Tag Archives: antibiotic resistance

Teaching molecular and synthetic biology in grades K-12

This* story actually started in 2018 with an August 1, 2018 Harvard University news release (h/t Aug. 1, 2018 news item on phys.org) by Leslie Brownell announcing molecular and synthetic biology educational kits that been tested in the classroom. (In 2019, a new kit was released but more about that later.)

As biologists have probed deeper into the molecular and genetic underpinnings of life, K-12 schools have struggled to provide a curriculum that reflects those advances. Hands-on learning is known to be more engaging and effective for teaching science to students, but even the most basic molecular and synthetic biology experiments require equipment far beyond an average classroom’s budget, and often involve the use of bacteria and other substances that can be difficult to manage outside a controlled lab setting.

Now, a collaboration between the Wyss Institute at Harvard University, MIT [Massachusetts Institute of Technology], and Northwestern University has developed BioBits, new educational biology kits that use freeze-dried cell-free (FD-CF) reactions to enable students to perform a range of simple, hands-on biological experiments. The BioBits kits introduce molecular and synthetic biology concepts without the need for specialized lab equipment, at a fraction of the cost of current standard experimental designs. The kits are described in two papers published in Science Advances [2018].

“The main motivation in developing these kits was to give students fun activities that allow them to actually see, smell, and touch the outcomes of the biological reactions they’re doing at the molecular level,” said Ally Huang, a co-first author on both papers who is an MIT graduate student in the lab of Wyss Founding Core Faculty member Jim Collins, Ph.D. “My hope is that they will inspire more kids to consider a career in STEM [science, technology, engineering, and math] and, more generally, give all students a basic understanding of how biology works, because they may one day have to make personal or policy decisions based on modern science.”

Synthetic and molecular biology frequently make use of the cellular machinery found in E. coli bacteria to produce a desired protein. But this system requires that the bacteria be kept alive and contained for an extended period of time, and involves several complicated preparation and processing steps. The FD-CF reactions pioneered in Collins’ lab for molecular manufacturing, when combined with innovations from the lab of Michael Jewett, Ph.D. at Northwestern University, offer a solution to this problem by removing bacteria from the equation altogether.

“You can think of it like opening the hood of a car and taking the engine out: we’ve taken the ‘engine’ that drives protein production out of a bacterial cell and given it the fuel it needs, including ribosomes and amino acids, to create proteins from DNA outside of the bacteria itself,” explained Jewett, who is the Charles Deering McCormick Professor of Teaching Excellence at Northwestern University’s McCormick School of Engineering and co-director of Northwestern’s Center for Synthetic Biology, and co-corresponding author of both papers. This collection of molecular machinery is then freeze-dried into pellets so that it becomes shelf-stable at room temperature. To initiate the transcription of DNA into RNA and the translation of that RNA into a protein, a student just needs to add the desired DNA and water to the freeze-dried pellets.

The researchers designed a range of molecular experiments that can be performed using this system, and coupled each of them to a signal that the students can easily detect with their sense of sight, smell, or touch. The first, called BioBits Bright, contains six different freeze-dried DNA templates that each encode a protein that fluoresces a different color when illuminated with blue light. To produce the proteins, students simply add these DNA templates and water to the FD-CF machinery and put the reactions in an inexpensive incubator (~$30) for several hours, and then view them with a blue light illuminator (~$15). The students can also design their own experiments to produce a desired collection of colors that they can then arrange into a visual image, a bit like using a Light Brite ©. “Challenging the students to build their own in vitro synthetic programs also allows educators to start to talk about how synthetic biologists might control biology to make important products, such as medicines or chemicals,” explained Jessica Stark, an NSF Graduate Research Fellow in the Jewett lab at Northwestern University who is co-first author on both papers.

