Visualizing ribonuclease digestion of RNA-like polymers produced by hot wet-dry cycles.

Polymerization of nucleotides under prebiotic conditions simulating the early Earth has been extensively studied. Several independent methods have been used to verify that RNA-like polymers can be produced by hot wet-dry cycling of nucleotides. However, it has not been shown that these RNA-like polymers are similar to biological RNA with 3'-5' phosphodiester bonds. In the results described here, RNA-like polymers were generated from 5'-monophosphate nucleosides AMP and UMP. To confirm that the polymers resemble biological RNA, ribonuclease A should catalyze hydrolysis of the 3'-5' phosphodiester bonds between pyrimidine nucleotides to each other or to purine nucleotides, but not purine-purine nucleotide bonds. Here we show AFM images of specific polymers produced by hot wet-dry cycling of AMP, UMP and AMP/UMP (1:1) solutions on mica surfaces, before and after exposure to ribonuclease A. AMP polymers were unaffected by ribonuclease A but UMP polymers disappeared. This indicates that a major fraction of the bonds in the UMP polymers is indeed 3'-5' phosphodiester bonds. Some of the polymers generated from the AMP/UMP mixture also showed clear signs of cleavage. Because ribonuclease A recognizes the ester bonds in the polymers, we show for the first time that these prebiotically produced polymers are in fact similar to biological RNA but are likely to be linked by a mixture of 3'-5' and 2'-5' phosphodiester bonds.

Cascade autohydrolysis of Alzheimer's Aß peptides.

Protein/peptide self-assembly into amyloid structures associates with major neurodegenerative disorders such as Alzheimer's disease (AD). Soluble assemblies (oligomers) of the Aß peptide and their aggregates are perceived as neurotoxic species in AD. While screening for synthetic cleavage agents that could break down such aberrant assemblies through hydrolysis, we observed that the assemblies of Aß oligopeptides, containing the nucleation sequence Aß14-24 (H14QKLVFFAEDV24), could act as cleavage agents by themselves. Autohydrolysis showed a common fragment fingerprint among various mutated Aß14-24 oligopeptides, Aß12-25-Gly and Aß1-28, and full-length Aß1-40/42, under physiologically relevant conditions. Primary endoproteolytic autocleavage at the Gln15-Lys16, Lys16-Leu17 and Phe19-Phe20 positions was followed by subsequent exopeptidase self-processing of the fragments. Control experiments with homologous d-amino acid enantiomers Aß12-25-Gly and Aß16-25-Gly showed the same autocleavage pattern under similar reaction conditions. The autohydrolytic cascade reaction (ACR) was resilient to a broad range of conditions (20-37 °C, 10-150 µM peptide concentration at pH 7.0-7.8). Evidently, assemblies of the primary autocleavage fragments acted as structural/compositional templates (autocatalysts) for self-propagating autohydrolytic processing at the Aß16-21 nucleation site, showing the potential for cross-catalytic seeding of the ACR in larger Aß isoforms (Aß1-28 and Aß1-40/42). This result may shed new light on Aß behaviour in solution and might be useful in the development of intervention strategies to decompose or inhibit neurotoxic Aß assemblies in AD.

Visualizing RNA polymers produced by hot wet-dry cycling.

It is possible that the transition from abiotic systems to life relied on RNA polymers that served as ribozyme-like catalysts and for storing genetic information. The source of such polymers is uncertain, but previous investigations reported that wet-dry cycles simulating prebiotic hot springs provide sufficient energy to drive condensation reactions of mononucleotides to form oligomers and polymers. The aim of the study reported here was to verify this claim and visualize the products prepared from solutions composed of single mononucleotides and 1:1 mixture of two mononucleotides. Therefore, we designed experiments that allowed comparisons of all such mixtures representing six combinations of the four mononucleotides of RNA. We observed irregular stringy patches and crystal strands when wet-dry cycling was performed at room temperature (20 °C). However, when the same solutions were exposed to wet-dry cycles at 80 °C, we observed what appeared to be true polymers. Their thickness was consistent with RNA-like products composed of covalently bonded monomers, while irregular strings and crystal segments of mononucleotides dried or cycled at room temperature were consistent with structures assembled and stabilized by weak hydrogen bonds. In a few instances we observed rings with short polymer attachments. These observations are consistent with previous claims of polymerization during wet-dry cycling. We conclude that RNA-like polymers and rings could have been synthesized non-enzymatically in freshwater hot springs on the prebiotic Earth with sizes sufficient to fold into ribozymes and genetic molecules required for life to begin.

