RamanSPy: An Open-Source Python Package for Integrative Raman Spectroscopy Data Analysis.

Raman spectroscopy is a nondestructive and label-free chemical analysis technique, which plays a key role in the analysis and discovery cycle of various branches of science. Nonetheless, progress in Raman spectroscopic analysis is still impeded by the lack of software, methodological and data standardization, and the ensuing fragmentation and lack of reproducibility of analysis workflows thereof. To address these issues, we introduce RamanSPy, an open-source Python package for Raman spectroscopic research and analysis. RamanSPy provides a comprehensive library of tools for spectroscopic analysis that supports day-to-day tasks, integrative analyses, the development of methods and protocols, and the integration of advanced data analytics. RamanSPy is modular and open source, not tied to a particular technology or data format, and can be readily interfaced with the burgeoning ecosystem for data science, statistical analysis, and machine learning in Python. RamanSPy is hosted at https://github.com/barahona-research-group/RamanSPy, supplemented with extended online documentation, available at https://ramanspy.readthedocs.io, that includes tutorials, example applications, and details about the real-world research applications presented in this paper.

Selective Macrocyclization of Unprotected Peptides with an Ex Situ Gaseous Linchpin Reagent.

Peptide cyclization has dramatic effects on a variety of important properties, enhancing metabolic stability, limiting conformational flexibility, and altering cellular entry and intracellular localization. The hydrophilic, polyfunctional nature of peptides creates chemoselectivity challenges in macrocyclization, especially for natural sequences without biorthogonal handles. Herein, we describe a gaseous sulfonyl chloride-derived reagent that achieves amine-amine, amine-phenol, and amine-aniline crosslinking via a minimalist linchpin strategy that affords macrocyclic urea or carbamate products. The cyclization reaction is metal-mediated, and involves a novel application of sulfine species that remains unexplored in aqueous or biological contexts. The aqueous method delivers unique cyclic or bicyclic topologies directly from a variety of natural bioactive peptides without the need for protecting-group strategies.

Commercial Optical and Acoustic Sensor Performances under Varying Turbidity, Illumination, and Target Distances.

Acoustic and optical sensing modalities represent two of the primary sensing methods within underwater environments, and both have been researched extensively in previous works. Acoustic sensing is the premier method due to its high transmissivity in water and its relative immunity to environmental factors such as water clarity. Optical sensing is, however, valuable for many operational and inspection tasks and is readily understood by human operators. In this work, we quantify and compare the operational characteristics and environmental effects of turbidity and illumination on two commercial-off-the-shelf sensors and an additional augmented optical method, including: a high-frequency, forward-looking inspection sonar, a stereo camera with built-in stereo depth estimation, and color imaging, where a laser has been added for distance triangulation. The sensors have been compared in a controlled underwater environment with known target objects to ascertain quantitative operation performance, and it is shown that optical stereo depth estimation and laser triangulation operate satisfactorily at low and medium turbidites up to a distance of approximately one meter, with an error below 2 cm and 12 cm, respectively; acoustic measurements are almost completely unaffected up to two meters under high turbidity, with an error below 5 cm. Moreover, the stereo vision algorithm is slightly more robust than laser-line triangulation across turbidity and lighting conditions. Future work will concern the improvement of the stereo reconstruction and laser triangulation by algorithm enhancement and the fusion of the two sensing modalities.

Revealing Population Heterogeneity in Vesicle-Based Nanomedicines Using Automated, Single Particle Raman Analysis.

The intrinsic heterogeneity of many nanoformulations is currently challenging to characterize on both the single particle and population level. Therefore, there is great opportunity to develop advanced techniques to describe and understand nanomedicine heterogeneity, which will aid translation to the clinic by informing manufacturing quality control, characterization for regulatory bodies, and connecting nanoformulation properties to clinical outcomes to enable rational design. Here, we present an analytical technique to provide such information, while measuring the nanocarrier and cargo simultaneously with label-free, nondestructive single particle automated Raman trapping analysis (SPARTA). We first synthesized a library of model compounds covering a range of hydrophilicities and providing distinct Raman signals. These compounds were then loaded into model nanovesicles (polymersomes) that can load both hydrophobic and hydrophilic cargo into the membrane or core regions, respectively. Using our analytical framework, we characterized the heterogeneity of the population by correlating the signal per particle from the membrane and cargo. We found that core and membrane loading can be distinguished, and we detected subpopulations of highly loaded particles in certain cases. We then confirmed the suitability of our technique in liposomes, another nanovesicle class, including the commercial formulation Doxil. Our label-free analytical technique precisely determines cargo location alongside loading and release heterogeneity in nanomedicines, which could be instrumental for future quality control, regulatory body protocols, and development of structure-function relationships to bring more nanomedicines to the clinic.

