Mouse Models for the Study of SARS-CoV-2 Infection

Mice are an invaluable resource for studying virus-induced disease. They are a small, genetically modifiable animal for which a large arsenal of genetic and immunologic tools is available for evaluation of pathogenesis and potential vaccines and therapeutics. SARS-CoV-2, the betacoronavirus responsible for the COVID-19 pandemic, does not naturally replicate in wild-type mice, due to structural differences between human and mouse ACE2, the primary receptor for SARS-CoV-2 entry into cells. However, several mouse strains have been developed that allow for SARS-CoV-2 replication and clinical disease. Two broad strategies have primarily been deployed for developing mouse strains susceptible to COVID-19-like disease: adding in the human ACE2 gene and adapting the virus to the mouse ACE2 receptor. Both approaches result in mice that develop several of the clinical and pathologic hallmarks of COVID-19, including acute respiratory distress syndrome and acute lung injury. In this review, we describe key acute pulmonary and extrapulmonary pathologic changes seen in COVID-19 patients that mouse models of SARS-CoV-2 infection ideally replicate, the essential development of mouse models for the study of Severe Acute Respiratory Syndrome and Middle Eastern Respiratory Syndrome and the basis of many of the models of COVID-19, and key clinical and pathologic features of currently available mouse models of SARS-CoV-2 infection.

Coronaviruses are widespread and infect several different species. In humans, coronaviruses historically cause primarily mild respiratory diseases, such as the common cold. However, in 2003 a novel coronavirus emerged, Severe Acute Respiratory Syndrome coronavirus (SARS-CoV), which caused severe respiratory disease with high mortality.¹⁸ Since then, 2 other highly pathogenic coronaviruses from the same betacoronavirus genus have emerged: Middle Eastern Respiratory Syndrome coronavirus (MERS-CoV) in 2012, and most recently, Severe Acute Respiratory Syndrome coronavirus 2 (SARS-CoV-2).^18,133,134 Spread of SARS-CoV-2, which causes coronavirus disease 2019 (COVID-19), has resulted in a once-in-a-lifetime pandemic, and as of May 2021, more than 160 million cases and more than 3.3 million deaths have been reported worldwide.¹²³Much like SARS-CoV, SARS-CoV-2 has 4 structural proteins—spike (S), envelope (E), membrane (M), and nucleocapsid (N)—and 8 accessory proteins. The S protein includes a receptor-binding domain, which is essential for the virus to bind to and subsequently infect a host cell.¹²⁴ SARS-CoV and SARS-CoV-2 both use angiotensin-converting enzyme 2 (ACE2) as the primary receptor and transmembrane protease serine 2 as a cofactor,^42,56,60 In contrast, the receptor for MERS-CoV is dipeptidyl peptidase 4 (DPP4).⁹¹ Because of amino acid changes in the S protein, SARS-CoV-2 binds ACE2 with a higher affinity than does SARS-CoV,¹²⁴ which may explain the greater human infectivity of SARS-CoV-2.^42,102 ACE2 is expressed throughout the body, allowing SARS-CoV-2 to potentially infect multiple organs, including the lung, heart, kidney, liver, intestines, and brain. Importantly, ACE2 is expressed on the apical surfaces of epithelial cells in these organs, permitting infection from direct viral contact with those cells.³⁷ As its name suggests, ACE2 is a critical component of the renin–angiotensin cascade, limiting vasoconstriction and promoting vasodilation by converting angiotensin II to angiotensin 1–7. ACE2 in the lung is hypothesized to reduce lung inflammation; SARS-CoV and SARS-CoV-2 potentially could exacerbate lung inflammation by altering this pathway.⁹⁸A thorough understanding of disease pathogenesis and any potential vaccines or therapeutics for any emerging pathogen is facilitated by studying animal models. For example, the first FDA-approved treatment for COVID-19 was remdesivir. This drug initially was granted emergency-use authorization for COVID-19 patients in part because of demonstrated therapeutic efficacy against MERS-CoV infection in a mouse model.¹⁰³ An ideal animal model of COVID-19 captures the wide spectrum of disease phenotypes attributed to this multifaceted disease, including organ-specific pathology and systemic changes such as hypercoagulation and cytokine storms. Animal models rarely capture every aspect of human disease, requiring the use of multiple models in order to replicate the numerous features of viral infection and disease and thereby build a comprehensive picture of what happens in human patients.SARS-CoV-2 naturally infects numerous animal species, including mink on farms, big cats in zoos and sanctuaries, and domestic dogs and cats.^{28,36,68,72,81,87,104,107} Although mink are particularly vulnerable to severe SARS-CoV-2 disease and can transmit virus to humans, they are rarely used in research studies.^68,81,87 Mice, hamsters, ferrets, and nonhuman primates are more widely used as experimental models of SARS-CoV-2 infection.⁶⁸ Hamsters replicate human disease well, are small, and are widely available, making them an attractive choice.^12,68,105 However, hamsters are less commonly used in research than are mice, and the availability of strains and reagents necessary for studying genetic and immune drivers of disease is limited for hamsters. Ferrets are frequently used as models for respiratory viruses, particularly influenza, primarily because they disperse and are susceptible to these viruses via airborne droplets, whereas rodents do not. However, ferrets do not develop severe symptoms or generate high virus titers in the lungs after SARS-CoV-2 infection.^52,68,95,104 NHPs have similar immune responses to humans and are invaluable for safety studies for preclinical trials but are costly to use and require significant infrastructure to maintain studies.^66,68,130 Mice provide a valuable model because they are widely available and relatively inexpensive, and a large arsenal of genetic and immunologic tools is available for mice. The use of mice to study SARS-CoV-2 infection provides the potential for a broader understanding of several different aspects of this complex disease.     

Hamsters as a Model of Severe Acute Respiratory Syndrome Coronavirus-2

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the cause of coronavirus disease 2019 (COVID-19), rapidly spread across the world in late 2019, leading to a pandemic. While SARS-CoV-2 infections predominately affect the respiratory system, severe infections can lead to renal and cardiac injury and even death. Due to its highly transmissible nature and severe health implications, animal models of SARS-CoV-2 are critical to developing novel therapeutics and preventatives. Syrian hamsters (Mesocricetus auratus) are an ideal animal model of SARS-CoV-2 infections because they recapitulate many aspects of human infections. After inoculation with SARS-CoV-2, hamsters become moribund, lose weight, and show varying degrees of respiratory disease, lethargy, and ruffled fur. Histopathologically, their pulmonary lesions are consistent with human infections including interstitial to broncho-interstitial pneumonia, alveolar hemorrhage and edema, and granulocyte infiltration. Similar to humans, the duration of clinical signs and pulmonary pathology are short lived with rapid recovery by 14 d after infection. Immunocompromised hamsters develop more severe infections and mortality. Preclinical studies in hamsters have shown efficacy of therapeutics, including convalescent serum treatment, and preventatives, including vaccination, in limiting or preventing clinical disease. Although hamster studies have contributed greatly to our understanding of the pathogenesis and progression of disease after SARS-CoV-2 infection, additional studies are required to better characterize the effects of age, sex, and virus variants on clinical outcomes in hamsters. This review aims to describe key findings from studies of hamsters infected with SARS-CoV-2 and to highlight areas that need further investigation.