Effect of pH on the Surface Layer of Molecular Crystals at the Solid-Liquid Interface.

Dissolution of solid matter into aqueous solution is one of the most challenging physicochemical aspects related to drug development. While influenced by several parameters, the effect of pH remains the most important one to be fully understood. The dissolution process is essentially controlled by activity at the surface of the molecular crystals, which is difficult to characterize experimentally. To address this, a combination of in situ atomic force microscopy (AFM) with molecular dynamics (MD) simulation is reported. AFM allows for direct visualization of the crystal surface of basic and acidic model compounds (carvedilol and ibuprofen) in contact with an aqueous medium with varying pH. A dramatic increase in surface mobility in the solid-liquid interface could be observed experimentally as a function of pH. The in situ AFM approach opens up for a more detailed understanding of the behavior of particulate matter in solution with importance at different levels, ranging from engineering aspects related to crystallization, and biological considerations related to bioavailability of the final drug product.

Controlling the fractal dimension in self-assembly of terpyridine modified insulin by Fe2+ and Eu3+ to direct in vivo effects.

Metal ion-induced self-assembly (SA) of proteins into higher-order structures can provide new, dynamic nano-assemblies. Here, the synthesis and characterization of a human insulin (HI) analog modified at LysB29 with the tridentate chelator 2,2':6',2''-terpyridine (Tpy) is described. SA of this new insulin analog (LysB29Tpy-HI) in the presence of the metal ions Fe2+ and Eu3+ at different concentrations was studied in solution by fluorescence luminescence and CD spectroscopy, dynamic light scattering, and small-angle X-ray scattering, while surface assembly was probed by AFM. Unique oligomerization was observed in solution, as Fe2+ yielded small magenta-colored discrete non-native assemblies, while Eu3+ caused the formation of large fractal assemblies. Binding of both metal ions to Tpy was demonstrated spectroscopically, and emission lifetime experiments revealed a distinct Eu3+ coordination geometry that included two water molecules. SAXS suggested that LysB29Tpy-HI with Fe2+ oligomerized to a discrete, roughly octameric species, while LysB29Tpy-HI with Eu3+ gave very large assemblies that could be modelled as fractals. The fractal dimensionality increased with the Eu3+ concentration. We propose that this is a consequence of Eu3+ binding to both Tpy and to free carboxylic acid groups on the insulin surface. LysB29Tpy-HI maintained insulin receptor affinity, and showed extended blood glucose lowering and plasma concentration after subcutaneous injection in rats. The combination of metal ion directed SA and native SA provides control of nano-scale fractal dimensionality and points towards use in therapeutics.

Direct evidence for eoarchean iron metabolism?

Metasedimentary rocks from Isua, West Greenland (> 3,700 million years old) contain carbonaceous compounds, compatible with a biogenic origin (Hassenkam, Andersson, Dalby, Mackenzie, & Rosing, 2017; Ohtomo, Kakegawa, Ishida, Nagase, & Rosing, 2014; Rosing, 1999). The metamorphic mineral assemblage with garnet and quartz intergrowths contains layers of carbonaceous inclusions contiguous with carbon-rich sedimentary beds in the host rock. Previous studies (Hassenkam et al., 2017; Ohtomo et al., 2014; Rosing, 1999) on Isua rocks focused on testing the biogenic origin of the carbonaceous material, but here we searched for evidence which could provide new insights into the nature of the life that generated this carbonaceous material. We studied material trapped in inclusions armoured within quartz grains inside garnet porphyroblasts by non-destructive ptychographic X-ray nanotomography (PXCT). The 3D electron density maps generated by PXCT were correlated with maps from X-ray fluorescence tomography and micro-Raman spectroscopy. We found that the material trapped inside inclusions in the quartz grains consist of disordered carbon material encasing domains of iron-rich carbonaceous material. These results corroborate earlier claims (Hassenkam et al., 2017; Ohtomo et al., 2014; Rosing, 1999) for biogenic origins and are compatible with relics of metamorphosed biological material originally containing high iron/carbon ratios, comparable to ratios found in most extant organisms. These iron-rich domains represent the oldest evidence for organic iron complexes in the geologic record and are consistent with Fe-isotopic evidence for metabolic iron fractionation in > 3,700 Ma Isua banded iron formation (Czaja et al., 2013; Whitehouse & Fedo, 2007).