Lignocellulose Conversion via Catalytic Transformations Yields Methoxyterephthalic Acid Directly from Sawdust.

Poly(ethylene terephthalate) polyester represents the most common class of thermoplastic polymers widely used in the textile, bottling, and packaging industries. Terephthalic acid and ethylene glycol, both of petrochemical origin, are polymerized to yield the polyester. However, an earlier report suggests that polymerization of methoxyterephthalic acid with ethylene glycol provides a methoxy-polyester with similar properties. Currently, there are no established biobased synthetic routes toward the methoxyterephthalic acid monomer. Here, we show a viable route to the dicarboxylic acid from various tree species involving three catalytic steps. We demonstrate that sawdust can be converted to valuable aryl nitrile intermediates through hydrogenolysis, followed by an efficient fluorosulfation-catalytic cyanation sequence (>90%) and then converted to methoxyterephthalic acid by hydrolysis and oxidation. A preliminary polymerization result indicates a methoxy-polyester with acceptable thermal properties.

Fundamentals and Applications of Raman-Based Techniques for the Design and Development of Active Biomedical Materials.

Raman spectroscopy is an analytical method based on light-matter interactions that can interrogate the vibrational modes of matter and provide representative molecular fingerprints. Mediated by its label-free, non-invasive nature, and high molecular specificity, Raman-based techniques have become ubiquitous tools for in situ characterization of materials. This review comprehensively describes the theoretical and practical background of Raman spectroscopy and its advanced variants. The numerous facets of material characterization that Raman scattering can reveal, including biomolecular identification, solid-to-solid phase transitions, and spatial mapping of biomolecular species in bioactive materials, are highlighted. The review illustrates the potential of these techniques in the context of active biomedical material design and development by highlighting representative studies from the literature. These studies cover the use of Raman spectroscopy for the characterization of both natural and synthetic biomaterials, including engineered tissue constructs, biopolymer systems, ceramics, and nanoparticle formulations, among others. To increase the accessibility and adoption of these techniques, the present review also provides the reader with practical recommendations on the integration of Raman techniques into the experimental laboratory toolbox. Finally, perspectives on how recent developments in plasmon- and coherently-enhanced Raman spectroscopy can propel Raman from underutilized to critical for biomaterial development are provided.

Spectral Unmixing for Label-Free, In-Liquid Characterization of Biomass Microstructure and Biopolymer Content by Coherent Raman Imaging.

Characterization of lignocellulosic biomass microstructure with chemical specificity and under physiological conditions could provide invaluable insights to our understanding of plant tissue development, microstructure, origins of recalcitrance, degradation, and solubilization. However, most methods currently available are either destructive, are not compatible with hosting a physiological environment, or introduces exogenous probes, complicating their use for studying changes in microstructure and mechanisms of plant development, recalcitrance, or degradation in situ. To address these challenges, we here present a multi-modal chemically specific imaging technique based on coherent anti-Stokes Raman scattering (CARS) microspectroscopy with simplex maximization and entropy-based spectral unmixing enabling label-free, chemically specific characterization of plant microstructure in liquid. We describe how spatial drift of samples suspended in liquid can introduce artifacts in spectral unmixing procedures for single-frequency CARS and propose a mitigative strategy toward these effects using simultaneously acquired forward-scattered CARS signals and epi-detected autofluorescence. We further apply the technique for chemical and microstructural characterization of untreated and liquid hot water pretreated rapeseed straw by CARS and show how the framework can be extended for 3D imaging with chemical specificity. Finally, we provide examples of the intricate chemical and microstructural details recovered by this hybrid imaging technique, including discerning between primary and secondary cell walls, localization of aqueous components to cell lumina, and the presence of funnel-type pits in samples ofBrassica napus.

Influence of protein source (cricket, pea, whey) on amino acid bioavailability and activation of the mTORC1 signaling pathway after resistance exercise in healthy young males.

