A One Health Perspective for Defining and Deciphering Escherichia coli Pathogenic Potential in Multiple Hosts

E. coli is one of the most common species of bacteria colonizing humans and animals. The singularity of E. coli’s genus and species underestimates its multifaceted nature, which is represented by different strains, each with different combinations of distinct virulence factors. In fact, several E. coli pathotypes, or hybrid strains, may be associated with both subclinical infection and a range of clinical conditions, including enteric, urinary, and systemic infections. E. coli may also express DNA-damaging toxins that could impact cancer development. This review summarizes the different E. coli pathotypes in the context of their history, hosts, clinical signs, epidemiology, and control. The pathotypic characterization of E. coli in the context of disease in different animals, including humans, provides comparative and One Health perspectives that will guide future clinical and research investigations of E. coli infections.

Escherichia coli (E. coli) is the most common bacterial model used in research and biotechnology. It is an important cause of morbidity and mortality in humans and animals worldwide, and animal hosts can be involved in the epidemiology of infections.^{240,367,373,452,727} The adaptive and versatile nature of E. coli argues that ongoing studies should receive a high priority in the context of One Health involving humans, animals, and the environment.^{240,315,343,727} Two of the 3 E. coli pathogens associated with death in children with moderate-to-severe diarrhea in Asia and Africa are classified into 2 E. coli pathogenic groups (also known as pathotypes or pathovars): enterotoxigenic E. coli (ETEC) and enteropathogenic E. coli (EPEC).³⁶⁷ In global epidemiologic studies, ETEC and EPEC rank among the deadliest causes of foodborne diarrheal illness and are important pathogens for increasing disability adjusted life years.^355,382,570 Furthermore, in humans, E. coli is one of the top-ten organisms involved in coinfections, which generally have deleterious effects on health.²⁷⁰ETEC is also an important etiologic agent of diarrhea in the agricultural setting.¹⁸³ E. coli-associated extraintestinal infections, some of which may be antibiotic-resistant, have a tremendous impact on human and animal health. These infections have a major economic impact on the poultry, swine, and dairy industries.^{70,151,168,681,694,781,797} The pervasive nature of E. coli, and its capacity to induce disease have driven global research efforts to understand, prevent, and treat these devastating diseases. Animal models for the study of E. coli infections have been useful for pathogenesis elucidation and development of intervention strategies; these include zebrafish, rats, mice, Syrian hamsters, guinea pigs, rabbits, pigs, and nonhuman primates.^{27,72,101,232,238,347,476,489,493,566,693,713,744,754} Experiments involving human volunteers have also been important for the study of infectious doses associated with E. coli-induced disease and of the role of virulence determinants in disease causation.^{129,176,365,400,497,702,703} E. coli strains (or their lipopolysaccharide) have also been used for experimental induction of sepsis in animals; the strains used for these studies, considered EPEC, are not typically involved in systemic disease.^{140,205,216,274,575,782}This article provides an overview of selected topics related to E. coli, a common aerobic/facultative anaerobic gastrointestinal organism of humans and animals.^{14,277,432,477,716} In addition, we briefly review: history, definition, pathogenesis, prototype (archetype or reference) strains, and features of the epidemiology and control of specific pathotypes. Furthermore, we describe cases attributed to different E. coli pathotypes in a range of animal hosts. The review of scientific and historical events regarding the discovery and characterization of the different E. coli pathotypes will increase clinical awareness of E. coli, which is too often regarded merely as a commensal organism, as a possible primary or co- etiologic agent during clinical investigations. As Will and Ariel Durant write in The Lessons of History: “The present is the past rolled up for action, and the past is the present unrolled for understanding”.     

Kinase Partner Protein Plays a Key Role in Controlling the Speed and Shape of Pollen Tube Growth in Tomato

The rapid and responsive growth of a pollen tube requires delicate coordination of membrane receptor signaling, Rho-of-Plants (ROP) GTPase activity switching, and actin cytoskeleton assembly. The tomato (Solanum lycopersicum) kinase partner protein (KPP), is a ROP guanine nucleotide exchange factor (GEF) that activates ROP GTPases and interacts with the tomato pollen receptor kinases LePRK1 and LePRK2. It remains unclear how KPP relays signals from plasma membrane-localized LePRKs to ROP switches and other cellular machineries to modulate pollen tube growth. Here, we biochemically verified KPP’s activity on ROP4 and showed that KPP RNA interference transgenic pollen tubes grew slower while KPP-overexpressing pollen tubes grew faster, suggesting that KPP functions as a rheostat for speed control in LePRK2-mediated pollen tube growth. The N terminus of KPP is required for self-inhibition of its ROPGEF activity, and expression of truncated KPP lacking the N terminus caused pollen tube tip enlargement. The C-terminus of KPP is required for its interaction with LePRK1 and LePRK2, and the expression of a truncated KPP lacking the C-terminus triggered pollen tube bifurcation. Furthermore, coexpression assays showed that self-associated KPP recruited actin-nucleating Actin-Related Protein2/3 (ARP2/3) complexes to the tip membrane. Interfering with ARP2/3 activity reduced the pollen tube abnormalities caused by overexpressing KPP fragments. In conclusion, KPP plays a key role in pollen tube speed and shape control by recruiting the branched actin nucleator ARP2/3 complex and an actin bundler to the membrane-localized receptors LePRK1 and LePRK2.