Coronavirus disease 2019 (COVID-19) is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), a novel betacoronavirus that was first detected in Wuhan, China at the end of 2019.³¹ Coronavirus infections predominantly present with either respiratory or gastrointestinal manifestations, depending on the strain and host. While many coronavirus infections result in mild clinical symptoms, SARS-CoV-2 is highly pathogenic and poses significant health concerns.^31,58,78 Although initial clinical signs are attributed to the respiratory system, severe infections result in systemic complications, such as acute cardiac and renal injury, secondary infections, and shock.^31,58SARS-CoV-2 relies on a structural surface spike glycoprotein to establish infection. The spike protein binds to the angiotensin-converting enzyme 2 (ACE2) receptor on host cells to gain entry in a receptor-mediated fashion. This interaction facilitates both human-to-human transmission and cross-species infection.⁷⁷ Species tropism is determined by the presence of ACE2 residues that recognize the SARS-CoV-2 spike protein. Animals permissive for SARS-CoV-2 infection include cats, ferrets, pigs, nonhuman primates, select genetically modified mice, and hamsters.^5,7,23,37,67 Susceptible species can be both intermediate hosts and sources of infection of SARS-CoV-2 for humans.⁷⁷ Rodents, such as mice and hamsters, are ideal models for the study of COVID-19 due to their small size, ready availability, low cost of care, SPF status, and in-depth characterization across a variety of translational models, including past and present betacoronavirus infections.^60,61 Although transgenic mice expressing human ACE2 are susceptible to SARS-CoV-2 infection, Syrian hamsters (Mesocricetus auratus) naturally express ACE2 residues that recognize the SARS-CoV-2 spike protein.^5,46,84 As such, Syrian hamsters are a valuable animal model for studying COVID-19.Syrian hamsters, commonly referred to as golden hamsters, belong to the family Cricetidae and have a natural geographic range of arid southeast Europe and Asia Minor. Additional members of the Cricetidae family used in biomedical research include Chinese hamsters (Cricetulus griseus), European hamsters (Cricetus cricetus), Armenian hamsters (Cricetulus migratorius), and dwarf hamsters (Phodopus species). Unless otherwise noted, any mention of hamsters in this overview refers to Syrian hamsters. Laboratory hamsters primarily originated from one Syrian litter captured in 1930. Progeny of this litter were first imported into the United States in 1938.⁵⁰ Outbred Syrian hamsters are widely available; recently developed transgenic hamsters are increasingly used in biomedical research and may provide unique insight into SARS-CoV-2 infections.^22,44 Syrian hamsters have a rich history in biomedical research and can be used to model cancer and infectious, metabolic, cardiovascular, and respiratory diseases.⁵⁰Hamsters play an important role in SARS-CoV-2 studies. This is due, in part, to their susceptibility to the first described highly pathogenic coronavirus infection in the 21st century, severe acute respiratory syndrome (SARS-CoV). SARS-CoV emerged in late 2002 in Southern China. Although individuals in more than 20 countries contracted SARS-CoV, the spread was quickly contained, with the last reported case in July 2003.^16,40 After experimental infection with SARS-CoV, hamsters developed high viral loads in the lungs and nasal turbinates.^{15,32,56,62,69} Pulmonary pathology included inflammation, cell necrosis, and consolidation without clinical signs of disease.⁶¹ Based on their susceptibility to SARS-CoV and natural expression of ACE2 capable of recognizing the SARS-CoV-2 spike protein, hamsters have been a preferred model of SARS-CoV-2. Hamster studies have replicated key aspects of SARS-CoV-2 infections in humans, including viral replication, transmission, and pathology. Furthermore, hamsters are a model organism for developing and testing novel preventions and therapeutics. However, using hamsters in biomedical research has several key limitations, including the lack of reagents, especially antibodies, suitable for use with hamster tissue and the relatively few established transgenic hamsters compared to mice. The purpose of this review is to describe key findings of hamster models of SARS-CoV-2 and to highlight gaps in our current understanding that will require further investigation.     

Viral and Host Attributes Underlying the Origins of Zoonotic Coronaviruses in Bats

Factors affecting the origins of zoonotic coronaviruses in batsWith a presumed origin in bats, the COVID-19 pandemic has been a major source of morbidity and mortality in the human population, and the causative agent, SARS-CoV-2, aligns most closely at the genome level with the bat coronaviruses RaBtCoV4991/RaTG13 and RmYN02. The ability of bats to provide reservoirs of numerous viruses in addition to coronaviruses remains an active area of research. Unique aspects of the physiology of the chiropteran immune system may contribute to the ability of bats to serve as viral reservoirs. The coronavirus spike protein plays important roles in viral pathogenesis and the immune response. Although much attention has focused on the spike receptor-binding domain, a unique aspect of SARS-CoV-2 as compared with its closest relatives is the presence of a furin cleavage site in the S1–S2 region of the spike protein. Proteolytic activation is likely an important feature that allows SARS-CoV-2—and other coronaviruses—to overcome the species barriers and thus cause human disease. The diversity of bat species limits the ability to draw broad conclusions about viral pathogenesis, but comparisons across species and with reference to humans and other susceptible mammals may guide future research in this regard.

The COVID-19 pandemic remains a global public health emergency. The virus causing the pandemic (SARS-CoV-2) was identified in late 2019, after an outbreak of respiratory disease in Wuhan, China.⁸⁷ Early sequencing aligned SARS-CoV-2 with the previously detected RaBtCoV/4991.^13,20 SARS-CoV-2 was then further sequenced to yield the full genome (reported as RaTG13⁹⁵), defining SARV-CoV-2 as being a probable bat-origin zoonotic coronavirus. Severe acute respiratory syndrome coronavirus (SARS-CoV), which emerged in 2002, and Middle East respiratory syndrome coronavirus (MERS-CoV), which emerged in 2012, are also considered to be of bat origin.^46,51,71 At least 2 other commonly circulating coronaviruses in humans also likely originated in bats: HCoV-229E and HCoV-NL63.^15,33 However, the human coronaviruses (HCoV) OC43 and HKU1 have alternatively been suggested to have emerged from rodents via cattle and from rodents via an unknown intermediate, respectively.^47,73 Swine acute diarrhea syndrome coronavirus, a cause of significant mortality in piglets, emerged in 2016 in the Guangdong province of China from the bat virus Rhinolophus bat HKU2.^22,59,94 The swine enteric coronavirus porcine epidemic diarrhea virus is also considered to have bat origins.³¹ Numerous other viral diseases of human concern, including Nipah and Hendra viruses and ebolavirus, moved into human populations from bats.^14,25,41 Bats are also a common reservoir for the lethal rabies virus.^9,64 The ability of bats to harbor and spread these viruses continues to be an active area of study, integrating surveillance, ecology, disease forecasting, and basic virology to protect human and animal populations.     

Overview of Nonhuman Primate Models of SARS-CoV-2

COVID-19, the disease caused by the SARS-CoV-2 betacoronavirus, was declared a pandemic by the World Health Organization on March 11, 2020. Since then, SARS-CoV-2 has triggered a devastating global health and economic emergency. In response, a broad range of preclinical animal models have been used to identify effective therapies and vaccines. Current animal models do not express the full spectrum of human COVID-19 disease and pathology, with most exhibiting mild to moderate disease without mortality. NHPs are physiologically, genetically, and immunologically more closely related to humans than other animal species; thus, they provide a relevant model for SARS-CoV-2 investigations. This overview summarizes NHP models of SARS-CoV-2 and their role in vaccine and therapeutic development.