PURPOSE: New dietary proteins are currently introduced to replace traditional animal protein sources. However, not much is known about their bioaccessibility and ability to stimulate muscle protein synthesis compared to the traditional protein sources. We aimed to compare effects of ingesting a protein bolus (0.25 g/kg fat free mass) of either cricket (insect), pea, or whey protein on circulating amino acid levels and activation of the mTORC1 signaling pathway in the skeletal muscle at rest and after exercise. METHODS: In a randomized parallel controlled trial, young males (n = 50) performed a one-legged resistance exercise followed by ingestion of one of the three protein sources. Blood samples were collected before and in the following 4 h after exercise. Muscle biopsies were obtained at baseline and after 3 h from the non-exercised and exercised leg. RESULTS: Analysis of blood serum showed a significantly higher concentration of amino acids after ingestion of whey protein compared to cricket and pea protein. No difference between protein sources in activation of the mTORC1 signaling pathway was observed either at rest or after exercise. CONCLUSION: Amino acid blood concentration after protein ingestion was higher for whey than pea and cricket protein, whereas activation of mTORC1 signaling pathway at rest and after exercise did not differ between protein sources. TRIAL REGISTRATION NUMBER: Clinicaltrials.org ID NCT04633694.

Direct formation and site-selective elaboration of methionine sulfoximine in polypeptides.

Sulfoximines are emerging moieties for medicinal and biological chemistry, due in part to their efficacy in selective inhibition of amide-forming enzymes such as γ-glutamylcysteine synthetase. While small-molecule sulfoximines such as methionine sulfoximine (MSO) and its derivatives are well studied, structures with methionine sulfoximine residues within complex polypeptides have been generally inaccessible. This paper describes a straightforward means of late-stage one-step oxidation of methionine residues within polypeptides to afford NH-sulfoximines. We also present chemoselective subsequent elaboration, most notably by copper(ii)-mediated N-H cross-coupling at methionine sulfoximine residues with arylboronic acid reagents. This development serves as a strategy to incorporate diverse sulfoximine structures within natural polypeptides, and also identifies the methionine sulfoximine residue as a new site for bioorthogonal, chemoselective bioconjugation.

Novel methods to characterise spatial distribution and enantiomeric composition of usnic acids in four Icelandic lichens.

Usnic acid is an antibiotic metabolite produced by a wide variety of lichenized fungal lineages. The enantiomers of usnic acid have been shown to display contrasting bioactivities, and hence it is important to determine their spatial distribution, amounts and enantiomeric ratios in lichens to understand their roles in nature and grasp their pharmaceutical potential. The overall aim of the study was to characterise the spatial distribution of the predominant usnic acid enantiomer in lichens by combining spatial imaging and chiral chromatography. Specifically, separation and quantification of usnic acid enantiomers in four common lichens in Iceland was performed using a validated chiral chromatographic method. Molecular dynamics simulation was carried out to rationalize the chiral separation mechanism. Spatial distribution of usnic acid in the lichen thallus cross-sections were analysed using Desorption Electrospray Ionization-Imaging Mass Spectrometry (DESI-IMS) and fluorescence microscopy. DESI-IMS confirmed usnic acid as a cortical compound, and revealed that usnic acid can be more concentrated around the algal vicinity. Fluorescence microscopy complemented DESI-IMS by providing more detailed distribution information. By combining results from spatial imaging and chiral separation, we were able to visualize the distribution of the predominant usnic acid enantiomer in lichen cross-sections: (+)-usnic acid in Cladonia arbuscula and Ramalina siliquosa, and (-)-usnic acid in Alectoria ochroleuca and Flavocetraria nivalis. This study provides an analytical foundation for future environmental and functional studies of usnic acid enantiomers in lichens.

Noble Metal Nanoparticle Biosensors: From Fundamental Studies toward Point-of-Care Diagnostics.