The delivery of nonmotile sperm to the embryo sac via a pollen tube is a key innovation that allowed flowering plants to carry out sexual reproduction without the need for water (Friedman, 1993; Lord and Russell, 2002). Both the speed and signal responsiveness of pollen tube growth are critical for successful fertilization (Johnson et al., 2019). The typical shape of a growing pollen tube cell protruding from a pollen grain is a cylinder with a dome-shaped tip (Geitmann, 2010). Maintaining such a typical tube shape during pollen tube growth is fundamental to support its ability for fast growth (Michard et al., 2017), and a plasticity range of tubular growth rates allows a pollen tube to optimize directional growth along its journey from the stigma to the ovule (Luo et al., 2017). The pollen tube cell extends mainly by tip growth, requiring huge amounts of secretion/exocytosis at the tip (McKenna et al., 2009; Grebnev et al., 2017). The newly secreted cell wall at the tip is mainly composed of esterified pectin, which is expandable, whereas cell wall remodeling at the lateral region (including pectin deesterification and callose deposition) limits expansion (Grebnev et al., 2017). The tip width of a growing pollen tube actually reflects the size of the secretion zone capped by an expandable membrane and cell wall, as a collective result of multiple pollen tube growth machineries (Luo et al., 2017).The tip-localized exocytosis of a growing pollen tube is supported by a spatiotemporal tightly controlled actin cytoskeleton network (Hepler, 2016). The actin cytoskeleton configuration in a pollen tube includes highly dynamic fine actin filaments in the apical and subapical regions and parallel longitudinal actin bundles in the shank region (Qu et al., 2017). Various actin-binding proteins, such as actin nucleation factors, actin-severing proteins, and actin-bundling factors, are responsible for organizing the dynamic actin cytoskeleton network (Ren and Xiang, 2007). For example, the actin-bundling proteins fimbrin and LIM (Lin-1, isl1, Mec3) domain-containing proteins function in shank-localized actin bundles in pollen tubes (Zhang et al., 2019). For another example, the actin nucleator formin (formin3 in Arabidopsis [Arabidopsis thaliana] and formin1 in lily [Lilium longiflorum]) functions in actin polymerization in the pollen tube tip (Li et al., 2017; Lan et al., 2018). The branched actin nucleator Actin-Related Protein2/3 (ARP2/3) complex is an evolutionarily conserved, seven-subunit complex consisting of the actin-related proteins ARP2 and ARP3 (Machesky et al., 1994). The ARP2/3 complex initiates the formation of branches on the side of preexisting actin filaments, locally creating a force-generating branched actin network that underlies cellular protrusion and movement (Blanchoin et al., 2000; Amann and Pollard, 2001; Molinie and Gautreau, 2018). The phenotypes of mutants in ARP2/3 in the moss Physcomitrella patens (Harries et al., 2005; Perroud and Quatrano, 2006), in Arabidopsis (Le et al., 2003; Li et al., 2003; Mathur et al., 2003; Brembu et al., 2004; Deeks et al., 2004), in maize (Zea mays; Frank and Smith, 2002), and in tomato (Solanum lycopersicum; Chang et al., 2019) demonstrated the broad importance of the ARP2/3 complex and its activation during cellular morphogenesis, including tip-growing cells. Perhaps surprisingly, in Arabidopsis, null ARP2/3 alleles are transmitted normally through pollen and there is no obvious root hair phenotype (Le et al., 2003; Djakovic et al., 2006).These cell growth machineries are tightly coordinated by multiple signaling pathways, including membrane-localized receptor kinases and Rho-of-Plants (ROP) GTPases (Li et al., 2018). The tomato pollen-specific and membrane-localized receptor kinases LePRK1 and LePRK2 mediate signaling during pollen tube growth (Muschietti et al., 1998). LePRK2 perceives several extracellular growth-stimulating factors, including a Cys-rich extracellular protein (Late-Anther-Specific52 [LAT52]), a Leu-rich repeat protein from pollen, and two pistil/stigma molecules, Style Interactor for LePRKs and Stigma-Specific Protein1 (Tang et al., 2002, 2004; Wengier et al., 2003, 2010), which increase the speed of pollen tube growth (Zhang et al., 2008b; Huang et al., 2014). LePRK2 antisense and RNA interference (RNAi) pollen tubes grow slower (Zhang et al., 2008b), consistent with a positive role for LePRK2 in regulating the speed of pollen tube growth. LePRK1 binds LePRK2 (Wengier et al., 2003), but LePRK1 plays a negative role in pollen tube growth by controlling a switch from a fast tubular mode to a slow blebbing mode (Gui et al., 2014). LePRK1 RNAi pollen tubes burst more often than wild-type pollen tubes, implicating a role for LePRK1 in maintaining plasma membrane integrity (Gui et al., 2014). An Arabidopsis paralog of these LePRKs, PRK6, also localized on the tip membrane, perceives Arabidopsis attraction cues from the female, AtLURE1s, to guide pollen tube growth (Takeuchi and Higashiyama, 2016; Zhang et al., 2017).Rho family small guanine nucleotide-binding proteins called ROPs or RACs, which can switch between a GDP-bound inactive form and a GTP-bound active form, are regulators of polar growth in pollen tubes (Cheung and Wu, 2008; Yang, 2008). In Arabidopsis, ROP1-dependent signaling controls tip growth. Active ROP1 defines a cap region in the apical plasma membrane as an exocytosis zone (Luo et al., 2017). Overexpression of ROP1 or of a constitutively active version resulted in pollen tube tip swelling (i.e. increased tip width) and slower growth (i.e. reduced tube length), while overexpressing a dominant negative version of ROP1 inhibited pollen tube growth (i.e. shorter but normal width tubes). The size of the pollen tube tip reflects the aggregate activity of membrane-associated ROP at the tip (McKenna et al., 2009; Luo et al., 2017). Tomato ROPs have been reported to be associated with the LePRK1-LePRK2 complex (Wengier et al., 2003) and therefore presumably play similar roles as the Arabidopsis homologs in pollen tube growth, yet their biological roles have not been directly investigated.Guanine nucleotide exchange factors (GEFs) activate ROPs by promoting the conversion of ROP/RAC GTPases from a GDP-bound inactive form to a GTP-bound active form. Plants possess a plant-specific ROPGEF family whose members contain a highly conserved GEF catalytic domain, the PRONE (plant-specific ROP nucleotide exchanger) domain (Berken et al., 2005; Gu et al., 2006). The intracellular portions of LePRK1 and LePRK2 interact with Kinase Partner Protein (KPP; Kaothien et al., 2005), whose Arabidopsis homologs were later shown to belong to the PRONE-type ROPGEF family (Berken et al., 2005; Gu et al., 2006). Pollen tubes overexpressing nearly full-length KPP (missing eight amino acids at the N terminus) developed swollen tips with abnormal cytoplasmic streaming and F-actin arrangements (Kaothien et al., 2005). An Arabidopsis homolog of receptor kinase, AtPRK2a (also named AtPRK2), interacts with AtROPGEF12 (Zhang and McCormick, 2007) and with AtROPGEF1 (Chang et al., 2013) to affect ROP activity. Based on the in vitro catalytic activity of full-length and truncated AtROPGEF1, an autoinhibition conferred by the C-terminal variable region was proposed (Gu et al., 2006). AtROPGEF12 was also shown to interact with the guidance receptor kinase PRK6 (Takeuchi and Higashiyama, 2016).Increased expression of full-length KPP increased the speed of pollen tube growth without significantly affecting pollen tube shape. We show biochemically that the PRONE domain of KPP does have ROPGEF activity on several class I ROPs, with highest activity on ROP4. The N-terminal domain of KPP inhibits its own GEF activity, while its C-terminal domain enhances its own GEF activity. The C-terminal domain of KPP is also required for its interactions with LePRK1, LePRK2, and an actin-bundling protein, Pollen-expressed LIM2a (PLIM2a), while the C-terminal domain alone is sufficient to bind LePRK1 but insufficient to bind LePRK2. Furthermore, self-associated KPP colocalized with the actin nucleation proteins ARP2/3 complex during pollen tube growth and enriched the membrane localization of ARP2/3 in the pollen tube. Interfering with ARP2/3 activation by coexpressing a dominant negative version of ARP2 reduced the speed of pollen tube growth and alleviated the defects caused by the overexpression of truncated KPP. CK-666, a specific small molecule inhibitor of ARP2/3 activation, canceled the promotive effect of full-length KPP on the speed of pollen tube growth. These results indicate that during pollen germination and tube growth, KPP not only links pollen receptor kinase and ROP signaling but also links the actin network to the pollen tube plasma membrane, thereby directly affecting the cellular morphology and efficiency of pollen tube growth.     