AFM Images of Viroid-Sized Rings That Self-Assemble from Mononucleotides through Wet-Dry Cycling: Implications for the Origin of Life.

It is possible that early life relied on RNA polymers that served as ribozyme-like catalysts and for storing genetic information. The source of such polymers is uncertain, but previous investigations reported that wet-dry cycles simulating prebiotic hot springs provide sufficient energy to drive condensation reactions of mononucleotides to form oligomers. The aim of the study reported here was to visualize the products by atomic force microscopy. In addition to globular oligomers, ring-like structures ranging from 10-200 nm in diameter, with an average around 30-40 nm, were abundant, particularly when nucleotides capable of base pairing were present. The thickness of the rings was consistent with single stranded products, but some had thicknesses indicating base pair stacking. Others had more complex structures in the form of short polymer attachments and pairing of rings. These observations suggest the possibility that base-pairing may promote polymerization during wet-dry cycling followed by solvation of the rings. We conclude that RNA-like rings and structures could have been synthesized non-enzymatically on the prebiotic Earth, with sizes sufficient to fold into ribozymes and genetic molecules required for life to begin.

Probing the Secondary Structure of Individual Aß40 Amorphous Aggregates and Fibrils by AFM-IR Spectroscopy.

Structural characterization of aggregates and fibrils of the Aß protein is pivotal to the molecular-level elucidation of Alzheimer's disease (AD). AFM-IR spectroscopy provides nanoscale resolution, and thus allows the interrogation of individual aggregates and fibrils. During aggregation of Aß, we observed mainly disordered Aß at t=15 min, but substantial structural diversity including the co-existence of parallel and antiparallel ß-sheets within a large amorphous aggregate at t=2 hours, while fibrils exhibited the expected signature of parallel ß-sheets at t=1 week. The resonance observed for parallel ß-sheets at t=2 hours coincides with that observed for fibrils (at 1634 cm-1 ), thus indicating that fibril-like species exist within the large aggregates. Therefore, nucleation might occur within such species, in analogy to current theories of protein crystallization in which nucleation occurs within large protein clusters. Cu2+ perturbs Aß aggregation, catalysing rapid formation of amorphous aggregates with diverse secondary structure, but inhibiting fibril growth.

A free-floating mucin layer to investigate the effect of the local microenvironment in lungs on mucin-nanoparticle interactions.

Respiratory tract mucus represents an important barrier for pulmonary drug delivery. Understanding of mucin-nanoparticle interactions is a prerequisite for rational design of inhalable nanoparticles. In the present study, in order to establish a reliable quartz crystal microbalance with dissipation (QCM-D) approach to reveal the effect of the lung microenvironment on the mucin-nanoparticle interactions, we investigated the intrinsic features of the mucin layers immobilized onto sensors via chemical conjugation or physical adsorption by using atomic force microscopy (AFM) and QCM-D. Our results demonstrated that the covalently-grafted mucin layer responded more sensitively than the physically-adsorbed mucin layer to the local microenvironment shifting from PBS (pH 7.35 and ionic strength 30 mM) to PBS (pH 6.25 and ionic strength 150 mM) and resulted in a softer mucin layer with more hydrophobic areas exposed. Furthermore, using the QCM-D approach with the covalently-grafted mucin layer, we demonstrated the significant influence of the local microenvironment on the interaction of mucin with poly (lactic-co-glycolic acid)-based nanoparticles with different surface hydrophilicity. The present work underlines the QCM-D approach with a covalently-grafted mucin layer as a potent tool to elucidate the potential influence of local microenvironment on mucin-nanoparticle interactions. STATEMENT OF SIGNIFICANCE: Studying interactions between nanoengineered materials and biological systems plays a vital role in development of biomedical applications of nanoengineered materials. In this work, by employing a more biologically relevant, 'free-floating' mucin layer model, we demonstrate the significant impact of the lung microenvironment on the nature and the extent of the interaction between the mucin and the nanoparticles with different surface hydrophilicity. To the best of our knowledge, this is the first work describing the nanoscale properties of immobilized mucin layers and investigating the mucin-nanoparticle interactions with emphasis on the impact of local microenvironment in lungs. Thus, it is expected to have important consequences in rational design of inhalable nanoparticle delivery systems.

Nanoscale chemical mapping of oxygen functional groups on graphene oxide using atomic force microscopy-coupled infrared spectroscopy.