Noble metal nanoparticles (NMNPs) have become firmly established as effective agents to detect various biomolecules with extremely high sensitivity. This ability stems from the collective oscillation of free electrons and extremely large electric field enhancement under exposure to light, leading to various light-matter interactions such as localized surface plasmon resonance (LSPR) and surface-enhanced Raman scattering. A remarkable feature of NMNPs is their customizability by mechanisms such as particle etching, growth, and aggregation/dispersion, yielding distinct color changes and excellent opportunities for colorimetric biosensing in user-friendly assays and devices. They are readily functionalized with a large variety of capping agents and biomolecules, with resultant bioconjugates often possessing excellent biocompatibility, which can be used to quantitatively detect analytes from physiological fluids. Furthermore, they can possess excellent catalytic properties that can achieve significant signal amplification through mechanisms such as the catalytic transformation of colorless substrates to colored reporters. The various excellent attributes of NMNP biosensors have put them in the spotlight for developing high-performance in vitro diagnostic (IVD) devices that are particularly well-suited to mitigate the societal threat that infectious diseases pose. This threat continues to dominate the global health care landscape, claiming millions of lives annually. NMNP IVDs possess the potential to sensitively detect infections even at very early stages with affordable and field-deployable devices, which will be key to strengthening infectious disease management. This has been the major focal point of current research, with a view to new avenues for early multiplexed detection of infectious diseases with portable devices such as smartphones, especially in resource-limited settings.In this Account, we provide an overview of our original inspiration and efforts in NMNP-based assay development, together with some more sophisticated IVD assays by ourselves and many others. Our work in the area has led to our recent efforts in developing IVDs for high-profile infectious diseases, including Ebola and HIV. We emphasize that integration with digital platforms represents an opportunity to establish and efficiently manage widespread testing, tracking, epidemiological intelligence, and data sharing backed by community participation. We highlight how digital technologies can address the limitations of conventional diagnostic technologies at the point of care (POC) and how they may be used to abate and contain the spread of infectious diseases. Finally, we focus on more recent integrations of noble metal nanoparticles with Raman spectroscopy for accurate, noninvasive POC diagnostics with improved sensitivity and specificity.

Single Particle Automated Raman Trapping Analysis of Breast Cancer Cell-Derived Extracellular Vesicles as Cancer Biomarkers.

Extracellular vesicles (EVs) secreted by cancer cells provide an important insight into cancer biology and could be leveraged to enhance diagnostics and disease monitoring. This paper details a high-throughput label-free extracellular vesicle analysis approach to study fundamental EV biology, toward diagnosis and monitoring of cancer in a minimally invasive manner and with the elimination of interpreter bias. We present the next generation of our single particle automated Raman trapping analysis─SPARTA─system through the development of a dedicated standalone device optimized for single particle analysis of EVs. Our visualization approach, dubbed dimensional reduction analysis (DRA), presents a convenient and comprehensive method of comparing multiple EV spectra. We demonstrate that the dedicated SPARTA system can differentiate between cancer and noncancer EVs with a high degree of sensitivity and specificity (>95% for both). We further show that the predictive ability of our approach is consistent across multiple EV isolations from the same cell types. Detailed modeling reveals accurate classification between EVs derived from various closely related breast cancer subtypes, further supporting the utility of our SPARTA-based approach for detailed EV profiling.

A Nickel(II)-Mediated Thiocarbonylation Strategy for Carbon Isotope Labeling of Aliphatic Carboxamides.

A series of pharmaceutically relevant small molecules and biopharmaceuticals bearing aliphatic carboxamides have been successfully labeled with carbon-13. Key to the success of this novel carbon isotope labeling technique is the observation that 13 C-labeled NiII -acyl complexes, formed from a 13 CO insertion step with NiII -alkyl intermediates, rapidly react in less than one minute with 2,2'-dipyridyl disulfide to quantitatively form the corresponding 2-pyridyl thioesters. Either the use of 13 C-SilaCOgen or 13 C-COgen allows for the stoichiometric addition of isotopically labeled carbon monoxide. Subsequent one-pot acylation of a series of structurally diverse amines provides the desired 13 C-labeled carboxamides in good yields. A single electron transfer pathway is proposed between the NiII -acyl complexes and the disulfide providing a reactive NiIII -acyl sulfide intermediate, which rapidly undergoes reductive elimination to the desired thioester. By further optimization of the reaction parameters, reaction times down to only 11 min were identified, opening up the possibility of exploring this chemistry for carbon-11 isotope labeling. Finally, this isotope labeling strategy could be adapted to the synthesis of 13 C-labeled liraglutide and insulin degludec, representing two antidiabetic drugs.

Clinically-suspected cast nephropathy: A retrospective, national, real-world study.

