Seed germination is controlled by several plant hormones, which are researched intensely. However, not much was known about the processes that need to take place to allow the hormones to function. How is energy in the seed made available? How can energy metabolism be started early and efficiently? An international team of researchers has now been looking into these questions.

Using a new type of fluorescent biosensors, the researchers observed, in living seed cells, both energy metabolism and the so-called redox metabolism, which relies in sulphur. The researchers discovered that when the seeds came into contact with water, energy metabolism was established in a matter of minutes, and the plant cells’ “power stations” — known as mitochondria — activated their respiration. The researchers also found out which molecular switches are activated to enable energy to be released efficiently — with the so-called thiol-redox switches playing a central role.

“By looking into the very early processes of germination control, we can gain a better understanding of the mechanisms driving seed germination,” says Prof. Markus Schwarzländer from the University of Münster (Germany), who led the study. “In future we could think about how such switches could be used in crop biotechnology.” The results of the study could be of relevance in farming, when seeds need to keep their germination vigour for as long as possible on the one hand, but should also germinate in synch and with minimal losses on the other hand. The study has been published in the journal PNAS (Proceedings of the National Academy of Sciences).

Background and method

In order to be able to observe the activities taking place in the energy metabolism, the researchers visualized under the microscope adenosine triphosphate (ATP), the general currency for energy in the cell, and Nicotinamide adenine dinucleotide phosphate (NADPH), the electron energy, in the mitochondria. They compared seeds from thale cress: both dry seeds and seeds “imbibed” with water.

To find out whether the redox switches are important for kick-starting germination, the researchers deactivated specific proteins using genetic methods and then compared the reaction shown by the modified seeds with that of the unmodified ones. The researchers allowed the seeds to age artificially in the laboratory, and they saw that the seeds germinated much less actively if they lacked the relevant proteins.

The researchers’ next step involved so-called redox proteome analysis, i.e. they examined the relevant redox proteins in their entirety with the use of biochemical methods. For this purpose, they isolated active mitochondria and flash-froze them in order to be able to study this state directly where the process was taking place. The researchers then used mass spectrometry methods to identify several so-called cysteine-peptides which are important for resource efficiency in energy metabolism.

“The process could be likened to the traffic control system of a large city. Before the rush hour — i.e. germination — starts, which puts large quantities of metabolites ‘on the road’, the traffic light and routing systems need to be switched on in the morning; and here this is done by the thiol redox switches,” explains lead author Dr. Thomas Nietzel, who carried out most of the experiments as part of his PhD at the Institute of Crop Science and Resource Conservation at the University of Bonn and later as a postdoctoral researcher at the Institute of Biology and Biotechnology of Plants at the University of Münster.

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One of the most consequential developments in the history of biological evolution occurred approximately 2 billion years ago with the appearance of the first eukaryotes — unicellular organisms that contain a distinct nucleus. This first eukaryotic lineage would subsequently give rise to all higher organisms including plants and animals, but its origins remain obscure. Some years ago, microbiologists analyzed DNA sequences from marine sediments, which shed new light on the problem. These sediments were recovered from a hydrothermal vent at a site known as Loki’s Castle (named for the Norse god of fire) on the Mid-Atlantic Ridge in the Arctic Ocean. Sequencing of the DNA molecules they contained revealed that they were derived from a previously unknown group of microorganisms.

Although the cells from which the DNA originated could not be isolated and characterized directly, the sequence data showed them to be closely related to the Archaea. The researchers therefore named the new group Lokiarchaeota.

Archaea, together with the phylum Bacteria, are the oldest known lineages of single-celled organisms. Strikingly, the genomes of the Lokiarchaeota indicated that they might exhibit structural and biochemical features that are otherwise specific to eukaryotes. This suggests that the Lokiarchaeota might be related to the last common ancestor of eukaryotes. Indeed, phylogenomic analysis of the Lokiarchaeota DNA from Loki’s Castle strongly suggested that they were derived from descendants of one of the last common ancestors of Eukaryota and Archaea. Professor William Orsi of the Department of Earth and Environmental Sciences at LMU, in cooperation with scientists at Oldenburg University and the Max Planck Institute for Marine Microbiology, has now been able to examine the activity and metabolism of the Lokiarchaeota directly. The results support the suggested relationship between Lokiarchaeota and eukaryotes, and provide hints as to the nature of the environment in which the first eukaryotes evolved. The new findings appear in the journal Nature Microbiology.

