Synthesis and VCD Spectroscopic Characterization of a Series of Azacryptands from a Chiral Valine-Based Derivative of Tris(2-aminoethyl)amine (TREN).

Utilizing experimental and computational vibrational circular dichroism (VCD) spectroscopy, we explored the conformational preferences of a series of chiral C3 -symmetric octaazacryptands with tris(2-aminoethyl)-amine head groups derived from valine. While the spectra of the smallest azacryptand with p-phenyl linkers and its elongated derivative with p-biphenyls linker were found to match well with the computed spectra, the computed conformational preferences of the m-biphenyl-based azacryptand did not seem to reflect the conformations dominating in chloroform solution. A detailed analysis revealed that structural changes resulting in a collapsed cage structure gave a notably better match with the experiment. It could subsequently be concluded from the VCD analysis, that the octaazacryptands prefer a collapsed structure, which is not predicted by density functional theory (DFT) calculations as the global minimum structures. These findings are expected to have consequences also for future studies on inclusion complexes of such azacryptands.

Solvation of serine-based model peptides and the role of the intramolecular OH·O hydrogen bond in interpreting VCD spectra.

Infrared and vibrational circular dichroism (VCD) spectra are occasionally very sensitive to solute-solvent interactions and show distinct spectral changes when strong solute-solvent hydrogen bonds give rise to conformational changes. In this regard, small peptides are ideal model systems to investigate such solvent effects on IR and VCD spectra as they possess several hydrogen bonding donor sites. In the present study, we investigate serine and serine-phenylalanine, which both are N-protected with Boc and C-capped with n-propylamine. Compared to previously studied model peptides, the serine residue introduces a strong hydrogen bonding site that competes with the amides for intra- and intermolecular interactions. For both compounds, we computationally found that the intramolecular OH·O interactions are preferentially broken by DMSO, but it was not sufficient to model only this particular interaction. Instead, depending on the conformer family, it was necessary to consider different numbers of solvent molecules in the computed structures and the experimental spectra were found to be best described by assuming mixed solvation states. Our analyses show that the IR and VCD spectra of molecules with multiple hydrogen bonding cannot be simulated by simply solvating all donor sites as this would neglect the presence of important conformer families. In turn, these results stresss the need for novel routines to account for solvation in IR and VCD spectra, that help estimating the contributions of different solvations states to the conformational distribution.

Quantum Rifling: Protecting a Qubit from Measurement Back Action.

Quantum mechanics postulates that measuring the qubit's wave function results in its collapse, with the recorded discrete outcome designating the particular eigenstate that the qubit collapsed into. We show that this picture breaks down when the qubit is strongly driven during measurement. More specifically, for a fast evolving qubit the measurement returns the time-averaged expectation value of the measurement operator, erasing information about the initial state of the qubit while completely suppressing the measurement backaction. We call this regime quantum rifling, as the fast spinning of the Bloch vector protects it from deflection into either of its eigenstates. We study this phenomenon with two superconducting qubits coupled to the same probe field and demonstrate that quantum rifling allows us to measure either one of the qubits on demand while protecting the state of the other from measurement backaction. Our results allow for the implementation of selective readout multiplexing of several qubits, contributing to the efficient scaling up of quantum processors for future quantum technologies.

Towards understanding two-level-systems in amorphous solids: insights from quantum circuits.

Amorphous solids show surprisingly universal behaviour at low temperatures. The prevailing wisdom is that this can be explained by the existence of two-state defects within the material. The so-called standard tunneling model has become the established framework to explain these results, yet it still leaves the central question essentially unanswered-what are these two-level defects (TLS)? This question has recently taken on a new urgency with the rise of superconducting circuits in quantum computing, circuit quantum electrodynamics, magnetometry, electrometry and metrology. Superconducting circuits made from aluminium or niobium are fundamentally limited by losses due to TLS within the amorphous oxide layers encasing them. On the other hand, these circuits also provide a novel and effective method for studying the very defects which limit their operation. We can now go beyond ensemble measurements and probe individual defects-observing the quantum nature of their dynamics and studying their formation, their behaviour as a function of applied field, strain, temperature and other properties. This article reviews the plethora of recent experimental results in this area and discusses the various theoretical models which have been used to describe the observations. In doing so, it summarises the current approaches to solving this fundamentally important problem in solid-state physics.

