New-Onset Diabetes Mellitus After Transplantation in a Cynomolgus Macaque (Macaca fasicularis)

A 5.5-y-old intact male cynomolgus macaque (Macaca fasicularis) presented with inappetence and weight loss 57 d after heterotopic heart and thymus transplantation while receiving an immunosuppressant regimen consisting of tacrolimus, mycophenolate mofetil, and methylprednisolone to prevent graft rejection. A serum chemistry panel, a glycated hemoglobin test, and urinalysis performed at presentation revealed elevated blood glucose and glycated hemoglobin (HbA1c) levels (727 mg/dL and 10.1%, respectively), glucosuria, and ketonuria. Diabetes mellitus was diagnosed, and insulin therapy was initiated immediately. The macaque was weaned off the immunosuppressive therapy as his clinical condition improved and stabilized. Approximately 74 d after discontinuation of the immunosuppressants, the blood glucose normalized, and the insulin therapy was stopped. The animal''s blood glucose and HbA1c values have remained within normal limits since this time. We suspect that our macaque experienced new-onset diabetes mellitus after transplantation, a condition that is commonly observed in human transplant patients but not well described in NHP. To our knowledge, this report represents the first documented case of new-onset diabetes mellitus after transplantation in a cynomolgus macaque.Abbreviations: NODAT, new-onset diabetes mellitus after transplantationNew-onset diabetes mellitus after transplantation (NODAT, formerly known as posttransplantation diabetes mellitus) is an important consequence of solid-organ transplantation in humans.^{7-10,15,17,19,21,25-28,31,33,34,37,38,42} A variety of risk factors have been identified including increased age, sex (male prevalence), elevated pretransplant fasting plasma glucose levels, and immunosuppressive therapy.^{7-10,15,17,19,21,25-28,31,33,34,37,38,42} The relationship between calcineurin inhibitors, such as tacrolimus and cyclosporin, and the development of NODAT is widely recognized in human medicine.^{7-10,15,17,19,21,25-28,31,33,34,37,38,42} Cynomolgus macaques (Macaca fasicularis) are a commonly used NHP model in organ transplantation research. Cases of natural and induced diabetes of cynomolgus monkeys have been described in the literature;^14,43,45 however, NODAT in a macaque model of solid-organ transplantation has not been reported previously to our knowledge.     

Calcineurin B-Like Protein-Interacting Protein Kinase CIPK21 Regulates Osmotic and Salt Stress Responses in Arabidopsis

The role of calcium-mediated signaling has been extensively studied in plant responses to abiotic stress signals. Calcineurin B-like proteins (CBLs) and CBL-interacting protein kinases (CIPKs) constitute a complex signaling network acting in diverse plant stress responses. Osmotic stress imposed by soil salinity and drought is a major abiotic stress that impedes plant growth and development and involves calcium-signaling processes. In this study, we report the functional analysis of CIPK21, an Arabidopsis (Arabidopsis thaliana) CBL-interacting protein kinase, ubiquitously expressed in plant tissues and up-regulated under multiple abiotic stress conditions. The growth of a loss-of-function mutant of CIPK21, cipk21, was hypersensitive to high salt and osmotic stress conditions. The calcium sensors CBL2 and CBL3 were found to physically interact with CIPK21 and target this kinase to the tonoplast. Moreover, preferential localization of CIPK21 to the tonoplast was detected under salt stress condition when coexpressed with CBL2 or CBL3. These findings suggest that CIPK21 mediates responses to salt stress condition in Arabidopsis, at least in part, by regulating ion and water homeostasis across the vacuolar membranes.Drought and salinity cause osmotic stress in plants and severely affect crop productivity throughout the world. Plants respond to osmotic stress by changing a number of cellular processes (Xiong et al., 1999; Xiong and Zhu, 2002; Bartels and Sunkar, 2005; Boudsocq and Lauriére, 2005). Some of these changes include activation of stress-responsive genes, regulation of membrane transport at both plasma membrane (PM) and vacuolar membrane (tonoplast) to maintain water and ionic homeostasis, and metabolic changes to produce compatible osmolytes such as Pro (Stewart and Lee, 1974; Krasensky and Jonak, 2012). It has been well established that a specific calcium (Ca²⁺) signature is generated in response to a particular environmental stimulus (Trewavas and Malhó, 1998; Scrase-Field and Knight, 2003; Luan, 2009; Kudla et al., 2010). The Ca²⁺ changes are primarily perceived by several Ca²⁺ sensors such as calmodulin (Reddy, 2001; Luan et al., 2002), Ca²⁺-dependent protein kinases (Harper and Harmon, 2005), calcineurin B-like proteins (CBLs; Luan et al., 2002; Batistič and Kudla, 2004; Pandey, 2008; Luan, 2009; Sanyal et al., 2015), and other Ca²⁺-binding proteins (Reddy, 2001; Shao et al., 2008) to initiate various cellular responses.Plant CBL-type Ca²⁺ sensors interact with and activate CBL-interacting protein kinases (CIPKs) that phosphorylate downstream components to transduce Ca²⁺ signals (Liu et al., 2000; Luan et al., 2002; Batistič and Kudla, 2004; Luan, 2009). In several plant species, multiple members have been identified in the CBL and CIPK family (Luan et al., 2002; Kolukisaoglu et al., 2004; Pandey, 2008; Batistič and Kudla, 2009; Weinl and Kudla, 2009; Pandey et al., 2014). Involvement of specific CBL-CIPK pair to decode a particular type of signal entails the alternative and selective complex formation leading to stimulus-response coupling (D’Angelo et al., 2006; Batistič et al., 2010).Several CBL and CIPK family members have been implicated in plant responses to drought, salinity, and osmotic stress based on genetic analysis of Arabidopsis (Arabidopsis thaliana) mutants (Zhu, 2002; Cheong et al., 2003, 2007; Kim et al., 2003; Pandey et al., 2004, 2008; D’Angelo et al., 2006; Qin et al., 2008; Tripathi et al., 2009; Held et al., 2011; Tang et al., 2012; Drerup et al., 2013; Eckert et al., 2014). A few CIPKs have also been functionally characterized by gain-of-function approach in crop plants such as rice (Oryza sativa), pea (Pisum sativum), and maize (Zea mays) and were found to be involved in osmotic stress responses (Mahajan et al., 2006; Xiang et al., 2007; Yang et al., 2008; Tripathi et al., 2009; Zhao et al., 2009; Cuéllar et al., 2010).In this report, we examined the role of the Arabidopsis CIPK21 gene in osmotic stress response by reverse genetic analysis. The loss-of-function mutant plants became hypersensitive to salt and mannitol stress conditions, suggesting that CIPK21 is involved in the regulation of osmotic stress response in Arabidopsis. These findings are further supported by an enhanced tonoplast targeting of the cytoplasmic CIPK21 through interaction with the vacuolar Ca²⁺ sensors CBL2 and CBL3 under salt stress condition.     

Integrating Image-Based Phenomics and Association Analysis to Dissect the Genetic Architecture of Temporal Salinity Responses in Rice