The most likely scenario for the emergence of eukaryotes is that they arose from a symbiosis in which the host was an archaeal cell and the symbiont was a bacterium. According to this theory, the bacterial symbiont subsequently gave rise to the mitochondria — the intracellular organelles that are responsible for energy production in eukaryotic cells. One hypothesis proposes that the archaeal host was dependent on hydrogen for its metabolism, and that the precursor of the mitochondria produced it. This “hydrogen hypothesis” posits that the two partner cells presumably lived in an anoxic environment that was rich in hydrogen, and if they were separated from the hydrogen source they would have become more dependent on one another for survival potentially leading to an endosymbiotic event. “If the Lokiarchaeota, as the descendants of this putative ur-archaeon, are also dependent on hydrogen, this would support the hydrogen hypothesis,” says Orsi. “However, up to now, the ecology of these Archaea in their natural habitat was a matter of speculation.”

Orsi and his team have now, for the first time, characterized the cellular metabolism of Lokiarchaeota recovered from sediment cores obtained from the seabottom in an extensive oxygen-depleted region off the coast of Namibia. They did so by analyzing the RNA present in these samples. RNA molecules are copied from the genomic DNA, and serve as blueprints for the synthesis of proteins. Their sequences therefore reflect patterns and levels of gene activity. The sequence analyses revealed that Lokiarchaeota in these samples outnumbered bacteria by 100- to 1000-fold. “That strongly indicates that these sediments are a favorable habitat for them, promoting their activity,” says Orsi.

He and his colleagues were able to establish enrichment cultures from the Lokiarchaeota in the sediment samples in the laboratory. This enabled them to study the metabolism of these cells using stable carbon isotopes as markers. The results demonstrated that the microorganisms make use of a complex network of metabolic pathways. Moreover, the data confirmed that Lokiarchaea indeed use hydrogen for the fixation of carbon dioxide. This process enhances the efficiency of metabolism, and allows these species to maintain high levels of biochemical activity, in spite of the energy limited conditions of their anoxic natural habitat. “Our experimental evidence the hydrogen hypothesis for the first eukaryotic cell,” says Orsi. “Consequently, the earliest eukaryotes could have originated in oxygen-depleted and hydrogen-rich marine sediments, such as those in which modern Lokiarchaeota are particularly active today.”

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The findings are published in the December 23, 2019 online issue of the journal Nature Medicine. Martin Marsala, MD, professor in the Department of Anesthesiology at UC San Diego School of Medicine and a member of the Sanford Consortium for Regenerative Medicine, is senior author of the study.

ALS is a neurodegenerative disease that affects nerve cells in the brain and spinal cord. Motor neurons responsible for communicating movement are specifically harmed, with subsequent, progressive loss of muscle control affecting the ability to speak, eat, move and breathe. More than 5,000 Americans are diagnosed with ALS each year, with an estimated 30,000 persons currently living with the disease. While there are symptomatic treatments for ALS, there is currently no cure. The majority of patients succumb to the disease two to five years after diagnosis.

There are two types of ALS, sporadic and familial. Sporadic is the most common form, accounting for 90 to 95 percent of all cases. It may affect anyone. Familial ALS accounts for 5 to 10 percent of all cases in the United States, and is inherited. Previous studies show that at least 200 mutations of a gene called SOD1 are linked to ALS.

The SOD1 gene normally serves to provide instructions for making an enzyme called superoxide dismutase, which is widely used to break down superoxide radicals — toxic oxygen molecules produced as a byproduct of normal cell processes. Previous research has suggested that SOD1 gene mutations may result in ineffective removal of superoxide radicals or create other toxicities that cause motor neuron cell death, resulting in ALS.

