The investigation drew on data from an earlier project involving 1,460 older women who were randomly assigned to receive either calcium supplements or a placebo for five years. Researchers found that the supplements did not raise the likelihood of dementia in the long term.

“Calcium supplements are often recommended to prevent or manage osteoporosis,” said ECU PhD student Ms. Negar Ghasemifard.

About 20 percent of women over 70 live with osteoporosis, and calcium is widely advised to help prevent bone fractures.

“Previous research has raised concerns around the impacts that calcium supplements could have on cognitive health, particularly dementia. Results from our study provides reassurance to patients and clinicians regarding the safety of calcium supplements in the context of dementia risk for older women,” Ms. Ghasemifard said.

According to ECU Senior Research Fellow Dr. Marc Sim, even after adjusting for supplement use, diet, lifestyle factors, and genetic risk, the outcomes did not change.

“Previous research suggesting potential links between calcium supplement use and the risk for dementia was purely observational in nature. Our research, in comparison, consisted of a post-hoc analysis from a 5-year double-blind, placebo controlled randomized clinical trial on calcium supplements to prevent fracture. Whilst our study is still epidemiology, its design does reduce the likelihood of unmeasured confounding”

“Some 730 older women were given calcium supplements over five years, and a further 730 were given placebo. This study design offers more accurate data on dosage and duration, and we had a long follow-up period of 14.5 years, which strengthens our results,” Dr. Sim said.

Although the findings suggest calcium does not increase the risk of dementia in older women, particularly those over 80, further studies are still needed, said Professor Simon Laws, Director of ECU’s Centre for Precision Health.

“Whether this extrapolates to other demographics, such as men or even women commencing supplementation earlier in life, remains unknown. To confirm the current findings, particularly regarding brain health, and to address these population gaps, future clinical trials of calcium supplements, with or without vitamin D, would need to be undertaken. These should include specific and robust assessments of brain health as the primary outcome measures.”

Professor Blossom Stephan, a Dementia Australia Honorary Medical Advisor said the research highlighted a very important finding that provides reassurance to clinicians and patients about the long-term safety of calcium supplementation.

“Given calcium’s critical role in multiple physiological functions, including bone health, these results provide reassurance that long-term calcium supplementation did not increase dementia risk in older women,” she said.

A team of EMBL researchers and collaborators has now created a tool that takes single-cell analysis to a new level. It can capture both genomic variations and RNA within the same cell, offering greater accuracy and scalability than earlier technologies. This approach allows scientists to identify variations in non-coding regions of DNA, the areas most often linked to disease, giving them a new way to explore how genetic differences contribute to human health. With its precision and ability to process large numbers of cells, the tool marks a major step toward linking specific genetic variants with disease outcomes.

“This has been a long-standing problem, as current single-cell methods to study DNA and RNA in the same cell have had limited throughput, lacked sensitivity, and are complicated,” said Dominik Lindenhofer, the lead author on a new paper about SDR-Seq published in Nature Methods and a postdoctoral fellow in EMBL’s Steinmetz Group. “On a single-cell level, you could read out variants in thousands of cells, but only if they had been expressed — so only from coded regions. Our tool works, irrespective of where variants are located, yielding single-cell numbers that enable analysis of complex samples.”

The important difference between coding and non-coding regions

DNA contains both coding and non-coding regions. The coding parts function like instruction manuals, since their genes are expressed into RNA, which directs cells in building proteins essential to life.

Non-coding regions, on the other hand, contain regulatory elements that guide how cells grow and function. Over 95% of disease-linked DNA variants occur in these non-coding regions, yet existing single-cell methods have not had the sensitivity or scale to study them effectively. Until now, researchers were unable to observe DNA and RNA from the same cell on a large scale, limiting insight into how DNA variants affect gene activity and contribute to disease.

“In this non-coding space, we know there are variants related to things like congenital heart disease, autism, and schizophrenia that are vastly unexplored, but these are certainly not the only diseases like this,” Lindenhofer said. “We needed a tool to do that exploration to understand which variants are functional in their endogenous genomic context and understand how they contribute to disease progression.”