Salinity affects a significant portion of arable land and is particularly detrimental for irrigated agriculture, which provides one-third of the global food supply. Rice (Oryza sativa), the most important food crop, is salt sensitive. The genetic resources for salt tolerance in rice germplasm exist but are underutilized due to the difficulty in capturing the dynamic nature of physiological responses to salt stress. The genetic basis of these physiological responses is predicted to be polygenic. In an effort to address this challenge, we generated temporal imaging data from 378 diverse rice genotypes across 14 d of 90 mm NaCl stress and developed a statistical model to assess the genetic architecture of dynamic salinity-induced growth responses in rice germplasm. A genomic region on chromosome 3 was strongly associated with the early growth response and was captured using visible range imaging. Fluorescence imaging identified four genomic regions linked to salinity-induced fluorescence responses. A region on chromosome 1 regulates both the fluorescence shift indicative of the longer term ionic stress and the early growth rate decline during salinity stress. We present, to our knowledge, a new approach to capture the dynamic plant responses to its environment and elucidate the genetic basis of these responses using a longitudinal genome-wide association model.Nearly one-third of the 54 million ha of the highly saline soils in the world are located in South and Southeast Asia. Rice (Oryza sativa), which is the primary source of calories and protein for these two regions, is very sensitive to salinity stress, with even moderate salinity levels known to decrease yields by 50% (Zeng et al., 2002). Projected sea level rise due to climate change is expected to increase saltwater ingress in coastal rice-growing regions of South and Southeast Asia. Therefore, development of salt-tolerant rice cultivars is essential to maintain rice productivity in the salinity-affected regions globally.Salt tolerance, defined as the ability to maintain growth and productivity in saline conditions, is a complex polygenic trait that may be influenced by distinct physiological mechanisms (Munns et al., 1982; Munns and Termaat, 1986; Cheeseman, 1988; Munns and Tester, 2008; Horie et al., 2012; for a comprehensive review of genes involved in salinity tolerance in rice, see Negrão et al., 2011) At the cellular level, plants respond to saline conditions in two phases, namely an osmotic (shoot ion independent) and an ionic stress phase, which can occur in an overlapping manner with varying intensity during the course of salinity stress (Munns and Termaat, 1986; Munns, 2002; Munns and James, 2003; Munns and Tester, 2008; Horie et al., 2012). During the osmotic stress phase, which occurs soon after the onset of salinity, the reduction in external osmotic potential disrupts water uptake and impedes cell expansion, which, at the whole plant level, leads to reduced growth rate (Matsuda and Riazi, 1981; Munns and Passioura, 1984; Rawson and Munns, 1984; Azaizeh and Steudle, 1991; Fricke and Peters, 2002; Fricke, 2004; Boursiac et al., 2005). As salinity stress persists over several days and weeks, sodium ions (Na⁺) accumulate to toxic levels, resulting in cell death and precocious leaf senescence (Lutts and Bouharmont, 1996; Munns, 2002; Munns and James, 2003; Ghanem et al., 2008). This is typically observed during the ionic phase of the salinity response (Munns, 2002; Munns and James, 2003; Munns and Tester, 2008). Plants possess distinct mechanisms to adapt to these osmotic and ionic stresses that are controlled by a suite of genes (Apse et al., 1999; Carvajal et al., 1999; Halfter et al., 2000; Ishitani et al., 2000; Shi et al., 2000; Zeng and Shannon, 2000; Rus et al., 2001; Berthomieu et al., 2003; Martínez-Ballesta et al., 2003; Boursiac et al., 2005, 2008; Ren et al., 2005; Huang et al., 2006; Davenport et al., 2007; Obata et al., 2007; Székely et al., 2008; Horie et al., 2011; Rivandi et al., 2011; Asano et al., 2012; Munns et al., 2012; Latz et al., 2013; Schmidt et al., 2013; Campo et al., 2014; Choi et al., 2014; Liu et al., 2014). The genetic basis of temporal adaptive responses to salinity stress remains to be explored in rice and other crops. This is primarily due to challenges in capturing the dynamic physiological responses to salinity for a large number of genotypes in a nondestructive manner. Manual phenotyping to detect incremental changes in growth rate during the osmotic stress and slight shifts in leaf color due to ionic stress is difficult to quantify for a large number of genotypes.In rice, at least one major quantitative trait loci (QTL; saltol) for salinity tolerance has been characterized based on end point measurements of biomass, senescence/injury, and Na⁺ and K⁺ concentrations (Bonilla et al., 2002; Lin et al., 2004; Thomson et al., 2010). SHOOT K⁺ CONTENT1 (SKC1) is the causative gene underlying the saltol region. SKC1 encodes a Na⁺-selective high-affinity potassium transporter that regulates Na⁺/K⁺ homeostasis during salinity stress (Ren et al., 2005). High levels of Na⁺ displace cellular K⁺, an essential element for several enzymatic reactions and physiological processes (Gierth and Mäser, 2007). The ability to maintain cellular K⁺ levels during salinity through the action of Na⁺-selective potassium transporters or Na⁺/H⁺ antiporters is a well-characterized tolerance mechanism in cereals including rice (Ren et al., 2005; Sunarpi et al., 2005; Huang et al., 2006; Møller et al., 2009; Mian et al., 2011; Munns et al., 2012).Numerous studies have utilized conventional linkage mapping to identify QTL for morphological and physiological responses to salinity in rice using discrete end point measurements (Bonilla et al., 2002; Lin et al., 2004; Ren et al., 2005; Negrão et al., 2011; Wang et al., 2012). However, the physiological adaptation to saline conditions is a complex continuous process that develops over time. While some accessions will exhibit similar end point phenotypic values, the genetic and physiological mechanisms giving rise to the similar phenotypes may be very different and the growth trajectories throughout the experiment may be distinct. Although single time point studies have yielded important information regarding the genetic basis of salinity tolerance, such approaches are too simple to reveal the genetic architecture of stress adaptation. With the advent of high-throughput image-based phenotyping platforms, it is now feasible to quantify dynamic responses during the stress treatment for a large number of genotypes (Berger et al., 2010; Golzarian et al., 2011; Chen et al., 2014; Honsdorf et al., 2014).Image-based phenotyping has been combined with genome-wide association studies (GWAS) and linkage mapping to examine the genetic basis of complex developmental processes (Busemeyer et al., 2013; Moore et al., 2013; Topp et al., 2013; Slovak et al., 2014; Würschum et al., 2014; Yang et al., 2014; Bac-Molenaar et al., 2015). Moreover, the introduction of the time axis provides a better understanding of the physiological processes underlying complex stress and developmental responses compared with single end point measurements (Zhang et al., 2012; Moore et al., 2013; Brown et al., 2014; Chen et al., 2014; Slovak et al., 2014; Bac-Molenaar et al., 2015). However, to date, no studies have implemented an association mapping approach using image-derived phenotypes to address the genetic basis of dynamic stress responses in plants. Image-based phenotyping offers several advantages over conventional phenotyping: (1) quantitative measurements can be recorded over discrete time points to capture morphological and physiological responses in a nondestructive manner, and (2) the use of various types of spectral imaging address phenotypes that are not detectable to the human eye such as chlorophyll fluorescence and leaf water content. Integrating dynamic phenotypic data and association mapping has the potential to query genetic diversity across hundreds of accessions for complex traits and provides much higher resolution compared with conventional linkage mapping. Here, we explored the dynamic growth and chlorophyll responses to salinity of a diverse set of rice accessions using high-throughput visible and fluorescence imaging. To assess the genetic basis of plant growth in saline conditions, a logistic model was used to describe the temporal growth responses and was incorporated into the statistical framework necessary for association mapping. Coupled with temporal fluorescence imaging, we present, to our knowledge, new insights into the genetic architecture of osmotic and ionic responses during salinity stress in rice.     

Plasma membrane poration by opioid neuropeptides: a possible mechanism of pathological signal transduction

