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All endocytosis and exocytosis in the African trypanosome Trypanosoma brucei occurs at a single subdomain of the plasma membrane. This subdomain, the flagellar pocket, is a small vase-shaped invagination containing the root of the single flagellum of the cell. Several cytoskeleton-associated multiprotein complexes are coiled around the neck of the flagellar pocket on its cytoplasmic face. One of these, the hook complex, was proposed to affect macromolecule entry into the flagellar pocket lumen. In previous work, knockdown of T. brucei (Tb)MORN1, a hook complex component, resulted in larger cargo being unable to enter the flagellar pocket. In this study, the hook complex component TbSmee1 was characterised in bloodstream form T. brucei and found to be essential for cell viability. TbSmee1 knockdown resulted in flagellar pocket enlargement and impaired access to the flagellar pocket membrane by surface-bound cargo, similar to depletion of TbMORN1. Unexpectedly, inhibition of endocytosis by knockdown of clathrin phenocopied TbSmee1 knockdown, suggesting that endocytic activity itself is a prerequisite for the entry of surface-bound cargo into the flagellar pocket., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2023. Published by The Company of Biologists Ltd.)

Objectives: Gaucher disease's (GD) pathophysiology generates anesthetic concerns in total hip joint arthroplasty (THA), and due to its rareness, data on perioperative risks are scarce. This 22-year study at a large reference center addresses anesthetic management and perioperative outcomes in GD., Methods: This retrospective-cohort study assessed anesthetic success and safety in 30 THA patients, comparing them with a control-matched group. Data on clinical characteristics, perioperative events, and outcomes were collected. The primary outcome was the success rate of anesthesia induction performance at first attempt. Secondary outcomes were difficult intraoperative course and hemodynamic management, and the development of postoperative complications. The age, sex, weight, body mass index, and primary-to-revision hip arthroplasty ratio were similar in both groups., Results: There was no significant difference at all-type anesthesia first initiation attempt success. No particular preference by staff anesthetists for general anesthesia or neuraxial procedures was observed. The GD group showed a significantly higher mean of intraoperative packed Red Blood Cell units administered ((0.73 vs. 0.18); ( p = 0.038)), higher intraoperative and postoperative platelet transfusion incidence ((5/30 [16.7%] vs. 0/56 [0.00%]; p = 0.004) and (3/30 [10%] vs. 0/56 [0%]; p = 0.040)), and longer mean recovery room length of stay (426 ± 412 vs. 175 ± 140; p = 0.004). Postoperative complications were not significantly different.

Inositol phosphates (InsPs) are ubiquitous in all eukaryotes. However, since there are 63 possible different phosphate ester isomers, the analysis of InsPs is challenging. In particular, InsP 1 , InsP 2, and InsP 3 already amass 41 different isomers, of which some occur as enantiomers. Profiling of these "lower" inositol phosphates in mammalian tissues requires powerful analytical methods and reference compounds. Here, we report an analysis of InsP 2 and InsP 3 with capillary electrophoresis coupled to electrospray ionization mass spectrometry (CE-ESI-MS). Using this method, the bacterial effector RipBL1 was analyzed and found to degrade InsP 6 to Ins(1,2,3)P 3 , an understudied InsP 3 isomer. This new reference molecule then aided us in the assignment of the isomeric identity of an InsP 3 while profiling human samples: in urine and kidney stones, we describe for the first time the presence of defined and abundant InsP 3 isomers, namely Ins(1,2,3)P 3 , Ins(1,2,6)P 3 and/or Ins(2,3,4)P 3 ., Competing Interests: The authors declare no conflicts of interest., (This journal is © The Royal Society of Chemistry.)