Coronaviruses are enveloped, single-stranded, positive-sense, RNA viruses in the subfamily Orthocoronavirinae, family Coronaviridae, order Nidovirales. There are 4 coronavirus genera, that is, Alphacoronavirus and Betacoronavirus, which infect mammals; and Gammacoronavirus and Deltacoronavirus, which primarily infect birds, with some able to infect mammals.¹³³ From these natural reservoirs, coronaviruses may infect other animals and humans. Human transmission typically requires an intermediate host.Prior to the 2002 SARS-CoV epidemic, only 2 human coronaviruses (HCoVs) had been identified - an alphacoronavirus (HCoV-229E) transmitted from bats to humans by alpacas, and a betacoronavirus (HCoV-OC43) transmitted from rodents to humans by cattle.^16,18 In 2004, HCoV-NL63 (alphacoronavirus, bat reservoir) and in 2005, HCoV-HKU1 (betacoronavirus, rodent reservoir) were identified.^39,132 Together, these 4 HCoVs cause an estimated 15% to 30% of common cold cases in humans, but can cause severe infections in infants, juvenile children, and the elderly.^23,64 However, in 2002, a new betacoronavirus caused an epidemic that originated in China, resulting in 8,000 confirmed cases with a mortality rate of 9.6%. The virus was named SARS-CoV and was transmitted from bats to humans by a palm civet intermediate host.^59,63,83 Ten years later in June 2012, MERS-CoV, a novel betacoronavirus transmitted from bats to humans by dromedary camels, emerged in Saudi Arabia.^17,25 MERS-CoV was also responsible for a 2015 outbreak in South Korea. Although human-to-human transmission of MERS-CoV was limited, the virus resulted in more than 2,000 confirmed cases and a mortality rate of approximately 35%.⁹ Elderly people and those with comorbidities were more likely to develop severe disease.⁴³Seven y later, in December 2019, another novel betacoronavirus named SARS-CoV-2, emerged in Wuhan City, Hubei Province China.^19,26 The animal reservoir responsible for transmission to humans has not been definitively identified but has been reported to be bats.^4,143 In February 2020, the World Health Organization named the disease associated with SARS-CoV-2, Corona virus disease 19 (COVID-19) and declared it a pandemic on March 11, 2020.^22,62,95 COVID-19 causes fever and pneumonia that can progress to acute respiratory distress syndrome (ARDS), multiple organ dysfunction and failure, coagulopathy, and death.³¹ Common gross findings in human autopsy specimens include lung consolidation, pulmonary edema, increased lung weight, pleurisy, white mucous and pink froth in airways, and hemorrhage. Histopathologic changes of human COVID-19 follow a timeline relative to the onset of symptoms.⁸⁶ During early infection, microvascular damage, thrombi, exudate formation, and intra-alveolar fibrin deposits occur. Epithelial changes can be present at all stages of disease, specifically diffuse alveolar damage (DAD), which includes hyaline membrane formation, epithelial denudation and pneumocyte hyperplasia. Finally, interstitial fibrosis develops about 3 wk after symptom onset.¹¹⁰ The clinical presentation of those infected with SARS-CoV-2 ranges from mild to severe to critical in 81%, 14%, and 5% of cases, respectively.^135,145 Similar to SARS-CoV and MERS-CoV, severe disease from SARS-CoV-2 is more likely in elderly individuals or in those with comorbidities.^12,72,127 In a New York City hospital study, deaths among hospital patients at the study endpoint were 3.3% or lower in patients in their 40s or younger, 4.8% among those in their 50s, 6.4% in their 60s, 12.6% in their 70s, and 25.9% in their 80s or above. Age related death rates reported by China, Italy and France are similar to the United States. Reported rates of asymptomatic infection range from 4% to 32%; however,¹²⁷ a systematic review concluded that true asymptomatic infection could be uncommon.^8,82,111,127The contagiousness of an infectious disease is referred to as the R₀, or reproduction number, and indicates the average number of people who will contract a contagious disease from someone infected with that disease. SARS-CoV (R₀ of 1.5 to 1.9)^12,72,127 and MERS-CoV (R₀ of less than 1) have R₀ values lower than SARS-CoV-2 (initial R₀ was calculated to be 2.0 to 2.5, now revised upward to 5.7) and a lower fatality rate (2.3%).^84,97 As of December 26, 2020, 78,604,532 confirmed SARS-CoV-2 cases and 1,744,235 COVID-19 related deaths have been reported worldwide.¹²⁹ The global impact of COVID-19 has been catastrophic, with adverse effects on physical and mental health, an overwhelming need for health care resources, and increased poverty and economic insecurity.⁴⁷ Effective vaccines and therapeutics are key to controlling the SARS-CoV-2 pandemic. The success of these efforts depends in part on animal models that replicate human COVID-19 disease.^52,81,105The ideal animal model for SARS-CoV-2 should be permissive to infection, have the same receptors for viral entry as in humans, and replicate the full spectrum of human COVID-19 disease and pathology.¹⁰⁹ Several publications review and compare common animal models for SARS-CoV-2 and conclude that current models simulate mild infection with full recovery, but not severe COVID-19 disease.^{13,31,52,78,81,100,105} Disease features not expressed in current animal models include ARDS, coagulopathy, systemic sequelae, and mortality.³¹This review will focus on why NHPs provide a valuable model for SARS-CoV-2 research. Using NHPs has several drawbacks as compared with small animal models, including higher purchase cost, limited availability, higher housing cost, larger space requirement, and need for specialized staff. In addition, NHPs are outbred, leading to greater variation in results among individual animals, sometimes making data analysis and interpretation difficult.^40,68 The preexisting shortage of NHPs available for biomedical research, combined with the high demand for COVID-19 research and China’s ban on the sale, transport, and export of NHPs to curtail the spread of COVID-19 (instituted January 26, 2020) has affected NHP research globally.^{3,116,138,141} Nevertheless, the scientific benefits of using NHPs for SARS-CoV-2 research outweigh these drawbacks. NHPs are physiologically, genetically, and immunologically more closely related to humans than are small animals.⁶⁸ Furthermore, the main receptor for SAR-CoV-2 binding, angiotensin l converting enzyme 2 (ACE2), in catarrhines (apes, Asian monkeys, and African monkeys) is identical to the human ACE2 receptor.^24,74 Moreover, like most humans, macaques infected with SARS-CoV-2 develop mild to moderate respiratory disease. Thus, macaques offer a relevant model to study SARS-CoV-2 pathogenesis, therapeutics, vaccines, and the impact of age and other comorbidities on disease outcome.     

A Meta-Analysis of Rhesus Macaques (Macaca mulatta), Cynomolgus Macaques (Macaca fascicularis), African green monkeys (Chlorocebus aethiops), and Ferrets (Mustela putorius furo) as Large Animal Models for COVID-19

Animal models are at the forefront of biomedical research for studies of viral transmission, vaccines, and pathogenesis, yet the need for an ideal large animal model for COVID-19 remains. We used a meta-analysis to evaluate published data relevant to this need. Our literature survey contained 22 studies with data relevant to the incidence of common COVID-19 symptoms in rhesus macaques (Macaca mulatta), cynomolgus macaques (Macaca fascicularis), African green monkeys (Chlorocebus aethiops), and ferrets (Mustela putorius furo). Rhesus macaques had leukocytosis on Day 1 after inoculation and pneumonia on Days 7 and 14 after inoculation in frequencies that were similar enough to humans to reject the null hypothesis of a Fisher exact test. However, the differences in overall presentation of disease were too different from that of humans to successfully identify any of these 4 species as an ideal large animal of COVID-19. The greatest limitation to the current study is a lack of standardization in experimentation and reporting. To expand our understanding of the pathology of COVID-19 and evaluate vaccine immunogenicity, we must extend the unprecedented collaboration that has arisen in the study of COVID-19 to include standardization of animal-based research in an effort to find the optimal animal model.