Comparison of Insulins Glargine and Degludec in Diabetic Rhesus Macaques (Macaca mulatta) with CGM Devices

Treating and monitoring type 2 diabetes mellitus (T2DM) in NHP can be challenging. Multiple insulin and hypoglycemic therapies and management tools exist, but few studies demonstrate their benefits in a NHP clinical setting. The insulins glargine and degludec are long-acting insulins; their duration of action in humans exceeds 24 and 42 h, respectively. In the first of this study''s 2 components, we evaluated whether insulin degludec could be dosed daily at equivalent units to glargine to achieve comparable blood glucose (BG) reduction in diabetic rhesus macaques (Macaca mulatta) with continuous glucose monitoring (CGM) devices. The second component assessed the accuracy of CGM devices in rhesus macaques by comparing time-stamped CGM interstitial glucose values, glucometer BG readings, and BG levels measured by using an automated clinical chemistry analyzer from samples that were collected at the beginning and end of each CGM device placement. The CGM devices collected a total of 21,637 glucose data points from 6 diabetic rhesus macaques that received glargine followed by degludec every 24 h for 1 wk each. Ultimately, glucose values averaged 29 mg/dL higher with degludec than with glargine. Glucose values were comparable between the CGM device, glucometer, and chemistry analyzer, thus validating that CGM devices as reliable for measuring BG levels in rhesus macaques. Although glargine was superior to degludec when given at the same dose (units/day), both are safe and effective treatment options. Glucose values from CGM, glucometers, and chemistry analyzers provided results that were analogous to BG values in rhesus macaques. Our report further highlights critical clinical aspects of using glargine as compared with degludec in NHP and the benefits of using CGM devices in macaques.