An expansion of the BioBits Bright kit, called BioBits Explorer, includes experiments that engage the senses of smell and touch and allow students to probe their environment using designer synthetic biosensors. In the first experiment, the FD-CF reaction pellets contain a gene that drives the conversion of isoamyl alcohol to isoamyl acetate, a compound that produces a strong banana odor. In the second experiment, the FD-CF reactions contain a gene coding for the enzyme sortase, which recognizes and links specific segments of proteins in a liquid solution together to form a squishy, semi-solid hydrogel, which the students can touch and manipulate. The third module uses another Wyss technology, the toehold switch sensor, to identify DNA extracted from a banana or a kiwi. The sensors are hairpin-shaped RNA molecules designed such that when they bind to a “trigger” RNA, they spring open and reveal a genetic sequence that produces a fluorescent protein. When fruit DNA is added to the sensor-containing FD-CF pellets, only the sensors that are designed to open in the presence of each fruit’s RNA will produce the fluorescent protein.

The researchers tested their BioBits kits in the Chicago Public School system, and demonstrated that students and teachers were able to perform the experiments in the kits with the same success as trained synthetic biology researchers. In addition to refining the kits’ design so that they can one day provide them to classrooms around the world, the authors hope to create an open-source online database where teachers and students can share their results and ideas for ways to modify the kits to explore different biological questions.

“Synthetic biology is going to be one of the defining technologies of the century, and yet it has been challenging to teach the fundamental concepts of the field in K-12 classrooms given that such efforts often require expensive, complicated equipment,” said Collins, who is a co-corresponding author of both papers and also the Termeer Professor of Medical Engineering & Science at MIT. “We show that it is possible to use freeze-dried, cell-free extracts along with freeze-dried synthetic biology components to conduct innovative educational experiments in classrooms and other low-resource settings. The BioBits kits enable us to expose young kids, older kids, and even adults to the wonders of synthetic biology and, as a result, are poised to transform science education and society.

“All scientists are passionate about what they do, and we are frustrated by the difficulty our educational system has had in inciting a similar level of passion in young people. This BioBits project demonstrates the kind of out-of-the-box thinking and refusal to accept the status quo that we value and cultivate at the Wyss Institute, and we all hope it will stimulate young people to be intrigued by science,” said Wyss Institute Founding Director Donald Ingber, M.D., Ph.D., who is also the Judah Folkman Professor of Vascular Biology at Harvard Medical School (HMS) and the Vascular Biology Program at Boston Children’s Hospital, as well as Professor of Bioengineering at Harvard’s John A. Paulson School of Engineering and Applied Sciences (SEAS). “It’s exciting to see this project move forward and become available to biology classrooms worldwide and, hopefully some of these students will pursue a path in science because of their experience.”

Additional authors of the papers include Peter Nguyen, Ph.D., Nina Donghia, and Tom Ferrante from the Wyss Institute; Melissa Takahashi, Ph.D. and Aaron Dy from MIT; Karen Hsu and Rachel Dubner from Northwestern University; Keith Pardee, Ph.D., Assistant Professor at the University of Toronto; and a number of teachers and students in the Chicago school system including: Mary Anderson, Ada Kanapskyte, Quinn Mucha, Jessica Packett, Palak Patel, Richa Patel, Deema Qaq, Tyler Zondor, Julie Burke, Tom Martinez, Ashlee Miller-Berry, Aparna Puppala, Kara Reichert, Miriam Schmid, Lance Brand, Lander Hill, Jemima Chellaswamy, Nuhie Faheem, Suzanne Fetherling, Elissa Gong, Eddie Marie Gonzales, Teresa Granito, Jenna Koritsaris, Binh Nguyen, Sujud Ottman, Christina Palffy, Angela Patel, Sheila Skweres, Adriane Slaton, and TaRhonda Woods.

This research was supported by the Army Research Office, the National Science Foundation, the Air Force Research Laboratory Center of Excellence Grant, The Defense Threat Reduction Agency Grant, the David and Lucile Packard Foundation, the Camille Dreyfus Teacher-Scholar Program, the Wyss Institute at Harvard University, the Paul G. Allen Frontiers Group, The Air Force Office of Scientific Research, and the Natural Sciences and Engineering Council of Canada. [emphases mine]

Well, that list of funding agencies is quite interesting. The US Army and Air Force but not the Navy? As for what the Natural Sciences and Engineering Council of Canada is doing on that list, I can only imagine why.

This is what they were doing in 2018,

Now for the latest update, a May 7, 2019 news item on phys.org announces the BioBits Kits have been expanded,

How can high school students learn about a technology as complex and abstract as CRISPR? It’s simple: just add water.

