The research paper, “Introduced Herbivores Restore Late Pleistocene Ecological Functions” is the work of an 11-member international team led by Australian ecologist Erick Lundgren of the University of Technology, Sydney.
The authors pored over scientific literature; created a list of living and extinct herbivores over the last 126,000 years; and categorized them by their body size, anatomy, habitat, diet, and how their bodies digested the vegetation. Then they compared their lifestyles in overlapping regions.
Carroll, affiliated with the UC Davis Department of Entomology and Nematology, said one of the studies dealt with the abandoned hippos of Colombian drug lord Pablo Escobar (1949-1993), who purchased a male and three females in the 1980s from a California zoo and kept them in fields along the Magdalena River, northwestern Colombia. Without humans and other predators decimating them, the population today is 80 and is expected to reach 800 to 5000 by 2050.
The out-of-place hippos may be filling the exotic roles of extinct massive animals, such as giant llamas and rhinoceros-sized relatives, the ecologists said.
Said Carroll: “That paleontological analysis found that, amazingly, introduced herbivores– including Pablo Escobar's escaped Colombian hippos– often match the functional traits of extinct natives better than do those missing species' closest living native relatives. In this way, the ‘out-of-place' make the world more similar to the pre-extinction past. The ‘shoot-first- and-ask-questions later' approach as a maxim is as reckless as it sounds, and it's not going to sustain our life-saving drugs, nor the species we revere or ecosystems we rely on, into the future.”
“Many introduced herbivores restore trait combinations that have the capacity to influence ecosystem processes, such as wildfire and shrub expansion in drylands,” the team wrote.
As for feral hogs in North America, Carroll said their rooting increases tree growth and attracts bird flocks, like the ecological work of their extinct ancestors. Likewise, the feral horses and burros, known for their well-digging behavior, are replacing the original American horses, which went extinct 12,000 years ago.
In their abstract, the authors pointed out that humans “have caused extinctions of large-bodied mammalian herbivores over the past 100,000 years, leading to cascading changes in ecosystems. Conversely, introductions of herbivores have, in part, numerically compensated for extinction losses. However, the net outcome of the twin anthropogenic forces of extinction and introduction on herbivore assemblages has remained unknown. We found that a primary outcome of introductions has been the reintroduction of key ecological functions, making herbivore assemblages with nonnative species more similar to preextinction ones than native-only assemblages are. Our findings support calls for renewed research on introduced herbivore ecologies in light of paleoecological change and suggest that shifting focus from eradication to landscape and predator protection may have broader biodiversity benefits.”
Carroll, who also co-led an author group of the newly published “Coevolutionary Governance of Antibiotic and Pesticide Resistance” in the journal Trends in Ecology, said that the publications together “address both sides of the human-environment co-existence issue.”
“Reading the titles, you might not expect these two studies are two sides of the same coin,” Carroll said, “but for me they address both sides of the human-environment issue that most compels me: How can we create more workable, productive and respectful long-term relationships with other species? To help think about this as an evolutionary biologist, I divide the key challenges of human interactions with Nature into those that arise from competitor and parasite species that adapt too quickly for us to control, and those that arise in in our efforts to protect more valued species– like endangered large mammals– that adapt too slowly to survive human impacts.”
“Pesticide and drug resistance are nature's predictable resilience to our reliance on an escalating war of toxic eradication,” Carroll commented. “A broader understanding shows how we can develop our own behavior to instead cultivate susceptibility to control in species we fight, using both new and known practices for improved sanitation, locally diversified agriculture, and eating lower on the food chain to inflect their evolution in a positive direction. Similarly, after millennia of driving much of the Earth's giant mammal community to extinction, we need to step back from our reflex to extinguish the errant survivors to preserve a modern sense of what's natural, without stopping to consider how these new neighbors (commonly fading from their native lands) may restore ancient ecological functions our own ancestors extinguished not so long ago.”
Carroll emphasized that “neither of these studies dismisses the serious problems irruptive populations can cause for meeting our food, health and environmental needs, nor seeks to oversimplify complex challenges. But it's actually important to work against being limited by prejudicial generalizations that lead us to sort other species into ‘good' versus ‘bad' bins. This is a sensibility that ecologists in particular should strive to cultivate. To continue to feed and shelter ourselves and remain healthy while sharing the Earth with other species, we need to develop methods that respect the tremendous information and know-how inherent in each species. I want us to do a much better job of working with that intrinsic functional diversity and adaptive potential as our best resource for advancing resilient and biodiverse ecological systems into the future.”
