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X-ray fluorescence (XRF) imaging is a highly sensitive non-invasive imaging method for detection of small element quantities in objects, from human-sized scales down to single-cell organelles, using various X-ray beam sizes. Our aim was to investigate the cellular uptake and distribution of Q 10 , a highly conserved coenzyme with antioxidant and bioenergetic properties. Q 10 was labeled with iodine (I 2 -Q 10 ) and individual primary human skin cells were scanned with nano-focused beams. Distribution of I 2 -Q 10 molecules taken up inside the screened individual skin cells was measured, with a clear correlation between individual Q 10 uptake and cell size. Experiments revealed that labeling Q 10 with iodine causes no artificial side effects as a result of the labeling procedure itself, and thus is a perfect means of investigating bioavailability and distribution of Q 10 in cells. In summary, individual cellular Q 10 uptake was demonstrated by XRF, opening the path towards Q 10 multi-scale tracking for biodistribution studies.

The present academic work aims to contribute to an early diagnosis of neuralgic amyotrophy (NA) because of its high prevalence in the population. This disease is a neuromuscular syndrome with unclear etiology; it affects mostly the brachial plexus, causing acute pain in the affected shoulder, paralysis, and disabilities. Considering the importance of an early treatment that can modify the prognosis of the patient, knowing the last updates about the syndrome as its clinical presentation is important. Data analysis was conducted through an online non-systematic review that indicated the epidemiology, pathophysiology, and differential diagnosis and prognosis of NA. Knowledge of the clinical features of NA is not common; however, it is important in orthopedic practice because it requires differentiation from spine pathologies., Competing Interests: Conflicts of Interest: The authors declare that there are no relevant conflicts of interest., (Copyright © 2021 by The Japanese Society for Spine Surgery and Related Research.)

The reproducibility of stem cell research relies on the constant availability of quality-controlled cells. As the quality of human induced pluripotent stem cells (hiPSCs) can deteriorate in the course of a few passages, cell banking is key to achieve consistent results and low batch-to-batch variation. Here, we provide a cost-efficient route to generate master and working cell banks for basic research projects. In addition, we describe minimal protocols for quality assurance including tests for sterility, viability, pluripotency, and genetic integrity. © 2020 The Authors. Basic Protocol 1: Expansion of hiPSCs Basic Protocol 2: Cell banking of hiPSCs Support Protocol 1: Pluripotency assessment by flow cytometry Support Protocol 2: Thawing control: Viability and sterility Support Protocol 3: Potency, viral clearance, and pluripotency: Spontaneous differentiation and qRT-PCR Support Protocol 4: Identity: Short tandem repeat analysis., (© 2020 The Authors.)

Aims: Chronic tachypacing is commonly used in animals to induce cardiac dysfunction and to study mechanisms of heart failure and arrhythmogenesis. Human induced pluripotent stem cells (hiPSC) may replace animal models to overcome species differences and ethical problems. Here, 3D engineered heart tissue (EHT) was used to investigate the effect of chronic tachypacing on hiPSC-cardiomyocytes (hiPSC-CMs)., Methods and Results: To avoid cell toxicity by electrical pacing, we developed an optogenetic approach. EHTs were transduced with lentivirus expressing channelrhodopsin-2 (H134R) and stimulated by 15 s bursts of blue light pulses (0.3 mW/mm2, 30 ms, 3 Hz) separated by 15 s without pacing for 3 weeks. Chronic optical tachypacing did not affect contractile peak force, but induced faster contraction kinetics, shorter action potentials, and shorter effective refractory periods. This electrical remodelling increased vulnerability to tachycardia episodes upon electrical burst pacing. Lower calsequestrin 2 protein levels, faster diastolic depolarization (DD) and efficacy of JTV-519 (46% at 1 µmol/L) to terminate tachycardia indicate alterations of Ca2+ handling being part of the underlying mechanism. However, other antiarrhythmic compounds like flecainide (69% at 1 µmol/L) and E-4031 (100% at 1 µmol/L) were also effective, but not ivabradine (1 µmol/L) or SEA0400 (10 µmol/L)., Conclusion: We demonstrated a high vulnerability to tachycardia of optically tachypaced hiPSC-CMs in EHT and the effective termination by ryanodine receptor stabilization, sodium or hERG potassium channel inhibition. This new model might serve as a preclinical tool to test antiarrhythmic drugs increasing the insight in treating ventricular tachycardia., (© The Author(s) 2019. Published by Oxford University Press on behalf of the European Society of Cardiology.)

