Prolonged presence of SARS-CoV-2 in a COVID-19 case with rheumatoid arthritis taking iguratimod treated with ciclesonide.

We report a coronavirus disease 2019 (COVID-19) case with rheumatoid arthritis taking iguratimod. The patient who continued iguratimod therapy without dose reduction was treated with ciclesonide had an uneventful clinical course, but prolonged detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was observed after resolution of symptoms. The effects of disease-modifying antirheumatic drugs (DMARDs) and ciclesonide on clinical course and viral shedding remain unknown and warrant further investigation.

Functional glycine receptor in cultured human keratinocytes.

We previously demonstrated that a series of neural receptors, those play a crucial role in nerve system, also expressed in epidermal keratinocytes. We also demonstrated that topical application of glycine on hairless mice skin after the barrier disruption also accelerated the barrier recovery and it was blocked by the strychnine. Glycine is known as one of the most important inhibitory neurotransmitters in the mammalian central nervous system. Thus, we hypothesized that glycine receptor also functionally expressed in the epidermal keratinocytes. In the present study, we first studied the expression of glycine receptor message in cultured human keratinocytes. Then we demonstrate for the first time the existence of a functional receptor with electrophysiological properties of glycine receptors in cultured human epidermal keratinocytes. Finally, we demonstrated immune-histochemical study against anti-glycine receptor subunits in human skin. Results of the present study might indicate new target of the clinical dermatology.

Coculture system of keratinocytes and dorsal-root-ganglion-derived cells for screening neurotrophic factors involved in guidance of neuronal axon growth in the skin.

The density of peripheral nerve fibres is increased in atopic dermatitis. Moreover, reduction in the fibres in a mouse model of atopic dermatitis reduces scratching behaviour. Thus, regulation of nerve fibre extension could be an effective strategy to reduce itching in pruritus dermatosis. In this study, we established a new coculture system of keratinocytes and dorsal-root-ganglion-derived cells using an apparatus, AXIS(™) , which consists of two different channels connected via a set of microgrooves, through which signalling molecules and axons, but not living cells, can pass. When we seeded keratinocytes in one chamber, extension of nerve fibres was observed from dorsal root ganglion cells seeded in the other chamber. Addition of anti-BDNF antibody in the keratinocyte-seeded chamber significantly reduced the extension. Application of Semaphorin 3A also reduced the extension by approximately 50%. We suggest that this coculture system may be useful for screening of anti-itching drugs.

Frontiers in epidermal barrier homeostasis--an approach to mathematical modelling of epidermal calcium dynamics.

Intact epidermal barrier function is crucial for survival and is associated with the presence of gradients of both calcium ion concentration and electric potential. Although many molecules, including ion channels and pumps, are known to contribute to maintenance of these gradients, the mechanisms involved in epidermal calcium ion dynamics have not been clarified. We have established that a variety of neurotransmitters and their receptors, originally found in the brain, are expressed in keratinocytes and are also associated with barrier homeostasis. Moreover, keratinocytes and neurons show some similarities of electrochemical behaviour. As mathematical modelling and computer simulation have been employed to understand electrochemical phenomena in brain science, we considered that a similar approach might be applicable to describe the dynamics of epidermal electrochemical phenomena associated with barrier homeostasis. Such methodology would also be potentially useful to address a number of difficult problems in clinical dermatology, such as ageing and itching. Although this work is at a very early stage, in this essay, we discuss the background to our approach and we present some preliminary results of simulation of barrier recovery.

Low environmental humidity induces synthesis and release of cortisol in an epidermal organotypic culture system.

Dry environmental conditions induce a variety of skin pathologies and a recent report indicating that cortisol synthesis in epidermis was increased during wound healing led us to hypothesize that environmental dryness might induce increased cortisol secretion in epidermis. Therefore, we incubated a skin equivalent model under dry (relative humidity: less than 10%) and humid (relative humidity: approximately 100%) conditions for 48 hours and evaluated cortisol secretion and mRNA levels of cortisol-synthesizing enzyme (steroid 11ß-hydroxylase, CYP11B1) and IL-1ß. Cortisol secretion was increased threefold, and CYP11B1 and IL-1ß mRNAs were increased 38-fold and sixfold, respectively, in the dry condition versus the humid condition. Occlusion with a water-impermeable plastic membrane partially blocked the increases in cortisol secretion and CYP11B1 and IL-1ß mRNA expression in the dry condition. Thus, environmental dryness might induce increased cortisol secretion in epidermis of diseased skin characterized by epidermal barrier dysfunction, potentially influencing mental state and systemic physiology.

