Contrast Agents for Quantitative MicroCT of Lung Tumors in Mice

The identification and quantitative evaluation of lung tumors in mouse models is challenging and an unmet need in preclinical arena. In this study, we developed a noninvasive contrast-enhanced microCT (μCT) method to longitudinally evaluate and quantitate lung tumors in mice. Commercially available μCT contrast agents were compared to determine the optimal agent for visualization of thoracic blood vessels and lung tumors in naïve mice and in non-small-cell lung cancer models. Compared with the saline control, iopamidol and iodinated lipid agents provided only marginal increases in contrast resolution. The inorganic nanoparticulate agent provided the best contrast and visualization of thoracic vascular structures; the density contrast was highest at 15 min after injection and was stable for more than 4 h. Differential contrast of the tumors, vascular structures, and thoracic air space by the nanoparticulate agent enabled identification of tumor margins and accurate quantification. μCT data correlated closely with traditional histologic measurements (Pearson correlation coefficient, 0.995). Treatment of ELM4–ALK mice with crizotinib yielded 65% reduction in tumor size and thus demonstrated the utility of quantitative μCT in longitudinal preclinical trials. Overall and among the 3 agents we tested, the inorganic nanoparticulate product was the best commercially available contrast agent for visualization of thoracic blood vessels and lung tumors. Contrast-enhanced μCT imaging is an excellent noninvasive method for longitudinal evaluation during preclinical lung tumor studies.Abbreviations: μCT, microCT, HU, Hounsfield units, RECIST, response evaluation criteria in solid tumorsLung cancer is the leading cause of cancer death worldwide, and non-small–cell lung cancer is the most common form of lung cancer that is diagnosed.¹⁹ Various animal models have been used to mimic these cancers, understand their biology, and evaluate potential therapeutics.^14,20,27 Traditionally, the method to study the disease in vivo has been to inject human tumor derived cell lines subcutaneously in immunodeficient mice (xenografts) or to orthotopically implant tumors in tissues of interest. Xenografts, despite being of human origin, are not ideal models because tumor cell–stroma interactions cannot be reconstituted in the system. Moreover, the tumor microenvironment has been shown to be essential in predicting cancer cell survival, progression, metastasis, and response to therapy.^14,20,27 To overcome these issues, several investigators have propagated tumors orthotopically by either direct injection of tumor cells into the organ of choice, intravenous injection of tumor cell lines, or implantation of patient-derived tumor biopsy samples into immunodeficient mice. These models simulate the tumor microenvironment and bear a closer resemblance to clinical cancer than do xenografts.^11,27 In the past decade, tremendous progress has been made in development of genetically engineered mouse models that reconstitute facets of human disease in the organ of choice. In these models, the tumors are developed in immunocompetent hosts with intact tumor–stroma interactions, and the tumors can be controlled temporally and spatially.^7,14,25,26Despite the many advantages of genetically engineered and orthotopic models, their use has been limited, primarily due to the heterogeneity and technical difficulties associated with monitoring disease progression.¹⁷ Conventional optical imaging is not widely used with genetically engineered and orthotopic models, because these modalities provide low spatial resolution, limited tissue penetration,^1,17 and rarely accomdate reporter genes like luciferase or fluorescent proteins.³⁰ Nuclear imaging (for example, positron-emission tomography and MRI) has been used in evaluating lung tumor models,^7,13,33 but these modalities are not readily available in all facilities, require specialized laboratories (for example, radionucleotide synthesis), and do not achieve accurate quantification of nodular tumors.³³ X-ray CT has been used in human clinical practice to assess lung tumor nodules to predict likelihood of malignancy and to monitor the response of tumors to treatment.^18,23 Response Evaluation Criteria in Solid Tumors (RECIST) scores based on CT imaging data are used routinely in clinical trials and practice.³² Similarly, high-resolution microCT (μCT) scanners have been used successfully to image lung tumors^{6,15,17,24,25,34} in small animals such as rodents. The investigators in the aforementioned studies took advantage of the natural air–tissue contrast within the thorax to identify tumors. However, this method was unable to distinguish tumor and soft tissue from nearby vascular structures.^25,29 This limitation decreased the accuracy of tumor margin demarcation and tumor volumetric measurements.The goal of our study was to compare 3 commercially available μCT contrast agents (products containing iopamidol, iodinated lipid, and inorganic nanoparticulate) to evaluate and accurately quantify lung tumors in preclinical models. Our results showed that, among those we tested, the nanoparticulate product was the best contrast agent for tumor visualization and quantitation. In addition, we demonstrated the utility of contrast-enhanced μCT in a preclinical evaluation of the efficacy of crizotinib (PF-2341066), a small-molecule multikinase inhibitor,^9,10 in a genetically modified mouse model of lung cancer.     