Neuropeptides induce signal transduction across the plasma membrane by acting through cell-surface receptors. The dynorphins, endogenous ligands for opioid receptors, are an exception; they also produce non-receptor-mediated effects causing pain and neurodegeneration. To understand non-receptor mechanism(s), we examined interactions of dynorphins with plasma membrane. Using fluorescence correlation spectroscopy and patch-clamp electrophysiology, we demonstrate that dynorphins accumulate in the membrane and induce a continuum of transient increases in ionic conductance. This phenomenon is consistent with stochastic formation of giant (~2.7 nm estimated diameter) unstructured non-ion-selective membrane pores. The potency of dynorphins to porate the plasma membrane correlates with their pathogenic effects in cellular and animal models. Membrane poration by dynorphins may represent a mechanism of pathological signal transduction. Persistent neuronal excitation by this mechanism may lead to profound neuropathological alterations, including neurodegeneration and cell death.Neuropeptides are the largest and most diverse family of neurotransmitters. They are released from axon terminals and dendrites, diffuse to pre- or postsynaptic neuronal structures and activate membrane G-protein-coupled receptors. Prodynorphin (PDYN)-derived opioid peptides including dynorphin A (Dyn A), dynorphin B (Dyn B) and big dynorphin (Big Dyn) consisting of Dyn A and Dyn B are endogenous ligands for the κ-opioid receptor. Acting through this receptor, dynorphins regulate processing of pain and emotions, memory acquisition and modulate reward induced by addictive substances.^{1, 2, 3, 4} Furthermore, dynorphins may produce robust cellular and behavioral effects that are not mediated through opioid receptors.^{5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29} As evident from pharmacological, morphological, genetic and human neuropathological studies, these effects are generally pathological, including cell death, neurodegeneration, neurological dysfunctions and chronic pain. Big Dyn is the most active pathogenic peptide, which is about 10- to 100-fold more potent than Dyn A, whereas Dyn B does not produce non-opioid effects.^{16, 17, 22, 25} Big Dyn enhances activity of acid-sensing ion channel-1a (ASIC1a) and potentiates ASIC1a-mediated cell death in nanomolar concentrations^{30, 31} and, when administered intrathecally, induces characteristic nociceptive behavior at femtomolar doses.^{17, 22} Inhibition of endogenous Big Dyn degradation results in pathological pain, whereas prodynorphin (Pdyn) knockout mice do not maintain neuropathic pain.^{22, 32} Big Dyn differs from its constituents Dyn A and Dyn B in its unique pattern of non-opioid memory-enhancing, locomotor- and anxiolytic-like effects.²⁵Pathological role of dynorphins is emphasized by the identification of PDYN missense mutations that cause profound neurodegeneration in the human brain underlying the SCA23 (spinocerebellar ataxia type 23), a very rare dominantly inherited neurodegenerative disorder.^{27, 33} Most PDYN mutations are located in the Big Dyn domain, demonstrating its critical role in neurodegeneration. PDYN mutations result in marked elevation in dynorphin levels and increase in its pathogenic non-opioid activity.^{27, 34} Dominant-negative pathogenic effects of dynorphins are not produced through opioid receptors.ASIC1a, glutamate NMDA (N-methyl-d-aspartate) and AMPA (α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid)/kainate ion channels, and melanocortin and bradykinin B2 receptors have all been implicated as non-opioid dynorphin targets.^{5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 30, 31, 35, 36} Multiplicity of these targets and their association with the cellular membrane suggest that their activation is a secondary event triggered by a primary interaction of dynorphins with the membrane. Dynorphins are among the most basic neuropeptides.^{37, 38} The basic nature is also a general property of anti-microbial peptides (AMPs) and amyloid peptides that act by inducing membrane perturbations, altering membrane curvature and causing pore formation that disrupts membrane-associated processes including ion fluxes across the membrane.³⁹ The similarity between dynorphins and these two peptide groups in overall charge and size suggests a similar mode of their interactions with membranes.In this study, we dissect the interactions of dynorphins with the cell membrane, the primary event in their non-receptor actions. Using fluorescence imaging, correlation spectroscopy and patch-clamp techniques, we demonstrate that dynorphin peptides accumulate in the plasma membrane in live cells and cause a profound transient increase in cell membrane conductance. Membrane poration by endogenous neuropeptides may represent a novel mechanism of signal transduction in the brain. This mechanism may underlie effects of dynorphins under pathological conditions including chronic pain and tissue injury.     

Induction of COX-2-PGE2 synthesis by activation of the MAPK/ERK pathway contributes to neuronal death triggered by TDP-43-depleted microglia

Neuroinflammation is a striking hallmark of amyotrophic lateral sclerosis (ALS) and other neurodegenerative disorders. Previous studies have shown the contribution of glial cells such as astrocytes in TDP-43-linked ALS. However, the role of microglia in TDP-43-mediated motor neuron degeneration remains poorly understood. In this study, we show that depletion of TDP-43 in microglia, but not in astrocytes, strikingly upregulates cyclooxygenase-2 (COX-2) expression and prostaglandin E2 (PGE2) production through the activation of MAPK/ERK signaling and initiates neurotoxicity. Moreover, we find that administration of celecoxib, a specific COX-2 inhibitor, greatly diminishes the neurotoxicity triggered by TDP-43-depleted microglia. Taken together, our results reveal a previously unrecognized non-cell-autonomous mechanism in TDP-43-mediated neurodegeneration, identifying COX-2-PGE2 as the molecular events of microglia- but not astrocyte-initiated neurotoxicity and identifying celecoxib as a novel potential therapy for TDP-43-linked ALS and possibly other types of ALS.Amyotrophic lateral sclerosis (ALS) is an adult-onset neurodegenerative disease characterized by the degeneration of motor neurons in the brain and spinal cord.¹ Most cases of ALS are sporadic, but 10% are familial. Familial ALS cases are associated with mutations in genes such as Cu/Zn superoxide dismutase 1 (SOD1), TAR DNA-binding protein 43 (TARDBP) and, most recently discovered, C9orf72. Currently, most available information obtained from ALS research is based on the study of SOD1, but new studies focusing on TARDBP and C9orf72 have come to the forefront of ALS research.^{1, 2} The discovery of the central role of the protein TDP-43, encoded by TARDBP, in ALS was a breakthrough in ALS research.^{3, 4, 5} Although pathogenic mutations of TDP-43 are genetically rare, abnormal TDP-43 function is thought to be associated with the majority of ALS cases.¹ TDP-43 was identified as a key component of the ubiquitin-positive inclusions in most ALS patients and also in other neurodegenerative diseases such as frontotemporal lobar degeneration,^{6, 7} Alzheimer''s disease (AD)^{8, 9} and Parkinson''s disease (PD).^{10, 11} TDP-43 is a multifunctional RNA binding protein, and loss-of-function of TDP-43 has been increasingly recognized as a key contributor in TDP-43-mediated pathogenesis.^{5, 12, 13, 14}Neuroinflammation, a striking and common hallmark involved in many neurodegenerative diseases, including ALS, is characterized by extensive activation of glial cells including microglia, astrocytes and oligodendrocytes.^{15, 16} Although numerous studies have focused on the intrinsic properties of motor neurons in ALS, a large amount of evidence showed that glial cells, such as astrocytes and microglia, could have critical roles in SOD1-mediated motor neuron degeneration and ALS progression,^{17, 18, 19, 20, 21, 22} indicating the importance of non-cell-autonomous toxicity in SOD1-mediated ALS pathogenesis.Very interestingly, a vital insight of neuroinflammation research in ALS was generated by the evidence that both the mRNA and protein levels of the pro-inflammatory enzyme cyclooxygenase-2 (COX-2) are upregulated in both transgenic mouse models and in human postmortem brain and spinal cord.^{23, 24, 25, 26, 27, 28, 29} The role of COX-2 neurotoxicity in ALS and other neurodegenerative disorders has been well explored.^{30, 31, 32} One of the key downstream products of COX-2, prostaglandin E2 (PGE2), can directly mediate COX-2 neurotoxicity both in vitro and in vivo.^{33, 34, 35, 36, 37} The levels of COX-2 expression and PGE2 production are controlled by multiple cell signaling pathways, including the mitogen-activated protein kinase (MAPK)/ERK pathway,^{38, 39, 40} and they have been found to be increased in neurodegenerative diseases including AD, PD and ALS.^{25, 28, 32, 41, 42, 43, 44, 45, 46} Importantly, COX-2 inhibitors such as celecoxib exhibited significant neuroprotective effects and prolonged survival or delayed disease onset in a SOD1-ALS transgenic mouse model through the downregulation of PGE2 release.²⁸Most recent studies have tried to elucidate the role of glial cells in neurotoxicity using TDP-43-ALS models, which are considered to be helpful for better understanding the disease mechanisms.^{47, 48, 49, 50, 51} Although the contribution of glial cells to TDP-43-mediated motor neuron degeneration is now well supported, this model does not fully suggest an astrocyte-based non-cell autonomous mechanism. For example, recent studies have shown that TDP-43-mutant astrocytes do not affect the survival of motor neurons,^{50, 51} indicating a previously unrecognized non-cell autonomous TDP-43 proteinopathy that associates with cell types other than astrocytes.Given that the role of glial cell types other than astrocytes in TDP-43-mediated neuroinflammation is still not fully understood, we aim to compare the contribution of microglia and astrocytes to neurotoxicity in a TDP-43 loss-of-function model. Here, we show that TDP-43 has a dominant role in promoting COX-2-PGE2 production through the MAPK/ERK pathway in primary cultured microglia, but not in primary cultured astrocytes. Our study suggests that overproduction of PGE2 in microglia is a novel molecular mechanism underlying neurotoxicity in TDP-43-linked ALS. Moreover, our data identify celecoxib as a new potential effective treatment of TDP-43-linked ALS and possibly other types of ALS.     