Correlating Decoherence in Transmon Qubits: Low Frequency Noise by Single Fluctuators.

We report on long-term measurements of a highly coherent, nontunable superconducting transmon qubit, revealing low-frequency burst noise in coherence times and qubit transition frequency. We achieve this through a simultaneous measurement of the qubit's relaxation and dephasing rate as well as its resonance frequency. The analysis of correlations between these parameters yields information about the microscopic origin of the intrinsic decoherence mechanisms in Josephson qubits. Our results are consistent with a small number of microscopic two-level systems located at the edges of the superconducting film, which is further confirmed by a spectral noise analysis.

BRIP1 loss-of-function mutations confer high risk for familial ovarian cancer, but not familial breast cancer.

BACKGROUND: Germline mutations in the BRIP1 gene have been described as conferring a moderate risk for ovarian cancer (OC), while the role of BRIP1 in breast cancer (BC) pathogenesis remains controversial. METHODS: To assess the role of deleterious BRIP1 germline mutations in BC/OC predisposition, 6341 well-characterized index patients with BC, 706 index patients with OC, and 2189 geographically matched female controls were screened for loss-of-function (LoF) mutations and potentially damaging missense variants. All index patients met the inclusion criteria of the German Consortium for Hereditary Breast and Ovarian Cancer for germline testing and tested negative for pathogenic BRCA1/2 variants. RESULTS: BRIP1 LoF mutations confer a high OC risk in familial index patients (odds ratio (OR) = 20.97, 95% confidence interval (CI) = 12.02-36.57, P < 0.0001) and in the subgroup of index patients with late-onset OC (OR = 29.91, 95% CI = 14.99-59.66, P < 0.0001). No significant association of BRIP1 LoF mutations with familial BC was observed (OR = 1.81 95% CI = 1.00-3.30, P = 0.0623). In the subgroup of familial BC index patients without a family history of OC there was also no apparent association (OR = 1.42, 95% CI = 0.70-2.90, P = 0.3030). In 1027 familial BC index patients with a family history of OC, the BRIP1 mutation prevalence was significantly higher than that observed in controls (OR = 3.59, 95% CI = 1.43-9.01; P = 0.0168). Based on the negative association between BRIP1 LoF mutations and familial BC in the absence of an OC family history, we conclude that the elevated mutation prevalence in the latter cohort was driven by the occurrence of OC in these families. Compared with controls, predicted damaging rare missense variants were significantly more prevalent in OC (P = 0.0014) but not in BC (P = 0.0693) patients. CONCLUSIONS: To avoid ambiguous results, studies aimed at assessing the impact of candidate predisposition gene mutations on BC risk might differentiate between BC index patients with an OC family history and those without. In familial cases, we suggest that BRIP1 is a high-risk gene for late-onset OC but not a BC predisposition gene, though minor effects cannot be excluded.

Passive On-Chip Superconducting Circulator Using a Ring of Tunnel Junctions.

We present the design of a passive, on-chip microwave circulator based on a ring of superconducting tunnel junctions. We investigate two distinct physical realizations, based on Josephson junctions (JJs) or quantum phase slip elements (QPS), with microwave ports coupled either capacitively (JJ) or inductively (QPS) to the ring structure. A constant bias applied to the center of the ring provides an effective symmetry breaking field, and no microwave or rf bias is required. We show that this design offers high isolation, robustness against fabrication imperfections and bias fluctuations, and a bandwidth in excess of 500 MHz for realistic device parameters.

Nonreciprocity Realized with Quantum Nonlinearity.

Nonreciprocal devices are a key element for signal routing and noise isolation. Rapid development of quantum technologies has boosted the demand for a new generation of miniaturized and low-loss nonreciprocal components. Here, we use a pair of tunable superconducting artificial atoms in a 1D waveguide to experimentally realize a minimal passive nonreciprocal device. Taking advantage of the quantum nonlinear behavior of artificial atoms, we achieve nonreciprocal transmission through the waveguide in a wide range of powers. Our results are consistent with theoretical modeling showing that nonreciprocity is associated with the population of the two-qubit nonlocal entangled quasidark state, which responds asymmetrically to incident fields from opposing directions. Our experiment highlights the role of quantum correlations in enabling nonreciprocal behavior and opens a path to building passive quantum nonreciprocal devices without magnetic fields.

Human VKORC1 mutations cause variable degrees of 4-hydroxycoumarin resistance and affect putative warfarin binding interfaces.