Oxytocin is expressed in epidermal keratinocytes and released upon stimulation with adenosine 5'-[γ-thio]triphosphate in vitro.

Oxytocin is a neuropeptide produced primarily in the hypothalamus and is best known for its roles in parturition and lactation. It also influences behaviour, memory and mental state. Recent studies have suggested a variety of roles for oxytocin in peripheral tissues, including skin. Here we show that oxytocin is expressed in human skin. Immunohistochemical studies showed that oxytocin and its carrier protein, neurophysin I, are predominantly localized in epidermis. RT-PCR confirmed the expression of oxytocin in both skin and cultured epidermal keratinocytes. We also show that oxytocin is released from keratinocytes after application of adenosine 5'-[γ-thio]triphosphate (ATPγS, a stable analogue of ATP) in a dose-dependent manner. The ATPγS-induced oxytocin release was inhibited by removal of extracellular calcium, or by the P2X receptor antagonist 2',3'-O-(2,4,6-trinitrophenyl)adenosine 5'-triphosphate (TNP-ATP). These results suggest that oxytocin is produced in human epidermal keratinocytes and is released in response to calcium influx via P2X receptors.

Multiple Secondary Healthcare-Associated Infections Due to Carbapenem-Resistant Organisms in a Critically Ill COVID-19 Patient on Extensively Prolonged Venovenous Extracorporeal Membrane Oxygenation Support-A Case Report.

Patients with severe Coronavirus disease 2019 (COVID-19) are at high risk for secondary infection with multidrug-resistant organisms (MDROs). Secondary infections contribute to a more severe clinical course and longer intensive care unit (ICU) stays in patients with COVID-19. A man in his 60s was admitted to the ICU at a university hospital for severe COVID-19 pneumonia requiring mechanical ventilation. His respiratory condition worsened further due to persistent bacteremia caused by imipenem-non-susceptible Klebsiella aerogenes and he required VV-ECMO. Subsequently, he developed a catheter-related bloodstream infection (CRBSI) due to Candida albicans, ventilator-associated pneumonia (VAP) due to multidrug-resistant Pseudomonas aeruginosa (MDRP), and a perianal abscess due to carbapenem-resistant K. aerogenes despite infection control procedures that maximized contact precautions and the absence of MDRO contamination in the patient's room environment. He was decannulated from VV-ECMO after a total of 72 days of ECMO support, and was eventually weaned off ventilator support and discharged from the ICU on day 138. This case highlights the challenges of preventing, diagnosing, and treating multidrug-resistant organisms and healthcare-associated infections (HAIs) in the critical care management of severe COVID-19. In addition to the stringent implementation of infection prevention measures, a high index of suspicion and a careful evaluation of HAIs are required in such patients.

In Silico Analysis of ACE Inhibitory Peptides from Chloroplast Proteins of Red Alga Grateloupia asiatica.

Inhibition of angiotensin I-converting enzyme (ACE) is one of the key factors to repress high blood pressure. Although many studies have been reported that seaweed protein hydrolysates showed the ACE inhibitory activity, the comprehensive understanding of the relationship was still unclear. In this study, we employed chloroplast genome for in silico analysis and compared it with in vitro experiments. We first extracted water-soluble proteins (WSP) from red alga Grateloupia asiatica, which contained mainly PE, PC, APC, and Rbc, and prepared WSP hydrolysate by thermolysin, resulting that the hydrolysate showed ACE inhibitory activity. Then, we determined the complete chloroplast genome of G. asiatica (187,518 bp: 206 protein-coding genes, 29 tRNA, and 3 rRNA) and clarified the amino acid sequences of main WSP, i.e., phycobiliproteins and Rubisco, to perform in silico analysis. Consequently, 190 potential ACE inhibitory peptides existed in the main WSP sequences, and 21 peptides were obtained by in silico thermolysin digestion. By comparing in vitro and in silico analyses, in vitro ACE inhibitory activity was correlated to the IC50 value from in silico digestion. Therefore, in silico approach provides insight into the comprehensive understanding of the potential bioactive peptides from seaweed proteins.

Highly sensitive and high-throughput analysis of plant hormones using MS-probe modification and liquid chromatography-tandem mass spectrometry: an application for hormone profiling in Oryza sativa.