Grain Unloading of Arsenic Species in Rice

Rice (Oryza sativa) is the staple food for over half the world''s population yet may represent a significant dietary source of inorganic arsenic (As), a nonthreshold, class 1 human carcinogen. Rice grain As is dominated by the inorganic species, and the organic species dimethylarsinic acid (DMA). To investigate how As species are unloaded into grain rice, panicles were excised during grain filling and hydroponically pulsed with arsenite, arsenate, glutathione-complexed As, or DMA. Total As concentrations in flag leaf, grain, and husk, were quantified by inductively coupled plasma mass spectroscopy and As speciation in the fresh grain was determined by x-ray absorption near-edge spectroscopy. The roles of phloem and xylem transport were investigated by applying a ± stem-girdling treatment to a second set of panicles, limiting phloem transport to the grain in panicles pulsed with arsenite or DMA. The results demonstrate that DMA is translocated to the rice grain with over an order magnitude greater efficiency than inorganic species and is more mobile than arsenite in both the phloem and the xylem. Phloem transport accounted for 90% of arsenite, and 55% of DMA, transport to the grain. Synchrotron x-ray fluorescence mapping and fluorescence microtomography revealed marked differences in the pattern of As unloading into the grain between DMA and arsenite-challenged grain. Arsenite was retained in the ovular vascular trace and DMA dispersed throughout the external grain parts and into the endosperm. This study also demonstrates that DMA speciation is altered in planta, potentially through complexation with thiols.Paddy rice (Oryza sativa) is particularly effective, compared to other cereals, at accumulating arsenic (As) in shoot and grain (Williams et al., 2007b). Rice is the staple food for over half the world''s population (Fageria, 2007) and rice represents a significant dietary source of inorganic As, a class 1, nonthreshold carcinogen, particularly in Southeast Asia (Meharg et al., 2009). Inorganic As levels in rice grain are problematic even where soil As is at background levels, derived from geogenic sources (Lu et al., 2009; Meharg et al., 2009). However, widespread pollution of paddy soils with As, leading to further elevation of grain As, has occurred in some regions due to base and precious mining (Liao et al., 2005; Zhu et al., 2008), irrigation of paddies with As-elevated groundwaters (e.g. Meharg and Rahman, 2003; Williams et al., 2006), and the use of arsenical pesticides (Williams et al., 2007a). Unlike other cereal grains, paddy rice cultivation is dependent of soils being anaerobic, and it is this anoxia that gives rise to elevated As concentrations in the plant. Anaerobic soil conditions lead to the mobilization of As as arsenite, where under aerobic systems arsenate dominates (Xu et al., 2008). Arsenite is efficiently assimilated by rice roots through silicic acid transport pathway (Ma et al., 2008).Knowledge of As metabolism and partitioning within plants, particularly rice, is still developing rapidly (Zhao et al., 2009). Several studies have now shown that As in rice vegetative tissue and grain is predominantly speciated as inorganic As and the methylated species dimethylarsinic acid (DMA), with variable, though low, levels of monomethyl arsonic acid (MMA; Abedin et al., 2002a; Williams et al., 2005, 2006; Norton et al., 2009). Arsenate is an analog of phosphate and competes with phosphate for rice root uptake (Abedin et al., 2002a) while arsenite is taken up by rice roots via silicic acid transporters (Ma et al., 2008). Abedin et al. (2002b) demonstrated that the methylated species DMA and MMA are also taken up by rice plants although at a much slower rate than inorganic As, with the protonated neutral forms also transported through silicic acid pathway (Li et al., 2009). Arsenate is reduced to arsenite within the rice root (Xu et al., 2008; Zhao et al., 2009), which then enters the xylem via a silicic acid/arsenite effluxer (Ma et al., 2008; Zhao et al., 2009). Arsenite may be detoxified through complexation with thiol-rich peptides including phytochelatins (PCs) and glutathione followed by sequestration into vacuoles (Bleeker et al., 2006; Raab et al., 2007b; Zhao et al., 2009). Raab et al. (2007a) found that while methylated As species are taken up by rice roots much less efficiently than inorganic species, they appear to be translocated within the plant more efficiently. The comparative contributions of xylem and phloem transport, in translocation of As to the grain, are unknown.The main species within rice grain, along with DMA, are inorganic As, particularly arsenite, which may be complexed with thiols (Williams et al., 2005; Lombi et al., 2009). Nutrients are unloaded into the grain from the ovular vascular trace (OVT) into the nucellar tissue and from there are uploaded, via the apoplast into the filial tissue (the aleurone and the endosperm; Krishnan and Dayanandan, 2003). Lombi et al. (2009) recently suggested that this may represent a physiological barrier that As species cross with differential efficiency. However, the transport and unloading of As to/into the grain, which are key processes in terms of human exposure to this contaminant, are far from being fully understood.This study investigated the differential efficiency with which important As species are translocated and unloaded into the rice grain and the comparative contributions of phloem and xylem transport. Rice panicles were excised below the flag leaf node during grain development, 10 DPA, and treated to a hydroponically administered 48-h pulse of arsenite, arsenate, arsenite glutathione, or DMA. Total As concentrations in flag leaf, grain, and husk samples for each treatment were quantified by inductively coupled plasma mass spectroscopy (ICP-MS), and As speciation in the fresh grain was determined by x-ray absorption near-edge spectroscopy (XANES) analysis. To evaluate the contributions of phloem versus xylem transport, a stem-girdling treatment was applied, using steam to destroy phloem cells in a second set of panicles prior to a pulse of either DMA or arsenite. The spatial unloading of As species into the developing grain was examined by synchrotron x-ray fluorescence (XRF) mapping, and fluorescence microtomography for the DMA and arsenite treatments.     

Focus on Ethylene: Ethylene Biosynthesis Is Promoted by Very-Long-Chain Fatty Acids during Lysigenous Aerenchyma Formation in Rice Roots

