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Category Archives: Georgia Stem Cells

CIRI calls for safety advice revamp after health concerns raised by 3D printing emission research – 3D Printing Industry

Posted: October 13, 2022 at 2:32 am

The Chemical Insights Research Institute (CIRI), a non-profit arm of safety research specialist Underwriters Laboratories, has found that 3D printing emissions can be damaging to human health, even in small quantities.

In a recent CIRI toxicity study, researchers found that exposure to the fumes created when printing ABS or PLA filaments, can contribute to airway cellular injury and inflammation. Based on their research, the scientists say that operating extrusion 3D printers from a safe distance, as well as ventilation and filtration mitigation strategies, should be discussed in the safety guidance around these machines.

Getting to the bottom of emissions

According to the researchers behind the paper, Dr. Christa Wright, now formerly of Georgia State University, and CIRI graduate student Jennifer Jeon, their study was prompted by concerns over the harmfulness of fumes emitted by certain printers.

Specifically, as FFF systems heat, cool and manipulate filaments, the scientists believed that the chemical compounds released into the air could be hazardous to operators. Given the growing popularity of 3D printers in STEM educational settings, the pair therefore said it was essential to find out which materials and conditions caused most risk, as well as the consequences of breathing in fumage.

To find out, the researchers cultured small airway epithelial cells in a dish before exposing them to the particulate matter generated via the 3D printing of ABS and PLA, and examining their responses. The team then measured particle emissions over three hours and fed them into a Multiple Path Particle Dosimetry model, capable of determining the impact of prolonged exposure to these, to users lungs.

The CIRI studys toxicity findings

Carried out in July 2021, the researchers experiments confirmed their fears about the damage emissions could be doing to 3D printer users lung cells. Those samples exposed to high doses of ABS printing fumes exhibited a 49.5% decline in viability, and even the dishes further away or in adjacent rooms were affected, albeit to a lesser extent.

The team then turned to metabolomic profiling, a process in which cells can be assessed for high levels of metabolites, the metabolism byproducts associated with cell injury and inflammation. In the case of PLA and ABS, testing showed that fumes emitted when printing these caused cells to be altered, with prostaglandins, compounds that target injuries or infections, being impacted in particular.

Interestingly, while the scientists research showed that both PLA and ABS fumes can be associated with a decline in airway cell viability, oxidative stress, an increase in DNA damage, and high levels of metabolites, it found that ABS had higher toxicity, as particles collected further from the printer caused more damage to viability.

In light of their findings, the researchers arent calling for anything too drastic, like reviewing the 3D printing of ABS or PLA in STEM settings. Instead, the duo say issues like proximity and opportunities to disperse harmful fumes should be discussed in machine safety guidance, to educate users on potential dangers.

ABS filament emissions may be more biologically active than PLA, the team say in their paper. Although contaminants were found at the high school site, ABS filament emissions and associated exposures contributed to a significant decline in small airway epithelial viability, oxidative stress, an increase in double stranded DNA breaks, and high levels of the metabolites associated with cellular injury.

AM emissions: a growing field of research

While its relatively well-known that the fumes emitted by certain materials during 3D printing could be harmful, the extent of this danger still isnt fully understood. Just last year, fume and particle extraction technology developer BOFA International emission research revealed the impact of particulates and gasses emitted, and suggested measures users can take to protect themselves from harm.

Scientists at the US Environmental Protection Agency (EPA) have also previously tried to assess the impact of 3D printing ABS that has been reinforced with carbon nanofibers. Through its 3D printing emission study, the agency sought to develop fresh literature on the topic, designed to help users understand potential issues.

In the past, Underwriters Laboratories itself has published UL standards for 3D printing emissions, to enable manufacturers to mitigate any indoor air pollution risks. Back in 2019, the organization issued ANSI/CAN/UL 2904, a document its VP of Standards Philip Piqueira said would advance the availability of low-emission printers and print media for use in the global marketplace.

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Featured image shows researchers working at Underwriters Laboratories CIRI institute. Photo via Underwriters Laboratories.

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Global Adult Stem Cells Market | Expected to Reach USD 9.45 Billion and Compound Annual Growth Rate (CAGR) is – openPR

Posted: October 4, 2022 at 2:34 am

Adult Stem Cells Market

Adult Stem Cells Market is projected to grow from USD 6.94 Billion in 2021 to USD 9.45 Billion by 2028, at a CAGR of 7.5% from 2022 to 2028.

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The cloudy connection between fragile X and cancer – Spectrum

Posted: October 4, 2022 at 2:34 am

About 20 years ago, Claudia Bagni and her team made a curious discovery: FMR1, the gene underlying most cases of fragile X syndrome, was strongly expressed in human cancer tissues.

At first, Bagni brushed it off as a fluke. FMR1 had no known roles in processes related to cancer, such as cell proliferation. But over time, she started to see evidence for a connection build. According to her own findings and those of other scientists, as well as a handful of small epidemiological studies and case reports, it seemed that people with fragile X, the most common form of inherited intellectual disability, might be protected from cancer.

Those initial clinical reports marked for our group the beginning of a new, completely unexplored [research] area connecting neuroscience and cancer biology, says Bagni, now director of fundamental neurosciences at the University of Lausanne in Switzerland.

In the past decade, Bagni and others have shown that several different types of cancer including colon cancer, liver cancer, pancreatic cancer and melanoma involve increased levels of FMRP, the protein encoded by FMR1. In a mouse model of breast cancer, having high levels of FMRP in tumors is linked to the spread of the cancer to other areas of the body, including the lungs, according to a 2013 report. And the list keeps growing.

Bagnis latest study, published last month in Cell Death & Disease, identified high levels of FMRP in tumors from people with glioblastoma, an aggressive cancer of the brain or spinal cord. The patients FMRP levels tracked not only with tumor growth but with their overall survival. Whats more, overexpressing FMRP in glioblastoma stem-like cells, which are thought to be involved in tumor initiation, also boosted cell proliferation in a lab dish.

The new results reinforce FMRPs link to cancer and the idea that people with fragile X syndrome may have an unusually low risk for it, says Randi Hagerman, medical director of the MIND Institute at the University of California, Davis, who was not involved in the work. People with fragile X, who often have autism as well, have mutations that silence FMR1 and prevent cells throughout the body from producing FMRP.

[The new work] also suggests a new treatment for glioblastoma, which is a terrible kind of cancer, Hagerman says.