A Northwestern University-led team has developed BioBits, a suite of hands-on educational kits that enable students to perform a range of biological experiments by adding water and simple reagents to freeze-dried cell-free reactions. The kits link complex biological concepts to visual, fluorescent readouts, so students know—after a few hours and with a single glance—the results of their experiments.

A May 7, 2019 Northwestern University news release (also on EurekAlert and received via email) by Amanda Morris, which originated the news item, provides more details,

After launching BioBits last summer, the researchers are now expanding the kit to include modules for CRISPR [clustered regularly interspaced short palindromic repeats] and antibiotic resistance. A small group of Chicago-area teachers and high school students just completed the first pilot study for these new modules, which include interactive experiments and supplementary materials exploring ethics and strategies.

“After we unveiled the first kits, we next wanted to tackle current topics that are important for society,” said Northwestern’s Michael Jewett, principal investigator of the study. “That led us to two areas: antibiotic resistance and gene editing.”

Called BioBits Health, the new kits and pilot study are detailed in a paper published today (May 7 [2019]) in the journal ACS Synthetic Biology.

Jewett is a professor of chemical and biological engineering in Northwestern’s McCormick School of Engineering and co-director of Northwestern’s Center for Synthetic Biology. Jessica Stark, a graduate student in Jewett’s laboratory, led the study.

Test in a tube

Instead of using live cells, the BioBits team removed the essential cellular machinery from inside the cells and freeze-dried them for shelf stability. Keeping cells alive and contained for an extended period of time involves several complicated, time-consuming preparation and processing steps as well as expensive equipment. Freeze-dried cell-free reactions bypass those complications and costs.

“These are essentially test-tube biological reactions,” said Stark, a National Science Foundation graduate research fellow. “We break the cells open and use their guts, which still contain all of the necessary biological machinery to carry out a reaction. We no longer need living cells to demonstrate biology.”

This method to harness biological systems without intact, living cells became possible over the last two decades thanks to multiple innovations, including many in cell-free synthetic biology by Jewett’s lab. Not only are these experiments doable in the classroom, they also only cost pennies compared to standard high-tech experimental designs.

“I’m hopeful that students get excited about engineering biology and want to learn more,” Jewett said.

Conquering CRISPR

One of the biggest scientific breakthroughs of the past decade, CRISPR (pronounced “crisper”) stands for Clustered Regularly Interspaced Short Palindromic Repeats. The powerful gene-editing technology uses enzymes to cut DNA in precise locations to turn off or edit targeted genes. It could be used to halt genetic diseases, develop new medicines, make food more nutritious and much more.

BioBits Health uses three components required for CRISPR: an enzyme called the Cas9 protein, a target DNA sequence encoding a fluorescent protein and an RNA molecule that targets the fluorescent protein gene. When students add all three components — and water — to the freeze-dried cell-free system, it creates a reaction that edits, or cuts, the DNA for the fluorescent protein. If the DNA is cut, the system does not glow. If the DNA is not cut, the fluorescent protein is made, and the system glows fluorescent.

“We have linked this abstract, really advanced biological concept to the presence or absence of a fluorescent protein,” Stark said. “It’s something students can see, something they can visually understand.”

The curriculum also includes activities that challenge students to consider the ethical questions and dilemmas surrounding the use of gene-editing technologies.

“There is a lot of excitement about being able to edit genomes with these technologies,” Jewett said. “BioBits Health calls attention to a lot of important questions — not only about how CRISPR technology works but about ethics that society should be thinking about. We hope that this promotes a conversation and dialogue about such technologies.”

Reducing resistance

Jewett and Stark are both troubled by a prediction that, by the year 2050, drug-resistant bacterial infections could outpace cancer as a leading cause of death. This motivated them to help educate the future generation of scientists about how antibiotic resistance emerges and inspire them to take actions that could help limit the emergence of resistant bacteria.
In this module, students run two sets of reactions to produce a glowing fluorescent protein — one set with an antibiotic resistance gene and one set without. Students then add antibiotics. If the experiment glows, the fluorescent protein has been made, and the reaction has become resistant to antibiotics. If the experiment does not glow, then the antibiotic has worked.