Since the discovery of warfarin-sensitive vitamin K 2,3-epoxide reductase complex subunit 1 (VKORC1), 26 human VKORC1 (hVKORC1) missense mutations have been associated with oral anticoagulant resistance (OACR). Assessment of warfarin resistance using the "classical" dithiothreitol-driven vitamin K 2,3-epoxide reductase (VKOR) assay has not reflected clinical resistance phenotypes for most mutations. Here, we present half maximal inhibitory concentrations (IC50) results for 21 further hVKORC1 mutations obtained using a recently validated cell-based assay (J Thromb Haemost 11(5):872). In contrast to results from the dithiothreitol-driven VKOR assay, all mutations exhibited basal VKOR activity and warfarin IC50 values that correspond well to patient OACR phenotypes. Thus, the present assay is useful for functional investigations of VKORC1 and oral anticoagulant inhibition of the vitamin K cycle. Additionally, we modeled hVKORC1 on the previously solved structure of a homologous bacterial enzyme and performed in silico docking of warfarin on this model. We identified one binding site delineated by 3 putative binding interfaces. These interfaces comprise linear sequences of the endoplasmic reticulum-lumenal loop (Ser52-Phe55) and the first (Leu22-Lys30) and fourth (Phe131-Thr137) transmembrane helices. All known OACR-associated hVKORC1 mutations are located in or around these putative interfaces, supporting our model.

Confirmation of warfarin resistance of naturally occurring VKORC1 variants by coexpression with coagulation factor IX and in silico protein modelling.

BACKGROUND: VKORC1 has been identified some years ago as the gene encoding vitamin K epoxide reductase (VKOR) - the target protein for coumarin derivates like warfarin or phenprocoumon. Resistance against warfarin and other coumarin-type anticoagulants has been frequently reported over the last 50 years in rodents due to problems in pest control as well as in thrombophilic patients showing variable response to anticoagulant treatment. Many different mutations have already been detected in the VKORC1 gene leading to warfarin resistance in rats, mice and in humans. Since the conventional in vitro dithiothreitol (DTT)-driven VKOR enzymatic assay often did not reflect the in vivo status concerning warfarin resistance, we recently developed a cell culture-based method for coexpression of VKORC1 with coagulation factor IX and subsequent measurement of secreted FIX in order to test warfarin inhibition in wild-type and mutated VKORC1. RESULTS: In the present study, we coexpressed wild-type factor IX with 12 different VKORC1 variants which were previously detected in warfarin resistant rats and mice. The results show that amino acid substitutions in VKORC1 maintain VKOR activity and are associated with warfarin resistance. When we projected in silico the amino acid substitutions onto the published three-dimensional model of the bacterial VKOR enzyme, the predicted effects matched well the catalytic mechanism proposed for the bacterial enzyme. CONCLUSIONS: The established cell-based system for coexpression of VKORC1 and factor IX uses FIX activity as an indicator of carboxylation efficiency. This system reflects the warfarin resistance status of VKORC1 mutations from anticoagulant resistant rodents more closely than the traditional DTT-driven enzyme assay. All mutations studied were also predicted to be involved in the reaction mechanism.

Human vitamin K 2,3-epoxide reductase complex subunit 1-like 1 (VKORC1L1) mediates vitamin K-dependent intracellular antioxidant function.

Human vitamin K 2,3-epoxide reductase complex subunit 1-like 1 (VKORC1L1), expressed in HEK 293T cells and localized exclusively to membranes of the endoplasmic reticulum, was found to support both vitamin K 2,3-epoxide reductase (VKOR) and vitamin K reductase enzymatic activities. Michaelis-Menten kinetic parameters for dithiothreitol-driven VKOR activity were: K(m) (µM) = 4.15 (vitamin K(1) epoxide) and 11.24 (vitamin K(2) epoxide); V(max) (nmol·mg(-1)·hr(-1)) = 2.57 (vitamin K(1) epoxide) and 13.46 (vitamin K(2) epoxide). Oxidative stress induced by H(2)O(2) applied to cultured cells up-regulated VKORC1L1 expression and VKOR activity. Cell viability under conditions of no induced oxidative stress was increased by the presence of vitamins K(1) and K(2) but not ubinquinone-10 and was specifically dependent on VKORC1L1 expression. Intracellular reactive oxygen species levels in cells treated with 2,3-dimethoxy-1,4-naphthoquinone were mitigated in a VKORC1L1 expression-dependent manner. Intracellular oxidative damage to membrane intrinsic proteins was inversely dependent on VKORC1L1 expression and the presence of vitamin K(1). Taken together, our results suggest that VKORC1L1 is responsible for driving vitamin K-mediated intracellular antioxidation pathways critical to cell survival.