Deciphering barcodes that track single cells

To perform single-cell DNA-RNA sequencing (SDR-seq), researchers used tiny oil-water droplets, each containing a single cell, allowing them to analyze DNA and RNA simultaneously. This method enabled them to examine thousands of cells in a single experiment and directly link genetic changes to patterns of gene activity. Developing this technology required overcoming major challenges and brought together teams from EMBL’s Genome Biology and Structural and Computational Biology units, the Stanford University School of Medicine, and Heidelberg University Hospital.

Collaborators from EMBL’s Judith Zaugg and Kyung-Min Noh groups developed a way to preserve delicate RNA by “fixing” the cells, while computational biologists in Oliver Stegle’s group designed a specialized program to decode the complex DNA barcoding system needed for data analysis. Although this decoding software was built for this specific project, the team believes it could prove valuable for many other studies.

Researchers from Wolfgang Huber’s and Sasha Dietrich’s groups at EMBL and Universitätsklinikum Heidelberg were already examining B-cell lymphoma samples for other studies. These patient samples, rich in genetic variation, provided an ideal test case for the new technology. Using these samples, Lindenhofer observed how variations in DNA were linked to disease processes and found that cancer cells with more variants showed stronger activation signals that support tumor growth.

“We are using these small reaction chambers to read out DNA and RNA in the same single cell,” Lindenhofer said. “This lets us accurately tell whether a variant is on one or both copies of a gene and measure its effects on gene expression in the same single cells. With the B-cell lymphoma cells, we were able to show that depending on the variant makeup of cells, they had different propensities to belong to distinct cellular states. We could also see that increasing variants in a cell actually were associated with a more malignant B-cell lymphoma state.”

The many opportunities from a single-cell sequencing tool

The SDR-seq tool now offers genomic biologists scale, precision, and speed to help better understand genetic variants. While it could eventually play a role in treating a broad range of complex diseases, it may first help in developing better screening tools for diagnosis.

“We have a tool that can link variants to disease,” said Lars Steinmetz, a senior author on the paper, an EMBL group leader, and a genetics professor at Stanford University School of Medicine. “This capability opens up a wide range of biology that we can now discover. If we can discern how variants actually regulate disease and understand that disease process better, it means we have a better opportunity to intervene and treat it.”

To explore why liver cancer responds poorly to immunotherapy, scientists at the Salk Institute examined how the immune system interacts with the liver. Using both mouse models and human tumor samples, they discovered that certain bile acids — molecules produced by the liver to aid digestion — can interfere with cancer-fighting immune cells known as T cells.

The team pinpointed several bile acids linked to weakened T cell function and faster tumor growth. By blocking the production of these acids, they were able to slow or stop tumor progression. One bile acid, called ursodeoxycholic acid (UDCA), had the opposite effect, enhancing T cell activity in the liver. When researchers increased UDCA levels through dietary supplements, liver tumors in mice shrank. Because UDCA supplements are already approved for other liver diseases, scientists believe they could potentially make immunotherapy more effective for liver cancer patients.

The study, published in Science, sheds light on why immune cells behave differently depending on the tumor’s location and identifies new molecular targets to strengthen liver cancer therapies.

“How do organ-specific properties and processes influence the immune response?” asks Professor Susan Kaech, senior author of the study and director of Salk’s NOMIS Center for Immunobiology and Microbial Pathogenesis. “Livers have a particularly unique environment, but we didn’t really understand how it was affecting the immune and cancer cells. By investigating these liver-specific features, we have identified several potential ways to regulate bile acids, improve T cell performance, and enhance patient outcomes.”

The liver generates more than 100 types of bile acids, which travel through the intestines to help digest fats. To combat liver cancer, T cells must function effectively within this chemically rich environment. Past studies have linked high bile acid levels to poor health and cancer progression, but researchers had not previously distinguished the effects of individual bile acids.