Carroll and his wife, UC Davis ecologist Jenella Loye, own Carroll-Loye Biological Research, Davis. They engage in public health and environmental entomology and natural product development.
(Editor's Note: The lead author of Coevolutionary Governance of Antibiotic and Pesticide Resistance is Peter Søgaard Jørgensen, who during his University of Copenhagen graduate work, spent a year at Davis studying soapberry bug host adaptation in California with Scott Carroll. The duo led the multi-year international "Living with Resistance" pursuit at the National Science Foundation's National Socio-Environmental Synthesis Center. Carroll served as the senior author.)
DAVIS--Newly published research in the Proceedings of the National Academy of Sciences (PNAS) indicates that a drug discovered and developed in the laboratory of Bruce Hammock,UC Davis Department of Entomology and Nematology, may have a major role in preventing and treating llnesses associated with obesity.
More than 43 percent of adults in the United States are obese, according to the Center for Disease Control and Prevention (CDC). Obesity increases the risk of coronary artery disease, stroke, type 2 diabetes, and certain kinds of cancer.
The drug, a soluble epoxide hydrolase (sEH) inhibitor, appears to regulate “obesity-induced intestinal barrier dysfunction and bacterial translocation,” the 12-member team of researchers from UC Davis, University of Massachusetts and University of Michigan discovered. The same non-opioid drug is being investigated in human clinical safety trials in Texas to see if it blocks chronic pain associated with diseases such as spinal cord injury, diabetes and inflammatory bowel disease.
The research, funded by multiple federal grants, is titled “Soluble Epoxide Hydrolase Is an Endogenous Regulator of Obesity-Induced Intestinal Barrier Dysfunction and Bacterial Translocation.”
“Obesity usually causes the loss of tight junctions and leaky gut,” said first author Yuxin Wang, a postdoctoral researcher who joined the Hammock lab in 2019 from the Department of Food Sciences, University of Massachusetts, Amherst. “In normal conditions, the gut mucosal barrier is like a defender to protect us from the ‘dirty things' in the lumen, such as bacteria and endotoxin. For obese individuals, the defender loses some function and leads to more ‘bad things' going into the circulation system, causing systemic or other organ disorders.”
Although intestinal dysfunction and other problems enhancing bacterial translocation underlies many human diseases, “the mechanisms remain largely unknown,” said Wang, who holds a doctorate in biochemistry and molecular biology from the Chinese Academy of Sciences. “What we found is sEH inhibition can repair the defender function (barrier function), decrease the ‘bad things' going into the blood (bacteria translocation), and reduce inflammation of fat.”
“Our research shows that sEH is a novel endogenous regulator of obesity-induced intestinal barrier dysfunction and bacterial translocation,” said corresponding author Guodong Zhang, a former researcher in the Hammock lab and now with the Food Science Department and Molecular and Cellular Biology Graduate Program at the University of Massachusetts. “To date, the underlying mechanisms for obesity-induced intestinal barrier dysfunction remain poorly understood. Therefore, our finding provides a novel conceptual approach to target barrier dysfunction and its resulting disorders with clinical/transitional importance.”
Corresponding author Hammock, a distinguished UC Davis professor who holds a joint appointment with the Department of Entomology and Nematology and the Comprehensive Cancer Center, praised Zhang's “amazing record while he was a postgraduate at UC Davis, and now in Food Science Department at the University of Massachusetts, where he recently received tenure.”
Zhang mentored two co-authors of the paper: Yuxin and Weicang Wang, both formerly of the Department of Food Science, University of Massachusetts and now with the Hammock lab.
“I feel so lucky that Yuxin and Weicang have joined my laboratory,” Hammock said. “The drugs studied in this PNAS paper are now in human clinical trials and on a path to replace opioid analgesics for pain treatment. I hope the continuing work of Guodong, Weicang and Yuxin will evaluate them as treatments for a variety of inflammatory bowel diseases.”