Risk assessment and genetic counseling in families with Duchenne muscular dystrophy.

The Duchenne Muscular dystrophy (DMD) is the most frequent muscle disorder in childhood caused by mutations in the Xlinked dystrophin gene (about 65% deletions, about 7% duplications, about 26% point mutations and about 2% unknown mutations). The clinically milder Becker muscular dystrophy (BMD) is allelic to DMD. About 33% of all patients are due to de novo mutations and germ line mosaicism is frequently observed. While in earlier studies equal mutation rates in males and females had been reported, a breakdown by mutation types can better explain the sex ratio of mutations: Point mutations and duplications arise preferentially during spermatogenesis whereas deletions mostly arise in oogenesis. With current analytical methods, the underlying mutation can be identified in the great majority of cases and be used for carrier detection. However, in families with no mutation carrier available, the genetic model to be used for counselling of relatives can be quite complex.

Endogenous regeneration after collagenase-induced knee joint damage in the adult newt Notophthalmus viridescens.

OBJECTIVES: To determine whether adult newts (Notophthalmus viridescens) are able to repair experimentally-induced joint damage in order to generate a model system for the study of endogenous joint regeneration. METHODS: Joint instability and articular cartilage lesions of the knee joint of adult newts (N viridescens) were induced by intra-articular injection of collagenase. The changes over time were analysed clinically, by MRI, histologically and by reverse transcription PCR to detect selected relevant markers. RESULTS: After rapid onset of disease with joint luxation, loss of proteoglycans and cartilage volume, the signs ameliorated continuously by regeneration of the affected joint compartments. The majority of joints were morphologically intact and functionally operative after 10 weeks. Upregulation of chondrogenic key genes, homogenous expression levels of factors implicated in cartilage homeostasis and limb regeneration as well as the distribution of the blastemal marker 22/18 in both treated and untreated knees suggest that joint regeneration in adult newts only partially invokes pathways of embryological organogenesis. CONCLUSIONS: Newts are able to regenerate articular cartilage injuries and to restore tissue integrity and function after induction of damage using a procedure known to induce experimental osteoarthritis in murine models. Further analysis of the underlying molecular mechanisms may contribute to the development of novel treatment approaches in joint failure.

Screening of the ryanodine 1 gene for malignant hyperthermia causative mutations by high resolution melt curve analysis.

BACKGROUND: A diagnosis of malignant hyperthermia (MH) can be determined by performing an in vitro (muscle) contracture test (IVCT) or by identifying a known MH causative mutation in the ryanodine receptor 1 gene (RYR1). Genetic diagnosis has an advantage over IVCT because it is less invasive. Direct sequencing of the very large RYR1 coding region (15.117 bases) is a laborious and expensive task. In this study, we applied the High Resolution Melting (HRM) curve analysis as a tool to screen the entire coding region of the gene. METHODS: Genomic DNA was extracted from peripheral blood samples in a cohort of 16 MH-susceptible patients diagnosed by the IVCT. The total coding region of RYR1 was divided and amplified by polymerase chain reaction in 131 DNA fragments and the melting profiles were compared with those of control samples. HRM curves were evaluated by Rotor-Gene Q software and visual inspection. Fragments showing aberrant melting profiles were sequenced to identify the underlying sequence variation. RESULTS: A subset of 520 of 2520 DNA fragments (21%) showed significantly aberrant melting profiles. Upon sequencing, 131 known polymorphisms and 17 known or suspected mutations were found in 13 of 16 MH-susceptible patients (81%). Thus, the workload of sequencing was reduced by 79%. CONCLUSION: HRM curve analysis is a sensitive and cost-effective tool for the identification of nucleotide sequence variants in complex genes such as the RYR1 gene.

Microbubble-mediated sonothrombolysis with BR38 of a venous full blood thrombus in a rat embolic stroke model.

