First-of-its-kind integrated dataset enables genes-to-ecosystems research

DOE national laboratory scientists led by Oak Ridge National Laboratory have developed the first tree dataset of its kind, bridging molecular information about the poplar tree microbiome to ecosystem-level processes.
Illustration Credit: Andy Sproles/ORNL, U.S. Dept. of Energy

The first-ever dataset bridging molecular information about the poplar tree microbiome to ecosystem-level processes has been released by a team of Department of Energy scientists led by Oak Ridge National Laboratory. The project aims to inform research regarding how natural systems function, their vulnerability to a changing climate, and ultimately how plants might be engineered for better performance as sources of bioenergy and natural carbon storage.

The data, described in Nature Publishing Group’s Scientific Data, provides in-depth information on 27 genetically distinct variants, or genotypes, of Populus trichocarpa, a poplar tree of interest as a bioenergy crop. The genotypes are among those that the ORNL-led Center for Bioenergy Innovation previously included in a genome-wide association study linking genetic variations to the trees’ physical traits. ORNL researchers collected leaf, soil and root samples from poplar fields in two regions of Oregon — one in a wetter area subject to flooding and the other drier and susceptible to drought. 

Details in the newly integrated dataset range from the trees’ genetic makeup and gene expression to the chemistry of the soil environment, analysis of the microbes that live on and around the trees and compounds the plants and microbes produce.

The dataset “is unprecedented in its size and scope,” said ORNL Corporate Fellow Mitchel Doktycz, section head for Bioimaging and Analytics and project co-lead. “It is of value in answering many different scientific questions.” By mining the data with machine learning and statistical approaches, scientists can better understand how the genetic makeup, physical traits and chemical diversity of Populus relate to processes such as cycling of soil nitrogen and carbon, he said. 

Autism and ADHD are linked to disturbed gut flora very early in life

The researchers have found links between the gut flora in babies first year of life and future diagnoses.
Photo Credit: Cheryl Holt

Disturbed gut flora during the first years of life is associated with diagnoses such as autism and ADHD later in life. This is according to a study led by researchers at the University of Florida and Linköping University and published in the journal Cell.

The study is the first forward-looking, or prospective, study to examine gut flora composition and a large variety of other factors in infants, in relation to the development of the children's nervous system. The researchers have found many biological markers that seem to be associated with future neurological development disorders, such as autism spectrum disorder, ADHD, communication disorder and intellectual disability.

“The remarkable aspect of the work is that these biomarkers are found at birth in cord blood or in the child’s stool at one year of age over a decade prior to the diagnosis,” says Eric W Triplett, professor at the Department of Microbiology and Cell Science at the University of Florida, USA, one of the researchers who led the study.

Discovery could end global amphibian pandemic

Panamanian golden frog
Photo Credit: Brian Gratwicke/U.S. Fish & Wildlife Service

A fungus devastating frogs and toads on nearly every continent may have an Achilles heel. Scientists have discovered a virus that infects the fungus, and that could be engineered to save the amphibians.

The fungus, Batrachochytrium dendrobatidis or Bd, ravages the skin of frogs and toads, and eventually causes heart failure. To date it has contributed to the decline of over 500 amphibian species, and 90 possible extinctions including yellow-legged mountain frogs in the Sierras and the Panamanian golden frog. 

A new paper in the journal Current Biology documents the discovery of a virus that infects Bd, and which could be engineered to control the fungal disease.

The UC Riverside researchers who found the virus are excited about the implications of their discovery. In addition to helping them learn about how fungal pathogens rise and spread, it offers the hope of ending what they call a global amphibian pandemic. 

“Frogs control bad insects, crop pests, and mosquitoes. If their populations all over the world collapse, it could be devastating,” said UCR microbiology doctoral student and paper author Mark Yacoub. 

“They’re also the canary in the coal mine of climate change. As temperatures get warmer, UV light gets stronger, and water quality gets worse, frogs respond to that. If they get wiped out, we lose an important environmental signal,” Yacoub said. 