The combinatorial phosphorylation of myo-inositol results in the generation of different inositol phosphates (InsPs), of which phytic acid (InsP6) is the most abundant species in eukaryotes. InsP6 is also an important precursor of the higher phosphorylated inositol pyrophosphates (PP-InsPs), such as InsP7 and InsP8, which are characterized by a diphosphate moiety and are also ubiquitously found in eukaryotic cells. While PP-InsPs regulate various cellular processes in animals and yeast, their biosynthesis and functions in plants has remained largely elusive because plant genomes do not encode canonical InsP6 kinases. Recent work has shown that Arabidopsis (Arabidopsis thaliana) INOSITOL (1,3,4) TRIPHOSPHATE 5/6 KINASE1 (ITPK1) and ITPK2 display in vitro InsP6 kinase activity and that, in planta, ITPK1 stimulates 5-InsP7 and InsP8 synthesis and regulates phosphate starvation responses. Here we report a critical role of ITPK1 in auxin-related processes that is independent of the ITPK1-controlled regulation of phosphate starvation responses. Those processes include primary root elongation, root hair development, leaf venation, thermomorphogenic and gravitropic responses, and sensitivity to exogenously applied auxin. We found that the recombinant auxin receptor complex, consisting of the F-Box protein TRANSPORT INHIBITOR RESPONSE1 (TIR1), ARABIDOPSIS SKP1 HOMOLOG 1 (ASK1), and the transcriptional repressor INDOLE-3-ACETIC ACID INDUCIBLE 7 (IAA7), binds to anionic inositol polyphosphates with high affinity. We further identified a physical interaction between ITPK1 and TIR1, suggesting a localized production of 5-InsP7, or another ITPK1-dependent InsP/PP-InsP isomer, to activate the auxin receptor complex. Finally, we demonstrate that ITPK1 and ITPK2 function redundantly to control auxin responses, as deduced from the auxin-insensitive phenotypes of itpk1 itpk2 double mutant plants. Our findings expand the mechanistic understanding of auxin perception and suggest that distinct inositol polyphosphates generated near auxin receptors help to fine-tune auxin sensitivity in plants., (© The Author(s) 2022. Published by Oxford University Press on behalf of American Society of Plant Biologists.)

Background: Sjogren's syndrome (SS) is a rare chronic autoimmune disease involving exocrine glands presenting with sicca syndrome, recurrent parotitis and other extraglandular stigmata. SS is well characterized in the adult population with classification criteria; however, primary SS presenting in childhood is poorly defined and rare in males. Recurrent parotitis is the most common presenting symptom in children with primary SS; however, clinical phenotype in children appears more variable than in adults. The lungs are a common extraglandular location for manifestations of primary SS. However, interstitial lung disease (ILD) is rare in children with primary SS. There are only four published reports of ILD associated with primary SS in female children. Here, we present a very rare case of primary SS in a pediatric male with pulmonary manifestations and review of the literature on ILD in childhood-onset primary SS., Case Presentation: A 14-year-old White male with a history of chronic severe asthma, recurrent parotitis and idiopathic intracranial hypertension was referred to pediatric rheumatology for evaluation of a positive ANA. In early childhood, he was diagnosed with persistent asthma recalcitrant to therapy. At age 8, he developed recurrent episodes of bilateral parotitis despite multiple treatments with sialoendoscopy. At age 14, respiratory symptoms significantly worsened prompting reevaluation. Lab workup was notable for positive ANA and Sjogren's Syndrome A and B antibodies. Pulmonary function tests showed only a mild obstructive process. Computed tomography of chest was significant for small airway disease, and lung biopsy was positive for mild interstitial lymphocytic inflammation presenting a conflicting picture for ILD. The constellation of findings led to the diagnosis of primary SS with associated pulmonary manifestations. He was treated with hydroxychloroquine, mycophenolate mofetil and oral corticosteroids with resolution of symptoms., Conclusions: Primary SS is a rare disease in the pediatric population that is poorly characterized. This case is the very rare presentation of childhood-onset primary SS with pulmonary manifestations in a male patient. ILD associated with primary SS is also very rare with only four pediatric patients reported in the literature. Collaborative effort is needed to develop pediatric specific diagnostic and treatment guidelines in this rare condition., (© 2022. The Author(s).)

Plant-parasitic nematodes are a major threat to crop production in all agricultural systems. The scarcity of classical resistance genes highlights a pressing need to find new ways to develop nematode-resistant germplasm. Here, we sequence and assemble a high-quality phased genome of the model cyst nematode Heterodera schachtii to provide a platform for the first system-wide dual analysis of host and parasite gene expression over time, covering all major parasitism stages. Analysis of the hologenome of the plant-nematode infection site identified metabolic pathways that were incomplete in the parasite but complemented by the host. Using a combination of bioinformatic, genetic, and biochemical approaches, we show that a highly atypical completion of vitamin B5 biosynthesis by the parasitic animal, putatively enabled by a horizontal gene transfer from a bacterium, is required for full pathogenicity. Knockout of either plant-encoded or now nematode-encoded steps in the pathway significantly reduces parasitic success. Our experiments establish a reference for cyst nematodes, further our understanding of the evolution of plant-parasitism by nematodes, and show that congruent differential expression of metabolic pathways in the infection hologenome represents a new way to find nematode susceptibility genes. The approach identifies genome-editing-amenable targets for future development of nematode-resistant crops., (© 2022. The Author(s).)