BACKGROUND: Early recanalization of an occluded vessel is associated with a better clinical outcome in acute ischemic stroke. Intravenous thrombolysis using recombinant tissue plasminogen activator (rt-PA) is only available in a minority of patients and often fails to reopen the occluded vessel. Mechanical recanalization is more effective in this matter but only available for selected patients when a thrombectomy centre can be reached. Therefore, sonothrombolysis might represent an alternative or complementary approach. Here, we tested microbubble-mediated sonothrombolysis (mmSTL) in a thromboembolic stroke model for middle cerebral artery occlusion (MCAO) in rats. METHODS: Sixty-seven male Wistar rats underwent MCAO using an autologous full blood thrombus and were randomly assigned to four groups receiving rt-PA, mmSTL, a combination of both, or a placebo. Diagnostic workup included neurological examination, assessment of infarct size, and presence of intracerebral haemorrhage by magnetic resonance imaging (MRI) and presence of microbleedings in histological staining. RESULTS: Neurological examination revealed no differences between the treatment groups. In all treatment groups, there was a reduction in infarct size 24 hours after MCAO as compared to the placebo (P≤0.05), but there were no differences between the active treatment groups (P>0.05) (placebo 0.75±0.10 cm3; mmSTL 0.43±0.07 cm3; rt-PA 0.4±0.07 cm3; mmSTL + rt-PA 0.27±0.08 cm3). Histological staining displayed intracerebral microbleedings in all animals. The frequency of gross bleeding detected by MRI did not differ between the groups (placebo 3; mmSTL 4; rt-PA 2; mmSTL + rt-PA 2; P>0.05) and was not associated with worse performance in clinical testing (P>0.05). There were no statistical differences in the mortality between the groups (P>0.05). CONCLUSIONS: Our study showed the efficacy and safety of mmSTL with or without rt-PA in an embolic rat stroke model using a continuous full blood thrombus. Sonothrombolysis might be useful for patients who need to be transported to a thrombectomy centre or for those with distal vessel occlusion.

Mutations in VKORC1 cause warfarin resistance and multiple coagulation factor deficiency type 2.

Coumarin derivatives such as warfarin represent the therapy of choice for the long-term treatment and prevention of thromboembolic events. Coumarins target blood coagulation by inhibiting the vitamin K epoxide reductase multiprotein complex (VKOR). This complex recycles vitamin K 2,3-epoxide to vitamin K hydroquinone, a cofactor that is essential for the post-translational gamma-carboxylation of several blood coagulation factors. Despite extensive efforts, the components of the VKOR complex have not been identified. The complex has been proposed to be involved in two heritable human diseases: combined deficiency of vitamin-K-dependent clotting factors type 2 (VKCFD2; Online Mendelian Inheritance in Man (OMIM) 607473), and resistance to coumarin-type anticoagulant drugs (warfarin resistance, WR; OMIM 122700). Here we identify, by using linkage information from three species, the gene vitamin K epoxide reductase complex subunit 1 (VKORC1), which encodes a small transmembrane protein of the endoplasmic reticulum. VKORC1 contains missense mutations in both human disorders and in a warfarin-resistant rat strain. Overexpression of wild-type VKORC1, but not VKORC1 carrying the VKCFD2 mutation, leads to a marked increase in VKOR activity, which is sensitive to warfarin inhibition.

Functional properties of RYR1 mutations identified in Swedish patients with malignant hyperthermia and central core disease.

BACKGROUND: A diagnosis of malignant hyperthermia susceptibility by in vitro contraction testing can often only be performed at specialized laboratories far away from where patients live. Therefore, we have designed a protocol for genetic screening of the RYR1-cDNA and for functional testing of newly identified ryanodine receptor 1 (RYR1) gene variants in B lymphocytes isolated from peripheral blood samples drawn at local primary care centers. METHODS: B lymphocytes were isolated for the extraction of RYR1-mRNA and genomic DNA and for establishment of lymphoblastoid B cell lines in 5 patients carrying yet unclassified mutations in the RYR1. The B lymphoblastoid cell lines were used to study resting cytoplasmic calcium concentration, the peak calcium transient induced by the sarco(endo)plasmic reticulum Ca-ATPase inhibitor thapsigargin, and the dose-dependent calcium release induced by the ryanodine receptor agonist 4-chloro-m-cresol. RESULTS: It was possible to extract mRNA for cDNA synthesis and to create B lymphocyte clones from all samples. All B lymphoblastoid cell lines carrying RYR1 candidate mutations showed significantly increased resting cytoplasmic calcium levels as well as a shift to lower concentrations of 4-chloro-m-cresol inducing calcium release compared with controls. CONCLUSIONS: Peripheral blood samples are stable regarding RNA and DNA extraction and establishment of lymphoblastoid B cell lines after transportation at ambient temperature over large distances by ordinary mail. Functional tests on B cells harboring the newly identified amino acid substitutions indicate that they alter intracellular Ca2+ homeostasis and are most likely causative of malignant hyperthermia.