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.     

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.     

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.     

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.     

Comparison of Biomarkers of Oxidative Stress and Cardiovascular Disease in Humans and Chimpanzees (Pan troglodytes)

In the oxidative stress hypothesis of aging, the aging process is the result of cumulative damage by reactive oxygen species. Humans and chimpanzees are remarkably similar; but humans live twice as long as chimpanzees and therefore are believed to age at a slower rate. The purpose of this study was to compare biomarkers for cardiovascular disease, oxidative stress, and aging between male chimpanzees and humans. Compared with men, male chimpanzees were at increased risk for cardiovascular disease because of their significantly higher levels of fibrinogen, IGF1, insulin, lipoprotein a, and large high-density lipoproteins. Chimpanzees showed increased oxidative stress, measured as significantly higher levels of 5-hydroxymethyl-2-deoxyuridine and 8-iso-prostaglandin F_2α, a higher peroxidizability index, and higher levels of the prooxidants ceruloplasmin and copper. In addition, chimpanzees had decreased levels of antioxidants, including α- and β-carotene, β-cryptoxanthin, lycopene, and tocopherols, as well as decreased levels of the cardiovascular protection factors albumin and bilirubin. As predicted by the oxidative stress hypothesis of aging, male chimpanzees exhibit higher levels of oxidative stress and a much higher risk for cardiovascular disease, particularly cardiomyopathy, compared with men of equivalent age. Given these results, we hypothesize that the longer lifespan of humans is at least in part the result of greater antioxidant capacity and lower risk of cardiovascular disease associated with lower oxidative stress.Abbreviations: 5OHmU, 5-hydroxymethyl-2-deoxyuridine; 8isoPGF_2α, 8-iso-prostaglandin F_2α; HDL, high-density lipoprotein; IGF1, insulin-like growth factor 1; LDL, low-density lipoprotein; ROS, reactive oxygen speciesAging is characterized as a progressive reduction in the capacity to withstand the stresses of everyday life and a corresponding increase in risk of mortality. According to the oxidative stress hypothesis of aging, much of the aging process can be accounted for as the result of cumulative damage produced by reactive oxygen species (ROS).^{6,21,28,41,97} Endogenous oxygen radicals (that is, ROS) are generated as a byproduct of normal metabolic reactions in the body and subsequently can cause extensive damage to proteins, lipids, and DNA.^6,41 Various prooxidant elements, in particular free transition metals, can catalyze these destructive reactions.⁶ The damage caused by ROS can be counteracted by antioxidant defense systems, but the imbalance between production of ROS and antioxidant defenses, over time, leads to oxidative stress and may contribute to the rate of aging.^28,97Oxidative stress has been linked to several age-related diseases including neurodegenerative diseases, ophthalmologic diseases, cancer, and cardiovascular disease.^21,28,97 Of these, cardiovascular disease remains the leading cause of adult death in the United States and Europe.⁷¹ In terms of cardiovascular disease, oxidative stress has been linked to atherosclerosis, hypertension, cardiomyopathy, and chronic heart failure in humans.^55,78,84 Increases in oxidant catalysts (prooxidants)—such as copper, iron, and cadmium—have been associated with hypertension, coronary artery disease, atherosclerosis, and sudden cardiac death.^98,102,106 Finally, both endogenous and exogenous antioxidants have been linked to decreased risk of cardiovascular disease, although the mechanisms behind this relationship are unclear.^11,52,53 However, the oxidative stress hypothesis of aging aims to explain not only the mechanism of aging and age-related diseases (such as cardiovascular disease) in humans but also the differences between aging rates and the manifestations of age-related diseases across species.The differences in antioxidant and ROS levels between animals and humans offer promise for increasing our understanding of human aging. Additional evidence supporting the oxidative stress hypothesis of aging has come from comparative studies linking differences in aging rates across taxa with both antioxidant and ROS levels.^{4,17-21,58,71,86,105} In mammals, maximum lifespan potential is positively correlated with both serum and tissue antioxidant levels.^{17,18,21,71,105} Research has consistently demonstrated that the rate of oxidative damage varies across species and is negatively correlated with maximum lifespan potential.^{4,19,20,58,71,86} However, few studies involved detailed comparisons of hypothesized biochemical indicators of aging and oxidative stress between humans and animals.⁶ This type of interspecies comparison has great potential for directly testing the oxidative stress hypothesis of aging.Much evolutionary and genetic evidence supports remarkable similarity between humans and chimpanzees.^95,100 Despite this similarity, humans have a lifespan of almost twice that of chimpanzees.^3,16,47 Most comparative primate aging research has focused on the use of a macaque model,^62,81,88 and several biochemical markers of age-related diseases have been identified in both humans and macaque monkeys.^{9,22,28,81,93,97} Several other species of monkeys have also been used in research addressing oxidative stress, antioxidant defenses, and maximum lifespan potential.^18,21,58,105 However, no study to date has examined biochemical indicators of oxidative stress and aging in chimpanzees and humans as a test of the oxidative stress hypothesis for aging. The purpose of this study is to compare biochemical markers for cardiovascular disease, oxidative stress, and aging directly between male chimpanzees and humans. Given the oxidative stress hypothesis for aging and the known role of oxidative stress in cardiovascular disease, we predict that chimpanzees will show higher levels of cardiovascular risk and oxidative stress than humans.     