Human research of disease presents a number of ethical dilemmas, prompting scientists to use animal models in their research with the primary goal of enhancing the understanding of a human disease or phenomenon. Animal models have been instrumental to our understanding of pathologies, the assessment of novel vaccines, and the testing of acute therapies. Of the past 222 Nobel prizes awarded in the physiology and medicine categories since 1901, all but 36 have been a direct result of animal-based research.³¹Insects, nematodes, fish, amphibians, and numerous mammals have enabled some of the most important advances in physiology and medicine since their introduction in disease research. Through genetic modification, surgical adaptation, xenografts, chemical induction, and infection models, these animals have been used to model human phenomena.³¹ However, although particular animal species are often chosen based on their ability to meet specific criteria in line with the research question, their size remains an important factor.^26,31Small animals are often preferred in laboratory settings for their ease of use, shorter life cycle, easier handling and care, and short gestation.⁵ Rodents are the most commonly used animal for the study of human diseases for these very reasons, although they frequently fail to fully mimic the clinical signs and significant pathologic hallmarks of human diseases.^11,18 For this reason, some researchers use large animal models. Nonhuman primates (NHPs), in particular, have been extremely useful in reproducing the clinical signs of human diseases due to their close phylogenetic relationship to humans and resulting genetic, behavioral, and biochemical similarities.¹⁴On March 11, 2020, the World Health Organization declared a SARS-CoV-2 pandemic. SARS-CoV-2 is a novel coronavirus causing symptoms similar to, but distinct from, those found in individuals infected with SARS-CoV, the coronavirus that caused the 2003 SARS pandemic. As of September 10, 2021, this coronavirus has infected 219 million individuals with the COVID-19 disease.¹⁰ Although vaccines have been developed and approved in record time, we still need to better understand the pathogenesis of the disease and the long-term implications of infections. To do this, and to increase our understanding of the immunogenicity of current vaccines, finding an animal that replicates the manifestation of COVID-19 in humans is imperative.Much of the research on COVID-19 thus far has been aided by previous SARS research. In both SARS-CoV and SARS-CoV-2 studies, mice^33,45 and hamsters^19,34 were small animal models of choice. Large animals such as ferrets, cats, pigs, chickens, dogs, and nonhuman primates have also been tested for their reproducibility of COVID-19, with varying degrees of success.^27,41,49 While a perfect animal model of this viral infection is unlikely, the need remains to identify at least one large animal species as a frontrunner in reproducibility of the human clinical signs and significant pathologies of SARS-CoV-2 infection.The need for a large animal model to study COVID-19 does not imply a replacement for murine models, but rather an adjunct. The closer phylogenetic relationship of humans to NHPs makes them excellent candidates for the study of this disease. Vaccine trials have already shown that the responses of NHPs are closer to those of humans than are those of mice.²³ This difference may be due to species differences in IgG antibody and T helper type 1 cell responses that influence virus-immune system interactions, which make small animal models problematic for studying SARS-CoV-2 infection and vaccine performance in humans.¹⁵ NHPs have potential high value as a model due to their homology to the human angiotensin‐converting enzyme‐2, which is the SARS-CoV-2 binding site.^23,28 After the outbreak, the World Health Organization (WHO) formed the WHO COVID-19 modelling ad-hoc expert grouping. The working group identified various NHP models, including rhesus macaques, cynomolgus macaques and African green monkeys, in addition to ferrets as being susceptible to SARS Co-V-2 isolates that would result in reproducible infection, with mild to moderate disease.⁵² Therefore, the present article is focused on summarizing the results of multiple studies on rhesus macaque, cynomolgus macaque, African green monkey, and ferret infection with SARS-CoV-2. To highlight the species that best replicate the human clinical and laboratory findings of COVID-19, we synthesized the results of 22 animal studies to provide a comprehensive analysis of what is known about their infections to date.     

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.     

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.     

Biology and Cellular Tropism of a Unique Astrovirus Strain: Murine Astrovirus 2

Biology and tropism of MuAstV2Murine astrovirus 2 (MuAstV2) is a novel murine astrovirus recently identified in laboratory and wild mice. MuAstV2 readily transmits between immunocompetent mice yet fails to transmit to highly immunocompromised mouse strains—a unique characteristic when contrasted with other murine viruses including other astroviruses. We characterized the viral shedding kinetics and tissue tropism of MuAstV2 in immunocompetent C57BL/6NCrl mice and evaluated the apparent resistance of highly immunocompromised NOD-Prkdc^em26Cd52Il2rg^em26Cd22/NjuCrl mice to MuAstV2 after oral inoculation. Temporal patterns of viral shedding were determined by serially measuring fecal viral RNA. Tissue tropism and viral load were characterized and quantified by using in-situ hybridization (ISH) targeting viral RNA. Cellular tropism was characterized by evaluating fluorescent colocalization of viral ISH with various immunohistochemical markers. We found a rapid increase of fecal viral RNA in B6 mice, which peaked at 5 d after inoculation (dpi) followed by cessation of shedding by 168 dpi. The small intestine had the highest percentage of hybridization (3.09% of tissue area) of all tissues in which hybridization occurred at 5 dpi. The thymus displayed the next highest degree of hybridization (2.3%) at 7 dpi, indicating extraintestinal viral spread. MuAstV2 RNA hybridization was found to colocalize with only 3 of the markers evaluated: CD3 (T cells), Iba1 (macrophages), and cytokeratin (enterocytes). A higher percentage of CD3 cells and Iba1 cells hybridized with MuAstV2 as compared with cytokeratin at 2 dpi (CD3, 59%; Iba1, 46%; cytokeratin, 6%) and 35 dpi (CD3, 14%; Iba1, 55%; cytokeratin, 3%). Neither fecal viral RNA nor viral hybridization was noted in NCG mice at the time points examined. In addition, mice of mixed genetic background were inoculated, and only those with a functioning Il2rg gene shed MuAstV2. Results from this study suggest that infection of, or interaction with, the immune system is required for infection by or replication of MuAstV2.