In rice (Oryza sativa) roots, lysigenous aerenchyma, which is created by programmed cell death and lysis of cortical cells, is constitutively formed under aerobic conditions, and its formation is further induced under oxygen-deficient conditions. Ethylene is involved in the induction of aerenchyma formation. reduced culm number1 (rcn1) is a rice mutant in which the gene encoding the ATP-binding cassette transporter RCN1/OsABCG5 is defective. Here, we report that the induction of aerenchyma formation was reduced in roots of rcn1 grown in stagnant deoxygenated nutrient solution (i.e. under stagnant conditions, which mimic oxygen-deficient conditions in waterlogged soils). 1-Aminocyclopropane-1-carboxylic acid synthase (ACS) is a key enzyme in ethylene biosynthesis. Stagnant conditions hardly induced the expression of ACS1 in rcn1 roots, resulting in low ethylene production in the roots. Accumulation of saturated very-long-chain fatty acids (VLCFAs) of 24, 26, and 28 carbons was reduced in rcn1 roots. Exogenously supplied VLCFA (26 carbons) increased the expression level of ACS1 and induced aerenchyma formation in rcn1 roots. Moreover, in rice lines in which the gene encoding a fatty acid elongase, CUT1-LIKE (CUT1L; a homolog of the gene encoding Arabidopsis CUT1, which is required for cuticular wax production), was silenced, both ACS1 expression and aerenchyma formation were reduced. Interestingly, the expression of ACS1, CUT1L, and RCN1/OsABCG5 was induced predominantly in the outer part of roots under stagnant conditions. These results suggest that, in rice under oxygen-deficient conditions, VLCFAs increase ethylene production by promoting 1-aminocyclopropane-1-carboxylic acid biosynthesis in the outer part of roots, which, in turn, induces aerenchyma formation in the root cortex.Aerenchyma formation is a morphological adaptation of plants to complete submergence and waterlogging of the soil, and facilitates internal gas diffusion (Armstrong, 1979; Jackson and Armstrong, 1999; Colmer, 2003; Voesenek et al., 2006; Bailey-Serres and Voesenek, 2008; Licausi and Perata, 2009; Sauter, 2013; Voesenek and Bailey-Serres, 2015). To adapt to waterlogging in soil, rice (Oryza sativa) develops lysigenous aerenchyma in shoots (Matsukura et al., 2000; Colmer and Pedersen, 2008; Steffens et al., 2011) and roots (Jackson et al., 1985b; Justin and Armstrong, 1991; Kawai et al., 1998), which is formed by programmed cell death and subsequent lysis of some cortical cells (Jackson and Armstrong, 1999; Evans, 2004; Yamauchi et al., 2013). In rice roots, lysigenous aerenchyma is constitutively formed under aerobic conditions (Jackson et al., 1985b), and its formation is further induced under oxygen-deficient conditions (Colmer et al., 2006; Shiono et al., 2011). The former and latter are designated constitutive and inducible lysigenous aerenchyma formation, respectively (Colmer and Voesenek, 2009). The gaseous plant hormone ethylene regulates adaptive growth responses of plants to submergence (Voesenek and Blom, 1989; Voesenek et al., 1993; Visser et al., 1996a,b; Lorbiecke and Sauter, 1999; Hattori et al., 2009; Steffens and Sauter, 2009; van Veen et al., 2013). Ethylene also induces lysigenous aerenchyma formation in roots of some gramineous plants (Drew et al., 2000; Shiono et al., 2008). The treatment of roots with ethylene or its precursor (1-aminocyclopropane-1-carboxylic acid [ACC]) stimulates aerenchyma formation in rice (Justin and Armstrong, 1991; Colmer et al., 2006; Yukiyoshi and Karahara, 2014), maize (Zea mays; Drew et al., 1981; Jackson et al., 1985a; Takahashi et al., 2015), and wheat (Triticum aestivum; Yamauchi et al., 2014a,b). Moreover, treatment of roots with inhibitors of ethylene action or ethylene biosynthesis effectively blocks aerenchyma formation under hypoxic conditions in maize (Drew et al., 1981; Konings, 1982; Jackson et al., 1985a; Rajhi et al., 2011).Ethylene biosynthesis is accomplished by two main successive enzymatic reactions: conversion of S-adenosyl-Met to ACC by 1-aminocyclopropane-1-carboxylic acid synthase (ACS), and conversion of ACC to ethylene by 1-aminocyclopropane-1-carboxylic acid oxidase (ACO; Yang and Hoffman, 1984). The activities of both enzymes are enhanced during aerenchyma formation under hypoxic conditions in maize root (He et al., 1996). Since the ACC content in roots of maize is increased by oxygen deficiency and is strongly correlated with ethylene production (Atwell et al., 1988), ACC biosynthesis is essential for ethylene production during aerenchyma formation in roots. In fact, exogenously supplied ACC induced ethylene production in roots of maize (Drew et al., 1979; Konings, 1982; Atwell et al., 1988) and wheat (Yamauchi et al., 2014b), even under aerobic conditions. Ethylene production in plants is inversely related to oxygen concentration (Yang and Hoffman, 1984). Under anoxic conditions, the oxidation of ACC to ethylene by ACO, which requires oxygen, is almost completely repressed (Yip et al., 1988; Tonutti and Ramina, 1991). Indeed, anoxic conditions stimulate neither ethylene production nor aerenchyma formation in maize adventitious roots (Drew et al., 1979). Therefore, it is unlikely that the root tissues forming inducible aerenchyma are anoxic, and that the ACO-mediated step is repressed. Moreover, aerenchyma is constitutively formed in rice roots even under aerobic conditions (Jackson et al., 1985b), and thus, after the onset of waterlogging, oxygen can be immediately supplied to the apical regions of roots through the constitutively formed aerenchyma.Very-long-chain fatty acids (VLCFAs; ≥20 carbons) are major constituents of sphingolipids, cuticular waxes, and suberin in plants (Franke and Schreiber, 2007; Kunst and Samuels, 2009). In addition to their structural functions, VLCFAs directly or indirectly participate in several physiological processes (Zheng et al., 2005; Reina-Pinto et al., 2009; Roudier et al., 2010; Ito et al., 2011; Nobusawa et al., 2013; Tsuda et al., 2013), including the regulation of ethylene biosynthesis (Qin et al., 2007). During fiber cell elongation in cotton ovules, ethylene biosynthesis is enhanced by treatment with saturated VLCFAs, especially 24-carbon fatty acids, and is suppressed by an inhibitor of VLCFA biosynthesis (Qin et al., 2007). The first rate-limiting step in VLCFA biosynthesis is condensation of acyl-CoA with malonyl-CoA by β-ketoacyl-CoA synthase (KCS; Joubès et al., 2008). KCS enzymes are thought to determine the substrate and tissue specificities of fatty acid elongation (Joubès et al., 2008). The Arabidopsis (Arabidopsis thaliana) genome has 21 KCS genes (Joubès et al., 2008). In the Arabidopsis cut1 mutant, which has a defect in the gene encoding CUT1 that is required for cuticular wax production (i.e. one of the KCS genes), the expression of AtACO genes and growth of root cells were reduced when compared with the wild type (Qin et al., 2007). Furthermore, expression of the AtACO genes was rescued by exogenously supplied saturated VLCFAs (Qin et al., 2007). These observations imply that VLCFAs or their derivatives work as regulatory factors for gene expression during some physiological processes in plants.reduced culm number1 (rcn1) was first identified as a rice mutant with a low tillering rate in a paddy field (Takamure and Kinoshita, 1985; Yasuno et al., 2007). The rcn1 (rcn1-2) mutant has a single nucleotide substitution in the gene encoding a member of the ATP-binding cassette (ABC) transporter subfamily G, RCN1/OsABCG5, causing an Ala-684Pro substitution (Yasuno et al., 2009). The mutation results in several mutant phenotypes, although the substrates of RCN1/OsABCG5 have not been determined (Ureshi et al., 2012; Funabiki et al., 2013; Matsuda et al., 2014). We previously found that the rcn1 mutant has abnormal root morphology, such as shorter root length and brownish appearance of roots, under stagnant (deoxygenated) conditions (which mimics oxygen-deficient conditions in waterlogged soils). We also found that the rcn1 mutant accumulates less of the major suberin monomers originating from VLCFAs in the outer part of adventitious roots, and this results in a reduction of a functional apoplastic barrier in the root hypodermis (Shiono et al., 2014a).The objective of this study was to elucidate the molecular basis of inducible aerenchyma formation. To this end, we examined lysigenous aerenchyma formation and ACC, ethylene, and VLCFA accumulation and their biosyntheses in rcn1 roots. Based on the results of these studies, we propose that VLCFAs are involved in inducible aerenchyma formation through the enhancement of ethylene biosynthesis in rice roots.     

Characterization of the Possible Roles for B Class MADS Box Genes in Regulation of Perianth Formation in Orchid