Diabetes is a group of metabolic diseases that cause hyperglycemia secondary to deficient insulin response, secretion, or both.⁴ Diabetes is categorized by the American Diabetes Association into 4 types: 1) type 1 diabetes mellitus, in which the pancreas is unable to produce insulin for glucose absorption; 2) type 2 diabetes mellitus (T2DM), when the body does not use insulin correctly; 3) gestational diabetes, in which the body is insulin-intolerant during pregnancy (or is first discovered then); and 4) other specific forms of diabetes in which the patient is particularly predisposed to becoming diabetic due to various comorbidities or to inadvertent induction caused by some medications.⁴ In 2018, 34.2 million (10.5%) Americans of all ages were diagnosed with diabetes.^22,23,30 Approximately 90% to 95% of Americans with diabetes have T2DM,²⁴ making T2DM the most common form of diabetes diagnosed in humans.T2DM is a multifactorial disease primarily determined by genetics, behavioral and environmental factors (for example, age, diet, sedentary lifestyle, obesity).^4,46,50,74 As a consequence of these factors, the pancreas increases insulin secretion to maintain normal glucose tolerance.⁷⁴ Over time, the high insulin demand causes pancreatic β-cell destruction, resulting in reduced production of insulin.^39,50,74 As β-cell destruction increases, hyperglycemia and T2DM develop. Insulin resistance and hyperglycemia are tolerated for a period of time^19,82,83 before clinical signs associated with T2DM develop (e.g., polydipsia, polyuria, polyphagia with concurrent weight loss).⁴ Once clinical signs develop, T2DM is most commonly diagnosed as a fasting blood glucose level (FBG) of 126 mg/dL or greater,^2,4 2-h plasma glucose value of 200 mg/d or greater during a 75-g oral glucose tolerance test,^2,4 and/or glycosylated hemoglobin (HbA1c) of 6.5% or greater.^2,4 Depending on the FBG, oral glucose tolerance test, and HbA1c results, various treatment options are recommended by the American Diabetes Association. Most importantly, lifestyle changes, including diet and exercise, are recommended as the first line of treatment, along with oral antihyperglycemic drugs such as metformin.^5,25,46 Treatment efficacy is evaluated with self-monitoring blood glucose or continuous glucose monitoring (CGM) devices.³ Human patients using CGM devices have achieved considerable reductions in HbA1c compared with patients not using them.³ As CGM devices have become more readily available, user friendly, and affordable, they have become an essential tool in managing T2DM.Similar to humans, most NHP affected by diabetes are diagnosed with T2DM.^80,83 NHP are predisposed to similar genetic, behavioral and environmental factors (e.g., age, diet, sedentary lifestyle, obesity);^{6,18,19,37,44,52,82,83} have similar pathophysiology;^38,81-83 are diagnosed via FBG,^39,83 HbA1c,^21,31,49,56 fructosamine,^20,83,87 and weight loss;^49,80,83,86 and are treated with exercise and diet modifications as a first line of treatment.^{11,19,39,53,79} Although the human and NHP conditions are similar, the treatment and management of T2DM is somewhat different, especially when NHP have restricted physical activity due to housing constraints.Previous studies indicate that daily dosing with insulin glargine achieves appropriate glycemic control in NHP.⁴⁸ Therefore, we implemented glargine, along with some diet modification, to improve glycemic control in our diabetic colony. Other noninsulin therapies, such as metformin, had been used, but compliance was low (for example, due to large pill size, unpleasant taste, etc.). However, achieving glycemic control using diet modification, insulin glargine treatment, monthly scheduled FBG, quarterly HbA1c, and regular weight monitoring was challenging in a large colony. Monthly FBG and fructosamine testing were performed due to affordability and practicality for NHP in a research setting. Given that fructosamine levels correlate with BG concentrations for the preceding 2 to 3 wk and HbA1c percentages relate to BG concentration over 1.5 to 3 mo,^49,87 HbA1C was selected over fructosamine for T2DM management in our colony. Determining which T2DM treatment and diagnostics are most effective can be difficult in large colonies of NHP. Therefore, improved treatment and management strategies would help to manage T2DM in NHP more efficiently.Insulin glargine is a long-acting insulin, with a half-life of 12 h and duration of action of 12 to 24 h in humans^40,55 and 12 h in dogs.^34,43,60 Once injected subcutaneously, insulin glargine forms a microprecipitate in the neutral pH environment, which delays and prolongs absorption in subcutaneous tissues.¹² Insulin degludec is a newer form of long-acting insulin, with a half-life of 25 h^41,63,62,77 and duration of action that exceeds 42 h in humans.^40,41,68,77 Insulin degludec forms a soluble and stable dihexamer in the pharmaceutical formulation, which includes phenol and zinc.^63,78 The phenol diffuses away, leading to the formation of a soluble depot in the form of long multihexamer chains in which zinc slowly diffuses from the end of the multihexamers, causing a gradual, continuous, and extended-release of monomers from the depot of the injection site.^63,78 Pharmacodynamic studies in humans, demonstrate that the “glucose-lowering effect” of insulin degluc⁴⁰ is evenly distributed over 24 h, allowing a more stable steady-state and improved wellbeing.⁷⁸ This approach could potentially reduce the number of hypoglycemic events and provide a less rigid daily injection schedule,⁵⁸ thus potentially making insulin degludec—compared with insulin glargine—a safer, alternative diabetes therapy.In addition to medical intervention, glycemic control is achieved through regular management and monitoring of BG. Self-monitoring blood glucose checks in humans^3,5 and glucose curves in animals¹⁰ are some of the management tools used to determine or evaluate therapy for T2DM patients. Telemetry systems like CGM devices are used to monitor interstitial glucose and have been used extensively in humans^3,17,33 and animals^{16,27,36,42,47,84,85} to monitor BG in real-time. Using CGM devices 1) reduces or eliminates the number of blood draws needed to collect FBG,⁶¹ 2) accurately assesses insulin therapy via a real-time glucose curve,^72,84,85 3) allows patients and clinicians to titrate treatment^61,73 as indicated, and 4) obtains continuous glucose data with reduced manipulation and subsequent decreased stress.^72,84,85 Therefore, CGM devices can be a safe and informative tool in monitoring spontaneous T2DM in NHP.Between 2015 and 2030, the prevalence of diabetes is predicted to increase by 54% to more than 54 million Americans affected by diabetes (i.e., diabetes mellitus types 1 and 2).⁷⁰ NHP are an essential model for human T2DM because of their similar pathophysiology, diagnostics, treatment, and management. As more people develop diabetes, novel therapies will continue to be developed. Studying new treatments and management tools in NHP can further human and NHP T2DM research to prevent the progression of T2DM and hopefully diminish projections for the number of future diabetes cases. Human medical literature, American Diabetes Association, and drug manufacturers all recommend giving equal doses (i.e., number of units/day) of long-acting insulins when changing from one long-acting insulin to degludec.^26,63,67 Therefore, we hypothesized that insulin degludec would provide effective glycemic control for rhesus macaques with T2DM when dosed at equivalent doses (that is, the same number of units/day) as insulin glargine. In addition, we hypothesized that CGM devices would provide accurate BG readings as compared with chemistry analyzer and glucometer BG readings, making it a more efficient and effective tool for measurement of BG levels in rhesus macaques with T2DM.     