Andreas Baumler, professor and vice chair of research in the UC Davis Department of Medical Microbiology and Immunology, who was not affiliated with the study, said: “Obesity-induced gut leakage and bacterial translocation can be ameliorated by targeting microbes with antibiotics, suggesting that the microbiota contributes to disease. However, the work by Zhang and co-workers suggest that rather than targeting the microbes themselves, obesity-induced gut leakage and bacterial translocation can be normalized by silencing a host enzyme, which identifies host metabolism as an alternative therapeutic target.”
In addition to Hammock, Zhang, Yuxin and her husband Weicang, the other eight co-authors on the team are:
- Jun Yang, Sung Hee Hwang, and Debin Wan of the Hammock lab, UC Davis Department of Entomology and Nematology, and UC Davis Comprehensive Cancer Center
- Kin Sing Stephen Lee, formerly of the Hammock lab, and Maris Cinelli, both of the Department of Pharmacology and Toxicology, Michigan State University, Lansing
- Katherine Sanidad and Hang Xiao, Department of Food Science and the Molecular and Cellular Biology Graduate Program, University of Massachusetts, Amherst
- Daeyoung Kim, Department of Mathematics and Statistics, University of Massachusetts, Amherst
The abstract: “Intestinal barrier dysfunction, which leads to translocation of bacteria or toxic bacterial products from the gut into bloodstream and results in systemic inflammation, is a key pathogenic factor in many human diseases. However, the molecular mechanisms leading to intestinal barrier defects are not well understood, and there are currently no available therapeutic approaches to target intestinal barrier function. Here we show that soluble epoxide hydrolase (sEH) is an endogenous regulator of obesity-induced intestinal barrier dysfunction. We find that sEH is overexpressed in the colons of obese mice. In addition, pharmacologic inhibition or genetic ablation of sEH abolishes obesity-induced gut leakage, translocation of endotoxin lipopolysaccharide or bacteria, and bacterial invasion-induced adipose inflammation. Furthermore, systematic treatment with sEH-produced lipid metabolites, dihydroxyeicosatrienoic acids, induces bacterial translocation and colonic inflammation in mice. The actions of sEH are mediated by gut bacteria-dependent mechanisms, since inhibition or genetic ablation of sEH fails to attenuate obesity-induced gut leakage and adipose inflammation in mice lacking gut bacteria. Overall, these results support that sEH is a potential therapeutic target for obesity-induced intestinal barrier dysfunction, and that sEH inhibitors, which have been evaluated in human clinical trials targeting other human disorders, could be promising agents for prevention and/or treatment.”
The research was funded by grants from the National Institute of Food and Agriculture, U.S. Department of Food and Agriculture (USDA); National Cancer Institute; USDA Hatch Grant; National Institute of Environmental Health Sciences (NIEHS) Superfund Research Program; and a National Science Foundation.
According to the CDC, many of obesity-related conditions that lead to diseases are preventable. In 2008, the estimated annual medical cost of obesity in the United States tallied $147 billion. The medical cost for obese individuals averaged $1,429 higher than those of normal weight.
Contact: Bruce Hammock, firstname.lastname@example.org
The scenario begins when aggregations of beetle larvae of Meloe franciscanus emit chemical signals that mimic the sex pheromones of female bees luring male digger bees to make contact. The Meloe larvae then attach to males bees on contact, Habropoda pallida, from California's Mojave Desert and H. miserabilis from the coastal dunes of Oregon.
During subsequent copulations, the larvae transfer from males to females, hitching a ride on female bees to their nests, where the larvae feed on the provisions and the bee egg, and emerge as adults the following winter, said Saul-Gershenz. The research paper, “Deceptive Signals and Behaviors of a Cleptoparasitic Beetle Show Local Adaptation to Different Host bee Species,” appears in the current edition of Proceedings of the National Academy of Sciences (PNAS).
In solving the puzzle, the scientists tested whether geographically isolated populations of M. franciscanus larvae--from Oregon's coast and California's Mojave Desert--use local adaptations to exploit their respective hosts, H. miserabilis and H. pallida.
“Interestingly, male H. miserabilis were attracted to conspecific females and to aggregations of local Meloe larvae, but not to male bees,” Saul-Gershenz said. “Importantly, male bees of both bee species were more attracted to local parasite larvae than larvae from the distant locale because the larvae tailored their pheromone-mimicking blends to the pheromones of their local hosts. Additionally, the larval aggregation adapted their perching height at each location to the patrolling height of local male bees.”