Astroviruses are nonenveloped, positive-sense, single-stranded RNA viruses with a star-like appearance—from which the name derives—when examined by transmission electron microscopy. First identified in 1975, astroviruses are commonly associated with gastrointestinal illness in children.^1,23 They demonstrate considerable diversity, and unique strains have been identified in numerous species through advances in molecular diagnostics.^{2,4-6,11-13,18,19,21,25,27,29,30,32,35-40} This broad distribution likely resulted from cross-species transmission and subsequent adaptation to the novel host.¹¹ Clinical presentation varies among species, although most infections are asymptomatic or limited to mild gastrointestinal illness.^4,9,11 Extraintestinal disease resulting in fatal encephalitis has been described in several species (including cows, mink, and immunocompromised people).^{3,19,22,26,35}Astroviral infection of mice was first described in 1985, when an unknown astrovirus was identified by electron microscopy in the feces of nude mice.¹⁷ Since then, astroviruses have been detected in many wild and laboratory mouse populations.^12,29,30,34 Despite their prevalence, studies have been limited and their effects on host biology remains largely unknown. Murine astrovirus (MuAstV) was identified through molecular sequencing in 2012 and has since been discovered to be enzootic in numerous research and production mouse colonies.^12,29,34 Whether the strain described in 1985 was MuAstV is unknown. Immunocompetent and immunodeficient mouse strains are both susceptible to MuAstV infection, although no clinical disease and only minimal pathology are observed.^7,47 Similar to astroviruses infecting other species, MuAstV infection is frequently localized to the gastrointestinal tract.⁴⁷ A recent study demonstrated MuAstV replication in goblet cells and altered mucus production within the gastrointestinal tract, highlighting the potential effect of the virus on select research studies despite the lack of clinical disease and pathology.⁸Our group previously reported the detection of a novel murine astrovirus, murine astrovirus 2 (MuAstV2), in a laboratory mouse colony.³¹ MuAstV2 is genetically distinct from MuAstV but is closely related to a strain recently reported in wild mice.^31,43 The MuAstV2 strain identified in the laboratory mouse colony shares 89.2% nucleotide identity to a strain detected in wild mice in New York City but less than 50% nucleotide identity to MuAstV, the strain commonly isolated from laboratory mice. In addition, MuAstV2 was found to share as much as 80.8% nucleotide similarity to an astrovirus strain isolated from urban brown rats (Rattus norvegicus) in Hong Kong.^5,31 Antibodies to MuAstV2 were inadvertently detected in laboratory colony mice when a serologic immunoassay for mouse thymic virus prepared from a murine T-cell line tested positive. Further analysis showed that the mice were negative for mouse thymic virus and that the T-cell line was contaminated with a novel astrovirus strain similar to MuAstV2, resulting in the positive test. The observation that MuAstV2 did not appear to infect highly immunocompromised mice via natural exposure or experimental inoculation was highly unusual.³¹ This finding is distinct from other murine viruses, including MuAstV, given that infection of immunocompromised mice leads to persistent infection and chronic virus shedding.^12,15,16,47We sought to further understand the biology of MuAstV2 by evaluating viral shedding kinetics and tissue tropism in immunocompetent mice and to further characterize the presumptive resistance to infection observed in highly immunocompromised mice. Temporal patterns of viral shedding were determined by serially measuring fecal viral RNA after oral inoculation. Tissue and cell tropism were characterized using in-situ hybridization (ISH) and immunohistochemistry during the course of infection. We hypothesized that MuAstV2 initially infects the gastrointestinal tract, as occurs with other astroviruses, but speculated that components of the immune system were required to support infection or replication or both. Furthermore, we sought to characterize the extraintestinal spread of MuAstV2.     

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.     

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.     

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.     

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.     

TRAF2 is a biologically important necroptosis suppressor

Tumor necrosis factor α (TNFα) triggers necroptotic cell death through an intracellular signaling complex containing receptor-interacting protein kinase (RIPK) 1 and RIPK3, called the necrosome. RIPK1 phosphorylates RIPK3, which phosphorylates the pseudokinase mixed lineage kinase-domain-like (MLKL)—driving its oligomerization and membrane-disrupting necroptotic activity. Here, we show that TNF receptor-associated factor 2 (TRAF2)—previously implicated in apoptosis suppression—also inhibits necroptotic signaling by TNFα. TRAF2 disruption in mouse fibroblasts augmented TNFα–driven necrosome formation and RIPK3-MLKL association, promoting necroptosis. TRAF2 constitutively associated with MLKL, whereas TNFα reversed this via cylindromatosis-dependent TRAF2 deubiquitination. Ectopic interaction of TRAF2 and MLKL required the C-terminal portion but not the N-terminal, RING, or CIM region of TRAF2. Induced TRAF2 knockout (KO) in adult mice caused rapid lethality, in conjunction with increased hepatic necrosome assembly. By contrast, TRAF2 KO on a RIPK3 KO background caused delayed mortality, in concert with elevated intestinal caspase-8 protein and activity. Combined injection of TNFR1-Fc, Fas-Fc and DR5-Fc decoys prevented death upon TRAF2 KO. However, Fas-Fc and DR5-Fc were ineffective, whereas TNFR1-Fc and interferon α receptor (IFNAR1)-Fc were partially protective against lethality upon combined TRAF2 and RIPK3 KO. These results identify TRAF2 as an important biological suppressor of necroptosis in vitro and in vivo.Apoptotic cell death is mediated by caspases and has distinct morphological features, including membrane blebbing, cell shrinkage and nuclear fragmentation.^{1, 2, 3, 4} In contrast, necroptotic cell death is caspase-independent and is characterized by loss of membrane integrity, cell swelling and implosion.^{1, 2, 5} Nevertheless, necroptosis is a highly regulated process, requiring activation of RIPK1 and RIPK3, which form the core necrosome complex.^{1, 2, 5} Necrosome assembly can be induced via specific death receptors or toll-like receptors, among other modules.^{6, 7, 8, 9} The activated necrosome engages MLKL by RIPK3-mediated phosphorylation.^{6, 10, 11} MLKL then oligomerizes and binds to membrane phospholipids, forming pores that cause necroptotic cell death.^{10, 12, 13, 14, 15} Unchecked necroptosis disrupts embryonic development in mice and contributes to several human diseases.^{7, 8, 16, 17, 18, 19, 20, 21, 22}The apoptotic mediators FADD, caspase-8 and cFLIP suppress necroptosis.^{19, 20, 21, 23, 24} Elimination of any of these genes in mice causes embryonic lethality, subverted by additional deletion of RIPK3 or MLKL.^{19, 20, 21, 25} Necroptosis is also regulated at the level of RIPK1. Whereas TNFα engagement of TNFR1 leads to K63-linked ubiquitination of RIPK1 by cellular inhibitor of apoptosis proteins (cIAPs) to promote nuclear factor (NF)-κB activation,²⁶ necroptosis requires suppression or reversal of this modification to allow RIPK1 autophosphorylation and consequent RIPK3 activation.^{2, 23, 27, 28} CYLD promotes necroptotic signaling by deubiquitinating RIPK1, augmenting its interaction with RIPK3.²⁹ Conversely, caspase-8-mediated CYLD cleavage inhibits necroptosis.²⁴TRAF2 recruits cIAPs to the TNFα-TNFR1 signaling complex, facilitating NF-κB activation.^{30, 31, 32, 33} TRAF2 also supports K48-linked ubiquitination and proteasomal degradation of death-receptor-activated caspase-8, curbing apoptosis.³⁴ TRAF2 KO mice display embryonic lethality; some survive through birth but have severe developmental and immune deficiencies and die prematurely.^{35, 36} Conditional TRAF2 KO leads to rapid intestinal inflammation and mortality.³⁷ Furthermore, hepatic TRAF2 depletion augments apoptosis activation via Fas/CD95.³⁴ TRAF2 attenuates necroptosis induction in vitro by the death ligands Apo2L/TRAIL and Fas/CD95L.³⁸ However, it remains unclear whether TRAF2 regulates TNFα-induced necroptosis—and if so—how. Our present findings reveal that TRAF2 inhibits TNFα necroptotic signaling. Furthermore, our results establish TRAF2 as a biologically important necroptosis suppressor in vitro and in vivo and provide initial insight into the mechanisms underlying this function.     