Fibrous Osteodystrophy,Chronic Renal Disease,and Uterine Adenocarcinoma in Aged Gray Mouse Lemurs (Microcebus murinus)

The gray mouse lemur (Microcebus murinus, GML) is a nocturnal, arboreal, prosimian primate that is native to Madagascar. Captive breeding colonies of GMLs have been established primarily for noninvasive studies on questions related to circadian rhythms and metabolism. GMLs are increasingly considered to be a strong translational model for neurocognitive aging due to overlapping histopathologic features shared with aged humans. However, little information is available describing the clinical presentations, naturally occurring diseases, and histopathology of aged GMLs. In our colony, a 9 y-old, male, GML was euthanized after sudden onset of weakness, lethargy, and tibial fracture. Evaluation of this animal revealed widespread fibrous osteodystrophy (FOD) of the mandible, maxilla, cranium, appendicular, and vertebral bones. FOD and systemic metastatic mineralization were attributed to underlying chronic renal disease. Findings in this GML prompted periodic colony-wide serum biochemical screenings for azotemia and electrolyte abnormalities. Subsequently, 3 additional GMLs (2 females and 1 male) were euthanized due to varying clinical and serum biochemical presentations. Common to all 4 animals were FOD, chronic renal disease, uterine adenocarcinoma (females only), cataracts, and osteoarthritis. This case study highlights the concurrent clinical and histopathologic abnormalities that are relevant to use of GMLs in the expanding field of aging research.

Within the past 5 y, recognition of the translational utility of the gray mouse lemur (Microcebus murinus, GML) has greatly expanded, in part due to the sequencing of its genome.²⁷ GMLs have been proposed as an animal model in the context of aging research,^14,35 most notably within the fields of Alzheimer disease and dementia^33,39 and circadian rhythms.^15,20 GMLs are nocturnal, arboreal, prosimian primates (family Cheirogaleidae) that are endemic to Madagascar. They are among the smallest primates, with a body weight of 49 to 80 g in the wild³⁷ (60 to 110 g in captivity) and have a life expectancy of approximately 8 to 10 y in captivity.¹⁴ A small number of captive breeding colonies have been established throughout Europe and the United States, many of which have arisen from a closed captive breeding colony at the Muséum National d''Histoire Naturelle (MNHN) in Brunoy, France.Despite an ever-growing interest in the GML as a model organism, clinical and pathologic case reports focusing on naturally occurring disease are rare for this species.^{1,4,10,16,17,20,28,31,34,38} Reports of spontaneous disease often focus on neoplasia^28,31,34 or on ocular abnormalities, which are accessible without invasive interventions.^1,4,12 Apart from age-related neurodegenerative disease and cognitive impairment,^{5,23,25,26,32,36} little is known about the natural disease predilection and histologic aging phenotypes of GMLs.In June 2017, a 9 y-old male GML was euthanized after the sudden onset of weakness, lethargy, and tibial fracture. Necropsy and histopathology revealed chronic renal disease, widespread fibrous osteodystrophy (FOD), and systemic metastatic mineralization. These findings prompted colony-wide serum biochemical screenings for potential underlying renal disease and subsequent metabolic bone disease within the population.Herein, we report the clinical, gross, and histologic multisystemic pathology of 4 aged GMLs. This is the first documentation of FOD secondary to chronic renal disease in GMLs in a captive research colony. In addition, we corroborate previous reports^31,34 of uterine adenocarcinoma in aged female GMLs. Together, these findings aid in providing appropriate clinical care to GMLs as their use in the field of aging research continues to expand.     

Inflammatory Responses with Left Ventricular Compromise after Induction of Myocardial Infarcts in Sheep (Ovis aries)

Ischemic myocardial disease is a major cause of death among humans worldwide; it results in scarring and pallor of the myocardium and triggers an inflammatory response that contributes to impaired left ventricular function. This response includes and is evidenced by the production of several inflammatory cytokines including TNFα, IL1β, IL4, IFNγ, IL10 and IL6. In the current study, myocardial infarcts were induced in 6 mo old male castrated sheep by ligation of the left circumflex obtuse marginal arteries (OM 1 and 2). MRI was used to measure parameters of left ventricular function that include EDV, ESV, EF, SVI, dp/dt max and dp/dt min at baseline and at 4 wk and 3 mo after infarct induction. We also measured serum concentrations of an array of cytokines. Postmortem histologic findings corroborate the existence of left ventricular myocardial injury and deterioration. Our data show a correlation between serum cytokine concentrations and the development of myocardial damage and left ventricular functional compromise.

Heart failure is a globally significant problem in both humans and lower animals.^3,18 The medical literature is replete with predisposing causes of heart disease,¹³ yet the prevalence of heart failure remained high.^4,5,16 Regardless of the cause of myocardial damage and subsequent left ventricular compromise, the literature indicated that the proinflammatory response that occurs after myocardial infarction is an important contributor to the deterioration of the myocardium^{1,9,12,14,17,18,20,21} Sheep and pigs are excellent translational models of human cardiology because their hearts bear many physiologic and anatomic similarities to the human heart.^4,8,15 The primary use of these models in cardiology is primarily to study myocardial infarction^5,13,16 and to a lesser extent, physiologic processes that develop after myocardial insult.Our study measured some of the major proinflammatory cytokines that contribute to myocardial damage. Most of these cytokines, including: TNFα, IL6, and IFNγ, are important correlates of myocardial ischemia that contribute to a decline in left ventricular myocardial function.^1,9,14 In our study, we detected left ventricular compromise as early as 4 wk after the infarction, while the proinflammatory response was recorded at 48 h after the infarct and peaked at 4 wk. Cardiac functional parameters began to decline early in the study consistent with the proinflammatory response. The cardiac functional parameters continued to decline until 3 mo, which was the termination of the study. These findings may support antiinflammatory intervention as an important adjunct of any therapeutic regimen.     