Inositol pyrophosphates (PP-InsPs), derivatives of inositol hexakisphosphate (phytic acid, InsP 6 ) or lower inositol polyphosphates, are energy-rich signaling molecules that have critical regulatory functions in eukaryotes. In plants, the biosynthesis and the cellular targets of these messengers are not fully understood. This is because, in part, plants do not possess canonical InsP 6 kinases and are able to synthesize PP-InsP isomers that appear to be absent in yeast or mammalian cells. This review will shed light on recent discoveries in the biosynthesis of these enigmatic messengers and on how they regulate important physiological processes in response to abiotic and biotic stresses in plants., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Riemer, Pullagurla, Yadav, Rana, Jessen, Kamleitner, Schaaf and Laha.)

Background: There is ongoing discussion whether operative fixation of partially stable lateral compression fractures of the pelvis is beneficial for the patient. Recent studies suggest that the pectineal ligament may act as a secondary stabilizer of the anterior pelvis ring. The purpose of this study was to investigate the influence of the pectineal ligament's integrity on the biomechanical stability and displacement in anterior pelvic ring fractures., Methods: In a biomechanical setup, a cyclic loading protocol was applied with sinusoidal axial force from 100 to 500 N on cadaver hemipelves with soft tissues (n = 5). After testing the native specimens ("No fracture"), increasing degrees of injury were created on the samples: 1. an osseous defect to the pubic ramus ("Bone #"), 2. cutting of all soft tissues including obturator membrane except for the pectineal ligament intact ("ObtM #"), 3. cutting of the pectineal ligament ("PectL #") - with the loading protocol being applied to each sample at each state of injury. Fracture motion and vertical displacement were measured using a digital image correlation system and opto-metric analysis., Results: No failure of the constructs was observed. Creating a pubic ramus fracture (p = 0.042) and cutting the pectineal ligament (p = 0.042) each significantly increased relative fracture movement. The mean change in absolute movement was 0.067 mm (range, 0.02 mm to 0.19 mm) for ObtM # and 0.648 mm (range, 0.07 mm to 2.93 mm), for PectL # in relation to Bone # (p = 0.043). Also for absolute vertical movement, there was a significant change when the pectineal ligament was cut (p = 0.043), while there was no such effect with cutting all other soft tissues including the obturator membrane., Conclusions: Based on the findings of this in vitro study, the pectineal ligament significantly contributes to the stability of the anterior pelvic ring. An intact pectineal ligament reduces fracture movement in presence of a pubic ramus fracture., Competing Interests: Conflict of interests and sources of funding The authors report no conflict of interest related to the paper entitled “The pectineal ligament is a secondary stabilizer in anterior pelvic ring fractures - a biomechanical study”., (Copyright © 2021. Published by Elsevier Ltd.)

In biomechanics, large finite element models with macroscopic representation of several bones or joints are necessary to analyze implant failure mechanisms. In order to handle large simulation models of human bone, it is crucial to homogenize the trabecular structure regarding the mechanical behavior without losing information about the realistic material properties. Accordingly, morphology and fabric measurements of 60 vertebral cancellous bone samples from three osteoporotic lumbar spines were performed on the basis of X-ray microtomography (μCT) images to determine anisotropic elastic parameters as a function of bone density in the area of pedicle screw anchorage. The fabric tensor was mapped in cubic bone volumes by a 3D mean-intercept-length method. Fabric measurements resulted in a high degree of anisotropy (DA = 0.554). For the Young's and shear moduli as a function of bone volume fraction (BV/TV, bone volume/total volume), an individually fit function was determined and high correlations were found (97.3 ≤ R 2 ≤ 99.1,p < 0.005). The results suggest that the mathematical formulation for the relationship between anisotropic elastic constants and BV/TV is applicable to current μCT data of cancellous bone in the osteoporotic lumbar spine. In combination with the obtained results and findings, the developed routine allows determination of elastic constants of osteoporotic lumbar spine. Based on this, the elastic constants determined using homogenization theory can enable efficient investigation of human bone using finite element analysis (FEA). Graphical Abstract Cancellous Bone with Fabric Tensor Ellipsoid representing anisotropy and principal axis (colored coordinate system) of given trabecular structure., (© 2021. The Author(s).)