“Because we’re using cell-free systems rather than organisms, we can demonstrate drug resistance in a way that doesn’t create drug-resistant bacteria,” Stark explained. “We can demonstrate these concepts without the risks.”

A supporting curriculum piece challenges students to brainstorm and research strategies for slowing the rate of emerging antibiotic resistant strains.

Part of something cool

After BioBits was launched in summer 2018, 330 schools from around the globe requested prototype kits for their science labs. The research team, which includes members from Northwestern and MIT, has received encouraging feedback from teachers, students and parents.

“The students felt like scientists and doctors by touching and using the laboratory materials provided during the demo,” one teacher said. “Even the students who didn’t seem engaged were secretly paying attention and wanted to take their turn pipetting. They knew they were part of something really cool, so we were able to connect with them in a way that was new to them.”

“My favorite part was using the equipment,” a student said. “It was a fun activity that immerses you into what top scientists are currently doing.”

###

The study, “BioBits Health: Classroom activities exploring engineering, biology and human health with fluorescent readouts,” was supported by the Army Research Office (award number W911NF-16-1-0372), the National Science Foundation (grant numbers MCB-1413563 and MCB-1716766), the Air Force Research Laboratory Center of Excellence (grant number FA8650-15-2-5518), the Defense Threat Reduction Agency (grant number HDTRA1-15-10052/P00001), the Department of Energy (grant number DE-SC0018249), the Human Frontiers Science Program (grant number RGP0015/2017), the David and Lucile Packard Foundation, the Office of Energy Efficiency and Renewable Energy (grant number DE-EE008343) and the Camille Dreyfus Teacher-Scholar Program. [emphases mine]

This is an image you’ll find in the abstract for the 2019 paper,

[downloaded from https://pubs.acs.org/doi/10.1021/acssynbio.8b00381]

Here are links and citations for the 2018 papers and the 2019 paper,

BioBits™ Explorer: A modular synthetic biology education kit by Ally Huang, Peter Q. Nguyen, Jessica C. Stark, Melissa K. Takahashi, Nina Donghia, Tom Ferrante, Aaron J. Dy, Karen J. Hsu, Rachel S. Dubner, Keith Pardee, Michael C. Jewett, and James J. Collins. Science Advances 01 Aug 2018: Vol. 4, no. 8, eaat5105 DOI: 10.1126/sciadv.aat5105

BioBits™ Bright: A fluorescent synthetic biology education kit by Jessica C. Stark, Ally Huang, Peter Q. Nguyen, Rachel S. Dubner, Karen J. Hsu, Thomas C. Ferrante, Mary Anderson, Ada Kanapskyte, Quinn Mucha, Jessica S. Packett, Palak Patel, Richa Patel, Deema Qaq, Tyler Zondor, Julie Burke, Thomas Martinez, Ashlee Miller-Berry, Aparna Puppala, Kara Reichert, Miriam Schmid, Lance Brand, Lander R. Hill, Jemima F. Chellaswamy, Nuhie Faheem, Suzanne Fetherling, Elissa Gong, Eddie Marie Gonzalzles, Teresa Granito, Jenna Koritsaris, Binh Nguyen, Sujud Ottman, Christina Palffy, Angela Patel, Sheila Skweres, Adriane Slaton, TaRhonda Woods, Nina Donghia, Keith Pardee, James J. Collins, and Michael C. Jewett. Science Advances 01 Aug 2018: Vol. 4, no. 8, eaat5107 DOI: 10.1126/sciadv.aat5107

BioBits Health: Classroom Activities Exploring Engineering, Biology, and Human Health with Fluorescent Readouts by Jessica C. Stark, Ally Huang, Karen J. Hsu, Rachel S. Dubner, Jason Forbrook, Suzanne Marshalla, Faith Rodriguez, Mechelle Washington, Grant A. Rybnicky, Peter Q. Nguyen, Brenna Hasselbacher, Ramah Jabri, Rijha Kamran, Veronica Koralewski, Will Wightkin, Thomas Martinez, and Michael C. Jewett. ACS Synth. Biol., Article ASAP
DOI: 10.1021/acssynbio.8b00381 Publication Date (Web): March 29, 2019

Copyright © 2019 American Chemical Society

Both of the 2018 papers appear to be open access while the 2019 paper is behind a paywall.