In their abstract, the scientists wrote: "Chemosensory signals play a key role in species recognition and mate location in both invertebrate and vertebrate species. Closely related species often produce similar but distinct signals by varying the ratios or components in pheromone blends to avoid interference in their communication channels and minimize cross- attraction among congeners. However, exploitation of reproductive signals by predators and parasites also may provide strong selective pressure on signal phenotypes. For example, bolas spiders mimic the pheromones of several moth species to attract their prey, and parasitic blister beetle larvae, known as triungulins, cooperatively produce an olfactory signal that mimics the sex pheromone of their female host bees to attract male bees, as the first step in being transported by their hosts to their nests.”
“In both cases, there is strong selection pressure on the host to discriminate real mates from aggressive mimics and, conversely, on the predator, parasite, or parasitoid to track and locally adapt to the evolving signals of its hosts,” the co-authors pointed out. “Here we show local adaptation of a beetle, Meloe franciscanus (Coleoptera: Meloidae), to the pheromone chemistry and mate location behavior of its hosts, two species of solitary bees in the genus Habropoda. We report that M. franciscanus' deceptive signal is locally host-adapted in its chemical composition and ratio of components, with host bees from each allopatric population preferring the deceptive signals of their sympatric parasite population. Furthermore, in different locales, the triungulin aggregations have adapted their perching height to the height at which local male bees typically patrol for females. "
Saul-Gershenz said that the study “provides strong evidence for two different but complementary types of local adaptation in geographically isolated populations of a parasitic insect.” Specifically, the beetles locally adapt their deceptive chemical signals to the differing pheromone blends of their local host species and “the local nest parasites are significantly more attractive to male bees than nonlocal parasites, using transplant experiments.” The scientists identified the attractant blends for the two host species and the compounds that the beetle larvae produce to attract their hosts. They also showed that the two parasite populations have evolved divergent host-matching behaviors.
“The blister beetle Meloe franciscanus has turned out to be an engaging research subject, commented Saul-Gershenz, who received her doctorate in entomology from UC Davis, studying with Neal Williams and Steve Nadler, professor and chair of the UC Davis Department of Entomology and Nematology. She is now an associate director of research, Wild Energy Initiative, John Muir Institute of the Environment, UC Davis. “The larvae cooperate with their siblings for a brief period; they mimic the pheromone of their hosts; they are locally adapted to different hosts both chemically and behaviorally; and their emergence times are plastic across their geographic range. It has been fantastic to unravel this species' puzzle.”
She credited the counsel of the “true native bee icons in my field"--Robbin Thorp, UC Davis distinguished emeritus professor of entomology; research entomologist Jim Cane, Agricultural Research Service of U.S. Department of Agriculture; research entomologist Tom Zavortink, Bohart Museum of Entomology and former professor and chair of the University of Francisco Department of Biology; blister beetle (Meloidae) expert John Pinto, UC Riverside emeritus professor; and emeritus entomologist Rick Westcott, Oregon Department of Agriculture.
Future plans? Saul-Gershenz and Millar will continue exploring chemical communication signals as reproductive isolating mechanisms and the effect of eavesdropping parasites, parasitoids and predators on these signals. “I also plan to continue collaborating with Dr. Rebecca Hernandez and her lab members (UC Davis Department of Land, Air and Water Resources, and the Wild Energy Initiative of the John Muir Institute of the Environment) on the intersection of utility-scale solar energy development and our wildlife resources,” Saul-Gershenz said. In addition, she will continue her research on the impact on native bee diversity and pollination services from utility-scale solar development in the western deserts.
The research drew funding from Sean and Anne Duffey and Hugh and Geraldine Dingle Research Fellowship, the Community Foundation's Desert Legacy Fund, California Desert Research, Disney Wildlife Conservation Fund, and UC Davis Department of Entomology and Nematology fellowships.
A newly published study by a team of scientists at Chiba University, Japan and at the University of California, Davis shows that inhibiting an enzyme, the soluble epoxide hydrolase (sEH), plays a key role in curbing the inflammation associated with the development and progression of Parkinson's disease, an age-related brain disorder that affects a million Americans, mostly 60 and over.