To investigate sepal/petal/lip formation in Oncidium Gower Ramsey, three paleoAPETALA3 genes, O. Gower Ramsey MADS box gene5 (OMADS5; clade 1), OMADS3 (clade 2), and OMADS9 (clade 3), and one PISTILLATA gene, OMADS8, were characterized. The OMADS8 and OMADS3 mRNAs were expressed in all four floral organs as well as in vegetative leaves. The OMADS9 mRNA was only strongly detected in petals and lips. The mRNA for OMADS5 was only strongly detected in sepals and petals and was significantly down-regulated in lip-like petals and lip-like sepals of peloric mutant flowers. This result revealed a possible negative role for OMADS5 in regulating lip formation. Yeast two-hybrid analysis indicated that OMADS5 formed homodimers and heterodimers with OMADS3 and OMADS9. OMADS8 only formed heterodimers with OMADS3, whereas OMADS3 and OMADS9 formed homodimers and heterodimers with each other. We proposed that sepal/petal/lip formation needs the presence of OMADS3/8 and/or OMADS9. The determination of the final organ identity for the sepal/petal/lip likely depended on the presence or absence of OMADS5. The presence of OMADS5 caused short sepal/petal formation. When OMADS5 was absent, cells could proliferate, resulting in the possible formation of large lips and the conversion of the sepal/petal into lips in peloric mutants. Further analysis indicated that only ectopic expression of OMADS8 but not OMADS5/9 caused the conversion of the sepal into an expanded petal-like structure in transgenic Arabidopsis (Arabidopsis thaliana) plants.The ABCDE model predicts the formation of any flower organ by the interaction of five classes of homeotic genes in plants (Yanofsky et al., 1990; Jack et al., 1992; Mandel et al., 1992; Goto and Meyerowitz, 1994; Jofuku et al., 1994; Pelaz et al., 2000, 2001; Theißen and Saedler, 2001; Pinyopich et al., 2003; Ditta et al., 2004; Jack, 2004). The A class genes control sepal formation. The A, B, and E class genes work together to regulate petal formation. The B, C, and E class genes control stamen formation. The C and E class genes work to regulate carpel formation, whereas the D class gene is involved in ovule development. MADS box genes seem to have a central role in flower development, because most ABCDE genes encode MADS box proteins (Coen and Meyerowitz, 1991; Weigel and Meyerowitz, 1994; Purugganan et al., 1995; Rounsley et al., 1995; Theißen and Saedler, 1995; Theißen et al., 2000; Theißen, 2001).The function of B group genes, such as APETALA3 (AP3) and PISTILLATA (PI), has been thought to have a major role in specifying petal and stamen development (Jack et al., 1992; Goto and Meyerowitz, 1994; Krizek and Meyerowitz, 1996; Kramer et al., 1998; Hernandez-Hernandez et al., 2007; Kanno et al., 2007; Whipple et al., 2007; Irish, 2009). In Arabidopsis (Arabidopsis thaliana), mutation in AP3 or PI caused identical phenotypes of second whorl petal conversion into a sepal structure and third flower whorl stamen into a carpel structure (Bowman et al., 1989; Jack et al., 1992; Goto and Meyerowitz, 1994). Similar homeotic conversions for petal and stamen were observed in the mutants of the AP3 and PI orthologs from a number of core eudicots such as Antirrhinum majus, Petunia hybrida, Gerbera hybrida, Solanum lycopersicum, and Nicotiana benthamiana (Sommer et al., 1990; Tröbner et al., 1992; Angenent et al., 1993; van der Krol et al., 1993; Yu et al., 1999; Liu et al., 2004; Vandenbussche et al., 2004; de Martino et al., 2006), from basal eudicot species such as Papaver somniferum and Aquilegia vulgaris (Drea et al., 2007; Kramer et al., 2007), as well as from monocot species such as Zea mays and Oryza sativa (Ambrose et al., 2000; Nagasawa et al., 2003; Prasad and Vijayraghavan, 2003; Yadav et al., 2007; Yao et al., 2008). This indicated that the function of the B class genes AP3 and PI is highly conserved during evolution.It has been thought that B group genes may have arisen from an ancestral gene through multiple gene duplication events (Doyle, 1994; Theißen et al., 1996, 2000; Purugganan, 1997; Kramer et al., 1998; Kramer and Irish, 1999; Lamb and Irish, 2003; Kim et al., 2004; Stellari et al., 2004; Zahn et al., 2005; Hernandez-Hernandez et al., 2007). In the gymnosperms, there was a single putative B class lineage that duplicated to generate the paleoAP3 and PI lineages in angiosperms (Kramer et al., 1998; Theißen et al., 2000; Irish, 2009). The paleoAP3 lineage is composed of AP3 orthologs identified in lower eudicots, magnolid dicots, and monocots (Kramer et al., 1998). Genes in this lineage contain the conserved paleoAP3- and PI-derived motifs in the C-terminal end of the proteins, which have been thought to be characteristics of the B class ancestral gene (Kramer et al., 1998; Tzeng and Yang, 2001; Hsu and Yang, 2002). The PI lineage is composed of PI orthologs that contain a highly conserved PI motif identified in most plant species (Kramer et al., 1998). Subsequently, there was a second duplication at the base of the core eudicots that produced the euAP3 and TM6 lineages, which have been subject to substantial sequence changes in eudicots during evolution (Kramer et al., 1998; Kramer and Irish, 1999). The paleoAP3 motif in the C-terminal end of the proteins was retained in the TM6 lineage and replaced by a conserved euAP3 motif in the euAP3 lineage of most eudicot species (Kramer et al., 1998). In addition, many lineage-specific duplications for paleoAP3 lineage have occurred in plants such as orchids (Hsu and Yang, 2002; Tsai et al., 2004; Kim et al., 2007; Mondragón-Palomino and Theißen, 2008, 2009; Mondragón-Palomino et al., 2009), Ranunculaceae, and Ranunculales (Kramer et al., 2003; Di Stilio et al., 2005; Shan et al., 2006; Kramer, 2009).Unlike the A or C class MADS box proteins, which form homodimers that regulate flower development, the ability of B class proteins to form homodimers has only been reported in gymnosperms and in the paleoAP3 and PI lineages of some monocots. For example, LMADS1 of the lily Lilium longiflorum (Tzeng and Yang, 2001), OMADS3 of the orchid Oncidium Gower Ramsey (Hsu and Yang, 2002), and PeMADS4 of the orchid Phalaenopsis equestris (Tsai et al., 2004) in the paleoAP3 lineage, LRGLOA and LRGLOB of the lily Lilium regale (Winter et al., 2002), TGGLO of the tulip Tulipa gesneriana (Kanno et al., 2003), and PeMADS6 of the orchid P. equestris (Tsai et al., 2005) in the PI lineage, and GGM2 of the gymnosperm Gnetum gnemon (Winter et al., 1999) were able to form homodimers that regulate flower development. Proteins in the euAP3 lineage and in most paleoAP3 lineages were not able to form homodimers and had to interact with PI to form heterodimers in order to regulate petal and stamen development in various plant species (Schwarz-Sommer et al., 1992; Tröbner et al., 1992; Riechmann et al., 1996; Moon et al., 1999; Winter et al., 2002; Kanno et al., 2003; Vandenbussche et al., 2004; Yao et al., 2008). In addition to forming dimers, AP3 and PI were able to interact with other MADS box proteins, such as SEPALLATA1 (SEP1), SEP2, and SEP3, to regulate petal and stamen development (Pelaz et al., 2000; Honma and Goto, 2001; Theißen and Saedler, 2001; Castillejo et al., 2005).Orchids are among the most important plants in the flower market around the world, and research on MADS box genes has been reported for several species of orchids during the past few years (Lu et al., 1993, 2007; Yu and Goh, 2000; Hsu and Yang, 2002; Yu et al., 2002; Hsu et al., 2003; Tsai et al., 2004, 2008; Xu et al., 2006; Guo et al., 2007; Kim et al., 2007; Chang et al., 2009). Unlike the flowers in eudicots, the nearly identical shape of the sepals and petals as well as the production of a unique lip in orchid flowers make them a very special plant species for the study of flower development. Four clades (1–4) of genes in the paleoAP3 lineage have been identified in several orchids (Hsu and Yang, 2002; Tsai et al., 2004; Kim et al., 2007; Mondragón-Palomino and Theißen, 2008, 2009; Mondragón-Palomino et al., 2009). Several works have described the possible interactions among these four clades of paleoAP3 genes and one PI gene that are involved in regulating the differentiation and formation of the sepal/petal/lip of orchids (Tsai et al., 2004; Kim et al., 2007; Mondragón-Palomino and Theißen, 2008, 2009). However, the exact mechanism that involves the orchid B class genes remains unclear and needs to be clarified by more experimental investigations.O. Gower Ramsey is a popular orchid with important economic value in cut flower markets. Only a few studies have been reported on the role of MADS box genes in regulating flower formation in this plant species (Hsu and Yang, 2002; Hsu et al., 2003; Chang et al., 2009). An AP3-like MADS gene that regulates both floral formation and initiation in transgenic Arabidopsis has been reported (Hsu and Yang, 2002). In addition, four AP1/AGAMOUS-LIKE9 (AGL9)-like MADS box genes have been characterized that show novel expression patterns and cause different effects on floral transition and formation in Arabidopsis (Hsu et al., 2003; Chang et al., 2009). Compared with other orchids, the production of a large and well-expanded lip and five small identical sepals/petals makes O. Gower Ramsey a special case for the study of the diverse functions of B class MADS box genes during evolution. Therefore, the isolation of more B class MADS box genes and further study of their roles in the regulation of perianth (sepal/petal/lip) formation during O. Gower Ramsey flower development are necessary. In addition to the clade 2 paleoAP3 gene OMADS3, which was previously characterized in our laboratory (Hsu and Yang, 2002), three more B class MADS box genes, OMADS5, OMADS8, and OMADS9, were characterized from O. Gower Ramsey in this study. Based on the different expression patterns and the protein interactions among these four orchid B class genes, we propose that the presence of OMADS3/8 and/or OMADS9 is required for sepal/petal/lip formation. Further sepal and petal formation at least requires the additional presence of OMADS5, whereas large lip formation was seen when OMADS5 expression was absent. Our results provide a new finding and information pertaining to the roles for orchid B class MADS box genes in the regulation of sepal/petal/lip formation.     