Using Multidetector Computed Tomography in a Swine Model to Assess the Effects of Sublingual Nitroglycerin and Intravenous Adenosine on Epicardial Coronary Arteries

This study examines the effects of intravenous infusion of adenosine and sublingual nitroglycerin on coronary angiograms obtained by current-generation multidetector computed tomography. We assessed coronary vasodilation at baseline and after intravenous adenosine (140 µg/kg/min) or sublingual nitroglycerin spray (800 µg) in 7 female swine (weight, 40.9 ± 1.4 kg) by using electrocardiogram-gated coronary angiography with a 64-detector scanner (rotation time, 400 ms; 120kV; 400 mA) and intravenous contrast (300 mg/mL iohexol, 4.5 mL/s, 2 mL/kg). Cross-sectional areas of segments in the left anterior descending, circumflex, and right coronary arteries were evaluated in oblique orthogonal views. Images were acquired at an average heart rate of 73 ± 11 beats per minute. Changes in aortic pressure were not significant with nitroglycerin but decreased (approximately 10%) with adenosine. Of the 76 segments analyzed (baseline range, 2 to 39 mm²), 1 distal segment could not be assessed after adenosine. Segment cross-sectional area increased by 11.3% with nitroglycerin but decreased by 9.6% during adenosine infusion. The results of the present study are consistent with the practice of using sublingual nitroglycerin to enhance visualization of epicardial vessels and suggest that intravenous adenosine may hinder coronary artery visualization. This study is the first repeated-measures electrocardiogram-gated CT evaluation to use the same imaging technology to assess changes in coronary cross-sectional area before and after treatment with a vasodilator. The nitroglycerin-associated changes in our swine model were modest in comparison with previously reported human studies.Abbreviations: LAD, left anterior descending artery; MDCT, multidetector computed tomographyRecent advances in multidetector computed tomography (MDCT) for coronary angiography, their increased availability, and the pending release of a new generation of CT scanners contribute to this methodology''s potential for revolutionizing the early diagnosis and functional assessment of coronary artery disease.^15,16,25 However, the benefits of methodologic advances do not diminish the need to validate and assess the safety, efficacy, and costs of technology.¹⁵ In this regard, the present study uses an animal model with coronary anatomy analogous to that of humans^11,43 to assess the effects of sublingual nitroglycerin and intravenous adenosine on coronary epicardial vessel visualization by using baseline imaging as a control. The radiation exposure associated with CT procedures precludes human studies that involve repeated measures on the same subjects.Sublingual nitroglycerin is hypothesized to enhance the visualization of epicardial coronary vessels due to its vasodilatory properties.^8-10 Nitroglycerin acts as a nitric oxide generator to induce relaxation of vascular smooth muscle independent of endothelial function.^1,18,20 Current evidence supporting improved coronary visualization with sublingual nitroglycerin is derived from clinical cross-sectional studies that compare results in different groups of patients with heterogeneous coronary lesions.^5,6,16,27,44 A recent survey reported that more than 80% of facilities in the United States routinely use nitroglycerin in cardiac MDCT angiography.²³In contrast to the action of nitroglycerin on epicardial vascular smooth muscle, intravenous adenosine frequently is used as a pharmacologic stress to affect vasodilatation within the coronary microcirculation.²⁰ Although current-generation CT scanners have limited capability to assess myocardial tissue perfusion, research efforts from advanced imaging centers suggest that future-generation CT scanners soon will permit myocardial perfusion imaging.^13,14,29 No studies to date have assessed epicardial vessel changes by MDCT with intravenous doses of adenosine appropriate for myocardial perfusion imaging.The objective of this study was to provide an animal model for MDCT studies that permits repeated measures of epicardial vessels without the limitations of cross-sectional designs or the complications of heterogeneity of vascular lesions within clinical populations. This study used routes of administration of nitroglycerin and adenosine that are consistent with clinical practice.     