The research, published today in the Proceedings of the National Academy of Sciences, is primarily the work of scientists in the labs of Kenji Hashimoto, a professor with the Division of Clinical Neuroscience, Chiba University Center for Forensic Mental Health, Chiba, Japan, and Bruce Hammock, UC Davis distinguished professor of entomology with a joint appointment in the UC Davis Comprehensive Cancer Center.
“Our research suggests that the sEH inhibitor may prevent the progression of Parkinson's disease (PD) as well as treat patients with dementia of Lewy bodies (DLB) if the sEH inhibitor is used in early phases of patients with these disorders,” said Hashimoto, whose career spans 30 years in the development of blood biomarkers and novel therapeutic drugs and includes more than 550 publications on the topic. “Both PD and DLB are chronic and progressive movement disorders. However, the precise causes of these diseases are largely unknown.”
Statistics indicate physicians diagnose 60,000 new cases of Parkinson's disease every year in the United States. The average age of onset is 60, and is more predominant among men.
Hammock said that the work by lead author Qian Ren and his colleagues in the Hashimoto lab “shows that markers and symptoms of Parkinson's disease in whole mice and in human cells with a mutation associated with Parkinson's disease can be treated with a small druglike molecule. By establishing this causal chain of events leading to Lewy body disorders we can better predict environmental chemicals that could predispose people to Parkinson's disease and possibly even treat the disease.”
The paper, titled “Soluble Epoxide Hydrolase Plays a Key Role in the Pathogenesis of Parkinson's Disease,” is co-authored by 14 scientists, including Professor Hammock and Jun Yang and Sung Hee Hwang, all part of the UC Davis Department of Entomology and Nematology and the UC Davis Comprehensive Cancer Center.
“Although there are many medications available to treat symptoms in PD, these do not prevent the progression of the disease, and, to date, no agent with a disease-modifying or neuroprotective indication for PD has been approved,” said Hashimoto. “Therefore, the development of new drugs possessing disease-modifying and /or neuroprotective properties is critical.”
In research studies involving mice, the scientists found “that sEH plays a key role in the inflammation associated with PD pathogenesis and the mechanisms that lead to the disease,” Hashimoto said. “The sEH inhibitor or deletion of the sEH gene protected against MPTP-induced neurotoxicity in mouse brain.” MPTP is an acronym for methyl-4-phenyl-1, 2, 3, 6 tetrahydropyridine, a relative of cyperquat and paraquat herbicides. “Our findings indicate that sEH inhibitors or epoxy fatty acids mimics may be promising prophylactic or therapeutic drugs for alpha-synuclein-related neurodegenerative disorders.”
Robert Higgins, emeritus professor of neuropathology at the UC Davis School of Veterinary Medicine, said: "I find it exciting that Ren and colleagues illustrate a promising path to a drug to prevent the progression of Parkinson's disease. It is impressive how far this work has come since we collaborated with Shirley Gee and the Hammock laboratory on developing a sheep model of Parkinson's disease in the early 1980s."
Neurosurgeon Cesar Borlongan of Morsani College of Medicine, University of South Florida, who was not involved in the study, praised the findings as advancing “our understanding of how Parkinson's disease evolves.” Describing Parkinson's disease as “a devastating brain disorder that mostly affects the aging population,” he said: “There is no cure, only relief from symptoms which include tremors, muscle rigidity, slurred speech, and freezing of gait.”
“While we know that a certain group of brain cells that produce dopamine are selectively destroyed in Parkinson's patients, what triggers this brain cell death remains poorly understood,” said Borlongan, a distinguished professor and vice chair for Research, Department of Neurosurgery and Brain Repair. “In their paper, the authors observed that a protein called soluble epoxide hydrolase (sEH) may be key to the demise of the brain dopamine cells. In small and large animal models of Parkinson's disease, and further confirmed in a group of PD patients, this protein is highly elevated in specific regions of the brain implicated in dopamine cell death.”
Borlongan pointed out that “Equally compelling evidence demonstrated that using a drug that inhibits sEH can reduce brain inflammation and levels of sEH and effectively lessen PD-associated toxicity in the animal models of the disease. Clinical trials of sEH inhibitors in heart and lung disease have been ongoing over the last decade, and may facilitate the entry of these drugs for PD. These results advance our understanding of how PD may evolve, but also point to its novel treatment.”