Biological pacemakers could be a promising alternative to electronic pacemakers and human induced pluripotent stem cell derived cardiomyocytes (hiPSC-CM) may represent a suitable source for implantable cells. To further unravel this potential a thorough understanding of pacemaker function with regard to coupling processes both in the physiological and in the graft-host context is required. Here we developed a 2-component cardiac organoid model with a hiPSC-CM embryoid body (EB) as trigger casted into a rat engineered heart tissue (EHT) as arrhythmic beating substrate. Contractility recordings revealed that the EB controlled the beating activity of the EHT, leading to a regular hiPSC-CM-like beating pattern instead of the irregular beating typically seen in rat EHT. Connectivity was observed with action potential (AP) measurements and calcium transients transmitting from the EB directly into the rat EHT. Immunohistochemistry and genetically labeled hiPSC-CMs demonstrated that EB-derived and rat cells intermingled and formed a transitional zone. Connexin 43 expression followed the same pattern as histological and computer models have indicated for the human sinoatrial node. In conclusion, hiPSC-CM EBs function as a biological pacemaker in a 2-component cardiac organoid model, which provides the possibility to study electrophysiological and structural coupling mechanisms underlying propagation of pacemaker activity., (Copyright © 2019. Published by Elsevier Ltd.)

Background: Cardiac repolarization abnormalities in drug-induced and genetic long-QT syndrome may lead to afterdepolarizations and life-threatening ventricular arrhythmias. Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) should help to overcome the limitations of animal models based on species differences in repolarization reserve. Here, we compared head-to-head the contribution of I Ks (long QT1) and I Kr (long QT2) on action potentials (APs) in human left ventricular (LV) tissue and hiPSC-CM-derived engineered heart tissue (EHT)., Methods: APs were measured with sharp microelectrodes in EHT from 3 different control hiPSC-CM lines and in tissue preparations from failing LV., Results: EHT from hiPSC-CMs showed spontaneous diastolic depolarization and AP generation that were sensitive to low concentrations of ivabradine. I Kr block by E-4031 prolonged AP duration at 90% repolarization with similar half-effective concentration in EHT and LV but larger effect size in EHT (+281 versus +110 ms in LV). Although I Kr block alone evoked early afterdepolarizations and triggered activity in 50% of EHTs, slow pacing, reduced extracellular K + , and blocking of I Kr , I Ks , and I K1 were necessary to induce early afterdepolarizations in LV. In accordance with their clinical safety, moxifloxacin and verapamil did not induce early afterdepolarizations in EHT. In both EHT and LV, I Ks block by HMR-1556 prolonged AP duration at 90% repolarization slightly in the combined presence of E-4031 and isoprenaline., Conclusions: EHT from hiPSC-CMs shows a lower repolarization reserve than human LV working myocardium and could thereby serve as a sensitive and specific human-based model for repolarization studies and arrhythmia, similar to Purkinje fibers. In both human LV and EHT, I Ks only contributed to repolarization under adrenergic stimulation., (© 2018 American Heart Association, Inc.)

Energy metabolism is a key aspect of cardiomyocyte biology. Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) are a promising tool for biomedical application, but they are immature and have not undergone metabolic maturation related to early postnatal development. To assess whether cultivation of hiPSC-CMs in 3D engineered heart tissue format leads to maturation of energy metabolism, we analyzed the mitochondrial and metabolic state of 3D hiPSC-CMs and compared it with 2D culture. 3D hiPSC-CMs showed increased mitochondrial mass, DNA content, and protein abundance (proteome). While hiPSC-CMs exhibited the principal ability to use glucose, lactate, and fatty acids as energy substrates irrespective of culture format, hiPSC-CMs in 3D performed more oxidation of glucose, lactate, and fatty acid and less anaerobic glycolysis. The increase in mitochondrial mass and DNA in 3D was diminished by pharmacological reduction of contractile force. In conclusion, contractile work contributes to metabolic maturation of hiPSC-CMs., (Copyright © 2018 The Author(s). Published by Elsevier Inc. All rights reserved.)

The proteinase 3 (PR3)-positive anti-neutrophil cytoplasmic autoantibody (ANCA)-associated vasculitis (AAV) granulomatosis with polyangiitis (GPA) has been associated with chronic nasal S. aureus carriage, which is a risk factor for disease relapse. The present study was aimed at comparing the genetic make-up of S. aureus isolates from PR3-ANCA-positive GPA patients with that of isolates from patients suffering from myeloperoxidase (MPO)-ANCA-positive AAV, and isolates from healthy controls. Based on a DNA microarray-based approach, we show that not only PR3-ANCA-positive GPA patients, but also MPO-ANCA-positive AAV patients mainly carried S. aureus types that are prevalent in the general population. Nonetheless, our data suggests that MPO-ANCA-associated S. aureus isolates may be distinct from healthy control- and PR3-ANCA-associated isolates. Furthermore, several genetic loci of S. aureus are associated with either PR3-ANCA- or MPO-ANCA-positive AAV, indicating a possible role for pore-forming toxins, such as leukocidins, in PR3-ANCA-positive GPA. Contrary to previous studies, no association between AAV and superantigens was detected. Our findings also show that a lowered humoral immune response to S. aureus is common for PR3-ANCA- and MPO-ANCA-positive AAV. Altogether, our observations imply that the presence or absence of particular virulence genes of S. aureus isolates from AAV patients contributes to disease progression and/or relapse.