But research on exactly how FMRP levels might influence a persons cancer risk is still in its infancy and how to investigate the connection to fragile X isnt straightforward, says Nien-Pei Tsai, associate professor of molecular and cellular biology at the University of Illinois Urbana-Champaign.

It needs much more work to say that people with fragile X syndrome have a lower risk of developing cancer, Tsai says, noting that FMRP is lost during development, and its long-term absence could lead to compensatory effects later on.

When compensatory effects are in place, the correlation between the levels of FMRP and cancer progression may not be the same anymore, he adds. There is no easy way to test how [fragile X syndrome] may reduce risk for cancer.

Epidemiology studies might seem like the most direct way to establish the cancer-fragile X connection. National registries, such as those in Sweden, Denmark and Finland, track the condition as well as newly diagnosed malignancies, says Sven Sandin, a biostatistician at the Karolinska Institutet in Stockholm, Sweden. With that information, he says, its not that tricky to do a straight-on analysis.

But, he notes, potential confounds abound: If we have an increase in fragile X diagnoses because of increasing awareness and testing, and if at the same time we diagnose more children with cancer, that by itself would create a spurious association, he says.

Its also difficult to definitively connect the dots through FMR1. The FMRP protein binds to hundreds of RNA molecules in the brain and other tissues, affecting numerous signaling pathways some of which help regulate brain development and are also implicated in cancer development.

The cancer connection is not unique to FMR1. More than 40 genes associated with autism, including PTEN, TSC1 and TSC2, have links to cancer. Studies suggest that some autistic people have decreased odds of developing cancer, although those with co-occurring intellectual disability or birth defects are at a higher risk of cancer early in life than are non-autistic people, according to an April report.

If cancer genes were not involved in autism, it would be a miracle: They are all over the place theyre involved in virtually every aspect of signal transduction that occurs in the organism, says Michael Wigler, professor of cancer research at Cold Spring Harbor Laboratory in New York. Theres no simple story here.

If cancer genes were not involved in autism, it would be a miracle: They are all over the place. Michael Wigler

Yet the link to fragile X seems particularly robust, based on reports collected over the past 20 years. Among 223 Danish people with fragile X syndrome, only three had cancer, according to a 2001 study. This proportion is about 70 percent lower than whats expected in the general population. Another study, conducted in Finland, found that 11 out of 302 people with fragile X had cancer about 20 percent lower than whats expected in the general population.

A third report described the case of a boy with fragile X who developed an inoperable form of glioblastoma, but who survived for at least eightyears after the cancer diagnosis, with the tumor growing at a lower-than-expected rate. Children with glioblastoma typically survive one to six years after diagnosis, and less than 20 percent survive five years after diagnosis.

A range of mechanisms could be at play: Two genes overexpressed in people with fragile X are related to tumor suppressors, a 2010 study found. And among the genes whose levels were decreased in more than half of people with the syndrome in that study, one encodes a component of the Wnt/beta-catenin pathway. This pathway regulates cell growth and has been implicated in both cancer and neurodevelopmental conditions such as autism. Bagnis latest work on glioblastoma also hints at Wnt involvement: Reductions in FMRP levels, she and her colleagues found, dampen Wnt signaling.

If youre trying to identify converging pathways, Wnt signaling could be one of them, says Peng Jin, professor of human genetics at Emory University in Atlanta, Georgia.

But FMRP also targets vimentin, a protein that is associated with increased metastatic potential, Hagerman says. Vimentin allows the cancer to invade tissues, and if FMRP is high, it suggests there might be metastases too.

And another FMRP target is MDM2, a protein that hinders the maturation of neurons and is expressed at increased levels in people with fragile X syndrome. A compound being tested in cancer clinical trials, nutlin-3, inhibits MDM2 and boosts the number of mature neurons in a fragile X mouse model. It can also reverse cognitive and behavioral difficulties in the model, according to a study published in May.

We were looking for molecular pathways that could be modulated to correct what is dysregulated in the mice, says lead investigator Xinyu Zhao, professor of neuroscience at the University of Wisconsin-Madison. It turns out that some of them are indeed cancer drug targets.

Singling out any of these potential contributors could prove difficult. FMRP appears to have certain functions in some types of cells and not in others, says Ethan Greenblatt, assistant professor of biochemistry and molecular biology at the University of British Columbia in Canada. It may also have cell type-specific functions in cancer, affecting only some cancers.

And whether children with certain neurodevelopmental conditions are at higher or lower risk for malignancies may depend on the nature of the molecules involved, Jin says.

FMRP acts as a brake to suppress protein translation, whereas other molecules could be involved in gene activation, he says. Different molecules can play distinct roles in regulating neuronal function as well as cell proliferation.

Ultimately, Wigler says, a link may exist between FMRP and only some malignancies, with no direct tie between cancer and fragile X syndrome or other neurodevelopmental conditions.

Regardless, looking at that tie could lead to fresh therapeutic targets for several conditions. Something that downregulates FMRP could help with glioblastoma and downregulation of FMRP may be really helpful for many other cancers, Hagerman says. And, Bagni adds, identifying molecular pathways downstream of FMRP that are dysregulated in cancer cells could also lead to more targeted treatments for fragile X.

Cite this article: https://doi.org/10.53053/XLXD4405

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CBD And CBG Show Promising Results In Treating Glioblastoma Brain Tumors – The Fresh Toast

Posted: June 22, 2022 at 2:17 am

By Nina Zdinjak

MGC Pharmaceuticals , a publicly-traded cannabis company, recently finalized pre-clinical trial research ofcannabinoids as a potential treatment for glioblastoma,anaggressive brain cancer.Theresults were positive,reported CityA.M.

The European-based bio-pharma company specializing in the production and development of phytomedicines said that the three-year in-vitro trial delivered outstanding results.

The research, conducted in collaboration with theNational Institute of Biology in Slovenia with30 biopsy samples from 18 patients,firstexamined how cannabinoids such as CBD can be used as a treatment for cancerous tumors. MGC also researched THC and later replaced it with cannabigerol, known as CBG, which has no known psychotropic effects.

The study took more than 5,800 cell tests to determine the most efficient concentration and ratios of CBD and CBGin the treatment formulation.