Presentation with severe acute kidney injury due to cast nephropathy (CN) is a medical emergency in multiple myeloma (MM), with high risk of dialysis-dependent renal failure and death. Accrual of patients with CN into interventional studies is difficult, while phase III trials exclude patients with severe renal insufficiency. Real-world data are warranted. We assessed 2252 patients from the population-based Danish Multiple Myeloma Registry (DMMR) who were diagnosed between 2013 and 2017. We identified 204 patients with clinically-suspected CN, defined as serum creatinine concentration >177 µmol/L and serum free light chain (sFLC) concentration >1000 mg/L at the time of diagnosis. The median age was 72 years. Thirty-one percent of patients presented with dialysis-dependent renal failure. Kidney biopsies were performed in 19% of patients and showed CN in 74% of cases. Despite prompt initiation of bortezomib-based therapy in 94% of patients, 33% of patients died in the first year after diagnosis. Compared with the rest of the patients in the DMMR with symptomatic MM, patients with clinically-suspected CN had worse overall survival (OS) irrespective of transplant eligibility. Achievement of renal recovery was associated with deep reductions of involved sFLC. Achievement of very good partial response or better in the first line of therapy and/or deep reduction of involved sFLC at 3 months after initiation of therapy were associated with superior OS. In conclusion, MM patients presenting with clinically-suspected CN have an alarmingly high one-year mortality when treated with current standards of care. Early and deep hematologic response is crucial for survival.

Direct Access to Isotopically Labeled Aliphatic Ketones Mediated by Nickel(I) Activation.

An extensive range of functionalized aliphatic ketones with good functional-group tolerance has been prepared by a NiI -promoted coupling of either primary or secondary alkyl iodides with NN2 pincer NiII -acyl complexes. The latter were easily accessed from the corresponding NiII -alkyl complexes with stoichiometric CO. This Ni-mediated carbonylative coupling is adaptable to late-stage carbon isotope labeling, as illustrated by the preparation of isotopically labelled pharmaceuticals. Preliminary investigations suggest the intermediacy of carbon-centered radicals.

Label free noninvasive spatially resolved NaCl concentration measurements using Coherent Anti-Stokes Raman Scattering microscopy applied to butter.

Imaging the microstructure of opaque composite foodstuffs and extracting quantitative chemical information about specific localized components is challenging. Herein, a method has been developed to determine spatially resolved concentrations of aqueous salt and applied to measure salt concentrations of water droplets in butter samples. This was done using Coherent Anti-Stokes Raman Scattering (CARS) microscopy which achieves non-invasive label free imaging based on visualization of specific chemical-bond vibrations. The concentration of salt in the dispersed water droplets in butter was determined based on the relative change in intensity of the CARS-signal at two distinct wavenumbers, which have been shown to be dependent on the inter-molecular coupling of water molecules and salt. The results provide the size and salt concentration distribution of the droplets in the samples. It is further shown that the average salt concentration in the whole sample can correctly be inferred from the concentration measured within the water droplets.

Multi-Phase Flow Metering in Offshore Oil and Gas Transportation Pipelines: Trends and Perspectives.

Multi-phase flow meters are of huge importance to the offshore oil and gas industry. Unreliable measurements can lead to many disadvantages and even wrong decision-making. It is especially important for mature reservoirs as the gas volume fraction and water cut is increasing during the lifetime of a well. Hence, it is essential to accurately monitor the multi-phase flow of oil, water and gas inside the transportation pipelines. The objective of this review paper is to present the current trends and technologies within multi-phase flow measurements and to introduce the most promising methods based on parameters such as accuracy, footprint, safety, maintenance and calibration. Typical meters, such as tomography, gamma densitometry and virtual flow meters are described and compared based on their performance with respect to multi-phase flow measurements. Both experimental prototypes and commercial solutions are presented and evaluated. For a non-intrusive, non-invasive and inexpensive meter solution, this review paper predicts a progress for virtual flow meters in the near future. The application of multi-phase flows meters are expected to further expand in the future as fields are maturing, thus, efficient utilization of existing fields are in focus, to decide if a field is still financially profitable.

Nonfasting Mild-to-Moderate Hypertriglyceridemia and Risk of Acute Pancreatitis.