The new approach involves injecting shRNA — an artificial RNA molecule capable of silencing or turning off a targeted gene — that is delivered to cells via a harmless adeno-associated virus. In the new research, single injections of the shRNA-carrying virus were placed at two sites in the spinal cord of adult mice expressing an ALS-causing mutation of the SOD1 gene, either just before disease onset or when the animals had begun showing symptoms.

Earlier efforts elsewhere had involved introducing the silencing vector intravenously or into cerebrospinal fluid in early symptomatic mice, but disease progression, while delayed, continued and the mice soon died. In the new study, the single subpial injection (delivered below the pia matter, the delicate innermost membrane enveloping the brain and spinal cord) markedly mitigated neurodegeneration in pre-symptomatic mice, which displayed normal neurological function with no detectable disease onset. The functional effect corresponded with near-complete protection of motor neurons and other cells, including the junctions between neurons and muscle fibers.

In adult mice already displaying ALS-like symptoms, the injection effectively blocked further disease progression and degeneration of motor neurons.

In both approaches, the affected mice lived without negative side effects for the length of the study.

“At present, this therapeutic approach provides the most potent therapy ever demonstrated in mouse models of mutated SOD1 gene-linked ALS,” said senior author Martin Marsala, MD, professor in the Department of Anesthesiology at UC San Diego School of Medicine.

“In addition, effective spinal cord delivery of AAV9 vector in adult animals suggests that the use of this new delivery method will likely be effective in treatment of other hereditary forms of ALS or other spinal neurodegenerative disorders that require spinal parenchymal delivery of therapeutic gene(s) or mutated-gene silencing machinery, such as in C9orf72 gene mutation-linked ALS or in some forms of lysosomal storage disease.”

The research team also tested the injection approach in adult pigs, whose spinal cord dimensions are similar to humans, for safety and efficacy. Using an injection device developed for use in adult humans, they found the procedure could be performed reliably and without surgical complications.

Marsala said next steps involve additional safety studies with a large animal model to determine the optimal, safe dosage of treatment vector. “While no detectable side effects related to treatment were seen in mice more than one year after treatment, the definition of safety in large animal species more similar to humans is a critical step in advancing this treatment approach toward clinical testing.”

Now, researchers have detailed a comprehensive interaction network map of how malaria traffics between human host cells. They published their results on Sept. 27 in iScience, a Cell Press journal.

The researchers focused on Plasmodium falciparum, the parasite that causes the most severe form of malaria. This parasite infects a host red blood cell, triggering the production of several proteins into the host cell’s cytoplasm — the bulk of the cell’s mechanics and the liquid in which they’re held, ultimately transforming the cell’s physical form. Not only does this transformation make the cells stick in place, out of the body’s immune response, it also helps the parasite travel to the surface of the cell and infect others. Together, the proteins work to proliferate the parasite, leading to the propagation of the malaria parasite.

“Our study sheds light on the highly complicated interplay between parasite and host proteins in the host cytoplasm,” said Kentaro Kato, professor in the Laboratory of Sustainable Animal Environment in Tohoku University Graduate School of Agricultural Science and paper authors. “The work provides a reliable dataset of the interactions connecting dozens of proteins the parasite exports to continue infecting the host cells.”

Previously, it was difficult to understand how the parasite works with the triggered proteins because the parasite was predicted to export about 400 proteins, yet another study found that proteins without the specific genetic sequence could also be exported to the cell’s cytoplasm. In this study, the researchers opted to focus on one of these proteins without the parasitic mark — skeleton-binding protein 1 (SBP1), which is known to be highly important for malaria to propagate. By studying a protein known to be related to malaria virulence, but that isn’t specifically triggered by the parasitic proteins, the researchers could narrow in on specific protein interactions to understand how the infection traffics within and beyond the host cells.

They used highly sensitive mass spectrometry to image the proteins interacting with SBP1 throughout the proliferation process, leading to the identification of several proteins specifically connected to transforming the host cell.

