen claras limitaciones: Aseguramiento de la Calidad, Bioseguridad y aspectos bioéticos, respeto de la confidencialidad y capacidad para brindar información adecuada al paciente. En este contexto, es imperativo repensar las Ciencias de la Salud en la importancia de la tecnología pero, también, y sobre todo, en el factor humano y en el rol social del Bioquímico integrante del equipo de salud.

In the hyper technological era, are we assisted by technology or assistants of technology? The intelligent machine, the ideal robot can perform all tasks in the laboratory, with equal or greater efficacy than man? We think not, because the machine can only have "calculating thinking". Instead, man has "reflexive thinking" and ethical judgment, which allow a fundamental critical discernment. The machine must assist professionals, but is the professional who must decide about questions relative to pre and post analysis, control of instruments in the analytical phase, and patient care. Simplification of analytical methods leads to the realization of delocalized analysis, or "point of care". Technical advances such as miniaturization, automation and computing favour delocalized analysis, which can take place in critical units, emergencies, surgeries or even outside the hospital setting. The speed of response in critical cases can justify the delocalized analysis, but there are clear limitations: Quality Assurance, biosafety and bioethical aspects: confidentiality and ability to provide adequate information to the patient. In this context, in the Health Sciences, it is imperative to rethink the importance of technology, but also, and especially, the human factor and the social role of the Biochemist as health team member.

Na era hipertecnológica, somos assistidos pela Tecnologia ou assistentes da Tecnologia? A máquina inteligente, o robô ideal: pode realizar todas as tarefas de laboratório com igual ou maior eficiência do que o homem? Acreditamos que não, visto que a máquina só pode ter "pensamento calculador". Contudo, o homem possui "pensamento reflexivo" e juízo ético que lhe permitem um discernimento crítico fundamental. A máquina deve assistir-nos, porém, é o profissional quem deve tomar as decisões a respeito das pré e pós-análises, do controle do instrumental na fase analítica e do atendimento do paciente. A simplificação dos métodos analíticos conduz à realização das análises deslocadas, "ao lado do paciente". Os avanços técnicos como a miniaturização, a automação e a informática favorecem o processo de deslocação que pode ter lugar em unidades críticas, plantões, salas de cirurgia, ou até mesmo, fora do ambiente hospitalar. A velocidade de resposta em casos críticos pode justificar as análises deslocadas, porém existem claras limitações: segurança na Qualidade, Biossegurança e aspectos bioéticos; a respeito da confidencialidade e capacidade para fornecer ao paciente informação adequada. Nesse contexto, é imperativo repensar as Ciências da Saúde não só na importância da Tecnologia, mas também, e principalmente, no fator humano e no papel social do Bioquímico como integrante da equipe de saúde.