In our third and final installment of the Promega qPCR Grant Recipient blog series, we highlight Dr. Sabrina Alves dos Reis, a trained immunotherapy researcher. Her work has focused on developing tools for more accessible cancer therapies using CAR-T cells. Here, we explore Dr. Alves dos Reis’ academic and scientific journeys, highlight influential mentorship and foreshadow her plans for the Promega qPCR grant funds.
Dr. Alves dos Reis’ career began with a strong affinity for biology. As an undergraduate student, she pursued a degree in biological science, where she developed a foundational understanding for designing and developing research projects. As her passion for science heightened, she decided to continue her journey in science, culminating in a PhD at the Fundação Oswaldo Cruz Institute in Rio de Janeiro, Brazil. Her research projects focused on the unexplored territory of adipose tissue as a site for Mycobacterium leprae—or leprosy bacillus—infection. She mentioned that this work piqued her curiosity for improving immunotherapies and laid the foundation for her future in cancer research.
Antimicrobial resistance (AMR) threatens the effective prevention and treatment of an ever-increasing range of infections. It’s a leading mortality factor worldwide, but the newly discovered antibiotic, clovibactin, may offer a pivotal solution. It effectively kills drug-resistant bacterial pathogens without detectable resistance—even multidrug-resistant “superbugs.”
Loss of life and serious illness from contamination of manufactured products that are consumed as food or used in medical procedures illustrate the need to prevent contamination events rather than merely detect them after the fact. High-profile news stories have described contamination events in compounding pharmacies (1), food processing and packaging plants (2) and medical device manufacturers (3). Although contamination in manufacturing settings can be physical, chemical, or biological, this article will focus environmental monitoring to determine the quality of a manufacturing facility with respect to microbial contamination.
To ensure that the products they produce and package are manufactured in a high-quality, contaminant-free environment, many industries are required to establish routine environmental monitoring programs. Samples are collected from all potential sources of contamination in the production environment including air, surfaces, water supplies and people. Routine monitoring is essential to detect trends such as increases in potential pathogens over time or the appearance of new species that have not been seen before so that contamination events can be prevented.
Because environmental monitoring requires identification to the level of the species, most environmental monitoring programs will collect samples and then send them off to a facility to be sequenced for genomic identification of any microbial species. Such genotypic analysis involves DNA sequencing of ribosomal RNA (rRNA) genes to determine the taxonomic classification of bacteria and fungi. In this method, informative sections of the rRNA genes are amplified by PCR; the PCR products sequenced; the sequence is compared to reference libraries; and the results interpreted to make a species-level identification for a given microbial isolate.
Mosquitos are the deadliest animal on earth—not because of the itchy bites they leave behind, but because of the diseases those bites can spread. Of these diseases, malaria, is the most widespread, killing 619,000 people in 2021 (1). Almost half of the world’s population live at risk of malaria (2). In humans, malaria is caused by certain species of single-cell micro-organisms belonging to the genus Plasmodium (3), which are transmitted by anopheline mosquitos.
Controlling malaria has proven challenging. Vaccines have yielded incomplete protection, and insecticides that once were successful at control mosquito populations are becoming less effective as the insects develop resistance. Finally, Plasmodium parasites themselves have developed resistance to leading anti-malaria drugs (2).
A New Weapon In The Fight Against Malaria
Approaches that target the disease-causing Plasmodium organisms—inside the mosquito and before they are transmitted to humans—could provide as effective way forward. In the past, researchers have explored leveraging genetically modified bacterium to kill or inhibit Plasmodium development within their mosquito host. However, using genetically altered bacteria makes wide-spread adoption of these techniques problematic. A recent study published in Science describes the discovery and early investigative results using a naturally occurring bacterial strain that inhibits Plasmodium spread (2). The bacteria, Delftia tsuruhatensis TC1, was isolated from a mosquito population that unexpectedly became resistant to Plasmodium infection (2).
Once the bacterium was identified as the cause of Plasmodium inhibition, the researchers tested how easily the bacteria was to introduce into naïve mosquitos and how effective it was at disrupting infection. To do this, they colonized female mosquitos by feeding them a sugar and bacterium solution and then Plasmodium-infected blood. Bacterial colonization occurred in almost all the mosquitos offered the sugar and bacterium food. Initially, bacterial colonization numbers were low, but they increased 100-fold following the blood meal.
Inhibiting Oocyte Formation Disrupts Cycle of Infection
Investigation into how D. tsuruhatensis inhibits Plasmodium infection showed that it inhibits oocyte formation within the gut, and this inhibition lasts for at least 16 days. Specifically, the inhibition is the result of a secreted compound called harmane, which is a small hydrophobic methylated b-carboline (2). When harmane is secreted in the guts of mosquitos it inhibits Plasmodium parasite development. The researchers further found that feeding harmane alone to mosquitos, or allowing it to be absorbed through direct contact produced the same results, but the inhibitory effects only lasted a few days (2).