PSI of the Colonial Alga Botryococcus braunii Has an Unusually Large Antenna Size

PSI is an essential component of the photosynthetic apparatus of oxygenic photosynthesis. While most of its subunits are conserved, recent data have shown that the arrangement of the light-harvesting complexes I (LHCIs) differs substantially in different organisms. Here we studied the PSI-LHCI supercomplex of Botryococccus braunii, a colonial green alga with potential for lipid and sugar production, using functional analysis and single-particle electron microscopy of the isolated PSI-LHCI supercomplexes complemented by time-resolved fluorescence spectroscopy in vivo. We established that the largest purified PSI-LHCI supercomplex contains 10 LHCIs (∼240 chlorophylls). However, electron microscopy showed heterogeneity in the particles and a total of 13 unique binding sites for the LHCIs around the PSI core. Time-resolved fluorescence spectroscopy indicated that the PSI antenna size in vivo is even larger than that of the purified complex. Based on the comparison of the known PSI structures, we propose that PSI in B. braunii can bind LHCIs at all known positions surrounding the core. This organization maximizes the antenna size while maintaining fast excitation energy transfer, and thus high trapping efficiency, within the complex.

The multisubunit-pigment-protein complex PSI is an essential component of the electron transport chain in oxygenic photosynthetic organisms. It utilizes solar energy in the form of visible light to transfer electrons from plastocyanin to ferredoxin.PSI consists of a core complex composed of 12 to 14 proteins, which contains the reaction center (RC) and ∼100 chlorophylls (Chls), and a peripheral antenna system, which enlarges the absorption cross section of the core and differs in different organisms (Mazor et al., 2017; Iwai et al., 2018; Pi et al., 2018; Suga et al., 2019; for reviews, see Croce and van Amerongen, 2020; Suga and Shen, 2020). For the antenna system, cyanobacteria use water-soluble phycobilisomes; green algae, mosses, and plants use membrane-embedded light-harvesting complexes (LHCs); and red algae contain both phycobilisomes and LHCs (Busch and Hippler, 2011). In the core complex, PsaA and PsaB, the subunits that bind the RC Chls, are highly conserved, while the small subunits PsaK, PsaL, PsaM, PsaN, and PsaF have undergone substantial changes in their amino acid sequences during the evolution from cyanobacteria to vascular plants (Grotjohann and Fromme, 2013). The appearance of the core subunits PsaH and PsaG and the change of the PSI supramolecular organization from trimer/tetramer to monomer are associated with the evolution of LHCs in green algae and land plants (Busch and Hippler, 2011; Watanabe et al., 2014).A characteristic of the PSI complexes conserved through evolution is the presence of “red” forms, i.e. Chls that are lower in energy than the RC (Croce and van Amerongen, 2013). These forms extend the spectral range of PSI beyond that of PSII and contribute significantly to light harvesting in a dense canopy or algae mat, which is enriched in far-red light (Rivadossi et al., 1999). The red forms slow down the energy migration to the RC by introducing uphill transfer steps, but they have little effect on the PSI quantum efficiency, which remains ∼1 (Gobets et al., 2001; Jennings et al., 2003; Engelmann et al., 2006; Wientjes et al., 2011). In addition to their role in light-harvesting, the red forms were suggested to be important for photoprotection (Carbonera et al., 2005).Two types of LHCs can act as PSI antennae in green algae, mosses, and plants: (1) PSI-specific (e.g. LHCI; Croce et al., 2002; Mozzo et al., 2010), Lhcb9 in Physcomitrella patens (Iwai et al., 2018), and Tidi in Dunaliela salina (Varsano et al., 2006); and (2) promiscuous antennae (i.e. complexes that can serve both PSI and PSII; Kyle et al., 1983; Wientjes et al., 2013a; Drop et al., 2014; Pietrzykowska et al., 2014).PSI-specific antenna proteins vary in type and number between algae, mosses, and plants. For example, the genomes of several green algae contain a larger number of lhca genes than those of vascular plants (Neilson and Durnford, 2010). The PSI-LHCI complex of plants includes only four Lhcas (Lhca1–Lhc4), which are present in all conditions analyzed so far (Ballottari et al., 2007; Wientjes et al., 2009; Mazor et al., 2017), while in algae and mosses, 8 to 10 Lhcas bind to the PSI core (Drop et al., 2011; Iwai et al., 2018; Pinnola et al., 2018; Kubota-Kawai et al., 2019; Suga et al., 2019). Moreover, some PSI-specific antennae are either only expressed, or differently expressed, under certain environmental conditions (Moseley et al., 2002; Varsano et al., 2006; Swingley et al., 2010; Iwai and Yokono, 2017), contributing to the variability of the PSI antenna size in algae and mosses.The colonial green alga Botryococcus braunii (Trebouxiophyceae) is found worldwide throughout different climate zones and has been targeted for the production of hydrocarbons and sugars (Metzger and Largeau, 2005; Eroglu et al., 2011; Tasić et al., 2016). Here, we have purified and characterized PSI from an industrially relevant strain isolated from a mountain lake in Portugal (Gouveia et al., 2017). This B. braunii strain forms colonies, and since the light intensity inside the colony is low, it is expected that PSI in this strain has a large antenna size (van den Berg et al., 2019). We provide evidence that B. braunii PSI differs from that of closely related organisms through the particular organization of its antenna. The structural and functional characterization of B. braunii PSI highlights a large flexibility of PSI and its antennae throughout the green lineage.     