Role of Retinoic Acid and Fibroblast Growth Factor 2 in Neural Differentiation from Cynomolgus Monkey (Macaca fascicularis) Embryonic Stem Cells

Retinoic acid is a widely used factor in both mouse and human embryonic stem cells. It suppresses differentiation to mesoderm and enhances differentiation to ectoderm. Fibroblast growth factor 2 (FGF2) is widely used to induce differentiation to neurons in mice, yet in primates, including humans, it maintains embryonic stem cells in the undifferentiated state. In this study, we established an FGF2 low-dose-dependent embryonic stem cell line from cynomolgus monkeys and then analyzed neural differentiation in cultures supplemented with retinoic acid and FGF2. When only retinoic acid was added to culture, neurons differentiated from FGF2 low-dose-dependent embryonic stem cells. When both retinoic acid and FGF2 were added, neurons and astrocytes differentiated from the same embryonic stem cell line. Thus, retinoic acid promotes the differentiation from embryonic stem cells to neuroectoderm. Although FGF2 seems to promote self-renewal in stem cells, its effects on the differentiation of stem cells are influenced by the presence or absence of supplemental retinoic acid.Abbreviations: EB, embryoid body; ES, embryonic stem; ESM, embryonic stem cell medium; FGF, fibroblast growth factor; GFAP, glial fibrillary acidic protein; LIF, leukemia inhibitory factor; MBP, myelin basic protein; RA, retinoic acid; SSEA, stage-specific embryonic antigen; TRA, tumor-related antigenPluripotent stem cells are potential sources of material for cell replacement therapy and are useful experimental tools for in vitro models of human disease and drug screening. Embryonic stem (ES) cells are capable of extensive proliferation and multilineage differentiation, and thus ES-derived cells are suitable for use in cell-replacement therapies.^18,23 Reported ES cell characteristics including tumorigenic potential, DNA methylation status, expression of imprinted genes, and chromatin structure were elucidated by using induced pluripotent stem cells.^2,11,17 Because the social expectations of regeneration medicine are growing, we must perform basic research with ES cells, which differ from induced pluripotent stem cells in terms of origin, differentiation ability, and epigenetic status.^2,8Several advances in research have been made by using mouse ES cells. Furthermore, primate ES cell lines have been established from rhesus monkeys (Macaca mulatta),²⁴ common marmosets (Callithrix jacchus),²⁵ cynomolgus monkeys (M. fascicularis),²⁰ and African green monkeys (Chlorocebus aethiops).¹⁹ Mouse and other mammalian ES cells differ markedly in their responses to the signaling pathways that support self-renewal.^8,28 Mouse ES cells require leukemia inhibitory factor (LIF)–STAT3 signaling.¹⁴ In contrast, primate ES cells do not respond to LIF. Fibroblast growth factor 2 (FGF2) appears to be the most upstream self-renewal factor in primate ES cells. FGF2 also exerts its effects through indirect mechanisms, such as the TGFβ–Activin–Nodal signaling pathway, in primate ES cells.²¹ In addition to the biologic similarities between monkeys and humans, ES cells derived from cynomolgus monkeys or human blastocysts have extensive similarities that are not apparent in mouse ES cells.^8,14,21,28 Numerous monkey ES cell lines are now available, and cynomolgus monkeys are an efficient model for developing strategies to investigate the efficacy of ES-cell–based medical treatments in humans.Several growth factors and chemical compounds, including retinoic acid (RA),^4,9,13,22,26 FGF2,^9,10,16,22 epidermal growth factor,^9,22 SB431542,^1,4,10 dorsomorphin,^10,27 sonic hedgehog,^{12,13,16,27,29} and noggin,^1,4,9,27 are essential for the differentiation and proliferation or maintenance of neural stem cells derived from primate ES cells. Of these factors, active RA signaling suppresses a mesodermal fate by inhibiting Wnt and Nodal signaling pathways during in vitro culture and leads to neuroectoderm differentiation in ES cells.^4,13,26 RA is an indispensable factor for the specialization to neural cells. FGF2 is important during nervous system development,¹² and FGF2 and RA both are believed to influence the differentiation to neural cells. The current study was done to clarify the mechanism of RA and FGF2 in the induction of differentiation along the neural lineage.We recently established a monkey ES cell line that does not need FGF2 supplementation for maintenance of the undifferentiated state. This ES cell line allowed us to study the role of differentiation to neural cells with RA and enabled us to compare ES cell differentiation in the context of supplementation with RA or FGF2 in culture. To this end, we established a novel cynomolgus monkey cell line derived from ES cells and maintained it in an undifferentiated state in the absence of FGF2 supplementation.     

Daytime Blue Light Enhances the Nighttime Circadian Melatonin Inhibition of Human Prostate Cancer Growth

Light controls pineal melatonin production and temporally coordinates circadian rhythms of metabolism and physiology in normal and neoplastic tissues. We previously showed that peak circulating nocturnal melatonin levels were 7-fold higher after daytime spectral transmittance of white light through blue-tinted (compared with clear) rodent cages. Here, we tested the hypothesis that daytime blue-light amplification of nocturnal melatonin enhances the inhibition of metabolism, signaling activity, and growth of prostate cancer xenografts. Compared with male nude rats housed in clear cages under a 12:12-h light:dark cycle, rats in blue-tinted cages (with increased transmittance of 462–484 nm and decreased red light greater than 640 nm) evinced over 6-fold higher peak plasma melatonin levels at middark phase (time, 2400), whereas midlight-phase levels (1200) were low (less than 3 pg/mL) in both groups. Circadian rhythms of arterial plasma levels of linoleic acid, glucose, lactic acid, pO₂, pCO₂, insulin, leptin, and corticosterone were disrupted in rats in blue cages as compared with the corresponding entrained rhythms in clear-caged rats. After implantation with tissue-isolated PC3 human prostate cancer xenografts, tumor latency-to-onset of growth and growth rates were markedly delayed, and tumor cAMP levels, uptake–metabolism of linoleic acid, aerobic glycolysis (Warburg effect), and growth signaling activities were reduced in rats in blue compared with clear cages. These data show that the amplification of nighttime melatonin levels by exposing nude rats to blue light during the daytime significantly reduces human prostate cancer metabolic, signaling, and proliferative activities.Abbreviations: A-V, arterial–venous difference, ipRGC, intrinsically photosensitive retinal ganglion cell, LA, linoleic acid, 13-HODE, 13-hydroxyoctadecadienoic acid, TFA, total fatty acidsLight profoundly influences circadian, neuroendocrine, and neurobehavioral regulation in all mammals and is essential to life on our planet.^{2,15,28, 40} The light–dark cycle entrains the master biologic clock, located in the suprachiasmatic nucleus of the brain, in an intensity-, duration-, and wavelength-dependent manner.^8-13 Photobiologic responses, including circadian rhythms of metabolism and physiology, are mediated by organic molecules called ‘chromophores,’ which are contained within a small subset of retinal cells, called the intrinsically sensitive retinal ganglion cells (ipRGC).^{16,29,31,36,41,49,53,59} In humans and rodents light quanta are detected by the chromophore melanopsin, which detects light quanta in principally the short-wavelength, blue-appearing portion of the spectrum (446 to 477 nm), and transmits its photic information via the retinohypothalamic tract to the ‘molecular clock’ of the suprachiasmatic nucleus. This region of the brain regulates the daily pineal gland production of the circadian neurohormone melatonin (N-acetyl-5-methoxytryptamine), which results in high levels produced at night and low levels during daytime.^38,54 The daily, rhythmic melatonin signal provides temporal coordination of normal behavioral and physiologic functions including chronobiologic rhythms of locomotor activity,² sleep-wake cycle,^2,14 dietary and water intake,^2,51 hormone secretion and metabolism.^5,44,47,61 Alterations in light intensity, duration, and spectral quality at a given time of day,^{8-13,17,19-22,24,61} such as occurs in night-shift workers exposed to light at night,^26,34,46,57 acutely suppresses endogenous melatonin levels in most mammalian species^{9,11,44,45,54,55} and may lead to various disease states, including metabolic syndrome^5,61 and carcinogenesis.^4-7,17,18Recent studies from our laboratory^{5,20,23-25,60,61} have demonstrated that relatively small changes in the spectral transmittance (color) of light passing through translucent amber (>590 nm), blue (>480 nm), and red-tinted (>640 nm) polycarbonate laboratory rodent cages, compared with standard polycarbonate clear cages (390 to 700 nm), during the light phase markedly influenced the normal nighttime melatonin signal and disrupted temporal coordination of metabolism and physiology.^19,24,61 Most notable was our discovery that, in both male and female pigmented nude rats maintained in blue-tinted rodent cages, nighttime melatonin levels were as much as 7 times higher than normal nighttime peak levels in animals maintained in all other cage types.¹⁹ An earlier study in human subjects diagnosed with midwinter insomnia coupled with low nighttime melatonin levels demonstrated that daily exposure to intense morning bright polychromatic light therapy for up to one week resulted in a restoration of nocturnal melatonin levels to those of control subjects.³⁵ In another study, exposure to blue-tinted (470 nm) LED light (100 lx) for approximately 20 min in the morning after 2 sleep-restricted (6 h) nights led to earlier onset of the melatonin surge at nighttime.³⁰In the United States alone this year, approximately 240,000 men will be diagnosed with prostate cancer, and nearly 30,000 will die from this disease (National Cancer Institute; www.cancer.gov/). Epidemiologic studies have shown that night shift work, which involves circadian disruption, including nocturnal melatonin suppression, markedly increases prostate cancer risk in men.^{26,34,46,57,58} Both in vitro and in vivo studies have demonstrated that melatonin inhibits human prostate cancer growth, including that of androgen-receptor–negative, castration-resistant PC3 human prostate cancer cells.^20,29,42,56 Cancer cells depend primarily on aerobic glycolysis (Warburg effect) over oxidative phosphorylation to meet their bioenergetic needs supporting biomass formation.⁵ The Warburg effect is characterized by increased cellular uptake of glucose and production of lactate despite an abundance of oxygen. Investigations have shown that signal transduction pathways that include AKT, MEK, NFκB, GS3Kβ, and PDK1 drive the Warburg effect.^5,61 In addition, cancer cells rely on increased uptake of the ω6 fatty acid linoleic acid (LA), which is prevalent in the western diet.^4-6 In most cancers, LA uptake occurs through a cAMP-dependent transport mechanism, and LA is metabolized to the mitogenic agent 13-hydroxyoctadecadienoic acid (13-HODE). In most tumors, 13-HODE plays an important role in enhancing downstream phosphorylation of ERK 1/2, AKT, and activation of the Warburg effect, thereby leading to increased cell proliferation and tumor growth.^4-6 Melatonin, the principal neurohormone of the pineal gland and whose production is regulated by the suprachiasmatic nucleus,^4,5 modulates processes of tumor initiation, progression, and growth in vivo.⁵ The circadian nocturnal melatonin signal not only inhibits LA uptake and metabolism, the Warburg effect in human cancer xenografts, and ultimately tumor growth, but it actually drives circadian rhythms in tumor metabolism, signal transduction activity, and cell proliferation. These effects are extinguished when melatonin production is suppressed by light exposure at night.⁵In the present investigation, we examined the hypothesis that the spectral transmittance (color) of short-wavelength (480 nm) bright light passing through blue-tinted standard laboratory rodent cages during the light phase not only amplifies the normal circadian nocturnal melatonin signal but also enhances the inhibition of the metabolism, signaling activity, and growth progression of human PC3 androgen-receptor–negative human prostate cancer xenografts in male nude rats.     