Since the advent of the generation of human induced pluripotent stem cells (hiPSCs), numerous protocols have been developed to differentiate hiPSCs into cardiomyocytes and then subsequently assess their ability to recapitulate the properties of adult human cardiomyocytes. However, hiPSC-derived cardiomyocytes (hiPSC-CMs) are often assessed in single-cell assays. A shortcoming of these assays is the limited ability to characterize the physiological parameters of cardiomyocytes, such as contractile force, due to random orientations. This protocol describes the differentiation of cardiomyocytes from hiPSCs, which occurs within 14 d. After casting, cardiomyocytes undergo 3D assembly. This produces fibrin-based engineered heart tissues (EHTs)-in a strip format-that generate force under auxotonic stretch conditions. 10-15 d after casting, the EHTs can be used for contractility measurements. This protocol describes parallel expansion of hiPSCs; standardized generation of defined embryoid bodies, growth factor and small-molecule-based cardiac differentiation; and standardized generation of EHTs. To carry out the protocol, experience in advanced cell culture techniques is required.

Pike, Robert M. (University of Texas Southwestern Medical School, Dallas), Mary L. Schulze, and Cleo H. Chandler. Agglutinating and precipitating capacity of rabbit anti-Salmonella typhosa gammaG and gammaM antibodies during prolonged immunization. J. Bacteriol. 92:880-886. 1966.-Antibody produced in rabbits immunized with acetone-dried typhoid bacilli was followed over a period of 445 days by agglutination and by quantitative precipitation. Repeated injections of vaccine resulted in suppression of antibody titers. Both gammaG and gammaM antibodies were rapidly increased by booster injections after rest periods during which titers had decreased to low levels. The O agglutinin titers and the amount of antibody protein, as determined by precipitation with endotoxin, generally were parallel, except in serum specimens in which unusually large proportions of the agglutinating activity were found in the gammaG fraction. These exceptions were explained by the greater agglutinating capacity of the gammaM. Endotoxin precipitated about 10 times as much antibody from gammaG preparations as it did from gammaM fractions of equivalent agglutinating strength. A much higher proportion of the serological activity, therefore, was found in the gammaG fractions when antibody was measured by precipitation than when agglutination was used as the measure of activity.

Brucella abortus L-forms were induced by 5.0 or 10.0 mug of penicillin/ml in a broth medium containing 0.3 m sucrose, and in a semisolid medium containing 10% calf serum and 20.0, 40.0, or 60.0 mug of penicillin/ml. After 96 hr of incubation, L-forms of various sizes and shapes were observed. Basic structures of the L-forms were similar whether induced in liquid or semisolid medium. L-forms had two "unit" membranes, each consisting of two outer dense layers separated by a lucent layer. A few large, irregularly shaped organisms in penicillin-treated broth cultures had additional surface material and were referred to as "transitional" forms. In contrast with L-forms, the bacterial cells were fairly uniform in size and shape, were smaller, and had a more complex cell wall structure. Small bodies limited by a "unit" membrane were present within and around numerous L-forms from liquid and semisolid medium cultures. Other internal membranous structures were also seen in some L-forms. Most Brucella L-forms described in this paper reverted to bacteria in the absence of penicillin and were structurally characteristic of unstable L-forms.

Thin sections of hamster kidney tissue cultures were examined by electron microscopy over a 7-day period after infection with Brucella abortus 3183. Numerous bacteria and structures resembling L-forms were present both intracellularly and extracellularly after the first 24 hr of infection. Most intracellular microorganisms were enclosed by a cytoplasmic membrane, but in a few instances no limiting membrane was detected. After 4 to 7 days, fewer microorganisms were present, and most normal-appearing bacteria were intracellular, particularly in antibiotic-treated cultures. Structures typical of Brucella L-forms were extracellular at the latter time intervals. Several structures were observed in cells from infected cultures whose relationship to the infecting organisms is not known. These consisted of various membranous structures within cytoplasmic vacuoles, myelin-like structures surrounding occasional intracellular organisms, and small bodies present within vacuoles and extracellularly. The latter structures observed throughout the experimental period appeared to occur more frequently as the duration of the infection increased.