No matter how harmane is introduced into the gut (directly or through bacterial colonization), the inhibition of oocyte formation results in a decrease in infectivity. Only one third (33%) of mice bitten by Plasmodium-infected, D. tsuruhatensis-colonized mosquitos become infected. This contrasts sharply with the 100% infection rate seen with mice bitten by non-colonized, Plasmodium-infected mosquitos (2). Further testing the researchers also showed that D. tsuruhatensis is not transferred during feeding, suggesting that that bacterium is unlikely to in introduced into mammals through colonized mosquitos.
To investigate how colonization and infection rates would correlate in a ‘real world’ environment, the researchers used a large (10 × 10 × 5 meter) enclosure that replicated the mosquitos’ natural environment. Once again, the mosquitos were colonized with D. tsuruhatensis through overnight feeding of the sugar and bacterium solution. They found ~75% of the mosquitos were colonized by D. tsuruhatensis in this time period.They also found that larvae reared in water seeded with D. tsuruhatensis experienced 100% colonization. In both scenarios, Plasmodium oocyte development was disrupted just as it had been in the laboratory-raise population (2).
Finally, the researchers found that D. tsuruhatensis colonization doesn’t occur between individuals between parent and offspring. For controlling Plasmodium, this means that inoculation with D. tsuruhatensis would require ongoing maintenance. However, it also decreases the risk of a contaminated strain being amplified uncontrollably if released, making it less risky.
Malaria mitigation and control requires a multipronged effort. Using naturally occurring, symbiotic, microbes such as D. tsuruhatensis is one approach that shows promise. There is still a lot of work to be done before this bacterium could be used outside of a controlled environment, including understanding how the bacterium might interact with other plants and animals from the same ecosystem.
According to the National Human Genome Research Institute, synthetic biology is “a field of science that involves redesigning organisms for useful purposes by engineering them to have new abilities”. Synthetic biology has a broad range of applications, from manufacturing pharmaceuticals and other biologically active chemicals and biofuels, to accelerating the adoption of plant-based burgers (1).
At the heart of the synthetic biology revolution is the rapid technological advancement—and accompanying drop in costs—of DNA oligonucleotide synthesis. Typically, synthetic biology researchers use oligonucleotides as building blocks to assemble genes of interest that are then introduced into, and expressed by, a different organism. For example, to create the plant-based Impossible Burger, the soy leghemoglobin gene (normally found in the root nodules of leguminous plants) was synthesized and expressed in yeast cells (1). This component gives the burger its meaty flavor and appearance of “bleeding” when cooked.
On June 15, 2023, we announced the winners of the 2023 Promega iGEM grant. Sixty-five teams submitted applications prior to the deadline with projects ranging from creating a biosensor to detect water pollution to solving limitations for CAR-T therapy in solid tumors. The teams are asking tough questions and providing thoughtful answers as they work to tackle global problems with synthetic biology solutions. Unfortunately, we could only award nine grants. Below are summaries of the problems this year’s Promega grant winners are addressing.
The UCSC iGEM team from the University of California–Santa Cruz is seeking a solution to mitigate the harmful algal blooms caused by Microcystisaeruginosa in Pinto Lake, which is located in the center of a disadvantaged community and is a water source for crop irrigation. By engineering an organism to produce microcystin degrading enzymes found in certain Sphingopyxis bacteria, the goal is to reduce microcystin toxin levels in the water. The project involves isolating the genes of interest, testing their efficacy in E. coli, evaluating enzyme production and product degradation, and ultimately transforming all three genes into a single organism. The approach of in-situ enzyme production offers a potential solution without introducing modified organisms into the environment, as the enzymes naturally degrade over time.
Endometriosis is a condition that affects roughly 190 million (10%) women of reproductive age worldwide. Currently, there is no treatment for endometriosis except surgery and hormonal therapy, and both approaches have limitations. The IISc-Bengaluru team at the Indian Institute of Science, Bengaluru, India, received 2023 Promega iGEM grant support to investigate the inflammatory nature of endometriosis by targeting IL-8 (interleukin-8) a cytokine. Research by other groups has snow that targeting IL-8 can reduce endometriotic tissue. This team will be attempting to create an mRNA vaccine to introduce mRNA for antibody against IL-8 into affected tissue. The team is devising a new delivery mechanism using aptides to maximize the delivery of the vaccine to the affected tissues.