The unambiguous determination of the chemical functionality over graphene oxide (GO) is important to unleash its potential applications. However, the mapping of oxygen functionalities distribution remains to be unequivocally determined because of highly inhomogeneous non-stoichiometric structures and ultra-thin layers of GO. In this study, we report an experimental observation of the spatial distribution of oxygen functional groups on monolayer and multilayer GO using AFM-IR, atomic force microscopy coupled with infrared spectroscopy. Overcoming conventional IR diffraction limit for several micrometers, the novel AFM-IR reaches high spatial resolution ∼20 nm and could detect IR absorption on ∼1 nm thickness of monolayer GO. With nanoscale chemical mapping, the distribution of different oxygen functional groups is distinguished with AFM-IR over the GO surface. It allows us to observe that these oxygen functional groups prefer to sit on the fold areas, in discrete domains and on the edges of GO, which gave more insights into its chemical nature. The determination of the position of functional groups through precise imaging contributes to our understanding of GO structure-properties relations and paves the way for targeted tethering of polymers, biomaterials, and other nanostructures.

PICH and TOP3A cooperate to induce positive DNA supercoiling.

All known eukaryotic topoisomerases are only able to relieve torsional stress in DNA. Nevertheless, it has been proposed that the introduction of positive DNA supercoiling is required for efficient sister-chromatid disjunction by Topoisomerase 2a during mitosis. Here we identify a eukaryotic enzymatic activity that introduces torsional stress into DNA. We show that the human Plk1-interacting checkpoint helicase (PICH) and Topoisomerase 3a proteins combine to create an extraordinarily high density of positive DNA supercoiling. This activity, which is analogous to that of a reverse-gyrase, is apparently driven by the ability of PICH to progressively extrude hypernegatively supercoiled DNA loops that are relaxed by Topoisomerase 3a. We propose that this positive supercoiling provides an optimal substrate for the rapid disjunction of sister centromeres by Topoisomerase 2a at the onset of anaphase in eukaryotic cells.

Molecular multifunctionality preservation upon surface deposition for a chiral single-molecule magnet.

The synthesis and characterization of a chiral, enneanuclear Mn(iii)-based, Single-Molecule Magnet, [Mn9O4(Me-sao)6(L)3(MeO)3(MeOH)3]Cl (1; Me-saoH2 = methylsalicylaldoxime, HL = lipoic acid) is reported. Compound 1 crystallizes in the orthorhombic P212121 space group and consists of a metallic skeleton describing a defect super-tetrahedron missing one vertex. The chirality of the [MnIII 9] core originates from the directional bridging of the Me-sao2- ligands via the -N-O- oximate moieties, which define a clockwise (1ΔΔ) or counter-clockwise (1ΛΛ) rotation in both the upper [MnIII 3] and lower [MnIII 6] subunits. Structural integrity and retention of chirality upon dissolution and upon deposition on (a) gold nanoparticles, 1@AuNPs, (b) transparent Au(111) surfaces, 1ΛΛ@t-Au(111); 1ΔΔ@t-Au(111), and (c) epitaxial Au(111) on mica surfaces, 1@e-Au(111), was confirmed by CD and IR spectroscopies, mass spectrometry, TEM, XPS, XAS, and AFM. Magnetic susceptibility and magnetization measurements demonstrate the simultaneous retention of SMM behaviour and optical activity, from the solid state, via dissolution, to the surface deposited species.

Isoenergic modification of whey protein structure by denaturation and crosslinking using transglutaminase.

Transglutaminase (TG) catalyzes formation of covalent bonds between lysine and glutamine side chains and has applications in manipulation of food structure. Physical properties of a whey protein mixture (SPC) denatured either at elevated pH or by heat-treatment and followed by TG catalyzed crosslinking, have been characterised using dynamic light scattering, size exclusion chromatography, flourescence spectroscopy and atomic force microscopy. The degree of enzymatic crosslinking appeared higher for pH- than for heat-denatured SPC. The hydrophobic surface properties depended on the treatment, thus heating caused the largest exposure of the hydrophobic core of SPC proteins, which was decreased by crosslinking. The particle size of the treated SPC samples increased upon crosslinking by TG. Moreover, the particle morphology depended on the type of denaturing treatment, thus heat-treated SPC contained fibrillar structures, while pH-denatured SPC remained globular as documented by using atomic force microscopy. Finally, the in vitro digestability of the different SPC samples was assessed under simulated gastric and intestinal conditions. Notably heat-treatment was found to lower the gastric digestion rate and enzymatic crosslinking reduced both the gastric and the intestinal rate of digestion. These characteristics of the various SPC samples provide a useful basis for design of isoenergic model foods applicable in animal and human studies on how food structure affects satiety.