“In this study, we developed an alternative approach to identify exported proteins involved in the trafficking complex and in the parasite protein exports,” Kato said. “The SBP1 interactions established in our study represent a powerful and invaluable platform to identify exported proteins related to severe malaria caused by Plasmodium falciparum.”

The research provided a comprehensive map of SBP1 interactions which shed light on the complex relationships and interplay between host and parasite proteins. The findings also pave the way for further study and discussion on the molecular mechanism of the infections that affect human red blood cells.

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Glioblastoma multiforme (GBM) remains the most difficult of all brain cancers to study and treat, largely because of tumor heterogeneity. Treatment approaches, like surgery, radiation and chemotherapy, along with newer personalized cellular therapies, have proven to slow tumor growth and keep patients disease-free for some periods of time; however, a cure remains elusive.

“While we’ve made important strides in glioblastoma research, preclinical and clinical challenges persist, keeping us from getting closer to more effective treatments,” said senior author Hongjun Song, PhD, Perelman Professor of Neuroscience in the Perelman School of Medicine at the University of Pennsylvania. “One hurdle is the ability to recapitulate the tumor to not only better understand its complex characteristics, but also to determine what therapies post-surgery can fight it in a timelier manner.”

Co-senior authors include Guo-li Ming, MD, PhD, Perelman Professor of Neuroscience, and Donald O’Rourke, MD, the John Templeton, Jr., MD Professor in Neurosurgery, and director of the GBM Translational Center of Excellence at Penn’s Abramson Cancer Center.

Lab-grown brain organoids — derived from human pluripotent stem cells or patient tissues and grown to a size no bigger than a pea — can recapitulate important genetic composition, brain cell type heterogeneity, and architecture, for example. These models are allowing researchers to recreate key features of patients’ diseased brains to help paint a clearer picture of their cancer, and allowing them to explore ways to best attack it.

What makes organoids so attractive in GBM is timing and the ability to maintain cell type and genetic heterogeneity. While existing in vitro models have added to researchers’ understanding of the biological mechanisms underlying the cancer, they have limitations. Unlike other models, which need more time to exhibit gene expression and other histological features that more closely represent the tumor, brain tumor organoids developed by the research group grow into use much more rapidly. That’s important because current treatment regimens are typically initiated one month following surgery, so having a road map sooner is more advantageous.

In the new study, the researchers removed fresh tumor specimens from 52 patients to “grow” corresponding tumor organoids in the lab. The overall success rate for generating glioblastoma organoids (GBOs) was 91.4 percent, with 66.7 percent of tumors expressing the IDH1 mutation, and 75 percent for recurrent tumors, within two weeks. These tumor glioblastoma organoids can also be biobanked and recovered later for analyses.

Genetic, histological, molecular analyses were also performed in 12 patients to establish that these new GBOs had largely retained features from the primary tumor in the patient.

Eight GBO samples were then successfully transplanted into adult mouse brains, which displayed rapid and aggressive infiltration of cancer cells and maintained key mutation expression up to three months later. Importantly, a major hallmark of GBM — the infiltration of tumor cells into the surrounding brain tissue — was observed in the mouse models.

To mimic post-surgery treatments, the researchers subjected GBOs to standard-of-care and targeted therapies, including drugs from clinical trials and chimeric antigen receptor T (CAR-T) cell immunotherapy. For each treatment, researchers showed that the organoid responses are different and effectiveness is correlated to their genetic mutations in patient tumors. This model opens the possibility for future clinical trials for personized treatment based on individual patient tumor responses to various different drugs.

Notably, the researchers observed a benefit in the organoids treated with CAR T therapies, which have been used in ongoing clinical trials to target the EGFRvIII mutation, a driver of the disease. In six GBOs, the researchers showed specific effect to patient GBOs with the EGFRvIII mutation with an expansion of CAR T cells and reduction in EGFRvIII expressing cells.

“These results highlight the potential for testing and treating glioblastomas with a personalized approach. The ultimate goal is to work towards a future where we can study a patient’s organoid and test which CAR T cell is going to be the best against their tumor, in real time.” O’Rourke said. “A shorter-term goal, given the heterogeneity of glioblastomas, is that in vitro testing of various therapeutic options may also help refine patient enrollment in clinical trials, by more accurately defining mutations and selecting the appropriate, available targeted therapies for each.”