IMPORTANCE: Severe hypertriglyceridemia is associated with increased risk of acute pancreatitis. However, the threshold above which triglycerides are associated with acute pancreatitis is unclear. OBJECTIVE: To test the hypothesis that nonfasting mild-to-moderate hypertriglyceridemia (177-885 mg/dL; 2-10 mmol/L) is also associated with acute pancreatitis. DESIGN, SETTING, AND PARTICIPANTS: This prospective cohort study examines individuals from the Copenhagen General Population Study in 2003 to 2015 and the Copenhagen City Heart Study initiated in 1976 to 1978 with follow-up examinations in 1981 to1983, 1991 to 1994, and in 2001 to 2003. Median follow-up was 6.7 years (interquartile range, 4.0-9.4 years); and includes 116 550 individuals with a triglyceride measurement from the Copenhagen General Population Study (n = 98 649) and the Copenhagen City Heart Study (n = 17 901). All individuals were followed until the occurrence of an event, death, emigration, or end of follow-up (November 2014), whichever came first. EXPOSURES: Plasma levels of nonfasting triglycerides. MAIN OUTCOMES AND MEASURES: Hazard ratios (HRs) for acute pancreatitis (n = 434) and myocardial infarction (n = 3942). RESULTS: Overall, 116 550 individuals were included in this study (median [interquartile range] age, 57 [47-66] years). Compared with individuals with plasma triglyceride levels less than 89 mg/dL (<1 mmol/L), the multivariable adjusted HRs for acute pancreatitis were 1.6 (95% CI, 1.0-2.6; 4.3 events/10 000 person-years) for individuals with triglyceride levels of 89 mg/dL to 176 mg/dL (1.00 mmol/L-1.99 mmol/L), 2.3 (95% CI, 1.3-4.0; 5.5 events/10 000 person-years) for 177 mg/dL to 265 mg/dL (2.00 mmol/L-2.99 mmol/L), 2.9 (95% CI, 1.4-5.9; 6.3 events/10 000 person-years) for 366 mg/dL to 353 mg/dL (3.00 mmol/L-3.99 mmol/L), 3.9 (95% CI, 1.5-10.0; 7.5 events/10 000 person-years) for 354 mg/dL-442 mg/dL (4.00 mmol/L-4.99 mmol/L), and 8.7 (95% CI, 3.7-20.0; 12 events/10 000 person-years) for individuals with triglyceride levels greater than or equal to 443 mg/dL (≥5.00 mmol/L) (trend, P = 6 × 10-8). Corresponding HRs for myocardial infarction were 1.6 (95% CI, 1.4-1.9; 41 events/10 000 person-years), 2.2 (95% CI, 1.9-2.7; 57 events/10 000 person-years), 3.2 (95% CI, 2.6-4.1; 72 events/10 000 person-years), 2.8 (95% CI, 2.0-3.9; 68 events/10 000 person-years), and 3.4 (95% CI, 2.4-4.7; 78 events/10 000 person-years) (trend, P = 6 × 10-31), respectively. The multivariable adjusted HR for acute pancreatitis was 1.17 (95% CI, 1.10-1.24) per 89 mg/dL (1 mmol/L) higher triglycerides. When stratified by sex, age, education, smoking, hypertension, statin use, study cohort, diabetes, body mass index (calculated as weight in kilograms divided by height in meters squared), alcohol intake, and gallstone disease, these results were similar with no statistical evidence of interaction. CONCLUSIONS AND RELEVANCE: Nonfasting mild-to-moderate hypertriglyceridemia from 177 mg/dL (2 mmol/L) and above is associated with high risk of acute pancreatitis, with HR estimates higher than for myocardial infarction.

The vasodilating effects of insulin and lactate are increased in precapillary arterioles in the porcine retina ex vivo.

PURPOSE: Disturbances in the diameter regulation of the larger retinal vessels are involved in the pathophysiology of a number of vision threatening retinal diseases, but the role of the smaller vessels has received less attention. Therefore, an experimental model was developed for studying diameter changes in retinal vessels at all branching levels secondary to pharmacological interventions ex vivo. METHODS: Porcine hemiretinas were mounted in a tissue chamber that allowed the control of temperature, pH and oxygen saturation. The chamber was positioned in a fluorescence microscope, and changes in the diameter of larger arterioles, precapillary arterioles and capillaries were studied after intravascular and extravascular application of the thromboxane analogue U46619, lactate, the glutamate agonist N-Methyl-D-aspartic acid (NMDA) and insulin. RESULTS: U46619 induced significant contraction of all vessel calibres after extravascular application, but had no significant effect on vessel diameters after intravascular application. In contracted vessels, lactate induced significant dilatation in all vessel calibres, and the effect was more pronounced after intravascular than after extravascular application where only precapillary arterioles dilated significantly. N-Methyl-D-aspartic acid (NMDA) induced similar dilatation in the three vessel types after intra- and extravascular application, whereas the dilating effect of insulin was significantly more pronounced in the precapillary arterioles than in the other vessel types. CONCLUSION: The diameter regulation in precapillary retinal arterioles differs from that of other retinal vascular branching levels and may play a special role in the pathophysiology of retinal vascular disease.