Should you be interested in acquiring a BioBits kit, you can check out the BioBits website. As for ‘conguering’ CRISPR, do we really need to look at it that way? Maybe a more humble appraoch could work just as well or even better, eh?

*’is’ removed from sentence on May 9, 2019.

CRISPR-Cas12a as a new diagnostic tool

Similar to Cas9, Cas12a is has an added feature as noted in this February 15, 2018 news item on ScienceDaily,

Utilizing an unsuspected activity of the CRISPR-Cas12a protein, researchers created a simple diagnostic system called DETECTR to analyze cells, blood, saliva, urine and stool to detect genetic mutations, cancer and antibiotic resistance and also diagnose bacterial and viral infections. The scientists discovered that when Cas12a binds its double-stranded DNA target, it indiscriminately chews up all single-stranded DNA. They then created reporter molecules attached to single-stranded DNA to signal when Cas12a finds its target.

A February 15, 2018 University of California at Berkeley (UC Berkeley) news release by Robert Sanders and which originated the news item, provides more detail and history,

CRISPR-Cas12a, one of the DNA-cutting proteins revolutionizing biology today, has an unexpected side effect that makes it an ideal enzyme for simple, rapid and accurate disease diagnostics.

blood in test tube

(iStock)

Cas12a, discovered in 2015 and originally called Cpf1, is like the well-known Cas9 protein that UC Berkeley’s Jennifer Doudna and colleague Emmanuelle Charpentier turned into a powerful gene-editing tool in 2012.

CRISPR-Cas9 has supercharged biological research in a mere six years, speeding up exploration of the causes of disease and sparking many potential new therapies. Cas12a was a major addition to the gene-cutting toolbox, able to cut double-stranded DNA at places that Cas9 can’t, and, because it leaves ragged edges, perhaps easier to use when inserting a new gene at the DNA cut.

But co-first authors Janice Chen, Enbo Ma and Lucas Harrington in Doudna’s lab discovered that when Cas12a binds and cuts a targeted double-stranded DNA sequence, it unexpectedly unleashes indiscriminate cutting of all single-stranded DNA in a test tube.

Most of the DNA in a cell is in the form of a double-stranded helix, so this is not necessarily a problem for gene-editing applications. But it does allow researchers to use a single-stranded “reporter” molecule with the CRISPR-Cas12a protein, which produces an unambiguous fluorescent signal when Cas12a has found its target.

“We continue to be fascinated by the functions of bacterial CRISPR systems and how mechanistic understanding leads to opportunities for new technologies,” said Doudna, a professor of molecular and cell biology and of chemistry and a Howard Hughes Medical Institute investigator.

DETECTR diagnostics

The new DETECTR system based on CRISPR-Cas12a can analyze cells, blood, saliva, urine and stool to detect genetic mutations, cancer and antibiotic resistance as well as diagnose bacterial and viral infections. Target DNA is amplified by RPA to make it easier for Cas12a to find it and bind, unleashing indiscriminate cutting of single-stranded DNA, including DNA attached to a fluorescent marker (gold star) that tells researchers that Cas12a has found its target.

The UC Berkeley researchers, along with their colleagues at UC San Francisco, will publish their findings Feb. 15 [2018] via the journal Science’s fast-track service, First Release.

The researchers developed a diagnostic system they dubbed the DNA Endonuclease Targeted CRISPR Trans Reporter, or DETECTR, for quick and easy point-of-care detection of even small amounts of DNA in clinical samples. It involves adding all reagents in a single reaction: CRISPR-Cas12a and its RNA targeting sequence (guide RNA), fluorescent reporter molecule and an isothermal amplification system called recombinase polymerase amplification (RPA), which is similar to polymerase chain reaction (PCR). When warmed to body temperature, RPA rapidly multiplies the number of copies of the target DNA, boosting the chances Cas12a will find one of them, bind and unleash single-strand DNA cutting, resulting in a fluorescent readout.

The UC Berkeley researchers tested this strategy using patient samples containing human papilloma virus (HPV), in collaboration with Joel Palefsky’s lab at UC San Francisco. Using DETECTR, they were able to demonstrate accurate detection of the “high-risk” HPV types 16 and 18 in samples infected with many different HPV types.