Plasmodium falciparum-infected erythrocyte knob density is linked to the PfEMP1 variant expressed.

UNLABELLED: Members of the clonally variant Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) family mediate adhesion of infected erythrocytes (IEs) to vascular receptors. PfEMP1 expression is normally confined to nanoscale knob protrusions on the IE surface membrane. To investigate the relationship between the densities of these IE surface knobs and the PfEMP1 variant expressed, we used specific antibody panning to generate three sublines of the P. falciparum clone IT4, which expresses the PfEMP1 variants IT4VAR04, IT4VAR32b, and IT4VAR60. The knob density in each subline was then determined by atomic force microscopy (AFM) and scanning electron microscopy (SEM) and compared to PfEMP1 and knob-associated histidine-rich protein (KAHRP) expression. Selection for uniform expression of IT4VAR04 produced little change in knob density, compared to unselected IEs. In contrast, selection for IT4VAR32b expression increased knob density approximately 3-fold, whereas IEs selected for IT4VAR60 expression were essentially knobless. When IT4VAR60(+) IEs were subsequently selected to express IT4VAR04 or IT4VAR32b, they again displayed low and high knob densities, respectively. All sublines expressed KAHRP regardless of the PfEMP1 expressed. Our study documents for the first time that knob density is related to the PfEMP1 variant expressed. This may reflect topological requirements to ensure optimal adhesive properties of the IEs. IMPORTANCE: Infections with Plasmodium falciparum malaria parasites are still responsible for many deaths, especially among children and pregnant women. New interventions are needed to reduce severe illness and deaths caused by this malaria parasite. Thus, a better understanding of the mechanisms behind the pathogenesis is essential. A main reason why Plasmodium falciparum malaria is more severe than disease caused by other malaria species is its ability to express variant antigens on the infected erythrocyte surface. These antigens are presented on membrane protrusions known as knobs. This study set out to investigate the interplay between different variant antigens on the surface of P. falciparum-infected erythrocytes and the density of the knobs on which the antigens are expressed. Such a direct analysis of this relationship has not been reported before but adds to the important understanding of the complexity of malaria antigen presentation.

Self-assembly and near perfect macroscopic alignment of fluorescent triangulenium salt in spin-cast thin films on PTFE.

Highly fluorescent, discotic trioxatriangulenium dyes were aligned by simple spin-casting on substrates with friction transferred PTFE layers. The fluorescent crystalline thin films show near perfect macroscopic alignment on centimeter large areas directly from spin-casting. Gracing Incidence X-ray Diffraction (GIXD) unambiguously allowed the determination of a long-range order unit cell as well as its orientation with respect to the PTFE fibers. Further analysis of the X-ray data, in conjunction with polarized absorption spectroscopy, suggest a lamellar packing model with alternating layers of alkyl chains and ionic dyes oriented parallel to the substrate. This structure results in a highly anisotropic electrostatic potential around the cationic chromophore, causing significant shifts in energy and orientation of the optical transitions. Thus, the optical properties of the material are, to a large extent, controlled by the position of the otherwise inert PF6(-) counterions. The bright fluorescence from the films is also polarized parallel to the PTFE alignment layer. Doping of the thin films with fluorescent energy acceptor traps shows that efficient exciton migration takes place in the thin films. The excellent exciton transfer capabilities, in conjunction with the perfect alignment, might be of interest in future applications in solar energy harvesting or as thin film sensors.

The density of knobs on Plasmodium falciparum-infected erythrocytes depends on developmental age and varies among isolates.