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.     

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.     

Paravascular pathways contribute to vasculitis and neuroinflammation after subarachnoid hemorrhage independently of glymphatic control

Subarachnoid hemorrhage (SAH) is a devastating disease with high mortality. The mechanisms underlying its pathological complications have not been fully identified. Here, we investigate the potential involvement of the glymphatic system in the neuropathology of SAH. We demonstrate that blood components rapidly enter the paravascular space following SAH and penetrate into the perivascular parenchyma throughout the brain, causing disastrous events such as cerebral vasospasm, delayed cerebral ischemia, microcirculation dysfunction and widespread perivascular neuroinflammation. Clearance of the paravascular pathway with tissue-type plasminogen activator ameliorates the behavioral deficits and alleviates histological injury of SAH. Interestingly, AQP4^−/− mice showed no improvements in neurological deficits and neuroinflammation at day 7 after SAH compared with WT control mice. In conclusion, our study proves that the paravascular pathway dynamically mediates the pathological complications following acute SAH independently of glymphatic control.Cerebral aneurysm rupture causes subarachnoid hemorrhage (SAH), which is associated with a high mortality due to its secondary complications, including hemorrhage, hydrocephalus and delayed cerebral ischemia (DCI).^{1, 2, 3} Therapeutic interventions against the secondary complications, especially DCI, are yet limited, as the pathological mechanism underlying that is not fully understood.^{2, 3, 4, 5, 6, 7} Current hypotheses of the development of the secondary complications mainly include cerebral vasospasm (CVS) and the microcirculation disturbance, as well as parenchymal arterial lesions, microthrombosis and neuroinflammation.^{1, 2, 4, 7, 8, 9}Previous studies have shown that the blockade of cerebral lymphatic drainage deteriorated the secondary cerebral ischemia after SAH, suggesting that the cerebral lymphatic drainage pathway could be involved in the pathological mechanism of SAH.^{10, 11} However, the central nervous system (CNS) was considered lack of a conventional lymphatic drainage system in the past. Recently, several studies have shown that the brain has in fact the proper lymphatic system, including sinus-associated lymphatic vessels and the glymphatic system (GS).^{12, 13, 14, 15} Sinus-associated lymphatic vessels express all of the molecular hallmarks of lymphatic endothelial cells, contain cerebrospinal fluid (CSF) and immune cells, and drain into the deep cervical lymph nodes.^{12, 13}There is a histologically defined space in the brain, the Virchow–Robin space, where the subarachnoid space meets the paravascular space (or perivascular space in somewhere, PVS).¹⁶ The GS is a specialized brain-wide anatomic structure locating at the PVS surrounding the brain vasculature, which is ensheathed with the astroglial endfeet and astroglial water channel aquaporin-4 (AQP4).^{14, 15} The GS facilitates the efficient lymphatic clearance of extracellular solutes and fluid in the brain through astroglial-mediated interstitial fluid bulk flow.¹⁴Impairment of GS involves neurological conditions including traumatic brain injuries,¹⁷ ischemic stroke¹⁸ and aged brain.¹⁹ Interestingly, brain imaging study with magnetic resonance imaging reported weakened GS perfusion following acute stroke or SAH.^{18, 20} However, little is known about whether the GS is involved in the secondary complications of SAH. Here, we examined the potential involvement of GS in SAH-associated pathology progression with in vivo two-photon microscopy and CLARITY technique.^{21, 22} Our data showed that subarachnoid blood flowed into the brain parenchyma rapidly through the PVS, causing CVS, vasculitis, widespread microinfraction and neuroinflammation in the animal model of SAH and SAH patients. Prevention of CVS with Fasudil²³ did not improve the neurological impairment nor alleviated the pathology, while the PVS clearance with tissue-type plasminogen activator (tPA) infusion improved the behavioral recovery and reduced neuroinflammation in the brain. Interestingly, AQP4^−/− mice showed no improvements in neurological deficits and neuroinflammation at day 7 after SAH compared with WT control mice. Our study therefore suggested that the paravacular pathway dynamically mediates the pathological complications following acute SAH independently of glymphatic control.     