Along with learning and memory, sensory experiences such as listening to music or appreciating a stunning view also have a similar effect on the brain. Incoming sensory information activates neurons in the cortex, causing long term modifications to the circuitry depending on what we experience. This process called experience dependent plasticity, is part of the reason why our brains develop differently due to unique individual life experiences.

But how do our brains convert relatively short-lasting neuronal activity into the long-term changes driven by our sensory experiences? The key lies in specialized proteins called activity-dependent transcription factors. Responding to neuronal activity, these factors activate genes within the cell helping to translate rapid incoming signaling into slower, lasting changes. Despite the importance of activity dependent transcription to development and long-term plasticity in the brain is evident, it was impossible to directly monitor transcription factors activity. This was mainly due to the lack of available tools to study the interaction between neuronal activity and transcription factor activation that occurs in an intact, living brain.

In a recently published study in Neuron, Scientists in the Yasuda Lab at the Max Planck Florida Institute for Neuroscience (MPFI) have designed and developed novel biosensors that allow the simultaneous study of both sensory evoked neuronal activity and transcription factor dynamics. Coupling the specialized techniques of 2-photon calcium imaging with 2-photon fluorescence lifetime imaging (2pFLIM), scientists for the first time ever will have the unique ability to investigate how transcription factors function in a living brain with single cell resolution.

“Transcription factor activity in the brain isn’t a static, but rather a very dynamic process that can occur on the order of hours to days after a sensory experience,” explains Dr. Tal Laviv, Research Fellow in the Yasuda Lab and leading author of the paper. “Traditional methods of studying these proteins involve freezing brain tissue at a single moment in time. So, while these approaches can tell you if a certain factor is activated or not, they aren’t good at capturing how experience shapes transcription factor activity over time. We wanted to develop a new way to study how this process is actually occurring in a living brain and chose to study CREB due to its strong involvement in plasticity, learning and memory.”

MPFI scientists started by creating sensitive and specific 2pFLIM biosensors designed to report the direct activity of cAMP response-element binding protein or “CREB” for short. Packaging their newly generated sensors using an adeno-associated viral strategy (AAVs), the team then expressed them in a population of neurons within the somatosensory cortex of mice. Changes in environment are known to activate this brain region and in response, the neurons within modulate numerous signaling molecules including CREB. But little is known about the temporal dynamics of CREB activation after a change to the environment. Using the 2pFLIM CREB sensor, the team chronically monitored CREB activity in the same population of neurons while mice experienced an enriched environment. The enriched environment caused a significant increase in overall CREB activity. Interestingly, when mice were removed from the enriched environment for an extended period of time, CREB activity returned to normal levels indicating sensory experience as a driver for the sustained activity.

Next, MPFI scientists sought to unravel how sensory experiences and neuronal activation shape CREB activity in the living brain. To do so, the team expressed the CREB sensor and a sensor of neuronal activity (calcium sensor) in the visual cortex of mice. Previous studies have shown that when mice are temporarily deprived of visual stimuli and placed in dark housing, neuronal plasticity was increased in the visual cortex. In the new study, visual cortical neurons were imaged in dark-reared mice during presentation of a visual stimuli. Both calcium and CREB dynamics in single cells were simultaneously imaged for prolonged periods of time. The results revealed a dynamic regulation of CREB activity in the visual cortex: Dark reared mice displayed dramatically increased levels of visually evoked CREB compared to mice raised in normal light/dark conditions. In addition, CREB activity levels were maintained for a period of at least one day in dark-reared mice. Intriguingly this elevated CREB activity was not due to elevated calcium levels within individual neurons, indicating that sensory experience can finely tune the sensitivity of activity dependent transcription in the living brain.

This unique approach can be broadly applied to many different types of transcription factors in the future and will allow an unprecedented opportunity to unravel the transcriptional dynamics underlying experience-dependent plasticity in the brain.

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