The results revealed the efficacy of cannabinoidsin treating glioblastoma, as well as the most efficient ratio of CBD:CBG in inhibiting the tumors viability, causing a cascade of biological processes resulting in the death of glioblastoma and stem cells,reported BusinessCann. This is important because glioblastoma stem cells are the primary cause of the diseases progressionand areresistant to standard treatments.Glioblastoma is the most common, fast-growing, and aggressive brain tumor. Composed of diverse cell types, the prognosis is always poor.

RELATED: CBD Restricts Tumor Growth In Cancer Patients, Finds New Study

The results of this trial are enormously exciting both for the company, and for the treatment of fatal cancerous tumors, saidRoby Zomer,co-founder and managing director at MGC Pharmaceuticals.

MGC Pharmas research has demonstrated the effect of naturally derived cannabinoid products on stage IV brain tumors without the use of toxic chemotherapy components. We are proud of the work achieved thus far and are looking forward to advancing our proprietary formulation to the next stage of clinical trials.

This is not the first successful research on the potential of CBD as a treatment for this type of brain tumor.

Researchers at theMedical College of Georgia at Augusta University, released astudyin December revealing thatinhaled CBD shrunkthe size of glioblastoma tumorsin an animal model.

The study suggested thatinhaled CBD reduces the size of glioblastomathrough the reduction of the essential support of its microenvironment. Researcherschose the inhaled approach to make sure the compound found in the cannabis plant reached the brain.

RELATED: CBG Mania: What Are the Best Ways To Use And Consume CBG?

We saw a significant reduction in the size of the tumor and its microenvironment was different, after only seven days of treatment,saidDr.Babak Baban,immunologist and associate dean for research at the Dental College of Georgia at Augusta University.

In eight days the aggressive brain tumor was formed in the brain of the mice, and the following day they started treating them with CBD via inhalation or placebo. The researchers discovered thatCBD altered the tumors ecosystem, or supportive tumor microenvironment, and restored levels of inflammation that attack glioblastoma.

RELATED: Cannabis-Based Sativex Mouth Spray To Treat Brain Tumors?

It is about immune balance, said Baban, the corresponding author of the study.

He explained that the human immune system is regularly fighting cancerous or precancerous cells, but when a tumor is completely established, it takes charge. This means that the tumor creates a state of more chronic inflammation that ends up protecting it from the immune system.

While further research is needed, these results are a great start as they offerhope to theapproximately250,000people worldwide struggling with this devastating condition.

This article originally appeared on Benzinga and has been reposted with permission.

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How Protein Nanoparticle Vaccines Have The Potential To Be Developed Into ‘Safer’ Covid-19 Vaccines: Study – ABP Live

Posted: June 4, 2022 at 2:30 am

New Delhi: A nanoparticle vaccine which combines two proteins that induce immune responses against SARS-CoV-2 has the potential to be developed into 'broader' and 'safe' Covid-19 vaccines, according to a new study.

The Covid-19 pandemic, or the SARS-CoV-2 pandemic, has claimed more than six million deaths since 2019, and is a public health burden worldwide. The rapid evolution of SARS-CoV-2 is characterised by the emergence of several significant variants, including Delta and Omicron.

The study, led by researchers in the Institute for Biomedical Sciences at Georgia State University, was recently published in the journal Small.

A nanoparticle-based vaccine is one in which the receptor-binding domain (RBD), which is a part of the spike protein of SARS-CoV-2, is attached to a protein designed to form nanometre-sized protein particles, or nanoparticles, according to a study by the US National Institutes of Health (NIH), which was published in the journal, Nature. SARS-CoV-2 attaches itself to cells using the spike protein.

These nanoparticles could be composed of lipids, metal and non-metal inorganics, several polymers, and virus-like particles which have been tested for research, according to a study published by the National Center for Biotechnology Information (NCBI), NIH. Virus-like-particles (VLP) are self-assembling nanoparticles lacking infectious nucleic acid.

Hyderabad-based Serum Institute of India's Covovax is a nanoparticle-based vaccine, manufactured by technology transfer from Novavax.

The spike protein (S) on SARS-CoV-2 is the preferred target antigen for vaccine development, based on its essential function and neutralising epitopes to combat the virus. An epitope is the part of an antigen molecule to which an antibody attaches itself.

The new study was conducted in mice, with the researchers investigating the immune response induced by two proteins, the spike protein and the relatively conserved stem subunit (S2) of the spike protein. The researchers found that the assembly of the two proteins into double-layered protein nanoparticles improves the immunogenicity of the proteins. Immunogenicity is defined as the ability of a molecule or substance to provoke an immune response.

In a statement released by Georgia State University, Dr Baozhong Wang, senior author of the study, said the entire S protein has been used as the major antigen in vaccines against the ongoing pandemic, adding that as the number of infections continues to rise, more and more variants have appeared and supplanted the ancestral virus.

Since the variants have superseded and replaced the ancestral virus, the efficacy and protection of current vaccines are under constant threat and need continuous improvement, Wang explained.

He further said that in contrast, the stem subunit of the spike protein is more conserved and has fewer mutations across lineages. He added that the stem could induce effective antibody neutralisation and vigorous antibody-dependent cellular cytotoxicity (ADCC) activity against multiple variants of S protein. ADCC, also known as antibody-dependent cell-mediated cytotoxicity, is a type of immune reaction in which a target cell or microbe is coated with antibodies and killed by certain types of white blood cells.

Wang explained that the work shows that the stabilised stem subunit could be a potential antigen for a SARS-CoV-2 universal vaccine against unpredictable variants.

According to the study, immunisation with the stem-induced balanced Immunoglobulin G (IgG) antibodies occurred through potent and broad ADCC activity, which is a type of immune reaction on which infected cells are coated with antibodies that recruit certain types of white blood cells to kill the infected cells. The study also found that the double-layered protein nanoparticles constructed from the stem and the full-length spike protein induced more robust ADCC and neutralising antibodies than the stem and spike protein, respectively.

The nanoparticles were observed to produce more potent and balanced serum IgG antibodies than the corresponding soluble protein mixture. The researchers also found that the immune responses are sustained for at least four months after the immunisation.

The stem induces a more balanced IgG isotype antibody. In immunology, antibodies or immunoglobulins are classified into several types called isotopes or classes. The double-layered nanoparticles not only have a more balanced IgG isotype antibody induced by the stem, but also long-lasting immune responses, and excellent safety profiles. As a result, the nanoparticles have the potential to be developed into broader SARS-CoV-2 vaccines, the authors noted in the study.