In plants, phosphate (P i ) homeostasis is regulated by the interaction of PHR transcription factors with stand-alone SPX proteins, which act as sensors for inositol pyrophosphates. In this study, we combined different methods to obtain a comprehensive picture of how inositol (pyro)phosphate metabolism is regulated by P i and dependent on the inositol phosphate kinase ITPK1. We found that inositol pyrophosphates are more responsive to P i than lower inositol phosphates, a response conserved across kingdoms. Using the capillary electrophoresis electrospray ionization mass spectrometry (CE-ESI-MS) we could separate different InsP 7 isomers in Arabidopsis and rice, and identify 4/6-InsP 7 and a PP-InsP 4 isomer hitherto not reported in plants. We found that the inositol pyrophosphates 1/3-InsP 7 , 5-InsP 7 , and InsP 8 increase several fold in shoots after P i resupply and that tissue-specific accumulation of inositol pyrophosphates relies on ITPK1 activities and MRP5-dependent InsP 6 compartmentalization. Notably, ITPK1 is critical for P i -dependent 5-InsP 7 and InsP 8 synthesis in planta and its activity regulates P i starvation responses in a PHR-dependent manner. Furthermore, we demonstrated that ITPK1-mediated conversion of InsP 6 to 5-InsP 7 requires high ATP concentrations and that Arabidopsis ITPK1 has an ADP phosphotransferase activity to dephosphorylate specifically 5-InsP 7 under low ATP. Collectively, our study provides new insights into P i -dependent changes in nutritional and energetic states with the synthesis of regulatory inositol pyrophosphates., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Background: Intervertebral fusions in cases of reduced bone density are a tough challenge. From a biomechanical point of view, most current studies have focused on the range of motion or have shown test setups for single-component tests. Definitive setups for biomechanical testing of the primary stability of a 360° fusion using a screw-rod system and cage on osteoporotic spine are missing. The aim of this study was to develop a test stand to provide information about the bone-implant interface under reproducible conditions., Methods: After pretesting with artificial bone, functional spine units were tested with 360° fusion in the transforaminal lumbar interbody fusion technique. The movement sequences were conducted in flexion/extension, right and left lateral bending, and right and left axial rotation on a human model with osteopenia or osteoporosis under permanent maximum load with 7.5 N-m., Results: During the testing of human cadavers, 4 vertebrae were fully tested and were inconspicuous even after radiological and macroscopic examination. One vertebra showed a subsidence of 2 mm, and 1 vertebra had a cage collapsed into the vertebra., Conclusions: This setup is suitable for biomechanical testing of cyclical continuous loads on the spine with reduced bone quality or osteoporosis. The embedding method is stable and ensures a purely single-level setup with different trajectories, especially when using the cortical bone trajectory. Optical monitoring provides a very accurate indication of cage movement, which correlates with the macroscopic and radiological results., (Copyright © 2021 Elsevier Inc. All rights reserved.)

The analysis of myo-inositol phosphates (InsPs) and myo-inositol pyrophosphates (PP-InsPs) is a daunting challenge due to the large number of possible isomers, the absence of a chromophore, the high charge density, the low abundance, and the instability of the esters and anhydrides. Given their importance in biology, an analytical approach to follow and understand this complex signaling hub is desirable. Here, capillary electrophoresis (CE) coupled to electrospray ionization mass spectrometry (ESI-MS) is implemented to analyze complex mixtures of InsPs and PP-InsPs with high sensitivity. Stable isotope labeled (SIL) internal standards allow for matrix-independent quantitative assignment. The method is validated in wild-type and knockout mammalian cell lines and in model organisms. SIL-CE-ESI-MS enables the accurate monitoring of InsPs and PP-InsPs arising from compartmentalized cellular synthesis pathways, by feeding cells with either [ 13 C 6 ]-myo-inositol or [ 13 C 6 ]-D-glucose. In doing so, we provide evidence for the existence of unknown inositol synthesis pathways in mammals, highlighting the potential of this method to dissect inositol phosphate metabolism and signalling.