The Fcp1-Wee1-Cdk1 axis affects spindle assembly checkpoint robustness and sensitivity to antimicrotubule cancer drugs

To grant faithful chromosome segregation, the spindle assembly checkpoint (SAC) delays mitosis exit until mitotic spindle assembly. An exceedingly prolonged mitosis, however, promotes cell death and by this means antimicrotubule cancer drugs (AMCDs), that impair spindle assembly, are believed to kill cancer cells. Despite malformed spindles, cancer cells can, however, slip through SAC, exit mitosis prematurely and resist killing. We show here that the Fcp1 phosphatase and Wee1, the cyclin B-dependent kinase (cdk) 1 inhibitory kinase, play a role for this slippage/resistance mechanism. During AMCD-induced prolonged mitosis, Fcp1-dependent Wee1 reactivation lowered cdk1 activity, weakening SAC-dependent mitotic arrest and leading to mitosis exit and survival. Conversely, genetic or chemical Wee1 inhibition strengthened the SAC, further extended mitosis, reduced antiapoptotic protein Mcl-1 to a minimum and potentiated killing in several, AMCD-treated cancer cell lines and primary human adult lymphoblastic leukemia cells. Thus, the Fcp1-Wee1-Cdk1 (FWC) axis affects SAC robustness and AMCDs sensitivity.The spindle assembly checkpoint (SAC) delays mitosis exit to coordinate anaphase onset with spindle assembly. To this end, SAC inhibits the ubiquitin ligase Anaphase-Promoting Complex/Cyclosome (APC/C) to prevent degradation of the anaphase inhibitor securin and cyclin B, the major mitotic cyclin B-dependent kinase 1 (cdk1) activator, until spindle assembly.¹ However, by yet poorly understood mechanisms, exceedingly prolonging mitosis translates into cell death induction.^{2, 3, 4, 5, 6, 7} Although mechanistic details are still missing on how activation of cell death pathways is linked to mitosis duration, prolongation of mitosis appears crucial for the ability of antimicrotubule cancer drugs (AMCDs) to kill cancer cells.^{2, 3, 4, 5, 6, 7} These drugs, targeting microtubules, impede mitotic spindle assembly and delay mitosis exit by chronically activating the SAC. Use of these drugs is limited, however, by toxicity and resistance. A major mechanism for resistance is believed to reside in the ability of cancer cells to slip through the SAC and exit mitosis prematurely despite malformed spindles, thus resisting killing by limiting mitosis duration.^{2, 3, 4, 5, 6, 7} Under the AMCD treatment, cells either die in mitosis or exit mitosis, slipping through the SAC, without or abnormally dividing.^{2, 3, 4} Cells that exit mitosis either die at later stages or survive and stop dividing or proliferate, giving rise to resistance.^{2, 3, 4} Apart from a role for p53, what dictates cell fate is still unknown; however, it appears that the longer mitosis is protracted, the higher the chances for cell death pathway activation are.^{2, 3, 4, 5, 6, 7}Although SAC is not required per se for killing,⁶ preventing SAC adaptation should improve the efficacy of AMCD by increasing mitosis duration.^{2, 3, 4, 5, 6, 7} Therefore, further understanding of the mechanisms by which cells override SAC may help to improve the current AMCD therapy. Several kinases are known to activate and sustain SAC, and cdk1 itself appears to be of primary relevance.^{1, 8, 9} By studying mitosis exit and SAC resolution, we recently reported a role for the Fcp1 phosphatase to bring about cdk1 inactivation.^{10, 11} Among Fcp1 targets, we identified cyclin degradation pathway components, such as Cdc20, an APC/C co-activator, USP44, a deubiquitinating enzyme, and Wee1.^{10, 11} Wee1 is a crucial kinase that controls the G2 phase by performing inhibitory phosphorylation of cdk1 at tyr-15 (Y15-cdk1). Wee1 is also in a feedback relationship with cdk1 itself that, in turn, can phosphorylate and inhibit Wee1 in an autoamplification loop to promote the G2-to-M phase transition.¹² At mitosis exit, Fcp1 dephosphorylated Wee1 at threonine 239, a cdk1-dependent inhibitory phosphorylation, to dampen down the cdk1 autoamplification loop, and Cdc20 and USP44, to promote APC/C-dependent cyclin B degradation.^{10, 11, 12} In this study we analysed the Fcp1 relevance in SAC adaptation and AMCD sensitivity.     

Adiponectin receptor-mediated signaling ameliorates cerebral cell damage and regulates the neurogenesis of neural stem cells at high glucose concentrations: an in vivo and in vitro study

In the central nervous system (CNS), hyperglycemia leads to neuronal damage and cognitive decline. Recent research has focused on revealing alterations in the brain in hyperglycemia and finding therapeutic solutions for alleviating the hyperglycemia-induced cognitive dysfunction. Adiponectin is a protein hormone with a major regulatory role in diabetes and obesity; however, its role in the CNS has not been studied yet. Although the presence of adiponectin receptors has been reported in the CNS, adiponectin receptor-mediated signaling in the CNS has not been investigated. In the present study, we investigated adiponectin receptor (AdipoR)-mediated signaling in vivo using a high-fat diet and in vitro using neural stem cells (NSCs). We showed that AdipoR1 protects cell damage and synaptic dysfunction in the mouse brain in hyperglycemia. At high glucose concentrations in vitro, AdipoR1 regulated the survival of NSCs through the p53/p21 pathway and the proliferation- and differentiation-related factors of NSCs via tailless (TLX). Hence, we suggest that further investigations are necessary to understand the cerebral AdipoR1-mediated signaling in hyperglycemic conditions, because the modulation of AdipoR1 might alleviate hyperglycemia-induced neuropathogenesis.Adiponectin secreted by the adipose tissue^{1, 2} exists in either a full-length or globular form.^{3, 4, 5, 6} Adiponectin can cross the blood–brain barrier, and various forms of adiponectin are found in the cerebrospinal fluid.^{7, 8, 9, 10, 11} Adiponectin exerts its effect by binding to the adiponectin receptor 1 (AdipoR1) and adiponectin receptor 2 (AdipoR2)^{12, 13} that have different affinities for the various circulating adiponectins.^{12, 14, 15, 16, 17} Several studies reported that both receptor subtypes are expressed in the central nervous system (CNS).^{7, 12, 18} As adiponectin modulates insulin sensitivity and inflammation,¹⁹ its deficiency induces insulin resistance and glucose intolerance in animals fed a high-fat diet (HFD).^{19, 20, 21} In addition, adiponectin can ameliorate the glucose homeostasis and increase insulin sensitivity.^{22, 23, 24} Adiponectin, which is the most well-known adipokine, acts mainly as an anti-inflammatory regulator,^{25, 26} and is associated with the onset of neurological disorders.²⁷ In addition, a recent study reported that adiponectin promotes the proliferation of hippocampal neural stem cells (NSCs).²⁸ Considering that adiponectin acts by binding to the adiponectin receptors, investigation of the adiponectin receptor-mediated signaling in the brain is crucial to understand the cerebral effects of adiponectin and the underlying cellular mechanisms.The prevalence of type II diabetes mellitus (DM2) and Alzheimer''s disease increases with aging.²⁹ According to a cross-sectional study, in people with DM2, the risk of dementia is 2.5 times higher than that in the normal population.^{30, 31} A study performed between 1980 and 2002 suggested that an elevated blood glucose level is associated with a greater risk for dementia in elderly patients with DM2.³² In addition, according to a 9-year-long longitudinal cohort study, the risk of developing Alzheimer''s disease was 65% higher in people with diabetes than in control subjects.³³ A community-based cohort study also reported that higher plasma glucose concentrations are associated with an increased risk for dementia, because the higher glucose level has detrimental effects on the brain.³¹ High blood glucose level causes mitochondria-dependent apoptosis,^{34, 35, 36} and aggravates diverse neurological functions.^{37, 38} Inflammation and oxidative stress, which are commonly observed in people with diabetes, inhibit neurogenesis.^{39, 40, 41} Similarly, neurogenesis is decreased in mice and rats with genetically induced type I diabetes.^{42, 43} In addition, diabetic rodents have a decreased proliferation rate of neural progenitors.^{43, 44} Furthermore, several studies suggested that an HFD leads to neuroinflammation, the impairment of synaptic plasticity, and cognitive decline.^{45, 46}Here, we investigated whether AdipoR1-mediated signaling is associated with cell death in the brain of mice on a HFD, and whether high glucose level modifies the proliferation and differentiation capacity of NSCs in vitro. Our study provides novel findings about the role of AdipoR1-mediated signaling in hyperglycemia-induced neuropathogenesis.     

