This blog originated as a press release from Notre Dame News.
As testing for the coronavirus continues throughout the United States, researchers have been closely watching results, particularly reported rates of false negatives.
According to the Radiological Society of North America, a reported 40 to 70 percent of coronavirus tests from throat swab samples returned false negatives at the onset of the epidemic. Given the highly infectious nature of this particular coronavirus, individuals receiving false negative results — told they do not carry the virus when in fact they do — could continue to infect others.
“It is very concerning,” said Hsueh-Chia Chang, the Bayer Professor of Chemical and Biomolecular Engineering at the University of Notre Dame. “In an overcrowded hospital, where there is only room to quarantine the COVID-19 carriers, false negatives would mean some carriers can continue to infect other patients and healthcare workers. This, unfortunately, is also true for other infectious viral diseases such as dengue and malaria, when there is an epidemic. False negatives are usually not an urgent problem, when every symptomatic patient can be quarantined and there are fewer people to infect — until an epidemic overcrowds our hospitals and we have only enough space to sequester the carriers.”
At Notre Dame, Chang’s research lab focuses on the development of new diagnostic and micro/nanofluidic devices that are portable, sensitive and fast. His work includes diagnostics with applications to DNA/RNA sensing. Current coronavirus tests are RNA-based.
Chang said technology his lab developed for other uses could easily be extended to apply to testing for the coronavirus.
“I had developed the technology for isolating cellular material such as vesicles and exosomes during liquid biopsies. They turn out to be the same size as the virus,” he said.
|Dr. Chang at the ERCC2 kickoff meeting in September 2019 discussing his work developing technology to isolate extracellular vesicles.|
The tests combine nanofiltration with immersed AC Electrospray (iACE) digital droplet isothermal polymerase chain reaction (PCR) technology. The nanofiltration part of the test would work to wash away inhibitors while the iACE would allow detection of a very small number of the coronavirus viral particles per sample, improving sensitivity during testing.
Detection can be inhibited at the molecular level, Chang explained. The current tests for coronavirus are PCR-based, a common method that replicates a small sample of RNA — from a nose or throat swab, for example —increasing the number of RNA exponentially in order to identify the presence of the virus and determine the stage of infection.
“The inhibitors, in this case molecules and ions, prevent the reaction from occurring even when the target virus is there, resulting in a false negative,” said Chang. “Our technology removes these inhibitors. There is also the question of yield. In removing the inhibitors, you do not want to lose the target virus as well, so they escape detection. Our technology achieves higher yield in retaining the virus. It extracts the target virus with higher yield and purity than current technology.”
His size-based nanotechnology is especially useful in this case. The coronavirus is between 60 and 140 nm in size. The inhibiting molecules, Chang explained, are smaller than 60 nm, which means he can effectively wash away those particles while retaining the virus.
“The issue is that such small particles often cause clogging and produce high pressure during tests, and break up virus particles, so they’re lost to detection. This is one cause for false negatives,” Chang said. “We already have a patented design that allows filtration of the virus from inhibitors without clogging and without breaking the target virus particles.”
Notre Dame has suspended laboratory research operations across campus with the exception of coronavirus-related research. Chang’s lab is one that received approval to remain operational. Researchers in his lab are not currently working with samples that contain the coronavirus, rather they are testing the technology against a lentivirus serum — a virus that is similar but safe to work with.
“I’m fortunate to have very passionate and capable postdoctoral and Ph.D. students that believe in these technologies and are willing to be in the lab during these trying times,” he said. “Their presence is completely voluntary. In fact, we reduced the number of researchers to three essential people even though several more had volunteered. They abide by very stringent social distancing and lab hygiene rules. They also work in shifts to minimize contact. Another research professor and I are in constant email and cellular communication with them. They are currently testing lentivirus in saliva samples and trying to get more data to back up the numbers.”
The numbers, so far, show that Chang’s test combining nanofiltration with iACE technology are 1,800 times more sensitive in tests run with the lentivirus.
If additional grant funding is approved for his research, Chang said he intends to work with the Centers for Disease Control and Prevention or other Food and Drug Administration approved labs to validate the technology with actual samples containing the coronavirus.
In a white paper outlining the research, Chang set milestones for the work with hopes —if approved — to begin manufacturing devices in six months. However, given the current state of the pandemic, Chang said realistically the technology would be used in cases of future epidemics and outbreaks.
“I think the country is realizing the need for better control of infectious epidemics,” he said. “We hope to develop technology that will help control future epidemics involving any virus or bacteria, not just in the U.S., but especially in the developing world.”
This blog originated as a press release from UCSD News.
Researchers at the University of California San Diego discovered that high blood levels of RNA produced by the PHGDH gene could serve as a biomarker for early detection of Alzheimer’s disease. The work could lead to the development of a blood test to identify individuals who will develop the disease years before they show symptoms.
The team published their findings in Current Biology.
The PHGDH gene produces RNA and proteins that are critical for brain development and function in infants, children, and adolescents. As people get older, the gene typically ramps down its production of these RNAs and proteins. The new study, led by Sheng Zhong, a professor of bioengineering at the UC San Diego Jacobs School of Engineering in collaboration with Dr. Edward Koo, a professor of neuroscience at the UC San Diego School of Medicine, suggests that overproduction of extracellular RNA (exRNA) by the PHGDH gene in the elderly could provide an early warning sign of Alzheimer’s disease.
“Several known changes associated with Alzheimer’s disease usually show up around the time of clinical diagnosis, which is a little too late. We had a hunch that there is a molecular predictor that would show up years before, and that’s what motivated this study,” Zhong said.