Dr Yao Ma, the first author of the study, said the stabilised, conserved S2 stem subunit demonstrated its potential as a universal SARS-CoV-2 vaccine candidate against unpredictable variants. The double-layered protein nanoparticles incorporating the full-length spike protein and the S2 stem induced robust and long-term immune responses, and exhibited a safety profile in the primary studies, Ma added. This provides an option for current SARS-CoV-2 vaccine development.

Ma further said that the pandemic is far from over, and new variants continue to emerge and pose a massive threat to human health. In order to avoid another pandemic with an unpredictable new variant, the updating of vaccines needs to keep pace with the times.

Nanoparticles help improve vaccine efficacy, by targeting desired antigen-presenting cells to improve immunisation strategies. They protect the antigen (foreign particle) from early proteolytic degradation (degradation of protein by hydrolytic enzymes), control antigen release, and facilitate antigen uptake.

Current vaccines cause cells in the body to make a version of the spike protein to elicit an immune response. Putting multiple copies of the RBD on nanoparticles enhances immune response, recent studies have found. The NIH researchers tested nanoparticle-based vaccines in monkeys and found that most of the monkeys had no virus in their lower respiratory tracts two days after exposure.

Nanoparticle-based vaccines contain a strand of genetic code that provides instructions for building a version of the spike protein, according to an article by the University of Pittsburgh Medical Center, US. Host cells build the protein when they see the genetic code.

The immune system begins building antibodies against the protein, which help the immune system fight the coronavirus. The body builds up an army of antibodies, which can fight off a Covid-19 infection before it causes disease.

Since mRNA is very fragile on its own, it would degrade in the body if it is injected simply as a strand. Hence, the genetic material is protected with a nanoparticle, which preserves the mRNA long enough to carry it into the bodys cells so that they can begin making the proteins.

The use of nanoparticles in vaccine formulations allows not only improved antigen stability and immunogenicity, but also targeted delivery and slow release, according to a article published in the journal, Elsevier.

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SpaceX capsule returns to Earth with first all-private space station crew Spaceflight Now – Spaceflight Now

Posted: May 2, 2022 at 2:22 am

SpaceXs Dragon Endeavour spacecraft splashes down in the Atlantic Ocean Monday to end Axioms Ax-1 mission. Credit: SpaceX

A SpaceX crew capsule splashed down in the Atlantic Ocean off the coast of Georgia Monday with a retired NASA astronaut and three wealthy businessmen, closing out an extended 17-day mission on the first fully commercial, non-government visit to the International Space Station.

Protected by a thermal shield, the Dragon Endeavour spacecraft plunged back into the atmosphere and withstood a scorching hot re-entry over the southeastern United States. Two drogue parachutes opened up, and then four main chutes unfurled to slow the capsule for a relatively gentle splashdown at 1:06 p.m. EDT (1706 GMT) Monday in rolling seas northeast of Jacksonville, Florida.

The splashdown capped 17 days in orbit for the four-man crew, led by commander Michael Lpez-Alegra, a retired NASA astronaut and now an employee of Axiom Space, the Houston-based company that managed the mission.

Lpez-Alegra was joined on the flight by Larry Connor, an investor and accomplished aerobatic pilot from Ohio, Canadian businessman Mark Pathy, and Israeli entrepreneur Eytan Stibbe, who became the second person from Israel to fly in space. Connor, Pathy, and Stibbe paid for their rides to orbit.

Dragon, SpaceX, we see splashdown and mains (parachutes) cut, radioed Sarah Gillis, SpaceXs crew operations resource engineer, from the companys mission control center in Hawthorne, California.

We concur, repliedLpez-Alegra, who returned from his fifth mission to space, totaling 275 days in orbit. He has now launched on three different types of vehicles NASAs space shuttle, Russias Soyuz spacecraft, and SpaceXs Dragon ferry ship.

On behalf of the entire SpaceX team, welcome back to planet Earth, Gillis said. The Axiom 1 mission marks the beginning of a new paradigm for human spaceflight. We hope you enjoyed the extra few days in space and thanks for choosing to fly SpaceX.

Lpez-Alegra said the crew was feeling well as the capsule bobbed in the Atlantic Ocean. After teams on fast boats secured the parachutes and inspected the Dragon capsule, SpaceXs recovery ship Megan, named for NASA astronaut Megan McArthur, pulled alongside the spacecraft and lifted it from the sea.

The recovery team then opened the hatch and helped each crew member from the spacecraft. All four stood and smiled, flashing a thumbs-up and walking albeit wobbly and with assistance to a medical evaluation room on the recovery ship.

While readjusting to gravity, private astronauts were expected to fly by helicopter back to shore, then travel to Orlando for more medical checks and to meet their families.

Axiom contracted with NASA and SpaceX for the all-private crew mission to the space station. NASA charged Axiom a daily rate for access to the stations life support system, communications network, and other equipment. NASA is paying some of that back to Axiom in exchange for the return of government freezers and experiment specimens on the Dragon spacecraft.

Axiom paid SpaceX for the ride to and from the station on the Dragon spacecraft, and the launch aboard a Falcon 9 rocket from the Kennedy Space Center.

The arrangement is the first of its kind. Previous visits by private astronauts, or space tourists, to the space station occurred on government-led missions on Russian Soyuz spaceships. Before Axioms flight, 11 people had traveled to the space station as paying passengers on Soyuz missions, but they all flew with a government cosmonaut commander.

More Axiom missions are planned to the station in the next few years, leading up to delivery of the companys own commercial module to the orbiting complex. That module will eventually detach from the International Space Station and become the centerpiece for Axioms privately-owned multi-element outpost in low Earth orbit.

Without those two partners, none of this would be possible, said Derek Hassmann, Axioms operations director, referring to NASA And SpaceX. So just an amazing first step thats leading up to our launch and activation of the Axiom station with the first module being planned for 2024.

I would say, overall, this has been just an amazing success, Hassmann told reporters Monday afternoon. The crew performed beyond expectations. The ground teams were tremendous.

The re-entry and splashdown Monday came about 16 hours after SpaceXs Dragon Endeavour spacecraft undocked from the space station. The capsule backed away from the complex at 9:10 p.m. EDT Sunday (0110 GMT Monday).

The Axiom mission, known as Ax-1, was supposed to last 10 days, with the crew spending eight days at the space station. The mission was extended one day because of timing conflict between the planned undocking of the Ax-1 mission and a previously-scheduled Russian spacewalk.