Blood Supply to the Chicken Femoral Head

The purpose of this study was to conduct a comprehensive evaluation of the vascular supply to the femoral head, including the vessels that give rise to the terminal perfusing branches. Using a casting agent, we highlighted the anatomy of the external iliac and ischiatic arteries with their associated branches after anatomic dissection of 24 hips from 12 Leghorn chickens. We confirmed published findings regarding perfusion of the femoral head and identified 3 previously undescribed arterial branches to this structure. The first branch (the acetabular branch of the femoralis artery) was supplied by the femoralis artery and directly perfused the acetabulum and femoral head. The second branch (the lateral retinacular artery) was a tributary of the femoralis artery that directly supplied the femoral head. Finally, we found that the middle femoral nutrient artery supplies a previously undescribed ascending intraosseous branch (the ascending branch of the middle femoral nutrient artery) that perfuses the femoral head. Precise understanding of the major vascular branches to the femoral head would allow for complete or selective ligation of its blood supply and enable the creation of a reproducible bipedal model of femoral head osteonecrosis.Like humans, chickens are bipedal animals that rely on the hip joint to absorb the majority of the body''s weight. This anatomy, in concert with their high activity level, makes chickens an attractive model for the study of osteonecrosis of the femoral head in humans. The vast majority of animal research on osteonecrosis of the femoral head has been performed on quadrupedal animals,^{3,4,10,19,25,26,28,29,31,36,37,41,51,52} thus limiting its application to bipedal species because most quadruped models fail to progress to end-stage mechanical collapse similar to that in humans.⁶Avascular necrosis is the death of bone that occurs from ischemia due to disruption of the vascular supply to bone through direct or indirect mechanisms.³⁸ Avascular necrosis should be differentiated from the broader term of osteonecrosis, which refers to bone death in general.³² Causes of femoral head osteonecrosis include direct and indirect disruption of vascular supply (traumatic injury, intravascular coagulation, extrinsic compression) as well as changes in cellular differentiation and cellular apoptosis.^{4,7,12,15,17,18,24,30-32,38,49,50} Accordingly, causes of osteonecrosis are both traumatic and nontraumatic.^16,31,32The arterial anatomy in the chicken hindlimb has been outlined by several authors.^{20,22,27,35,42,44,45} Briefly, the external iliac and ischiatic artery arise from the abdominal aorta to provide blood supply to the chicken hind limb. The external iliac artery has 2 main branches—the femoralis and femoral circumflex arteries—that distribute blood to the chicken hindlimb. The ischiatic artery provides 3 main branches: the trochanteric artery, superior femoral nutrient artery, and middle femoral nutrient artery. Although the terminal vascular supply to the femoral head of Leghorn and Broiler chickens has been described,^46,47 the origin of these terminal arteries with reference to the ischiatic and femoralis arteries and their respective branches has not been addressed. The current study will describe the blood vessels that feed these terminal branches to the chicken femoral head.     

A cellular screen identifies ponatinib and pazopanib as inhibitors of necroptosis

Necroptosis is a form of regulated necrotic cell death mediated by receptor-interacting serine/threonine-protein kinase 1 (RIPK1) and RIPK3. Necroptotic cell death contributes to the pathophysiology of several disorders involving tissue damage, including myocardial infarction, stroke and ischemia-reperfusion injury. However, no inhibitors of necroptosis are currently in clinical use. Here we performed a phenotypic screen for small-molecule inhibitors of tumor necrosis factor-alpha (TNF-α)-induced necroptosis in Fas-associated protein with death domain (FADD)-deficient Jurkat cells using a representative panel of Food and Drug Administration (FDA)-approved drugs. We identified two anti-cancer agents, ponatinib and pazopanib, as submicromolar inhibitors of necroptosis. Both compounds inhibited necroptotic cell death induced by various cell death receptor ligands in human cells, while not protecting from apoptosis. Ponatinib and pazopanib abrogated phosphorylation of mixed lineage kinase domain-like protein (MLKL) upon TNF-α-induced necroptosis, indicating that both agents target a component upstream of MLKL. An unbiased chemical proteomic approach determined the cellular target spectrum of ponatinib, revealing key members of the necroptosis signaling pathway. We validated RIPK1, RIPK3 and transforming growth factor-β-activated kinase 1 (TAK1) as novel, direct targets of ponatinib by using competitive binding, cellular thermal shift and recombinant kinase assays. Ponatinib inhibited both RIPK1 and RIPK3, while pazopanib preferentially targeted RIPK1. The identification of the FDA-approved drugs ponatinib and pazopanib as cellular inhibitors of necroptosis highlights them as potentially interesting for the treatment of pathologies caused or aggravated by necroptotic cell death.Programmed cell death has a crucial role in a variety of biological processes ranging from normal tissue development to diverse pathological conditions.^{1, 2} Necroptosis is a form of regulated cell death that has been shown to occur during pathogen infection or sterile injury-induced inflammation in conditions where apoptosis signaling is compromised.^{3, 4, 5, 6} Given that many viruses have developed strategies to circumvent apoptotic cell death, necroptosis constitutes an important, pro-inflammatory back-up mechanism that limits viral spread in vivo.^{7, 8, 9} In contrast, in the context of sterile inflammation, necroptotic cell death contributes to disease pathology, outlining potential benefits of therapeutic intervention.¹⁰ Necroptosis can be initiated by death receptors of the tumor necrosis factor (TNF) superfamily,¹¹ Toll-like receptor 3 (TLR3),¹² TLR4,¹³ DNA-dependent activator of IFN-regulatory factors¹⁴ or interferon receptors.¹⁵ Downstream signaling is subsequently conveyed via RIPK1¹⁶ or TIR-domain-containing adapter-inducing interferon-β,^{8, 17} and converges on RIPK3-mediated^{13, 18, 19, 20} activation of MLKL.²¹ Phosphorylated MLKL triggers membrane rupture,^{22, 23, 24, 25, 26} releasing pro-inflammatory cellular contents to the extracellular space.²⁷ Studies using the RIPK1 inhibitor necrostatin-1 (Nec-1) ²⁸ or RIPK3-deficient mice have established a role for necroptosis in the pathophysiology of pancreatitis,¹⁹ artherosclerosis,²⁹ retinal cell death,³⁰ ischemic organ damage and ischemia-reperfusion injury in both the kidney³¹ and the heart.³² Moreover, allografts from RIPK3-deficient mice are better protected from rejection, suggesting necroptosis inhibition as a therapeutic option to improve transplant outcome.³³ Besides Nec-1, several tool compounds inhibiting different pathway members have been described,^{12, 16, 21, 34, 35} however, no inhibitors of necroptosis are available for clinical use so far.^{2, 10} In this study we screened a library of FDA approved drugs for the precise purpose of identifying already existing and generally safe chemical agents that could be used as necroptosis inhibitors. We identified the two structurally distinct kinase inhibitors pazopanib and ponatinib as potent blockers of necroptosis targeting the key enzymes RIPK1/3.