Analysis of Gene Expression and Ultrastructure of Stifle Menisci from Juvenile and Adult Pigs

The origin of the age-associated degenerative processes in meniscal tissue is poorly understood and may be related to an imbalance of anabolic and catabolic metabolism. The aim of the current study was to compare medial menisci isolated from juvenile pigs and degenerated medial menisci from adult pigs in terms of gene expression profile and ultrastructure. Medial menisci were isolated from the knee joints of juvenile and adult pigs (n = 8 for each group). Degeneration was determined histologically according to a scoring system. In addition, the gene expression profiles of 14 genes encoding extracellular matrix proteins, catabolic matrix metalloproteinases and mediators of inflammation were analyzed. Changes in the ultrastructure of the collagen network of the meniscal tissue were analyzed by using transmission electron microscopy. The histologic analysis of menisci showed significantly higher grade of degeneration in tissue isolated from adult porcine knee joints compared with menisci isolated from juvenile knee joints. In particular, destruction of the collagen network was greater in adult menisci than in juvenile menisci. Degenerated menisci showed significantly decreased gene expression of COL1A1 and increased expression of MMP2, MMP13, and IL8. The menisci from adult porcine knee joints can serve as a model for meniscal degeneration. Degenerative changes were manifested as differences in histopathology, gene expression and ultrastructure of collagen network.Abbreviations: MMP, matrix metalloproteinase; SOX, sex-determining region box; VEGF, vascular endothelial growth factorMuch research is focused on the degeneration of connective tissue, particularly of the cartilage tissue that stabilizes the knee joint. The goal of the current study was to determine whether menisci in adult pigs show patterns of degeneration in the absence of previous major injuries, which might cause a secondary form of degeneration.In the mammalian knee joint, the incongruence between the femoral condyle and the tibial plateau is partially balanced by 2 C-shaped fibrocartilaginous menisci. These menisci absorb impact forces, help to distribute the mechanical load on the tibial plateau, and act as stabilizers of the knee joint.^35,54 Therefore, menisci are thought to play an important role in the development of osteoarthritis in knee joints,³⁹ as indicated by the results following meniscal resection.⁵⁸ Both injuries¹³ and degeneration⁴ of meniscal tissue increase the risk of osteoarthritis of the knee joint. Because the self-repair mechanisms of meniscal tissue appear to be inadequate,⁹ more than 1 million surgical interventions on menisci are performed every year in the United States.²⁷ In addition, degenerative changes in the meniscal tissue are believed to contribute to meniscal lesions.The main component of the meniscus is water (70%); and most of its dry weight is due to the protein collagen, mainly collagen I (98%).⁵⁷ The ultrastructure of meniscal tissue consists of collagen fibers that are orientated circumferentially in the superficial layers. The intermediate layer consists of tangentially orientated collagen fibers.¹⁵ Other proteins involved in the extracellular matrix of meniscal tissue include collagen II and the proteoglycan aggrecan.¹⁵In addition, biglycan and fibromodulin have been found in porcine menisci.⁴²The primary cells in meniscal tissue are chondrocytes which, in concert with fibroblasts, produce the extracellular matrix of fibrocartilage.⁵⁵ In addition, chondrocytes produce the lubricant lubricin,⁵⁶ which also is expressed in meniscal tissue.^24,49Meniscal degeneration can be understood as an imbalance between anabolic and catabolic processes, as it has already been shown for articular cartilage.² Changes in the gene expression of menisci from osteoarthritic knee joints including genes involved in immune and inflammatory responses as well as in tissue development have been previously shown.⁵² Furthermore, the production of the matrix proteins collagen types I, II, and III decreases during the progression of human meniscal degeneration.⁴³ Changes in the gene expression profile of anabolic genes (for example, collagen I, collagen II, aggrecan) and catabolic genes (for example, matrix metalloproteinases) are early indicators of osteoarthritis.^{2,14,22,26,34,51} Therefore, additional studies analyzing the gene expression profiles of healthy and degenerated menisci are warranted to evaluate whether such marker genes also exist for meniscal tissue. As a next step, the ultrastructure of degenerated meniscal tissue should be evaluated to analyze the influence of an altered gene expression profile on the extracellular matrix of menisci.In general, pigs are an appropriate animal model in biomedical research because of their similarities to humans in terms of anatomy and metabolism.^7,19,53 In meniscus research, goats³⁰ and sheeps are well-established animal models.^11,31,58 Nevertheless, the structure of porcine collagen is highly analogous to human collagen.³ This similarity was supported by the results of a comparison of the collagen in the hyaline cartilage among several species. ²⁵ Because pathogenesis for osteoarthritis is similar between human and animal tissue,³⁸ similarities in the pathophysiology of porcine and human meniscal tissue seem likely.The aim of the current study was to compare the medial menisci isolated from juvenile pigs with the degenerated medial menisci from adult pigs to determine whether differences in their gene expression profiles and ultrastructure are present.     