Scientists link certain gut bacteria to lower heart disease risk

Rod-shaped Oscillibacter sp. bacteria take up fluorescently labeled cholesterol (here shown in green).
Image Credit: Ahmed Mohamed 

Changes in the gut microbiome have been implicated in a range of diseases including type 2 diabetes, obesity, and inflammatory bowel disease. Now, a team of researchers at the Broad Institute of MIT and Harvard along with Massachusetts General Hospital has found that microbes in the gut may affect cardiovascular disease as well. In a study published in Cell, the team has identified specific species of bacteria that consume cholesterol in the gut and may help lower cholesterol and heart disease risk in people.

Members of Ramnik Xavier’s lab, Broad’s Metabolomics Platform, and collaborators analyzed metabolites and microbial genomes from more than 1,400 participants in the Framingham Heart Study, a decades-long project focused on risk factors for cardiovascular disease. The team discovered that bacteria called Oscillibacter take up and metabolize cholesterol from their surroundings, and that people carrying higher levels of the microbe in their gut had lower levels of cholesterol. They also identified the mechanism the bacteria likely use to break down cholesterol. The results suggest that interventions that manipulate the microbiome in specific ways could one day help decrease cholesterol in people. The findings also lay the groundwork for more targeted investigations of how changes to the microbiome affect health and disease.

“Our research integrates findings from human subjects with experimental validation to ensure we achieve actionable mechanistic insight that will serve as starting points to improve cardiovascular health,” said Xavier, who is a core institute member, director of the Immunology Program, and co-director of the Infectious Disease and Microbiome Program at the Broad. He is also a professor at Harvard Medical School and Massachusetts General Hospital.

Rice study identifies protein responsible for gas vesicle clustering in bacteria

Zongru Li (left) and George Lu
Photo Credit: Anna Stafford/Rice University

Gas vesicles are hollow structures made of protein found in the cells of certain microorganisms, and researchers at Rice University believe they can be programmed for use in biomedical applications.

“Inside cells, gas vesicles are packed in a beautiful honeycomb pattern. How this pattern is formed has never been thoroughly understood. We are presenting the first identification of a protein that can regulate this patterning, and we believe this will be a milestone in molecular microbiology,” said George Lu, assistant professor of bioengineering and a Cancer Prevention and Research Institute of Texas scholar.

Lu and colleagues have published their findings in a paper published in Nature Microbiology. The lead author is Zongru Li, a fourth-year bioengineering doctoral student in Lu’s Laboratory for Synthetic Macromolecular Assemblies.

“Gas vesicles are cylindrical tubes closed by conical end caps,” Li said. “They provide buoyancy within the cells of their native hosts.”

Researchers Identify Microbes That Help Plants Thwart Parasite

Sorghum crops in sub-Saharan Africa suffer heavy losses from the parasitic plant witchweed (Striga hermonthica). A new study shows how soil microbes can help protect sorghum from this pest and could be the basis for a soil probiotic treatment.
Photo Credit: Sabine

Bacteria that could help one of Africa’s staple crops resist a major pest have been identified by researchers at the University of California, Davis. Their findings, published in Cell Reports, could improve yields of sorghum, a mainstay of food and drink in West and East African countries.

About 20 percent of Africa’s sorghum crop is lost due to witchweed (Striga hermonthica), a parasitic plant that steals nutrients and water by latching onto the plant’s roots.

In a new study, UC Davis researchers show that soil microbes induce changes in sorghum roots that make the plant more resistant to infection by witchweed. They identified specific strains of bacteria that trigger these resistance traits and could be applied as a soil “probiotic” to improve sorghum yields in future.

“These microbes have great promise as soil additives that can help farmers grow sorghum successfully in sub-Saharan Africa,” said Siobhan Brady, a professor in the Department of Plant Biology and Genome Center and a senior author on the paper. 

Researchers a step closer to a cure for HIV

HIV, the AIDS virus (yellow), infecting a human cell
Image Credit: National Cancer Institute

A new study involving University of Bristol researchers has shown a virus-like particle (HLP) can effectively 'shock and kill' the latent HIV reservoir.

By 2030, the World Health Organization (WHO), the Global Fund and UNAIDS are hoping to end the human immunodeficiency virus (HIV) and AIDS epidemic. An international team of researchers led by Professor Eric Arts from the Schulich School of Medicine & Dentistry, Canada, and Dr Jamie Mann, Senior Lecturer at the University of Bristol, has brought us another step closer to meeting this goal, by finding an effective and affordable targeted treatment strategy for an HIV cure. 