BACKGROUND: The virulence of Plasmodium falciparum malaria is related to the parasite's ability to evade host immunity through clonal antigenic variation and tissue-specific adhesion of infected erythrocytes (IEs). The P. falciparum erythrocyte membrane protein 1 (PfEMP1) family expressed on dome-shaped protrusions called knobs on the IE surface is central to both. Differences in receptor specificity and affinity of expressed PfEMP1 are important for IE adhesiveness, but it is not known whether differences in the number and size of the knobs on which the PfEMP1 proteins are expressed also play a role. Therefore, the aim of this study was to provide detailed information on isolate- and time-dependent differences in knob size and density. METHODOLOGY/PRINCIPAL FINDINGS: We used atomic force microscopy to characterize knobs on the surface of P. falciparum-infected erythrocytes. Fourteen ex vivo isolates from Ghanaian children with malaria and 10 P. falciparum isolates selected in vitro for expression of a particular PfEMP1 protein (VAR2CSA) were examined. Knob density increased from ∼20 h to ∼35 h post-invasion, with significant variation among isolates. The knob density ex vivo, which was about five-fold higher than following long-term in vitro culture, started to decline within a few months of culture. Although knob diameter and height varied among isolates, we did not observe significant time-dependent variation in these dimensions. CONCLUSIONS/SIGNIFICANCE: The density of knobs on the P. falciparum-IE surface depends on time since invasion, but is also determined by the infecting isolate in a time-independent manner. This is the first study to quantitatively evaluate knob densities and dimensions on different P. falciparum isolates, to examine ex vivo isolates from humans, and to compare ex vivo and long-term in vitro-cultured isolates. Our findings contribute to the understanding of the interaction between P. falciparum parasites and the infected host.

A triptycene-based approach to solubilising carbon nanotubes and C60.

We describe herein the synthesis of a triptycene-based surfactant designed with the ability to solubilise single-walled carbon nanotubes (SWNTs) and C(60) in water through non-covalent interactions. Furthermore, an amphiphilic naphthalene-based surfactant with the same ability to solubilise SWNTs and C(60) has also been prepared. The compounds synthesised were designed with either two ionic or non-ionic tails to ensure a large number of supramolecular interactions with the solvent, thereby promoting strong solubilisation. The surfactants produced stable suspensions in which the SWNTs are dispersed and the surfactant/SWNT complexes formed are stable for more than one year. UV/Vis/NIR absorption spectroscopy, TEM and AFM were employed to probe the solubilisation properties of the dispersion of surfactants and SWNTs in water.

Mechanical properties of human patellar tendon at the hierarchical levels of tendon and fibril.

Tendons are strong hierarchical structures, but how tensile forces are transmitted between different levels remains incompletely understood. Collagen fibrils are thought to be primary determinants of whole tendon properties, and therefore we hypothesized that the whole human patellar tendon and its distinct collagen fibrils would display similar mechanical properties. Human patellar tendons (n = 5) were mechanically tested in vivo by ultrasonography. Biopsies were obtained from each tendon, and individual collagen fibrils were dissected and tested mechanically by atomic force microscopy. The Young's modulus was 2.0 ± 0.5 GPa, and the toe region reached 3.3 ± 1.9% strain in whole patellar tendons. Based on dry cross-sectional area, the Young's modulus of isolated collagen fibrils was 2.8 ± 0.3 GPa, and the toe region reached 0.86 ± 0.08% strain. The measured fibril modulus was insufficient to account for the modulus of the tendon in vivo when fibril content in the tendon was accounted for. Thus, our original hypothesis was not supported, although the in vitro fibril modulus corresponded well with reported in vitro tendon values. This correspondence together with the fibril modulus not being greater than that of tendon supports that fibrillar rather than interfibrillar properties govern the subfailure tendon response, making the fibrillar level a meaningful target of intervention. The lower modulus found in vitro suggests a possible adverse effect of removing the tissue from its natural environment. In addition to the primary work comparing the two hierarchical levels, we also verified the existence of viscoelastic behavior in isolated human collagen fibrils.

Tensile force transmission in human patellar tendon fascicles is not mediated by glycosaminoglycans.

Correct mechanical function of tendons is essential to human physiology and therefore the mechanical properties of tendon have been a subject of research for many decades now. However, one of the most fundamental questions remains unanswered: How is load transmitted through the tendon? It has been suggested that the proteoglycan-associated glycosaminoglycans (GAGs) found on the surface of the collagen fibrils may be an important transmitter of load, but existing results are ambiguous and have not investigated human tendons. We have used a small-scale mechanical testing system to measure the mechanical properties of fascicles from human patellar tendon at two different deformation rates before and after removal of GAGs by treatment with chondroitinase ABC. Efficiency of enzyme treatment was quantified using dimethylmethylene blue assay. Removal of at least 79% of the GAGs did not significantly change the tendon modulus, relative energy dissipation, peak stress, or peak strain. The effect of deformation rate was not modulated by the treatment either, indicating no effect on viscosity. These results suggest that GAGs cannot be considered mediators of tensile force transmission in the human patellar tendon, and as such, force transmission must either take place through other matrix components or the fibrils must be mechanically continuous at least to the tested length of 7 mm.