The discovery was made possible thanks to a technique developed by Zhong and colleagues that is sensitive enough to sequence tens of thousands of exRNAs in less than one drop of blood. The method, dubbed SILVER-SEQ, was used to analyze the exRNA profiles in blood samples of 35 elderly individuals 70 years and older who were monitored up to 15 years prior to death. The subjects consisted of 15 patients with Alzheimer’s disease; 11 “converters,” which are subjects who were initially healthy then later developed Alzheimer’s; and 9 healthy controls. Clinical diagnoses were confirmed by analysis of post-mortem brain tissue.
The results showed a steep increase in PHGDH exRNA production in all converters approximately two years before they were clinically diagnosed with Alzheimer’s. PHGDH exRNA levels were on average higher in Alzheimer’s patients. They did not exhibit an increasing trend in the controls, except for in one control that became classified as a converter.
The researchers note some uncertainty regarding the anomalous converter. Since the subject died sometime during the 15-year monitoring, it is unclear whether that individual would have indeed developed Alzheimer’s if he or she lived longer, Zhong said.
The team acknowledges additional limitations of the study.
“This is a retrospective study based on clinical follow-ups from the past, not a randomized clinical trial on a larger sample size. So we are not yet calling this a verified blood test for Alzheimer’s disease,” said co-first author Zixu Zhou, a bioengineering alumnus from Zhong’s lab who is now at Genemo Inc., a startup founded by Zhong. “Nevertheless, our data, which were from clinically collected samples, strongly support the discovery of a biomarker for predicting the development of Alzheimer’s disease.”
In addition to randomized trials, future studies will include testing if the PHGDH biomarker can be used to identify patients who will respond to drugs for Alzheimer’s disease.
The team is also open to collaborating with Alzheimer’s research groups that might be interested in testing and validating this biomarker.
“If our results can be replicated by other centers and expanded to more cases, then it suggests that there are biomarkers outside of the brain that are altered before clinical disease onset and that these changes also predict the possible onset or development of Alzheimer’s disease,” Koo said. “If this PDGDH signal is shown to be accurate, it can be quite informative for diagnosis and even treatment response for Alzheimer’s research.”
This study was performed in collaboration with Genemo Inc.
Yan Z*, Zhou Z*, Wu Q*, et al. Presymptomatic increase of an extracellular RNA in blood plasma associates with the development of Alzheimer’s disease. Curr Biol (2020) AOP. doi: 10.1016/j.cub.2020.02.084 PMID: 32220323.
*These authors contributed equally
Extracellular vesicles (EVs) regulate many processes in the healthy body. They also play a role in cancer, sending signals between cells in the tumor microenvironment. EVs can stimulate tumor cell migration, invasion, blood vessel growth, immune response, and cell survival, as well as metastasis. However, we know little about the cargo of these EVs that play such diverse roles. Analysis of vesicle cargo can shed light on the molecular mechanisms of vesicle biology and be helpful in disease diagnosis and prognosis.
I am lucky to be a member in Jan Lötvall’s lab in Gothenburg, Sweden, which pioneered the field of extracellular vesicles with the early discovery of exosomes shuttling RNA between cells. An exciting collaboration with Yong Song Gho from POSTECH in South Korea led us to develop a new approach to isolate vesicles from human tumor tissues. Using this technology, we were able to isolate and characterize subpopulations of extracellular vesicles from melanoma metastatic tissue. We just published our findings in the Journal of Extracellular Vesicles. Jan Lötvall also discussed them in a recent ERCC webinar.
Finding extracellular vesicles in tumor tissue with TEM
Our first challenge was to find vesicles in metastatic melanoma tumor tissues. Using transmission electron microscopy, we showed that the tumor microenvironment is a complex world composed of different types of cells and structures with vesicles present between them.
Transmission electron micrograph of melanoma metastatic tissue showing a large tumor cell and two lymphocytes. Black stain, possibly melanin, is clearly visible inside the melanoma cells, which are recognizable from their characteristic cell membrane. The higher magnification image shows vesicles (red arrows) in the extracellular space.
In this study, we performed a detailed proteomics analysis of EVs isolated from metastatic melanoma tissues from 27 patients. We identified numerous new EV proteins, including potential biomarkers for metastatic melanoma.
Tumor tissue vs. Cell lines
Studying extracellular vesicles in tumor tissues is important, because, compared to cell lines, tumor tissues more closely approximate the situation in vivo. EVs from tumor tissue are more likely to represent the full array of vesicle behaviors and populations in the tumor microenvironment. Furthermore, to develop a non-invasive test for cancer, we must use biofluids such as circulating plasma, where vesicles from all over the body intermingle. A proteomic snapshot of vesicles isolated directly from tumor tissue can help target the search for disease-specific biomarker in that complex mixture. We trust the tools and experiments developed in this work will contribute to our field’s understanding of EV function in complicated tissues such as the metastatic melanoma tumor.
Crescitelli R, Lässer C, Jang SC, Cvjetkovic A, Malmhäll C, Karimi N, Höög J.L, Johansson I, Fuchs J, Thorsell A, Gho YS, R, Olofsson Bagge R, Lötvall J Subpopulations of extracellular vesicles from human metastatic melanoma tissue identified by quantitative proteomics after optimized isolation. Journal of Extracellular Vesicles 9:1, 1722433 doi: 10.1080/20013078.2020.1722433.
This work was supported by the Swedish Research Council (K2014-85X-22504-01-3), the Swedish Heart and Lung Foundation (20120528), the Swedish Cancer Foundation (CAN2014/844), and the Knut och Alice Wallenberg Foundation (Wallenberg Centre for Molecular and Translational Medicine, University of Gothenburg, Sweden).