The nuclease activity of DNA2 promotes exonuclease 1–independent mismatch repair

The DNA mismatch repair (MMR) system is a major DNA repair system that corrects DNA replication errors. In eukaryotes, the MMR system functions via mechanisms both dependent on and independent of exonuclease 1 (EXO1), an enzyme that has multiple roles in DNA metabolism. Although the mechanism of EXO1-dependent MMR is well understood, less is known about EXO1-independent MMR. Here, we provide genetic and biochemical evidence that the DNA2 nuclease/helicase has a role in EXO1-independent MMR. Biochemical reactions reconstituted with purified human proteins demonstrated that the nuclease activity of DNA2 promotes an EXO1-independent MMR reaction via a mismatch excision-independent mechanism that involves DNA polymerase δ. We show that DNA polymerase ε is not able to replace DNA polymerase δ in the DNA2-promoted MMR reaction. Unlike its nuclease activity, the helicase activity of DNA2 is dispensable for the ability of the protein to enhance the MMR reaction. Further examination established that DNA2 acts in the EXO1-independent MMR reaction by increasing the strand-displacement activity of DNA polymerase δ. These data reveal a mechanism for EXO1-independent mismatch repair.

The mismatch repair (MMR) system has been conserved from bacteria to humans (1, 2). It promotes genome stability by suppressing spontaneous and DNA damage-induced mutations (1, 3, 4, 5, 6, 7, 8, 9, 10, 11). The key function of the MMR system is the correction of DNA replication errors that escape the proofreading activities of replicative DNA polymerases (1, 4, 5, 6, 7, 8, 9, 10, 12). In addition, the MMR system removes mismatches formed during strand exchange in homologous recombination, suppresses homeologous recombination, initiates apoptosis in response to irreparable DNA damage caused by several anticancer drugs, and contributes to instability of triplet repeats and alternative DNA structures (1, 4, 5, 7, 8, 9, 10, 11, 13, 14, 15, 16, 17, 18). The principal components of the eukaryotic MMR system are MutSα (MSH2-MSH6 heterodimer), MutLα (MLH1-PMS2 heterodimer in humans and Mlh1-Pms1 heterodimer in yeast), MutSβ (MSH2-MSH3 heterodimer), proliferating cell nuclear antigen (PCNA), replication factor C (RFC), exonuclease 1 (EXO1), RPA, and DNA polymerase δ (Pol δ). Loss-of-function mutations in the MSH2, MLH1, MSH6, and PMS2 genes of the human MMR system cause Lynch and Turcot syndromes, and hypermethylation of the MLH1 promoter is responsible for ∼15% of sporadic cancers in several organs (19, 20). MMR deficiency leads to cancer initiation and progression via a multistage process that involves the inactivation of tumor suppressor genes and action of oncogenes (21).MMR occurs behind the replication fork (22, 23) and is a major determinant of the replication fidelity (24). The correction of DNA replication errors by the MMR system increases the replication fidelity by ∼100 fold (25). Strand breaks in leading and lagging strands as well as ribonucleotides in leading strands serve as signals that direct the eukaryotic MMR system to remove DNA replication errors (26, 27, 28, 29, 30). MMR is more efficient on the lagging than the leading strand (31). The substrates for MMR are all six base–base mismatches and 1 to 13-nt insertion/deletion loops (25, 32, 33, 34). Eukaryotic MMR commences with recognition of the mismatch by MutSα or MutSβ (32, 34, 35, 36). MutSα is the primary mismatch-recognition factor that recognizes both base–base mismatches and small insertion/deletion loops whereas MutSβ recognizes small insertion/deletion loops (32, 34, 35, 36, 37). After recognizing the mismatch, MutSα or MutSβ cooperates with RFC-loaded PCNA to activate MutLα endonuclease (38, 39, 40, 41, 42, 43). The activated MutLα endonuclease incises the discontinuous daughter strand 5′ and 3′ to the mismatch. A 5'' strand break formed by MutLα endonuclease is utilized by EXO1 to enter the DNA and excise a discontinuous strand portion encompassing the mismatch in a 5''→3′ excision reaction stimulated by MutSα/MutSβ (38, 44, 45). The generated gap is filled in by the Pol δ holoenzyme, and the nick is ligated by a DNA ligase (44, 46, 47). DNA polymerase ε (Pol ε) can substitute for Pol δ in the EXO1-dependent MMR reaction, but its activity in this reaction is much lower than that of Pol δ (48). Although MutLα endonuclease is essential for MMR in vivo, 5′ nick-dependent MMR reactions reconstituted in the presence of EXO1 are MutLα-independent (44, 47, 49).EXO1 deficiency in humans does not seem to cause significant cancer predisposition (19). Nevertheless, it is known that Exo1^-/- mice are susceptible to the development of lymphomas (50). Genetic studies in yeast and mice demonstrated that EXO1 inactivation causes only a modest defect in MMR (50, 51, 52, 53). In agreement with these genetic studies, a defined human EXO1-independent MMR reaction that depends on the strand-displacement DNA synthesis activity of Pol δ holoenzyme to remove the mismatch was reconstituted (54). Furthermore, an EXO1-independent MMR reaction that occurred in a mammalian cell extract system without the formation of a gapped excision intermediate was observed (54). Together, these findings implicated the strand-displacement activity of Pol δ holoenzyme in EXO1-independent MMR.In this study, we investigated DNA2 in the context of MMR. DNA2 is an essential multifunctional protein that has nuclease, ATPase, and 5''→3′ helicase activities (55, 56, 57). Previous research ascertained that DNA2 removes long flaps during Okazaki fragment maturation (58, 59, 60), participates in the resection step of double-strand break repair (61, 62, 63), initiates the replication checkpoint (64), and suppresses the expansions of GAA repeats (65). We have found in vivo and in vitro evidence that DNA2 promotes EXO1-independent MMR. Our data have indicated that the nuclease activity of DNA2 enhances the strand-displacement activity of Pol δ holoenzyme in an EXO1-independent MMR reaction.     