We have developed a highly sensitive and high-throughput method for the simultaneous analysis of 43 molecular species of cytokinins, auxins, ABA and gibberellins. This method consists of an automatic liquid handling system for solid phase extraction and ultra-performance liquid chromatography (UPLC) coupled with a tandem quadrupole mass spectrometer (qMS/MS) equipped with an electrospray interface (ESI; UPLC-ESI-qMS/MS). In order to improve the detection limit of negatively charged compounds, such as gibberellins, we chemically derivatized fractions containing auxin, ABA and gibberellins with bromocholine that has a quaternary ammonium functional group. This modification, that we call 'MS-probe', makes these hormone derivatives have a positive ion charge and permits all compounds to be measured in the positive ion mode with UPLC-ESI-qMS/MS in a single run. Consequently, quantification limits of gibberellins increased up to 50-fold. Our current method needs <100 mg (FW) of plant tissues to determine phytohormone profiles and enables us to analyze >180 plant samples simultaneously. Application of this method to plant hormone profiling enabled us to draw organ distribution maps of hormone species in rice and also to identify interactions among the four major hormones in the rice gibberellin signaling mutants, gid1-3, gid2-1 and slr1. Combining the results of hormone profiling data with transcriptome data in the gibberellin signaling mutants allows us to analyze relationships between changes in gene expression and hormone metabolism.

Regulation of cytokinin biosynthesis, compartmentalization and translocation.

Cytokinins, a group of mobile phytohormones, play an important role in plant growth and development, and their activity is finely controlled by environmental factors in the control of morphogenic and metabolic adaptations. Inorganic nitrogen sources, such as nitrate, are a major factor regulating gene expression of adenosine phosphate-isopentenyltransferase (IPT), a key enzyme of cytokinin biosynthesis. Modulation of IPT and macronutrient transporter gene expression in response to nitrate, sulphate and phosphate, and cytokinin-dependent repression of the transporter genes suggest that cytokinins play a critical role in balancing acquisition and distribution of macronutrients. Biased distribution of trans-zeatin (tZ)-type cytokinins in xylem and N(6)-(Delta(2)-isopentenyl)adenine (iP)-type cytokinins in phloem saps suggest that, in addition to acting as local signals, cytokinins communicate acropetal and systemic long-distance signals, and that structural side chain variations mediate different biological messages. The compartmentalization of tZ- and iP-type cytokinins implies the involvement of a selective transport system. Recent studies have raised the possibility of subsets of the purine permease family as a transporter of cytokinin nucleobases and equilibrative nucleoside transporters (ENT) for cytokinin nucleosides. These biochemical and transgenic data suggest that AtENT6, an Arabidopsis ENT, could also participate in cytokinin nucleoside transport with a preference for iP riboside in vascular tissue.

Interactions between nitrogen and cytokinin in the regulation of metabolism and development.

Inorganic nitrogen is a substrate for nitrogen assimilation and also functions as a signal triggering widespread changes in gene expression that modulate metabolism and development. To integrate the actions of the nitrogen signal at the whole plant level, plants use multiple signaling routes that communicate internal and external nitrogen status. One route depends on nitrate itself and one uses cytokinin as a messenger. Recent genome-wide research has shown that the nitrate-specific signal regulates a wide variety of metabolic processes including nitrogen and carbon metabolism, and cytokinin biosynthesis. Cytokinin-mediated signaling is related to the control of development, protein synthesis and acquisition of macronutrients. The coordination and interaction of both regulatory pathways is important for normal plant growth under variable nitrogen supply conditions.

How does epidermal pathology interact with mental state?

The hypothesis is presented that human emotional state is influenced by epidermal pathology via the release from epidermal keratinocytes of a wide variety of chemical mediators (including neurotransmitters) that act on the brain. It has long been recognized that epidermal keratinocytes play a key role in the function of the stratum corneum as an impermeable barrier, and that skin diseases such as atopic dermatitis and psoriasis, which cause itching, sleep disturbance and concern over appearance, are associated with depression and anxiety. On the other hand, epidermal keratinocytes are known to produce and release multiple cytokines and chemical mediators in response to barrier impairment or insult, such as environmental dryness or UV radiation. Elevation of plasma cytokines is associated with depression in cancer patients. Serum levels of oxytocin and glucocorticoid have been shown to influence mental state, and a recent study showed that glucocorticoid is generated in injured epidermis. Thus, there are multiple plausible pathways through which changes in skin can affect emotional state.

Side-chain modification of cytokinins controls shoot growth in Arabidopsis.