Global pandemics, such as COVID-19, have taught us to abhor viruses. The emergence of new, highly infectious viruses is—rightfully so—a cause for concern. However, despite the average human body harboring 380 trillion viruses, most of them simply coexist with us and are harmless. When it comes to an ancient lineage of viruses within the realm Duplodnaviria, researchers are even using them as weapons in the battle against infectious diseases.
In 1915, Frederick William Twort, an English bacteriologist at the University of London, reported the discovery of an unusual “ultramicroscopic virus” (1). Twort was culturing vaccinia virus as part of an experiment to determine if he could prepare smallpox vaccines in vitro. These vaccines, made in calves, were typically contaminated with Staphylococcus bacteria. When Twort plated the vaccines, he found small, clear areas on the agar plates where the bacteria would not grow, and these clear areas were the source of his ultramicroscopic virus. Two years later, a French-Canadian microbiologist, Félix d’Hérelle, independently discovered a similar phenomenon when culturing Shigella bacteria from fecal samples of patients with bacillary dysentery. He called the new virus “un bactériophage obligatoire” (2). Shortly after his discovery, he found that bacteriophages (phages) could be used as powerful agents to treat a variety of bacterial infections, and the field of phage therapy was born (3).
Yellowstone National Park —located partially in Idaho, Montana and Wyoming—puts modern volcanic activity on full display. Near boiling, ominous pools of water in the form of geysers, mud pots, fumaroles (vents that release steam) and hot springs are all present and active in the park and visitors flock to the park to view a handful of thermal features every year during the peak summer visitor season. Coincidentally, this is when a large portion of scientific research also takes place at the park. Combining both the boardwalk paths that are open to all who visit the park and the expansive backcountry, Yellowstone is host to over 10,000 thermal features. These thermal features are fed by superheated water that travels through a complex groundwater system—think the pipes under your kitchen sink—where subsurface water collects gases and chemical compounds en route to the surface. As a result, near-boiling water that bubbles through to the surface is often rife with chemicals like sulfur, iron or magnesium. Early scientists thought of hot springs as uninhabitable, but as it turns out, these conditions are just the right environment for thermophilic (or “heat-loving”) bacteria to thrive.
Our world is a complex, interdependent system, and invertebrate pollinators such as honeybees play a pivotal role in its survival. Threats to populations numbers of pollinators like honeybees can be equated to threats to the overall health and survival of the ecosystem in which they live. Of the over 20,000 known bee species, one—the western honeybee (Apis mellifera)—acts as the single most frequent pollinator for crops worldwide (1). Found on every continent except Antarctica, the western honeybee owes its status as a top pollinator to its widespread geographic distribution, generalist foraging behavior and competence as pollinators (1).
Deadly American Foulbrood Disease
Honeybees are the most economically valuable pollinators and are threatened by several pathogens (2). Perhaps the biggest threat to honeybee colony health and survival is the bacterial disease, American Foulbrood (AFB; (3). Caused by the spore-forming, Gram+ bacteria, Paenibacillus larvae, the highly contagious AFB disease affects the young brood of colonies. When newly hatched larvae are fed spore-contaminated food, the spores germinate and replicate causing septicemia and death. P. larvae spores are incredibly resilient and can remain viable for decades (3). Each infected larva can produce over 1 billion new spores. Thus, a colony can produce large numbers of spores with just a few cases of symptomatic brood (4).
Roses, the universal symbol of love and affection, are one of the most popular ornamental flowering shrubs used by landscapers and home gardeners and account for almost half of the billion-dollar ornamental plant market. The growing prevalence of rose rosette disease poses a significant threat to these industries. This lethal disease is caused by the Rose rosette emaravirus (RRV) and transmitted by the tiny eriophyid mite, Phyllocoptes fructiphilus. Infection by RRV results in prolific growth of clustered and bunched plant shoots (witches’ broom), malformed flowers and leaves, malformed shoots and enlarged stems and abundant leaf growth and thorniness. This excessive growth depletes the plant’s energy, eventually causing death.
Emerging and Devastating Plant Viruses of the Genus Emaravirus
RRV is a single-stranded, segmented, negative-sense RNA virus belonging to the genus Emaravirus, a relatively new genus that was established in 2012. These emerging viruses can be devastating to trees, herbaceous woody plants and vines. At Texas A&M University, Dr. Jeanmarie Verchot’s lab is working to better characterize and understand these new viruses. In addition to threatening roses, these viruses cause damage to important agriculture crops such as wheat and pigeon peas. They also endanger sensitive ecosystems when they infect plants specialized to a particular habitat, as is the case with Palo verde broom virus infection of palo verde trees of the Sonoran Desert (1).
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