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.     

Distinct roles of RIP1–RIP3 hetero- and RIP3–RIP3 homo-interaction in mediating necroptosis

Necroptosis is mediated by a signaling complex called necrosome, containing receptor-interacting protein (RIP)1, RIP3, and mixed-lineage kinase domain-like (MLKL). It is known that RIP1 and RIP3 form heterodimeric filamentous scaffold in necrosomes through their RIP homotypic interaction motif (RHIM) domain-mediated oligomerization, but the signaling events based on this scaffold has not been fully addressed. By using inducible dimer systems we found that RIP1–RIP1 interaction is dispensable for necroptosis; RIP1–RIP3 interaction is required for necroptosis signaling, but there is no necroptosis if no additional RIP3 protein is recruited to the RIP1–RIP3 heterodimer, and the interaction with RIP1 promotes the RIP3 to recruit other RIP3; RIP3–RIP3 interaction is required for necroptosis and RIP3–RIP3 dimerization is sufficient to induce necroptosis; and RIP3 dimer-induced necroptosis requires MLKL. We further show that RIP3 oligomer is not more potent than RIP3 dimer in triggering necroptosis, suggesting that RIP3 homo-interaction in the complex, rather than whether RIP3 has formed homo polymer, is important for necroptosis. RIP3 dimerization leads to RIP3 intramolecule autophosphorylation, which is required for the recruitment of MLKL. Interestingly, phosphorylation of one of RIP3 in the dimer is sufficient to induce necroptosis. As RIP1–RIP3 heterodimer itself cannot induce necroptosis, the RIP1–RIP3 heterodimeric amyloid fibril is unlikely to directly propagate necroptosis. We propose that the signaling events after the RIP1–RIP3 amyloid complex assembly are the recruitment of free RIP3 by the RIP3 in the amyloid scaffold followed by autophosphorylation of RIP3 and subsequent recruitment of MLKL by RIP3 to execute necroptosis.Necroptosis is a type of programmed necrosis characterized by necrotic morphological changes, including cellular organelle swelling, cell membrane rupture,^{1, 2, 3} and dependence of receptor-interacting protein (RIP)1⁴ and RIP3.^{5, 6, 7} Physiological function of necroptosis has been illustrated in host defense,^{8, 9, 10, 11} inflammation,^{12, 13, 14, 15, 16} tissue injury,^{10, 17, 18} and development.^{19, 20, 21}Necroptosis can be induced by a number of different extracellular stimuli such as tumor necrosis factor (TNF). TNF stimulation leads to formation of TNF receptor 1 (TNFR1) signaling complex (named complex I), and complex II containing RIP1, TRADD, FAS-associated protein with a death domain (FADD), and caspase-8, of which the activation initiates apoptosis. If cells have high level of RIP3, RIP1 recruits RIP3 to form necrosome containing FADD,^{22, 23, 24} caspase-8, RIP1, and RIP3, and the cells undergo necroptosis.^{25, 26} Caspase-8 and FADD negatively regulates necroptosis,^{27, 28, 29, 30} because RIP1, RIP3, and CYLD are potential substrates of caspase-8.^{31, 32, 33, 34} Necrosome also suppresses apoptosis but the underlying mechanism has not been described yet. Mixed-lineage kinase domain-like (MLKL) is downstream of RIP3,^{35, 36} and phosphorylation of MLKL is required for necroptosis.^{37, 38, 39, 40, 41, 42}Apoptosis inducing complex (complex II) and necrosome are both supramolecular complexes.^{43, 44, 45} A recent study showed that RIP1 and RIP3 form amyloidal fibrils through their RIP homotypic interaction motif⁴⁶ (RHIM)-mediated polymerization, and suggested that amyloidal structure is essential for necroptosis signaling.⁴⁷ The RIP1–RIP3 heterodimeric amyloid complex is believed to function as a scaffold that brings signaling proteins into proximity to permit their activation. However, RIP1 and RIP3 also can each form fibrils on their own RHIM domains in vitro. It is unclear how the homo- and hetero-interactions are coordinated and organized on the amyloid scaffold to execute their functions in necroptosis. Here, we used inducible dimerization systems to study the roles of RIP1–RIP1, RIP1–RIP3, and RIP3–RIP3 interactions in necroptosis signaling. Our data suggested that it is the RIP1–RIP3 interaction in the RIP1–RIP3 heterodimeric amyloid complex that empowers to recruit other free RIP3; homodimerization of RIP3 triggers its autophosphorylation and only the phosphorylated RIP3 can recruit MLKL to execute necroptosis.     