Cytokinins (CKs), a class of plant hormones, are central regulators of plant growth and development. Based on numerous physiological and genetic studies, the quantitative regulation of cytokinin levels is the major mechanism regulating cytokinin action in diverse developmental processes. Here, we identified a different mechanism with which the physiological function of CK is modulated through side-chain modification (trans-hydroxylation). The trans-hydroxylation that forms trans-zeatin (tZ)-type CK from N(6)-(Δ(2)-isopentenyl)adenine (iP)-type CK is catalyzed by the cytochrome P450 enzymes CYP735A1 and CYP735A2 in Arabidopsis. Deficiency in trans-hydroxylation activity results in dramatic retardation of shoot growth without affecting total CK quantity, while augmentation of the activity enhances shoot growth. Application of exogenous tZ but not iP recovers the wild-type phenotype in the mutants, indicating that trans-hydroxylation modifies the physiological function of CK. We propose that the control of cytokinin function by side-chain modification is crucial for shoot growth regulation in plants.

Uniconazole, a cytochrome P450 inhibitor, inhibits trans-zeatin biosynthesis in Arabidopsis.

Cytokinin (CK) is a plant hormone that plays important regulatory roles in many aspects of plant growth and development. Although functions of CK and its biosynthesis pathway have been studied extensively, there is still no efficient biosynthesis inhibitor, which would be useful for studying CK from a chemical genetic approach. Here, CK biosynthesis inhibitor candidates were searched for using a systematic approach. In silico screening of candidates were carried out using genome-wide gene expression profiles and prediction of target sites using global CK accumulation profile analysis. As a result of these screenings, it was found that uniconazole, a well known inhibitor of cytochrome P450 monooxygenase, prevents the biosynthesis of trans-zeatin, and that its target is CYP735As in Arabidopsis.

Ryanodine receptors are expressed in epidermal keratinocytes and associated with keratinocyte differentiation and epidermal permeability barrier homeostasis.

Ryanodine receptors (RyRs) have an important role as calcium channels in the regulation of intracellular calcium levels in the nervous system and muscle. In the present study, we investigated the expression of RyR in human epidermis. Immunohistochemical studies and reverse transcription-PCR indicated the expression of RyR type 1, 2, and 3 proteins in epidermal keratinocytes. The expression level of each RyR subtype was higher in differentiating keratinocytes than in proliferative cells. We also demonstrated the functional expression of RyR by calcium imaging. In cultured human keratinocytes, application of the RyR agonist 4-chloro-m-cresol (CMC) induced elevation of the intracellular calcium concentration, and co-application of the RyR antagonist 1,1'-diheptyl-4,4'-bipyridinium dibromide (DHBP) blocked the elevation. Application of CMC accelerated keratinocyte differentiation in vitro. On the other hand, topical application of CMC after tape-stripping of hairless mouse skin delayed barrier recovery, whereas application of an RyR antagonist, dantrolene or DHBP, accelerated the barrier recovery. These results suggest that RyR expressed in epidermal keratinocytes is associated with both differentiation of keratinocytes and epidermal barrier homeostasis.

Auxin controls local cytokinin biosynthesis in the nodal stem in apical dominance.

In intact plants, the shoot apex grows predominantly and inhibits outgrowth of axillary buds. After decapitation of the shoot apex, outgrowth of axillary buds begins. This phenomenon is called an apical dominance. Although the involvement of auxin, which represses outgrowth of axillary buds, and cytokinin (CK), which promotes outgrowth of axillary buds, has been proposed, little is known about the underlying molecular mechanisms. In the present study, we demonstrated that auxin negatively regulates local CK biosynthesis in the nodal stem by controlling the expression level of the pea (Pisum sativum L.) gene adenosine phosphate-isopentenyltransferase (PsIPT), which encodes a key enzyme in CK biosynthesis. Before decapitation, PsIPT1 and PsIPT2 transcripts were undetectable; after decapitation, they were markedly induced in the nodal stem along with accumulation of CK. Expression of PsIPT was repressed by the application of indole-3-acetic acid (IAA). In excised nodal stem, PsIPT expression and CK levels also increased under IAA-free conditions. Furthermore, beta-glucuronidase expression, under the control of the PsIPT2 promoter region in transgenic Arabidopsis, was repressed by an IAA. Our results indicate that in apical dominance one role of auxin is to repress local biosynthesis of CK in the nodal stem and that, after decapitation, CKs, which are thought to be derived from the roots, are locally biosynthesized in the nodal stem rather than in the roots.