Background: Caveolin-1 has emerged as a critical regulator of signaling pathways involved in lung fibrosis and inflammation., Methods: Therefore, we investigated whether caveolin-1 is deficient in asthmatic patients and in a murine model of asthma., Results: Immunohistochemical analyses of endobronchial biopsies showed a remarkable loss of caveolin-1 in the lungs of asthmatic patients compared with controls. This loss was most evident in bronchial epithelial cells and associated with an increase in the expression of extracellular matrix proteins: collagen I, tenascin, and periostin. Cultured primary bronchial epithelial cells of asthmatics had lower caveolin-1 expression compared with control cells. In addition, caveolin-1 expression was significantly decreased in peripheral blood monocytes from asthma patients. The loss of caveolin-1 was also observed in a mouse model for asthma (mice sensitized and challenged with aspergillus fumigatus)., Conclusions: To our knowledge, this is the first demonstration that the regulatory protein caveolin-1 is reduced in patients with asthma., (© 2012 John Wiley & Sons A/S.)

Aim: The aim of the study is to analyse haemodialysis patients' prescriptions accordance with legal registrations in 64 chronic haemodialysis patients during a one-month follow-up period., Results: Patients are taking 9.6+/-3 different drugs. A mean of 2.4+/-1.3 prescriptions per patient disagree with the recommended drug use. The directions for use in this specific population are defective in 89%. Moreover, at least one drug-drug interaction is found in 78% of our patients., Conclusion: Only 8% of the studied hemodialysis patients benefit from prescriptions in agreement with the legal recordings, as a consequence of the lack of drug studies in this particular population. Therefore, clinicians have to rely on their own experience to establish their prescriptions, which can involve their responsibility in case of litigation. Furthermore, the frequency of drug-drug interactions in these polymedicated patients implies that a close pharmaceutical follow-up should be implemented.

A patient with BCR/ABL negative myeloproliferative syndrome with a 46,XY,del(3)(q21), t(4;15)(p16;q24) karyotype is described. Fluorescence in situ hybridization performed with chromosomes 4 and 15 painting probes confirmed a novel reciprocal (4;15) translocation. The absence of crkl tyrosine phosphorylation, no activation of the abl kinase as measured by autophosphorylation, and a normal-size abl transcript suggest an alternative mechanism for leukemogenesis to that operative in Ph positive BCR/ABL positive chronic myeloid leukemia. A number of genes potentially relevant to tumorigenesis, some involving the ras signaling pathway, map to the 4p16 and 15q24 chromosome regions.

A functional model for the aspartate/glutamate carrier of the inner mitochondrial membrane was established based on a kinetic evaluation of this transporter. Antiport kinetics were measured in proteoliposomes that contained partially purified carrier protein of definite transmembrane orientation (Dierks, T. and Krämer, R. (1988) Biochim. Biophys. Acta 937, 122-126). Bireactant initial velocity analyses of the counterexchange reaction were carried out varying substrate concentrations both in the internal and the external compartment. The kinetic patterns obtained were inconsistent with a pong-pong mechanism; rather they demonstrated the formation of a ternary complex as a consequence of sequential binding of one internal and one external substrate molecule to the carrier. Studies on transport activity in the presence of aspartate and glutamate in the same compartment (formally treated as substrate inhibition) clearly indicated that during exchange only one form of the carrier at either membrane surface exposes its binding sites, for which the two different substrates compete. In the deenergized state (pH 6.5) both substrates were translocated at about the same rate. Aspartate/glutamate antiport became asymmetric if a membrane potential was imposed, due to the electrogenic nature of the heteroexchange resulting from proton cotransport together with glutamate. Investigation of the electrical properties of aspartate/aspartate homoexchange led to the conclusion that the translocating carrier-substrate intermediate exhibits a transmembrane symmetry with respect to the (negative) charge, which again only is conceivable assuming a ternary complex. Thus, an antiport model is outlined that shows the functional complex of the carrier with two substrate molecules bound, one at either side of the membrane. The conformational change associated with the transition of both substrate molecules across the membrane then occurs in a single step. Furthermore the model implicates a distinct proton binding site, which is derived from the different influence of H+ concentration observed on transport affinity and transport velocity, respectively, when glutamate is used as a substrate.

We have shown that allantoin degradation in Saccharomyces cerevisiae proceeds exclusively through the intermediate formation of allantoic acid, urea, and allophanic acid. The number of reactions between allantoic acid and urea, however, remains obscure owing to our inability to isolate a mutant defective in ureidoglycolate hydrolase. Structural genes for the enzymes, allantoinase (dal1) and allantoicase (dal2) are located on chromosome IX promixal to the centromere in the order dal1-dal2-lysl.