Then persistent high winds in all seven SpaceXs splashdown zones near Florida kept the crew aloft through last week and the weekend. Mission managers were finally satisfied that conditions would be favorable for a return Monday, and they cleared Ax-1 to depart the station Sunday night.

During their time on the International Space Station, the Ax-1 astronauts brought the crew complement on the research lab up to 11 people, including five Americans, three Russians, one German, one Canadian, and one Israeli occupant. The Ax-1 crewspent the bonus time in orbit finishing up experiments and participating in more outreach events that didnt fit into the original flight plan, Hassmann said.

Despite nearly doubling their stay at the space staton, the paying passengers didnt rack up any late checkout fees.

The contact between Axiom and NASA included an equitable balance to cover potential delays in the undocking and return of the Ax-1 mission, said Stephanie Schierholz, a NASA spokesperson.

Knowing that International Space Station mission objectives like the recently conducted Russian spacewalk or weather challenges could result in a delayed undock, NASA negotiated the contract with a strategy that does not require reimbursement for additional undock delays, Schierholz said in a written statement.

There were no additional costs for any parties based on the extension of the mission, Hassmann said.

Axiom has not disclosed the price Connor, Pathy, and Stibbe paid for their flight to the space station. But NASAs inspector general has said a seat on a Dragon mission costs roughly $55 million.

The Ax-1 crew members trained for the mission in Houston and at SpaceXs headquarters in Hawthorne, California.

The first private astronaut mission is a harbinger of a transition from government-led spaceflight operations in low Earth orbit to a future generation of commercial space stations. NASA wants to help steer the space industry through the transition by providing the International Space Station as a testbed for markets that must be developed before companies can take launch and operate a revenue-earning destination in orbit.

A major goal for the U.S. space agency is to rely on commercial industry to develop the next orbiting outpost to replace the International Space Station. The Biden administration recently signaled it will support an extension of station operations though 2030, but by then the research labs oldest elements will have been in space 32 years, more than twice their original design lives.

The fraught relationship between the United States and Russia, the two largest ISS partners, in the wake of Russias invasion of Ukraine has also raised concerns about a replacement for the International Space Station.

Aside from Axiom, several other companies have plans to develop commercial space stations. In December, NASA selected Blue Origin, Nanoracks, and Northrop Grumman to advance their concepts for a commercial habitat and research facility in low Earth orbit.

Those companies are developing concepts for a standalone station, while Axiom will initially focus on a commercial add-on to the ISS.

The Ax-1 mission was a stepping stone toward that goal.

During their time in space, the Ax-1 crew worked with 26 science payloads and technology demonstration experiments, according to Hassmann. They also conducted more than 30 public outreach events in multiple languages.

The experiments on Ax-1 included investigations into self-assembling technology for future satellites and space habitats, the study cancer stem cells, and the test of a new Japanese air purification device. The crew members also served as experiment subjects for scientists to study how spaceflight affects the human body.

Connor partnered with Mayo Clinic and Cleveland Clinic on research looking at heart health and brain and spinal tissue. Pathy worked with the Montreal Childrens Hospital, Canadian research universities, and the Royal Canadian Geographic Society on technology demonstrations, a sleep study and chronic pain experiment, an eye health investigation, and Earth observations.

Stibbe worked with the Israel Space Agency, the Israeli Ministry of Innovation, Science, and Technology, and the Ramon Foundation, an organization established to honor the memory of Ilan Ramon, the first Israeli astronaut who died on the space shuttle Columbia in 2003. He alsocarried with him fragments from Ilan Ramons diary that survived the searing heat of re-entry after the fatal breakup of the shuttle Columbia over Texas in 2003.

The end of the Ax-1 mission clears the way for SpaceX to launch the next crew flight to the space station. Three NASA astronauts and a European Space Agency mission specialist are ready for liftoff from the Kennedy Space Center at 3:52 a.m. EDT (0752 GMT) Wednesday.

The four-person crew will ride a Falcon 9 rocket and SpaceXs Dragon Freedom spacecraft into orbit to begin NASAs Crew-4 mission. The expedition on the space station is scheduled to last approximately five months.

The launch of the Crew-4 mission was delayed to await the return of the Ax-1 mission. The Dragon Endeavour spaceship was docked to the same port needed for arrival of the Dragon Freedom capsule.

SpaceX engineers will analyze data from the Ax-1 mission to ensure there were no issues that might affect the launch of the Crew-4 flight Wednesday. Managers planned to meet for a launch readiness review early Thursday to formally give the go-ahead for liftoff of the Crew-4 mission.

The Ax-1 mission was SpaceXs sixth launch to carry people since a Dragon test flight took off in May 2020 with NASA astronauts Doug Hurley and Bob Behnken, ending a nearly nine-year gap in launching astronauts into orbit from U.S. soil.

NASA invested billions of dollars in helping SpaceX develop the human-rated Dragon spacecraft. SpaceX put up its own private funding in a cost-sharing arrangement with the federal government.

Benji Reed, senior director of SpaceXs human spaceflight programs, said Monday the companys fleet of four Crew Dragon spaceships could accommodate up to six astronaut missions per year.

Half a dozen crew flights per year would be great, or more, Reed said. AndIthink we can get to a place where we can sustain that. If theres a market for it, we can definitely do that.

Each Dragon capsule has four seats.In the long-term, SpaceX wants to retire the Falcon 9 and Dragon fleets in favor of the next-generation fully reusable Starship rocket, which could carry many more passengers into space. When asked Monday, Reed offered no estimate on when the Starship might be ready to fly people.

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Identifying Missing Links – and Why Certain Drugs Don’t Work – in Alzhiemer’s – BioSpace

Posted: April 6, 2022 at 2:26 am

Before the U.S. Food and Drug Administrations controversial approval of Biogens Aduhelm (aducanumab) for Alzheimers disease in June 2021, there were well over 200 failed clinical trials in the last twenty years for drugs to treat the disease. Part of the drugs controversy is the alleged negligible improvement in Alzheimers symptoms. At the highest doses, which can be risky, Aduhelm appears to slow the progression of the disease in patients if caught early enough.

Aducanumab is an antibody that clears beta-amyloid in the brain, one of two proteins that accumulate abnormally in the disease. Although beta-amyloid is associated with the disease, its not the only factor, and researchers are making progress on understanding Alzheimer's better and taking new approaches. Heres a look.