“Considering how T cell performance varies across different organs, tissues, and tumors puts us at a great vantage point for looking at ways to optimize cancer treatment,” says Siva Karthik Varanasi, former postdoctoral researcher in Kaech’s lab and current assistant professor at the University of Massachusetts Chan Medical School. “By taking this unique approach, we’re able to see that bile acids in the liver are hugely influencing T cells’ ability to do their job and therefore may be a useful therapeutic target.”

To better understand these effects, the Salk team first analyzed human liver cancer biopsies to identify which bile acids were present. They found elevated levels of conjugated bile acids and tested whether these compounds contributed to tumor growth. When they removed a protein called BAAT, which produces conjugated bile acids, the tumor load in mice dropped significantly. This suggests that adjusting BAAT activity in humans could improve their response to immunotherapy.

The researchers then examined 20 distinct bile acids to determine how each affected T cells. Most primary bile acids showed little influence, except for one called TCDCA, which triggered oxidative stress — a harmful molecular imbalance. Secondary bile acids had much stronger effects. One, called LCA, damaged T cell function by causing endoplasmic reticulum stress, while another, UDCA, boosted T cell performance and drew more immune cells to the liver. Increasing UDCA levels through supplementation effectively reduced tumor growth in mice, pointing to a promising strategy for enhancing immunotherapy in liver cancer.

Together, these results suggest that lowering BAAT and increasing UDCA could help control liver tumor growth and strengthen the immune system’s response to treatment.

“We’re already a huge step ahead when it comes to translating our findings to the clinic, because UDCA supplementation is already used to treat liver disease and could easily be tested in liver cancer next,” says Kaech, who also holds the NOMIS Chair at Salk. “We are really excited to also explore the role of the gut microbiome in all of this, since bile acids are a huge part of that picture — how can we manipulate ‘good’ and ‘bad’ bacteria in the microbiome to further regulate bile acid levels? How does the microbiome change during liver cancer? Could probiotics be a therapeutic approach?”

In addition to exploring dietary and microbiome manipulations that could help with liver cancer, the team is curious to see if other conditions could be treated by targeting BAAT. Already, they believe chronic liver disease and obesity may benefit from the same reduction of conjugated bile acids.

Other authors include Dan Chen, Melissa Johnson, Kathryn Lande, Michael LaPorta, Filipe Hoffmann, Thomas Mann, Eduardo Casillas, Kailash Mangalhara, Varsha Mathew, Ming Sun, Yagmur Farsakoglu, Timothy Chen, Bianca Parisi, Shaunak Deota, H. Kay Chung, Satchidananda Panda, April Williams, and Gerald Shadel of Salk; Jin Lee, Yingluo Liu, Cayla Miller, and Gen-Sheng Feng of UC San Diego; Souradipta Ganguly and Debanjan Dhar of UC San Diego and Sanford Burnham Prebys Medical Discovery Institute; Marcos Teneche, Aaron Havas, and Peter Adams of Sanford Burnham Prebys Medical Discovery Institute; Isaac Jensen and Donna Farber of Columbia University; Andrea Schietinger of Memorial Sloan Kettering Cancer Center, Weill Cornell Graduate School of Medical Sciences, and Parker Institute for Cancer Immunotherapy; and Mark Sundrud of Dartmouth College.

The work was supported by the National Institutes of Health (NCI CCSG: P30 014195, S10-OD023689, P30 AG068635, P30 CA014195, P01 AG073084, R01 CA240909-04, R21 AI151562, F31CA278581, CCSG Grant P30CA23100, R01DK137061, R01DK133930, DK120515, R01AI143821, R01AI164772, U01AI163063), Waitt Foundation, Helmsley Charitable Trust, Chapman Foundation, Cancer Research Institute, National Cancer Center, NOMIS Foundation, Salkexcellerators Fellowship, Damon Runyon Fellowship, Audrey Geisel endowed Chair of Biomedical Science, Altman Clinical Translational Research Institute (KL2TR001444), San Diego Digestive Diseases Research Center, and Dartmouth Cancer Center.