Metabolic Syndrome and Coronary Artery Disease in Ossabaw Compared with Yucatan Swine

Metabolic syndrome (MetS), a compilation of associated risk factors, increases the risk of type 2 diabetes and coronary artery disease (CAD, atherosclerosis), which can progress to the point of artery occlusion. Stents are the primary interventional treatment for occlusive CAD, and patients with MetS and hyperinsulinemia have increased restenosis. Because of its thrifty genotype, the Ossabaw pig is a model of MetS. We tested the hypothesis that, when fed high-fat diet, Ossabaw swine develop more features of MetS, greater native CAD, and greater stent-induced CAD than do Yucatan swine. Animals of each breed were divided randomly into 2 groups and fed 2 different calorie-matched diets for 40 wk: control diet (C) and high-fat, high-cholesterol atherogenic diet (H). A bare metal stent was placed in the circumflex artery, and pigs were allowed to recover for 3 wk. Characteristics of MetS, macrovascular and microvascular CAD, in-stent stenosis, and Ca²⁺ signaling in coronary smooth muscle cells were evaluated. MetS characteristics including, obesity, glucose intolerance, hyperinsulinemia, and elevated arterial pressure were elevated in Ossabaw swine compared to Yucatan swine. Ossabaw swine with MetS had more extensive and diffuse native CAD and in-stent stenosis and impaired coronary blood flow regulation compared with Yucatan. In-stent atherosclerotic lesions in Ossabaw coronary arteries were less fibrous and more cellular. Coronary smooth muscle cells from Ossabaw had impaired Ca²⁺ efflux and intracellular sequestration versus cells from Yucatan swine. Therefore, Ossabaw swine are a superior model of MetS, subsequent CAD, and cellular Ca²⁺ signaling defects, whereas Yucatan swine are leaner and relatively resistant to MetS and CAD.Abbreviations: CAD, coronary artery disease; CSM, coronary smooth muscle; IVGTT, intravenous glucose tolerance test; MetS, metabolic syndrome; SERCA, sarco–endoplasmic reticulum Ca²⁺ ATPase; ET1, endothelin 1; SOCE, store-operated Ca²⁺ entryAtherosclerotic coronary artery disease (CAD) is increased at least 2-fold in patients with metabolic syndrome (MetS)²⁷ and is accompanied by marked microvascular dysfunction that further impairs coronary blood flow.¹⁰ MetS generally is diagnosed by the presence of 3 or more of the following conditions: obesity, insulin resistance, glucose intolerance, dyslipidemia, and hypertension.^17,28 There is strong support for the role of the hyperinsulinemia component of MetS in increased restenosis after percutaneous coronary interventions.^74,75,84,85 Further, our group has shown that severe coronary microvascular dysfunction occurs in MetS.⁵ Because MetS (so-called ‘prediabetes’) affects as much as 27% of the United States population, is increasing dramatically in prevalence,⁹⁴ and can progress to type 2 diabetes, there is great need for basic research using animal models that accurately mimic MetS and the accompanying CAD. Clearly, there is need for study of MetS-induced CAD and in-stent stenosis and the underlying cellular and molecular mechanisms.Mice, rats, and swine are known to recapitulate MetS;^{3,12,36,60,71,72} however, none of these models fully reproduce the combined symptoms of MetS and CAD. Further, transgenic mouse models are simply not adequate for coronary vascular interventions using stents identical to those used in humans,^{18,23,38,55,57,79,83,86} a step that is essential for translation to the clinic. Yucatan and domestic swine are commonly used large animal models for study of cardiovascular disease due to their ability to mimic the neointimal formation and thrombosis observed in humans.⁸⁶ For example, several laboratories have produced severe CAD in swine,^{8,24,51,61,62,68,91} but through toxin-induced pancreatic β-cell ablation and feeding of an atherogenic diet, rather than as a natural development subsequent to MetS or diabetes. Currently, there is a paucity of large animal models that reproduce MetS and CAD.³Research on the obesity-prone Ossabaw miniature swine⁵⁹ clearly indicates that these animals develop MetS and cardiovascular disease when fed a high-calorie atherogenic diet,^{4,5,9,16,19,42,50,52,83,92} Female Ossabaw swine on this type of diet nearly doubled their percentage body fat in only 9 wk, showed insulin resistance, impaired glucose tolerance, dyslipidemia (profound increase in the ratio of low-density to high-density lipoprotein cholesterol, hypertriglyceridemia), hypertension, and early coronary atherosclerosis.¹⁶ These data contrast with those from male Yucatan miniature pigs, which did not develop MetS even after 20 wk on a comparable excess calorie atherogenic diet.^8,68,95 Yucatan swine do not develop MetS through diet manipulation, unlike Ossabaw swine, which consistently recapitulate all MetS characteristics. However, important differences in study design have not allowed direct comparison between Yucatan and Ossabaw swine.Cytosolic Ca²⁺ signaling is involved in ‘phenotypic modulation’ of coronary smooth muscle (CSM), as characterized by proliferation and migration in several in vitro cell culture models^33,35,89,90 and in vivo rodent models of the peripheral circulation (for example, reference 51). The Yucatan swine model of diabetic dyslipidemia shows altered Ca²⁺ extrusion,⁹⁶ Ca²⁺ sequestration by the sarcoplasmic reticulum,^32,34,98 and Ca²⁺ influx through voltage-gated Ca²⁺ channels.⁹⁸ Currently, Ca²⁺ signaling has not been compared directly between MetS Ossabaw and Yucatan swine CSM. Therefore, the purpose of the present study was to test the hypothesis that compared with Yucatan swine on calorie-matched standard chow (for example, Yucatan maintenance diet^8,95) and atherogenic diets, Ossabaw swine have a greater propensity to MetS and CAD with impaired coronary microvascular dysfunction and Ca²⁺ handling in CSM.