Screening Approach to Why Alzheimers Drugs Fail

A team at the University of California, San Diegodeveloped a drug screening method to help determine why Alzheimers drugs fail. The system analyzes disease mechanisms in human neurons. They published their research in Alzheimers & Dementia. The researchers note that sometimes drugs that clear amyloid dont improve the disease but make it worse. With that as the focus, their drug screening method evaluates what endotypes, or disease mechanisms, are modified in the patients neurons from the treatment. They note that although amyloid plaque formation is one endotype, others should be targeted, including differentiation of neurons to an early non-neuron state, neuronal gene suppression and loss of synaptic connection.

The key here is that we are using the endotypes that we discovered to see how current drugs fail, senior author Shankar Subramaniam, Ph.D., said. When drugs interact with human neurons, what endotypes do the drugs fix and what endotypes do they not fix in the process?

Their technique requires taking human-induced pluripotent stem cells derived from patients with familial Alzheimers disease and transforming them into neurons. They then treat these cells with drugs and leverage genetic sequencing to determine what endotypes change pre-and post-treatment. They also do the same on neurons derived from healthy people.

In a test of two experimental Alzheimers drugs targeting amyloid, the researchers found the drugs only improved some endotypes, such as the formation of amyloid plaques, partly fixing some, while not fixing others.

What we are seeing is that fixing amyloid plaque formation does not reverse the disease in any way, Subramaniam said. It turns out that this endotype is way downstream, so it is too late. Once neurons de-differentiate into non-neurons, they lose their synaptic connections, which leads to loss of memory and cognition and as a consequence, dementia.

Targeting Alzheimers Earlier in the Process

Although amyloid and a second protein, tau, are linked to Alzheimers, with amyloid generally occurring earlier and tau later in the disease, some researchers are focused earlier. Qin Wang, MD, Ph.D., with the new Alzheimers Therapeutics Discovery program at the Medical College of Georgia, described amyloid and tau as like a gun in the brain: beta amyloid is the trigger and tau is the bullet. Wang is looking at why and how they interact.

Theres a missing link there, Wang stated.

Earlier in Alzheimers, a group of neurons in a part of the brain called the locus coeruleus seem to die first. Those nerves interact with norepinephrine, a neurotransmitter. Norepinephrine is involved in various processes, including fight or flight, arousal, attention and memory. They have connections throughout the brain and, Wang said, seem to be hypersensitive to amyloid beta and tau.

These cells have a special receptor that can both release, and are involved in receiving norepinephrine. Once turned on, the receptor controls the protein that creates beta amyloid. But once beta amyloid is formed, it can attach to the same receptor in a different location and begin creating more beta amyloid in what Wang describes as a vicious cycle.

This is very important for the initial seeding of the (deposits) in the brain, Wang said.

Wang noted that drugs that target amyloid remove about 60% at best, which isnt nearly enough. Very little beta amyloid is needed to feed the cycle, with even 1% still sufficiently activates this detrimental pathway.

So Wang and her team are evaluating drugs that can prevent that cycle. Two are already on the market. One of them is an approved antidepressant, Idazoxan. Another is a blood pressure medication called clonidine that also activates the particular receptor in the brain.

Hopefully in humans, if we can block those receptors, we will see the oppositive effect, Wang said. We can see an improvement in their cognitive function.

NeuroNascents Neuron Regeneration Drug

NeuroNascent reported data from a Phase I trial of NNI-362 in Alzheimers disease. The drug demonstrated favorable safety and a significant decrease in a biomarker for Alzheimers disease. The drug appears to produce new neurons to replace lost neurons or improve their numbers in aging patients or neurodegenerative diseases, such as Alzheimers.

The drug is taken orally and data showed it to be well tolerated in healthy aged people after single and multiple daily dosing. Using a new test to detective Alzheimers biomarkers in blood plasma, the drug also significantly decreased p-tau181 levels compared to pre-treatment levels.

This exciting early data, demonstrating our novel allosteric oral therapy, NNI-362, can normalize the plasma p-tau181 a biomarker correlated with Alzheimers disease brain pathology and progression, supporting the need to further test NNI-362 in long-term trials, Judith Kelleher-Andersson, Ph.D., founder and chief executive officer of Neuronascent, said. Running of a Phase II trial in mild to moderate Alzheimers disease patients could assess longer-term amelioration of p-tau181 levels and to determine if this intervention could consequently improve quality of life for patient and caregiver.

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Axion BioSystems Acquires Live-Cell Imaging Innovator CytoSMART Technologies – Business Wire

Posted: March 25, 2022 at 1:59 am

ATLANTA--(BUSINESS WIRE)--Axion BioSystems, a leading life sciences tools company focused on advanced live-cell assay systems, announces the acquisition of the Netherlands-based CytoSMART Technologiesan innovator in kinetic live-cell imaging analysis. The collaboration positions Axion for significant expansion in the fields of stem cell research, immuno-oncology, cell-based therapies, and drug discovery.

Since Axion BioSystems inception, our focus has been on developing user-centric products that accelerate scientific discovery, with the specific goal of building innovative systems that monitor cellular activity without disturbing the underlying biology. The Maestro, our flagship bioelectronic assay (BEA) platform, has transformed neurological disease research over the past 14 years. Likewise, our most recent addition to the BEA portfolio is doing the same in groundbreaking fields like immunotherapy, said Tom OBrien, President & CEO of Axion BioSystems. In CytoSMART, we found a company with technology complementary to our own, as well as a partner with the same fundamental objectiveto provide customers with high-quality instruments and powerful software that rapidly advance live-cell research and cell therapy development. We welcome the entire CytoSMART team, and we are excited to add their expertise to our own.

Noninvasively studying the real-time biology of live cells allows scientists to obtain a more complete understanding of the complex processes underpinning health and disease. In their respective arenas, both Axion and CytoSMART have focused on the development of next-generation analysis tools for use in pharmaceutical, academic, and biotechnology research. Along with Axions recent acquisition of UK-based printed electronics manufacturer M-Solv Manufacturing Limited, this latest product portfolio expands the companys offerings and opportunities in a rapidly growing life sciences tools market.

According to Joffry Maltha, CEO of CytoSMART, the mutually beneficial partnership is a natural fit. Axions overall mission and corporate culture align with our efforts to provide scientists around the world with high-quality, easy-to-use products to advance their research goals. With Axions resources and experience, we look forward to accelerating our market growth and product innovation.