Comprehensive evaluation of player-surface interaction on artificial soccer turf.

The purpose of this study was to evaluate the traction characteristics of four different stud configurations on Fédération Internationale de Football Association (FIFA) 2-Star, third-generation artificial soccer turf. The investigated stud configurations were hard ground design, firm ground design, soft ground design, and an experimental prototype. The concept of this study combines performance, perception, biomechanical, and mechanical testing procedures. Twenty-five soccer players took part in the different testing procedures. Variables of this study were: running times, subjective rankings/ratings, ground reaction forces, and mechanical traction properties. Statistical discrimination between the four stud configurations was shown for performance, perception, and biomechanical testing (p < 0.05). Unsuited stud configurations for playing on artificial turf are characterized by less plain distributed and pronounced studs.

How much mutant protein is needed to cause a protein aggregate myopathy in vivo? Lessons from an exceptional desminopathy.

Myofibrillar myopathies are caused by mutations in desmin, alphaB-crystallin, myotilin, ZASP, and filamin C genes. Since the vast majority of myofibrillar myopathy causing mutations are heterozygous single amino acid substitutions or small in-frame deletions, the pathogenic role of mutant versus wild-type protein cannot be assessed in human skeletal muscle by standard immunodetection techniques. We report on an exceptional desminopathy due to a heterozygous c.735G>C mutation. Immunoblotting detected full-length 53 kDa desmin and a truncated 50 kDa variant in skeletal muscle from three affected patients of two different families. RT-PCR identified three desmin mRNA species encoding for wild-type and two mutant proteins, p.Glu245Asp and p.Asp214_Glu245del. Since previous functional studies on the p.Glu245Asp mutant showed biological properties identical to wild-type desmin, the truncated p.Asp214_Glu245del desmin is the disease-causing mutant. Semiquantitative RT-PCR established a fraction of the truncated desmin mRNA species in a range from 24% to 37%. Initial quantification of corresponding desmin proteins in the muscle biopsy of the index patient of one family indicated a fraction of only 10% of the truncated species. However, serial analyses of different sections from each muscle biopsy revealed a high intra- and interindividual variability of the truncated desmin protein level within a range from 5% to 43%. Desmin assembly studies in vitro have established clear-cut pathogenic ratios of mutant versus wild-type proteins. However, our findings point out a far more complex situation in human skeletal muscle. The heterogeneously distributed mutation load within and between individual specimens, which reflects local differences in the expression and/or turnover of the mutant protein in different areas containing multiple myonuclear domains, renders it impossible to define an exact pathogenic threshold of a specific mutant in vivo.

VKORC1 deficiency in mice causes early postnatal lethality due to severe bleeding.

Vitamin K hydroquinone is oxidised to the epoxide form (K>O) during vitamin K-dependent posttranslational gamma-glutamyl carboxylation resulting in biological active so called vitamin K-dependent proteins. In turn, K>O is reduced by the enzyme VKORC1 (vitamin K epoxide reductase complex component 1) to complete the vitamin K cycle. To investigate the biological role of VKORC1 in vivo, we generated VKORC1 knockout mice. Homozygous VKORC1-deficient mice developed normally until birth. Within 2-20 days after birth, the knockout mice died due to extensive, predominantly intracerebral haemorrhage. Bleeding resulted from a severe deficiency of gamma-carboxylated clotting factors. This lethal phenotype could be rescued by oral administration of vitamin K. Additionally, morphometric analysis of the limbs in VKORC1-deficient animals revealed reduced length of bone calcification relative to wild-type control mice. The observed phenotype of VKORC1 knockout mice excludes the existence of other enzymes with VKOR activity that can substitute to supply vitamin K hydroquinone required for maturation of blood clotting factors. Thus, our study underscores the essential role of VKORC1 in vitamin K-dependent gamma-glutamyl carboxylation.