In a first, the study published in Emerging Microbes and Infections demonstrated the team's patented therapeutic candidate. The HIV-virus-like-particle (HLP), is 100 times more effective than other candidate HIV cure therapeutics for people living with chronic HIV on combined antiretroviral therapy (cART). If successful in clinical trials, HLP could be used by millions of people living around the world to free them of HIV. This study was done using blood samples from people living with chronic HIV. 

HLPs are dead HIV particles hosting a comprehensive set of HIV proteins that increase immune responses without infecting a person. When compared with other potential cure approaches, HLP is an affordable biotherapeutic and can be administered by intramuscular injection – similar to the seasonal flu vaccine. 

Study explores severe hurricanes and coral reef sponge recolonization

For the study, scuba divers collected small samples of the thin purple morphotype sponges 14 and 22 months after the two Category 5 hurricanes in St. Thomas.
Photo Credit: Karli Hollister

Named for its ropy-looking long branches, Aplysina cauliformis, a coral reef sponge, provides a critical 3D habitat for marine organisms and helps to stabilize the foundation of coral reefs. However, these upright branching sponges are highly susceptible to breaking during storms, which increases sponge fragmentation and contributes to population clonality and inbreeding.

Many sponges can survive severe damage and undergo frequent fragmentation, which is considered a mechanism for asexual reproduction. While fragmentation is a commonly utilized reproductive strategy in rope sponges, they also can reproduce sexually by producing larvae. How and whether they recolonize following extreme weather events is critical for the restoration and resilience of coral reef ecosystems.

Hurricanes Irma and Maria – both in 2017 – were two rapid succession storms that provided researchers from Florida Atlantic University’s Harriet L. Wilkes Honors College and Harbor Branch Oceanographic Institute, and collaborators from the University of the Virgin Islands, the University of Mississippi and the University of Alabama, with a unique opportunity to address a priority concern – the resilience of coral reef sponge populations after severe hurricanes. 

Gut microbiota and antibiotics: Missing puzzle piece discovered

3D model of the small ribonucleic acid MasB.
Image Credit: Alexander Westermann/HIRI

HIRI scientists have identified a small RNA that influences the sensitivity of the intestinal commensal Bacteroides thetaiotaomicron to certain antibiotics.

The intricacies of how intestinal bacteria adapt to their environment have yet to be fully explored. Researchers from the Helmholtz Institute for RNA-based Infection Research (HIRI) in Würzburg and the University of California, Berkeley, USA, have now successfully closed a gap in this knowledge: They have identified a small ribonucleic acid (sRNA) that affects the susceptibility of the gut commensal Bacteroides thetaiotaomicron to specific antibiotics. The findings, published today in the journal Nature Microbiology, could serve as the foundation for novel therapies addressing intestinal diseases and combating antibiotic resistance. 

The gut, a complex ecosystem of numerous microorganisms, plays a pivotal role in human well-being. Factors like dietary changes, medications, or bile salts can influence the microbiota, impacting health. Among the prevalent intestinal bacteria in humans are Bacteroides thetaiotaomicron. These gut microbes play a role in breaking down polysaccharides during digestion, contributing to human health. Yet they can also promote infections when the ecosystem is disbalanced, such as after antibiotic treatment. However, the molecular mechanisms enabling these gut microbes to adapt to their environment remain largely unknown.

New vaccine against a highly fatal tropical disease – and potential bioterror weapon – demonstrates efficacy in animal studies

Burkholderia pseudomallei infecting a human cell. The bacteria (red) are polymerizing actin (green).
 Photo Credit: Courtesy of Christopher T. French.

In a mouse study, UCLA researchers tested a vaccine against the bacterium that causes melioidosis and found it was highly protective against the disease, which is endemic in many tropical areas, causing approximately 165,000 cases with 89,000 fatalities around the world each year. 

The bacterium, called Burkholderia pseudomallei, is spread through contact with contaminated soil and water through inhalation, ingestion or broken skin. It is so dangerous that it is categorized as a Tier 1 Select Agent of bioterrorism, and it can cause rapidly fatal pneumonia when inhaled in low doses. If aerosolized and unleashed in a terror attack, it could lead to widespread death.

To date there are no licensed vaccines against the bacterium, said senior author Dr. Marcus Horwitz, Distinguished Professor of Medicine, in the division of infectious diseases, and of Microbiology, Immunology and Molecular Genetics at the David Geffen School of Medicine at UCLA.