Ubiquitination of S4-RNase by S-LOCUS F-BOX LIKE2 Contributes to Self-Compatibility of Sweet Cherry ‘Lapins’

Recent studies have shown that loss of pollen-S function in S₄′ pollen from sweet cherry (Prunus avium) is associated with a mutation in an S haplotype-specific F-box4 (SFB4) gene. However, how this mutation leads to self-compatibility is unclear. Here, we examined this mechanism by analyzing several self-compatible sweet cherry varieties. We determined that mutated SFB4 (SFB4ʹ) in S4′ pollen (pollen harboring the SFB4ʹ gene) is approximately 6 kD shorter than wild-type SFB4 due to a premature termination caused by a four-nucleotide deletion. SFB4′ did not interact with S-RNase. However, a protein in S4′ pollen ubiquitinated S-RNase, resulting in its degradation via the 26S proteasome pathway, indicating that factors in S4′ pollen other than SFB4 participate in S-RNase recognition and degradation. To identify these factors, we used S₄-RNase as a bait to screen S4′ pollen proteins. Our screen identified the protein encoded by S₄-SLFL2, a low-polymorphic gene that is closely linked to the S-locus. Further investigations indicate that SLFL2 ubiquitinates S-RNase, leading to its degradation. Subcellular localization analysis showed that SFB4 is primarily localized to the pollen tube tip, whereas SLFL2 is not. When S₄-SLFL2 expression was suppressed by antisense oligonucleotide treatment in wild-type pollen tubes, pollen still had the capacity to ubiquitinate S-RNase; however, this ubiquitin-labeled S-RNase was not degraded via the 26S proteasome pathway, suggesting that SFB4 does not participate in the degradation of S-RNase. When SFB4 loses its function, S₄-SLFL2 might mediate the ubiquitination and degradation of S-RNase, which is consistent with the self-compatibility of S4′ pollen.

In sweet cherry (Prunus avium), self-incompatibility is mainly controlled by the S-locus, which is located at the end of chromosome 6 (Akagi et al., 2016; Shirasawa et al., 2017). Although the vast majority of sweet cherry varieties show self-incompatibility, some self-compatible varieties have been identified, most of which resulted from the use of x-ray mutagenesis and continuous cross-breeding (Ushijima et al., 2004; Sonneveld et al., 2005). At present, naturally occurring self-compatible varieties are rare (Marchese et al., 2007; Wünsch et al., 2010; Ono et al., 2018). X-ray-induced mutations that have given rise to self-compatibility include a 4-bp deletion (TTAT) in the gene encoding an SFB4′ (S-locus F-box 4′) protein, located in the S-locus and regarded as the dominant pollen factor in self-incompatibility. This mutation is present in the first identified self-compatible sweet cherry variety, ‘Stellar’, as well as in a series of its self-compatible descendants, including ‘Lapins’, ‘Yanyang’, and ‘Sweet heart’ (Lapins, 1971; Ushijima et al., 2004). Deletion of SFB3 and a large fragment insertion in SFB5 have also been identified in other self-compatible sweet cherry varieties (Sonneveld et al., 2005; Marchese et al., 2007). Additionally, a mutation not linked to the S-locus (linked instead to the M-locus) could also cause self-compatibility in sweet cherry and closely related species such as apricot (Prunus armeniaca; Wünsch et al., 2010; Zuriaga et al., 2013; Muñoz-Sanz et al., 2017; Ono et al., 2018). Much of the self-compatibility in Prunus species seems to be closely linked to mutation of SFB in the S-locus (Zhu et al., 2004; Muñoz-Espinoza et al., 2017); however, the mechanism of how this mutation of SFB causes self-compatibility is unknown.The gene composition of the S-locus in sweet cherry differs from that of other gametophytic self-incompatible species, such as apple (Malus domestica), pear (Pyrus spp.), and petunia (Petunia spp.). In sweet cherry, in addition to a single S-RNase gene, the S-locus contains one SFB gene, which has a high level of allelic polymorphism, and three SLFL (S-locus F-box-like) genes with low levels of, or no, allelic polymorphism (Ushijima et al., 2004; Matsumoto et al., 2008). By contrast, the apple, pear, and petunia S-locus usually contains one S-RNase and 16 to 20 F-box genes (Kakui et al., 2011; Okada et al., 2011, 2013; Minamikawa et al., 2014; Williams et al., 2014a; Yuan et al., 2014; Kubo et al., 2015; Pratas et al., 2018). The F-box gene, named SFBB (S-locus F-box brother) in apple and pear and SLF (S-locus F-box) in petunia, exhibits higher sequence similarity with SLFL than with SFB from sweet cherry (Matsumoto et al., 2008; Tao and Iezzoni, 2010). The protein encoded by SLF in the petunia S-locus is thought to be part of an SCF (Skp, Cullin, F-box)-containing complex that recognizes nonself S-RNase and degrades it through the ubiquitin pathway (Kubo et al., 2010; Zhao et al., 2010; Chen et al., 2012; Entani et al., 2014; Li et al., 2014, 2016, 2017; Sun et al., 2018). In sweet cherry, a number of reports have described the expression and protein interactions of SFB, SLFL, Skp1, and Cullin (Ushijima et al., 2004; Matsumoto et al., 2012); however, only a few reports have examined the relationship between SFB/SLFL and S-RNase (Matsumoto and Tao, 2016, 2019), and none has investigated whether the SFB/SLFL proteins participate in the ubiquitin labeling of S-RNase.Although the function of SFB4 and SLFL in self-compatibility is unknown, the observation that S4′ pollen tubes grow in sweet cherry pistils that harbor the same S alleles led us to speculate that S4′ pollen might inhibit the toxicity of self S-RNase. In petunia, the results of several studies have suggested that pollen tubes inhibit self S-RNase when an SLF gene from another S-locus haplotype is expressed (Sijacic et al., 2004; Kubo et al., 2010; Williams et al., 2014b; Sun et al., 2018). For example, when SLF2 from the S₇ haplotype is heterologously expressed in pollen harboring the S₉ or S₁₁ haplotype, the S₉ or S₁₁ pollen acquire the capacity to inhibit self S-RNase and break down self-incompatibility (Kubo et al., 2010). The SLF2 protein in petunia has been proposed to ubiquitinate S₉-RNase and S₁₁-RNase and lead to its degradation through the 26S proteasome pathway (Entani et al., 2014). If SFB/SLFL in sweet cherry have a similar function, the S4′ pollen would not be expected to inhibit self S₄-RNase, prompting the suggestion that the functions of SFB/SLFL in sweet cherry and SLF in petunia vary (Tao and Iezzoni, 2010; Matsumoto et al., 2012).In this study, we used sweet cherry to investigate how S4′ pollen inhibits S-RNase and causes self-compatibility, focusing on the question of whether the SFB/SLFL protein can ubiquitinate S-RNase, resulting in its degradation.     