About Axion BioSystems

Axion BioSystems is a leading life sciences tools company focused on innovative live-cell assays used to study the function of cells in vitro for drug discovery and disease modeling. The team at Axion BioSystems is dedicated to continuing the advancement of new technologies that accelerate research and further the understanding of biological complexity outside of the body. Axion BioSystems is headquartered in Atlanta, Georgia, USA, and has offices worldwide. Axion has more than 90 employees across its current locations. http://www.axionbiosystems.com

About CytoSMART

CytoSMART Technologies is a specialist in the development and manufacturing of live-cell imaging systems for life science laboratories. The company was founded in 2012 by a team of biologists and engineers who were convinced that a new generation of miniaturized microscopes, powered by artificial intelligence for image analysis, would allow biologists to make discoveries more efficiently and at scale. In 2018, CytoSMART was selected by Microsoft for its prestigious ScaleUp program. CytoSMARTs microscopy solutions are used in research laboratories worldwide. CytoSMART Technologies is headquartered in Eindhoven, the Netherlands, and has more than 50 employees. http://www.cytosmart.com

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Avra, Inc. Completes its Merger with Springs Rejuvenation, LLC, a Stem Cell and Anti-Aging Treatment Company – GlobeNewswire

Posted: December 24, 2021 at 2:23 am

ATLANTA, GA, Dec. 20, 2021 (GLOBE NEWSWIRE) -- viaNewMediaWire --Avra, Inc. (OTC PINK: AVRN), is pleased to announce that it has closed the merger with Springs Rejuvenation, LLC (https://springsrejuvenation.com). The surviving entity will be Springs Rejuvenation, Inc. (SPRINGS), a Chamblee, Georgia anti-aging and stem cell center focusing on stem cell therapy, facial rejuvenation, hair rejuvenation, non-surgical hair restoration, protein rich plasma (PRP) injections, and anti-aging treatments.

The merger documents have been filed with the State of Nevada. They have also been posted on OTC Markets along with the Consolidated financials in the last quarterly filing. On December 20, 2021, Avra, Inc. filed a Corporate Action with FINRA for a name and ticker change.

SPRINGS was founded and incorporated in the State of Georgia in May of 2019 by Dr. Charles Pereyra. The company currently has one facility located in Chamblee, Georgia. Dr. Pereyra, has assembled a very skilled team of doctors and support staff to expand the business. They include Dr. Juan P. Nieto M.D., Dr. Andrew Bernstein M.D., and Alyssa Stilwell, as executive director.

Dr. Pereyra is a practicing physician with multiple peer-reviewed publications. He earned an undergraduate degree from Cornell University, where his work with stem cells began, a Doctorate of Medicine (M.D.) from St. Georges University, and completed his residency training at New York Presbyterian Hospital Brooklyn.

Stem cell therapy is a form of regenerative medicine, designed to repair damaged cells within the body by reducing inflammation and modulating the immune system. This makes stem cell therapy a viable treatment option for a variety of medical conditions. Stem cell therapies are currently being researched throughout the country with many positive results. Showing profound outcomes in autoimmune, inflammatory, neurological, and orthopedic conditions; including Multiple Sclerosis, Lupus, Crohns disease, COPD, Parkinson's, ALS, Stroke, Congestive Heart Failure and more. Recently the FDA has begun to grant INDs for use of products containing stem cells for several conditions.

A primary goal of the merger is to allow SPRINGS to duplicate its model of high-quality stem cell treatments throughout the United States. SPRINGS is in the process of opening a facility in Austin, Texas, in conjunction with an existing clinic, and a third location in southeast Florida. The Company plans to open a total of ten new facilities in the next twelve months.

I am excited for SPRINGS Rejuvenation to take the next step with the Avra merger. Ive always been passionate about delivering the highest quality care to my patients and making sure they have the most cutting-edge medical treatments available. For over a decade a community of only a select few has benefited from use of stem cells. With this merger we will not only expand our reach to many communities, but also drive down the cost of stem cells for everyone, making access to these remarkable treatments much easier. Im incredibly excited for the future, said Dr. Charles Pereyra.

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From asthma to cancer to infertility, the new treatments, jabs and meds making us healthier… – The Sun

Posted: November 22, 2021 at 2:04 am

WHEN it comes to health, the news in recent times has been sombre.

It has been another rollercoaster year battling Covid, with the UK emerging from a third lockdown in spring.

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Millions of people have since had their jabs, and boosters are being rolled out as winter looms large.

But Covid is not the only big health story to come out of the past two years.

Behind the scenes, scientists around the world have been working on medical trials in the hope of finding cures for major illnesses.

And there have been dozens of major breakthroughs that could save billions of lives and change the way diseases are treated forever.

Just this month it emerged the vaccine for the human papillomavirus virus (HPV) could eradicate cervical cancer within the next few years.

From asthma to Alzheimers and cancer to infertility, CLARE OREILLY looks at the new treatments, vaccines and medicines that could put an end to some of the most common and deadly conditions.

CERVICAL cancer kills more than two women every day in the UK, claiming around 850 lives every year.

Yet a new study has found the disease could soon be a thing of the past.

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Kings College London scientists found the human papillomavirus (HPV) vaccine cut cases by 90 per cent.

The jab, which was first rolled out to teenage girls in the UK in 2008 then to boys in 2019, prevents HPV, which is responsible for nearly all cases of cervical cancer.

The study, in the Lancet, tracked women who received some of the first doses and found it prevented an estimated 17,200 pre-cancers and 450 cases in women in their twenties.

Cancer Research UKs chief executive Michelle Mitchell said: Its a historic moment to see the first study showing that the HPV vaccine has and will continue to protect women from cervical cancer.

A NEW antibody-based treatment developed by scientists in the UK and Germany could soon yield a vaccine to prevent Alzheimers.

The degenerative condition is thought to be caused by a type of protein that sticks to brain cells.

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The scientists were able to trigger the immune system to make antibodies, which targeted the protein before it was deposited.

Professor Mark Carr, who led a team at the University of Leicester, said: It has the real potential to provide an effective treatment for Alzheimers using a therapeutic antibody and highlights the potential of a simple vaccine.

Meanwhile, a year-long study has started in Norway where Alzheimers patients will receive a transfusion of blood taken from runners.

It is hoped the chemicals released in the blood after running have a rejuvenating effect to slow disease progression.

THREE new drugs are being put through trials in the hope they could end the misery of hot flushes for menopausal women.