“A safe and effective vaccine is needed to prevent this disease as melioidosis is often difficult to diagnose, requires very lengthy treatment lasting three to six months, and has a high fatality rate even in high resource settings,” Horwitz said. “Such a vaccine would be of great benefit to people living in endemic regions, travelers, and military personnel stationed in these areas, and it would also reduce the risk from an intentional release of B. pseudomallei in a bioterrorist attack.” 

Study reveals how pH affects the ability of ulcer bacteria to attach

Anna Åberg and Anna Arnqvist Björklund.
Photo Credit: Mattias Pettersson

A study by Anna Arnqvist's research group at Umeå University reveals molecular details about the gastric pathogen Helicobacter pylori's ability to bind to an inflamed stomach and how this is controlled by the stomach's pH. Increased understanding of how H. pylori bacteria can cause a persistent lifelong infection is an important piece of the puzzle in order to ultimately identify the characteristics that contribute to disease.

When the stomach becomes infected with the gastric pathogen Helicobacter pylori, the infection lasts for life if it is left untreated. The infection can cause peptic ulcer disease as well as stomach cancer. The environment within the stomach undergoes continuous changes, requiring the bacteria to adapt by adjusting the expression of certain proteins based on the prevailing conditions.

It is commonly assumed that the stomach has a low pH. However, the pH levels vary significantly, ranging from the highly acidic environment in the stomach lumen to largely neutral conditions at the outermost layer of the stomach epithelial cells, which is protected by a mucus layer. It is in the mucus layer or tightly attached to the outermost cell layer that most H. pylori bacteria are found. The expression of many genes is regulated in response to pH, causing the bacterium to produce varying amounts of proteins depending on the pH of its surroundings.

Alzheimer’s Drug Fermented with Help from AI and Bacteria Moves Closer to Reality

Photo-Illustration Credit: Martha Morales/The University of Texas at Austin

Galantamine is a common medication used by people with Alzheimer’s disease and other forms of dementia around the world to treat their symptoms. Unfortunately, synthesizing the active compounds in a lab at the scale needed isn’t commercially viable. The active ingredient is extracted from daffodils through a time-consuming process, and unpredictable factors, such as weather and crop yields, can affect supply and price of the drug. 

Now, researchers at The University of Texas at Austin have developed tools — including an artificial intelligence system and glowing biosensors — to harness microbes one day to do all the work instead. 

In a paper in Nature Communications, researchers outline a process using genetically modified bacteria to create a chemical precursor of galantamine as a byproduct of the microbe’s normal cellular metabolism.  Essentially, the bacteria are programmed to convert food into medicinal compounds.

“The goal is to eventually ferment medicines like this in large quantities,” said Andrew Ellington, a professor of molecular biosciences and author of the study. “This method creates a reliable supply that is much less expensive to produce. It doesn’t have a growing season, and it can’t be impacted by drought or floods.” 

Researchers discover a coral superhighway in the Indian Ocean

A coral reef in the Seychelles.
Photo Credit: Christophe Mason-Parker

Despite being scattered across more than a million square kilometers, new research has revealed that remote coral reefs across the Seychelles are closely related. Using genetic analyses and oceanographic modelling, researchers at Oxford University demonstrated for the first time that a network of ocean currents scatter significant numbers of larvae between these distant islands, acting as a ‘coral superhighway.’ These results have been published today in Nature Scientific Reports.

"This study couldn’t come at a timelier moment. The world is once again watching, as El Niño devastates coral reefs throughout the Indian Ocean. Now we know which reefs will be crucial to coral recovery, but we can’t pause in our commitment to reducing greenhouse gas emissions and stopping climate change."
Senior author of the study, Professor Lindsay Turnbull 
Department of Biology, University of Oxford

Dr April Burt (Department of Biology, University of Oxford, and Seychelles Islands Foundation), lead author of the study, said: ‘This discovery is very important because a key factor in coral reef recovery is larval supply. Although corals have declined alarmingly across the world due to climate change and a number of other factors, actions can be taken at local and national scale to improve reef health and resilience. These actions can be more effective when we better understand the connectivity between coral reefs by, for instance, prioritizing conservation efforts around coral reefs that act as major larval sources to support regional reef resilience.’