Qing Li, a professor in the Department of Molecular Biosciences and Bioengineering, College of Tropical Agriculture and Human Resources, University of Hawaii at Mānoa, who also was not involved in the study, called Parkinson's disease “a devastating neurodegenerative disorder that affects patients and caregivers alike with a significant economic burden in the United States and worldwide.”
This basic research drew support from several grants from Japan, including the Strategic Research Program for Brain Sciences, and at UC Davis, grants funded by the National Institute of Health's Institute of Environmental Health Sciences (NIH/NIEHS), and the NIEHS Superfund Program.
Hammock, a member of the National Academy of Sciences and the National Academy of Inventors, has directed the NIH/NIEH Superfund Program for more than 30 years.
Hammock said the soluble epoxide hydrolase inhibitors that inhibit the soluble epoxide hydrolase will soon enter human clinical trials supported by the NIH-NINDS Blueprint Program (NIH's Health's National Institute of Neurological Disorders and Stroke). “These drugs could provide relief for patients with a wide variety of inflammatory diseases,” he said.
The Hammock laboratory has published nearly 900 peer-reviewed papers on the sEH enzyme, discovered while Hammock and Sarjeet Gill (now of UC Riverside) were researching insect developmental biology and green insecticides at UC Berkeley. The work, begun in 1969, led to the discovery that many regulatory molecules are controlled as much by degradation as by biosynthesis, Hammock said. These epoxy fatty acid chemical mediators control blood pressure, fibrosis, immunity, tissue growth, and pain and inflammation.
To date, journals have published more than 17,000 peer-reviewed papers on the sEH enzyme and its inhibitors. Hammock credits the NIEHS for supporting his research in this area since the 1970s.
A Davis-based company, EicOsis, is developing inhibitors to sEH
to treat unmet medical needs in humans and animals. The company recently received a multi-million dollar grant from the NIH/NINDS Blueprint Program to move sEH inhibitors through phase I human clinical trials. “We are developing a non-opiate analgesic to treat the chronic pain often associated with diabetes,” said William Schmidt, vice president of clinical development at EicOsis. “Once we have investigational new drug status from the Food and Drug Administration and have finished our phase I trial, physicians will be able initiate their own trials with the EicOsis compound on Parkinson's disease and other Lewy body disorders.”
“We think that this research will lead to a very positive outcome to improve the lives of cystic fibrosis patients,” said co-author Bruce Hammock, distinguished professor in the UC Davis Department of Entomology and Nematology who holds a joint appointment with the UC Davis Comprehensive Cancer Center.
The research, published in the current edition of the Proceedings of the National Academy of Sciences, links a newly discovered class of bacterial enzymes to battling cystic fibrosis, a progressive, genetic disease characterized by persistent lung infections and inability to breathe normally.
Senior author Jennifer Bomberger of the Department of Microbiology and Molecular Genetics, University of Pittsburgh School of Medicine explained that the opportunistic bacterium, Pseudomonas aeruginosa, outcompetes other microorganisms in a cystic fibrosis patient's lungs and establishes a stronghold.
The scientific discovery could lead to new therapies that would interrupt or correct the bacterial sabotage, Hammock and Bomberger said.
“This paper is the outcome of an exciting and interdisciplinary project,” said Hammock, who directs the UC Davis Superfund Program financed by the National Institutes of Health's National Institute of Environmental Health Sciences (NIH-NIEHS).
“It started several years ago with the NIEHS Superfund Program funding both a group at Dartmouth and at UC Davis. A very productive and exciting collaboration resulted in looking at how to mitigate the effects of environmental chemicals on human health. Our collaborative work led to this joint publication which yields exciting hope for cystic fibrosis patients.”
Bomberger continues to work on the biology of the system while the Dartmouth and Davis groups have developed inhibitors of the action of CIF to stabilize pro-resolving mediators, reduce inflammation, and control periodic flare ups of bacterial infections.
"It will be key to devise a way to remove P. aeruginosa's ability to capitalize on the body's natural inflammatory response, without eliminating that response," said Bomberger. "Inflammation is happening for a reason—to clear infection. We just need it to temper the response when it is not effectively doing its job or is no longer needed."
Other co-authors of the paper include Hammock lab members Christophe Morisseau and Jun Yang, both from the UC Davis Department of Entomology and Nematology and UC Davis Comprehensive Cancer Center. .Institutions involved in the study also included the Harvard School of Medicine.