Hot flushes are thought to be caused by changes in hormone levels affecting the bodys temperature control.

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But the medicines fezolinetant, elinzanetant and pavinetant can block the receptors which are responsible for the common symptom.

London GP Dr Zoe Watson says it could be years before the treatment is available on the NHS, though.

She says: It looks interesting in theory, but there are question marks over its efficacy, its side-effect profile and its cost.

Certainly if it does this well then it could be extremely useful for women whose most troubling menopausal symptom is hot flushes.

However, menopause is much more than just hot flushes and halting periods."

A BRAND new injection could reverse spinal cord injuries and allow patients to walk again just four weeks after treatment.

Developed by a team at Northwestern University in the US, the jab encourages nerves to regrow.

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It gave paralysed mice the ability to walk and human trials are expected to begin next year.

For decades, this has remained a major challenge for scientists because our bodys central nervous system, which includes the brain and spinal cord, doesnt have any significant capacity to repair itself after injury.

Professor Samuel Stupp said: Our research aims to find a therapy that can prevent individuals from becoming paralysed after major trauma or disease.

We are going straight to the FDA [the US Food and Drug Administration] to get this approved for use in patients.

DEMENTIA affects around 850,000 people in the UK and costs 26.3billion a year, but scientists at Durham University have made a breakthrough.

They are working on a treatment that could boost memory and muscle control in patients with the killer disease.

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Using infrared light to zap the brain improved the memory and thought processing in trials of healthy people.

And the next step is to enlist dementia patients to test the therapy.

Its delivered by a specially equipped helmet, which beams invisible light waves into the brain and forces cells to boost levels, improving blood flow too.

Dr Paul Chazot, who led the study, said: While more research is needed, there are promising signs that therapy involving infrared light might also be beneficial for people living with dementia and this is worth exploring.

ANYONE with asthma knows how debilitating it can be to receive a diagnosis.

Yet more than five million people in the UK are asthmatic. But a brand new drug, already approved for use on the NHS, is set to transform the lives of many with the condition, making attacks less frequent and less severe.

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Dupilumab is prescribed to treat eczema and rhinosinusitis a type of sinusitis where the nasal cavity as well as sinuses become inflamed.

Its from a family of drugs used to treat Covid.

Currently only patients with very serious asthma who have had at least four severe asthma attacks in the last year and are ineligible for other biological treatments will be considered for a prescription.

But the drug is set to change the lives of many asthma sufferers across the country.

HALF of us will get cancer at some point in our lives. But new jab Survivin could change the landscape dramatically, scientists say.

The first clinical trials are already under way, and the injection works to boost the bodys immune system. It supercharges the immune cells, helping them seek out and destroy cancerous cells while leaving healthy cells alone.

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Currently there are 36 terminally ill patients taking part in the trials, which are focused on ovarian, prostate and lung cancers.

Michelle Mitchell, chief executive of Cancer Research UK, said: Just this month we heard the HPV vaccine has likely prevented hundreds of women from developing cervical cancer.

This is a new and exciting frontier in cancer medicine and if this trial and others are successful, we could see thousands more lives saved.

AROUND seven per cent of all men are affected by infertility.

And while treatments currently focus on solutions rather than cures, scientists at the University of Georgia, in the US, are looking to reverse male infertility altogether.

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The researchers have used primate embryonic stem cells the building blocks of all cells in the body to grow sperm cells in the earlier stages of development in a petri dish.

These spermatids, which lack a head and tail for swimming, were capable of fertilising a rhesus macaque egg in vitro.

Lead researcher and associate professor Dr Charles Easley says: This is a major breakthrough towards producing stem cell-based therapies to treat male infertility in cases where the men do not produce any viable sperm cells.

It is the first step that shows this technology is potentially translatable.

GETTING through the blood/brain barrier to target treatments for brain cancer is complex.

But now a team of scientists in Toronto, Canada, have found a way to use ultrasound beams.

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They help open the barrier and can help facilitate drug delivery, which could change the way the disease is treated.

A trial this year saw four women with breast cancer that had spread to their brains treated with magnetic resonance-guided focused ultrasound (MRgFUS).

It allowed the antibody therapy herceptin to pass into their brain tissue, and caused the tumours to shrink without damaging any healthy tissue.

Dr Nir Lipsman, who led the study, said: It has long been theorised that focused ultrasound can be used to enhance drug delivery, but this is the first time we have shown we can get drugs into the brain.

A DRUG taken in pill form is to be trialled to combat the deadliest form of cancer.Auceliciclib is already used to treat brain tumours.

But now scientists hope it can help fight pancreatic cancer, which is often first diagnosed when it is at a late stage.

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Professor Shudong Wang and her team at the University of South Australia are also working on new ways to detect the disease.

She said: Pancreatic cancer is extremely difficult to diagnose at an early stage because there are very few symptoms.

If it is caught early the malignant tumour can be surgically removed, but once it spreads into other organs it is lethal.

Chemotherapy and radiotherapy only buy patients a little extra time.

The team hopes the drug will be more effective and with fewer side-effects than current treatment options.

THE heroic scientists who developed the Covid vaccine did not stop there.

The team at the University of Oxford has also developed a malaria jab that will save billions of lives.

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A trial showed 77 per cent of volunteers who were vaccinated stayed malaria-free over the following 12 months.

More than 100 malaria vaccines have been developed in recent decades, but the Oxford jab is the first to have such a high success rate.

Halidou Tinto, professor of parasitology and the principal investigator on the trial, said: These are very exciting results showing unprecedented efficacy levels from a vaccine that has been well-tolerated in our trial programme.

We look forward to the upcoming phase III trial to demonstrate large-scale safety and efficacy data for a vaccine that is greatly needed.

A DRUG that repairs cancerous cells could revolutionise the way breast cancer is treated.

Patients given olaparib as part of a two-and-a-half year trial were 42 per cent less likely to see their cancer return.

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There was also a 43 per cent dEcrease in the risk of the disease spreading.

Until the breakthrough earlier this year, the drug was mainly used for late-stage cancers, but the new findings suggest it is effective as an early treatment.

Professor Andrew Tutt, professor of oncology at the Institute of Cancer Research who led the study, said: Women with early-stage breast cancer who have inherited BRCA1 or BRCA2 mutations are typically diagnosed at a younger age.

Up to now, there has been no treatment that specifically targets the unique biology of these cancers to reduce the rate of recurrence, beyond initial treatment such as surgery.

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