“Molecular Rosetta Stone” Reveals How our Microbiome Talks to Us

Bacteria in the gut convert bile acids produced by the liver into a wide array of new compounds. These molecules are akin to the language of the gut microbiome, allowing them to influence distant organ systems.
Photo Credit: Lakshmiraman Oza

Researchers from Skaggs School of Pharmacy and Pharmaceutical Sciences at the University of California San Diego have uncovered thousands of previously unknown bile acids, a type of molecule used by our gut microbiome to communicate with the rest of the body.

“Bile acids are a key component of the language of the gut microbiome, and finding this many new types radically expands our vocabulary for understanding what our gut microbes do and how they do it,” said senior author Pieter Dorrestein, Ph.D., professor at Skaggs School of Pharmacy and Pharmaceutical Sciences and professor of pharmacology and pediatrics at UC San Diego School of Medicine. “It’s like going from ‘See Spot Run’ to Shakespeare.”

The results, as described by study co-author and bile acids expert Lee Hagey, Ph.D, are akin to a molecular Rosetta stone, providing previously unknown insight into the biochemical language microbes use to influence distant organ systems.

Possible ‘Trojan Horse’ found for treating stubborn bacterial infections

Transmission electron microscope (TEM) image of the bacterial cell with an extracellular vesicle attached.
Image Credit: Courtesy of Washington State University

Bacteria can be tricked into sending death signals to stop the growth of their slimy, protective homes that lead to deadly infections, a new study demonstrates.

The discovery by Washington State University researchers could someday be harnessed as an alternative to antibiotics for treating difficult infections. Reporting in the journal Biofilm, the researchers used the messengers, which they named death extracellular vesicles (D-EVs), to reduce growth of the bacterial communities by up to 99.99% in laboratory experiments.

“Adding the death extracellular vesicles to the bacterial environment, we are kind of cheating the bacteria cells,” said Mawra Gamal Saad, first author on the paper and a graduate student in WSU’s Gene and Linda Voiland School of Chemical Engineering and Bioengineering. “The cells don’t know which type of EVs they are, but they take them up because they are used to taking them from their environment, and with that, the physiological signals inside the cells change from growth to death.”

Scientists assemble a richer picture of the plight and resilience of the foothill yellow-legged frog

Foothill yellow-legged frogs live in the flowing water of rivers and streams, so are especially vulnerable when these shrink to isolated pools.
Photo Credit: Brome McCreary / USGS

Up to only a few inches in length, with a lemon-hued belly, the foothill yellow-legged frog may seem unassuming. But its range once stretched from central Oregon to Baja California. In 2023, it was listed under the federal Endangered Species Act. Its rapidly decreasing range is due in part to a fungal pathogen called Batrachochytrium dendrobatidis, or Bd, that has devastated amphibians around the world.

A team of researchers, including UC Santa Barbara’s Andrea Adams, has conducted the most comprehensive study to date of disease dynamics in foothill yellow-legged frogs. The team’s data — sourced from both wild frogs and specimens in museum collections — enabled them to track patterns of infection across a large geographic range. In a study published in Royal Society Open Science, the researchers reveal that drought, rising temperatures and the increasing conversion of land for agriculture appear to be the largest factors driving Bd infection in this species.

The researchers aimed to assemble as much data as they could, both in space and time. They surveyed in the creeks and rivers of California and Oregon, where they swabbed wild yellow-legged frogs for the presence of Bd. It also led them into fluorescent-lit museum collections to sample specimens from as far back as the 1890s.

Research reveals new insights into marine plastic pollution

Photo Credit: Lucien Wanda

A groundbreaking study led by researchers at the University of Stirling has uncovered the crucial role of bacteria living on plastic debris.

The research also identifies rare and understudied bacteria that could assist in plastic biodegradation, offering new insights for tackling plastic pollution.

Plastic pollution is a worldwide problem, with up to two million tons estimated to enter oceans every year, damaging wildlife and ecosystems.

In a pioneering study, experts at the University of Stirling’s Faculty of Natural Sciences and the University of Mons (Belgium) analyzed the proteins in plastic samples taken from Gullane Beach in Scotland.

Unlike previous studies carried out in warmer climates that focus on the genetic potential of biofilms inhabiting plastics, this research led by Dr Sabine Matallana-Surget took a unique approach by analyzing the proteins expressed by active microorganisms.