The Effect of Body Region on Hair Cortisol Concentration in Common Marmosets (Callithrix jacchus)

Common marmosets (Callithrix jacchus) are a valuable research model for the study of neuroscience and the biologic impact of aging due to their adaptivity, physiologic characteristics, and ease of handling for experimental manipulations. Quantification of cortisol in hair provides a noninvasive, retrospective biomarker of hypothalamics-pituitary-adrenal (HPA) axis activity and information on animal wellbeing, including responses to environmental and social stimuli. To obtain valid and reliable measurements of long-term HPA activity, we investigated the variability of cortisol concentration in the hair depending on the body region of marmosets. Hair was collected from the back and tail of 9 adult common marmosets during annual health screenings (male n = 3; female n = 6) and these samples were analyzed for cortisol via methanol extraction and enzyme immunoassay. We found that hair cortisol concentration differed between the tail and back regions, with the tail samples having a significantly higher cortisol concentration. These results indicate intraindividual and interindividual comparisons of hair cortisol concentration should use hair obtained from the same body region in marmosets.

Common marmosets (Callithrix jacchus) are small, New World Primates endemic to Northeast Brazil. Marmosets are similar to humans in their physiology, neuroanatomy, cognition, and sociality.^11,16 They have become an important model in behavioral and biomedical research, including studies of the physiologic effects of aging and neurologic disease. Furthermore, their use in studies of aging or development includes their frequent use in longitudinal studies. Therefore, the ability to frequently and noninvasively measure glucocorticoid hormones as a marker of the response to environmental or social stimuli is an important aspect of longitudinal research in this species.Glucocorticoids are regulated by the hypothalamic-pituitary-adrenal (HPA) axis, a complex neural negative-feedback system that perceives environmental stimuli and regulates hormones accordingly. Cortisol is the dominant glucocorticoid hormone found in most mammals²⁵ and is involved in several physiologic processes, such as metabolizing sugar, fat, and protein stores into usable energy and the inhibition of swelling and inflammation.²¹ Quantification of cortisol concentration can therefore provide valuable insight into how an animal is responding to its environment or to an experimental manipulation and can also provide a valid, retrospective biomarker of HPA axis activity and individual health.^4,5,14,26Cortisol can be measured in blood, saliva, urine, feces, and hair. These tissue types differ in terms of the time frame reflected in the sample and the invasiveness of sample collection.²² Different tissues reflect different intervals with regard to when the cortisol was secreted from the adrenal cortex. Blood and saliva are considered to provide point measures because they indicate a time frame of minutes since the cortisol was secreted.²² Urine and feces are considered to be state measures; they reflect a time frame from hours to a day.²² Finally, hair incorporates a chronic time frame of weeks or months.²² Long-term HPA axis activity can be quantified retrospectively from hair, which accumulates its cortisol concentration over weeks or months, depending upon the length of hair analyzed²² and the growth dynamics of the hair.²⁰ Blood-born substances, such as cortisol, diffuse from blood capillaries into hair follicle cells.^7,27 Only cortisol molecules that are not bound to proteins diffuse into blood capillaries; therefore, hair cortisol concentration (HCC) represents unbound cortisol molecules.^7,27 Once cortisol molecules enter the follicle cells, they are deposited into the hair shaft.⁷ The cortisol deposition process only occurs at a specific time during hair growth, and cortisol deposition into the hair shaft over time is directly proportional to blood cortisol concentration.⁷The stage of growth of a hair follicle influences the cortisol deposition process. All hair has 3 stages of growth: the anagen, catagen, and telogen. The anagen is the active stage when growth occurs; in the catagen or intermediate stage, old hair shafts break down and newer shafts are produced in preparation for the next round of growth; lastly, the telogen stage is the resting phase of hair growth. Most cut or plucked hair samples are expected to be in the anagen phase, while shed hair is likely in the telogen phase.¹³ Cortisol deposition occurs during the anagen stage as active hair follicles require nutrients via the bloodstream to grow. This slow rate of deposition provides information on HPA activity over weeks or months.²²While hair cortisol concentration is increasingly used to assess retrospective HPA axis activity in captive and wild animals, a standard body region from which to obtain hair samples has not been determined. Studies report sampling from various regions, including the back of the neck, back, shoulder, chest, the base of tail, thigh, deltoid, and interscapular regions.^1,7-9 In this investigation, we sought to determine if HCC was affected by the body region from which the samples were taken in common marmosets. We compared HCC from 2 common and readily accessible locations of sample collection in common marmosets, the back and the tail.