Spontaneous Osteoblastic Osteosarcoma in a Mongolian Gerbil (Meriones unguiculatus)

Spontaneous neoplasms in Mongolian gerbils have an incidence of 20% to 26.8%, but osteosarcomas occur at a much lower rate. Here we report a 1-y-old Mongolian gerbil with a spontaneous osteosarcoma at the level of the proximal tibia, with metastases to the pectoral muscles and lungs. Grossly, the tibial mass obliterated the tibia and adjacent muscles, and an axillary mass with a bloody, cavitary center expanded the pectoral muscles. Microscopically, the tibial mass was an infiltrative, osteoblastic mesenchymal neoplasm, and the axillary mass was an anaplastic mesenchymal neoplasm with hemorrhage. The lung contained multiple metastatic foci. Immunohistochemistry for osteonectin was strongly positive in the tibial, axillary, and pulmonary metastases. Although osteosarcoma is the most common primary malignant bone neoplasm that occurs spontaneously in all laboratory and domestic animal species and humans, it arises less frequently than does other neoplasms. The current case of spontaneous osteoblastic osteosarcoma of the proximal tibia and metastases to the pectoral muscles and lung in a Mongolian gerbil is similar in presentation, histology, and predilection site of both osteoblastic and telangiectatic osteosarcomas in humans. In addition, this case is an unusual manifestation of osteosarcoma in the appendicular skeleton of a Mongolian gerbil.Mongolian gerbils are used frequently in biologic research,^{1,2,4,9,10,12-14} particularly in oncogenic studies and filariasis research studying Brugia malayi.² There have been several reports^{1,6,10,11,13-15} of spontaneous neoplasms, particularly in gerbils 2 y of age and older, typically occurring with the highest incidences in the skin, reproductive tract, and adrenal glands; however, neoplasms have also been reported in the thyroid, thymus, liver, kidney, pancreas, and bone.^{1,6,10,11,13-15} The incidence of spontaneous neoplasms occurring in the subfamily Gerbillinae ranges from 20% to 26.8%,^{1,6,10,11,13-15} depending on the study, age, and sex of the animals.With a lower incidence than those reported for other neoplasms, osteosarcomas in gerbils have been described in the ramus of the mandible and as an extraskeletal mass throughout the peritoneum.^10,11 The usual age of onset for osteosarcomas in Mongolian gerbils is approximately 3 y (36 to 39 mo); however, no tumor type has been reported at less than 2 y of age in this species.^10,11 Here we report a spontaneous osteosarcoma that occurred at the level of the proximal tibia, with metastases to the pectoral muscles and lung, in a 1-y-old Mongolian gerbil.