Their findings have unveiled a remarkable discovery of enzymes actively engaged in degrading plastic. Moreover, the team has pioneered new methodologies for enhanced predictions in marine microbiology research.

Mice study suggests metabolic diseases may be driven by gut microbiome, loss of ovarian hormones

Mice that received fecal implants from donors that had their ovaries removed gained more fat mass and had greater expression of liver genes associated with inflammation, Type 2 diabetes, fatty liver disease and atherosclerosis. The findings may shed light on the greater incidence of metabolic dysfunction in postmenopausal women. The team members included, from left: molecular and integrative physiology professor Erik R. Nelson; Kelly Swanson, the director of the Division of Nutritional Sciences and the Kraft Heinz Endowed Professor in Human Nutrition; and animal sciences professor Brett R. Loman.
  Photo Credit: Fred Zwicky

The gut microbiome interacts with the loss of female sex hormones to exacerbate metabolic disease, including weight gain, fat in the liver and the expression of genes linked with inflammation, researchers found in a new rodent study.

The findings, published in the journal Gut Microbes, may shed light on why women are at significantly greater risk of metabolic diseases such as obesity and Type 2 diabetes after menopause, when ovarian production of female sex hormones diminishes.

“Collectively, the findings demonstrate that removal of the ovaries and female hormones led to increased permeability and inflammation of the gut and metabolic organs, and the high-fat diet exacerbated these conditions,” said Kelly S. Swanson, the director of the Division of Nutritional Sciences and the Kraft Heinz Endowed Professor in Human Nutrition at the University of Illinois Urbana-Champaign who is a corresponding author of the paper.  “The results indicated that the gut microbiome responds to changes in female hormones and worsens metabolic dysfunction.”

Discovery about bacterial cell walls can lead to new antibiotics

Felipe Cava is Professor of Infection Biology, Department of Molecular Biology, Umeå University and affiliated group leader with the Laboratory for Molecular Infection Medicine Sweden (MIMS), Umeå Centre for Microbial Research (UCMR) and the Integrated Science Lab (Icelab) and SciLifeLab.
Photo Credit: Mattias Pettersson, simon ohman jonsson inhousebyran

Researchers at Umeå University in Sweden, led by Professor Felipe Cava, have identified a new family of enzymes that creates a unique type of cross-linking between the building blocks of bacterial cell walls. This discovery could help develop new antibiotics against infectious diseases.

Bacterial cell walls form mesh-like structures, shielding cells from rupturing under high internal pressure and safeguarding against external threats. The cell wall is comprised of sugar and amino acid molecules interconnected by various types of cross-links. These cross-links play a crucial role in providing strength and stability to the cell wall, while also enabling bacteria to adapt to diverse environments and stressors.

In a groundbreaking study recently published in the esteemed journal Nature Communications, researchers from Umeå University and international institutions have unveiled a novel family of enzymes responsible for generating a unique cross-linkage between L-alanine and meso-diaminopimelic acid. These amino acids are integral components of the peptide chains constituting the cell wall of numerous bacterial species. Termed LD1,3-transpeptidase, this enzyme has been identified across various groups of alpha and beta proteobacteria, including opportunistic pathogens such as Burkholderia and Achromobacter.

Scientists are unraveling the secrets of red and grey squirrel competition

Image Credit: Gemini Advance AI

In a first of its kind study, researchers have identified significant differences between the diversity of gut bacteria in grey squirrels compared to red squirrels which could hold the key to further understanding the ability of grey squirrels to outcompete red squirrels in the UK. 

New research, published in the Journal of Medical Microbiology, looked to understand more about the mechanisms by which grey squirrels are able to gain an advantage over red squirrels. Chris Nichols, Conservation Evidence Manager at the Woodland Trust, and co-author of the study, said:

“The more we know about grey squirrels, the more equipped we’re going to be in the future to tackle the threats they pose to red squirrels and our native trees, which is one of the biggest problems for forest conservation in the UK.”

Grey squirrels are an invasive, non-native species introduced from North America to sites in Great Britain and Ireland between 1876 to 1929. They out-compete the native red squirrels and multiply thanks to various traits including their ability to access a broader range of food sources including the bark of UK broadleaved trees. This causes significant damage to the trees, and is a behavior that, prior to this research, was not completely understood.