“This protein works as a robust tool to detect DNA from a variety of sources,” Chen said. “We want to push the limits of the technology, which is potentially applicable in any point-of-care diagnostic situation where there is a DNA component, including cancer and infectious disease.”

The indiscriminate cutting of all single-stranded DNA, which the researchers discovered holds true for all related Cas12 molecules, but not Cas9, may have unwanted effects in genome editing applications, but more research is needed on this topic, Chen said. During the transcription of genes, for example, the cell briefly creates single strands of DNA that could accidentally be cut by Cas12a.

The activity of the Cas12 proteins is similar to that of another family of CRISPR enzymes, Cas13a, which chew up RNA after binding to a target RNA sequence. Various teams, including Doudna’s, are developing diagnostic tests using Cas13a that could, for example, detect the RNA genome of HIV.

infographic about DETECTR system

(Infographic by the Howard Hughes Medical Institute)

These new tools have been repurposed from their original role in microbes where they serve as adaptive immune systems to fend off viral infections. In these bacteria, Cas proteins store records of past infections and use these “memories” to identify harmful DNA during infections. Cas12a, the protein used in this study, then cuts the invading DNA, saving the bacteria from being taken over by the virus.

The chance discovery of Cas12a’s unusual behavior highlights the importance of basic research, Chen said, since it came from a basic curiosity about the mechanism Cas12a uses to cleave double-stranded DNA.

“It’s cool that, by going after the question of the cleavage mechanism of this protein, we uncovered what we think is a very powerful technology useful in an array of applications,” Chen said.

Here’s a link to and a citation for the paper,

CRISPR-Cas12a target binding unleashes indiscriminate single-stranded DNase activity by Janice S. Chen, Enbo Ma, Lucas B. Harrington, Maria Da Costa, Xinran Tian, Joel M. Palefsky, Jennifer A. Doudna. Science 15 Feb 2018: eaar6245 DOI: 10.1126/science.aar6245

This paper is behind a paywall.

Treating bacterial infections without antibiotics in Switzerland

One of the major problems in medicine is the growing tolerance to antibiotics consequently there are research teams around the world attempting to find alternatives. A team in Switzerland has proposed a new means of eliminating bacteria without inducing tolerance according to a Nov. 2, 2014 news item on ScienceDaily,

Ever since the development of penicillin almost 90 years ago, antibiotics have remained the gold standard in the treatment of bacterial infections. However, the WHO has repeatedly warned of a growing emergence of bacteria that develop antibiotic resistance. Once antibiotics do no longer protect from bacterial infection, a mere pneumonia might be fatal.

A team of international scientists has tested a novel substance, which has been developed by Eduard Babiychuk and Annette Draeger from the Institute of Anatomy, University of Bern in Switzerland. This compound constitutes a novel approach for the treatment of bacterial infections: the scientists engineered artificial nanoparticles made of lipids, “liposomes” that closely resemble the membrane of host cells. These liposomes act as decoys for bacterial toxins and so are able to sequester and neutralize them. Without toxins, the bacteria are rendered defenseless and can be eliminated by the cells of the host’s own immune system. The study will be published in Nature Biotechnology Nov 2nd [2014].

A Nov. 3, 2014 news item on startupticker.ch provides more detail about the research and the startup which will be taking the research from the lab to the marketplace,

Artificial bait for bacterial toxins

In clinical medicine, liposomes are used to deliver specific medication into the body of patients. Here, the Bernese scientists have created liposomes which attract bacterial toxins and so protect host cells from a dangerous toxin attack. “We have made an irresistible bait for bacterial toxins. The toxins are fatally attracted to the liposomes, and once they are attached, they can be eliminated easily without danger for the host cells”, says Eduard Babiychuk who directed the study. “Since the bacteria are not targeted directly, the liposomes do not promote the development of bacterial resistance”, adds Annette Draeger. Mice which were treated with the liposomes after experimental, fatal septicemia survived without additional antibiotic therapy.

Treatment developed by the Swiss start-up LASCCO

The Technology transfer organisation of the Universities of Bern, Basel and Zurich “Unitectra” has filed a patent for this compound. The liposomal treatment is being developed as a new medicine named “CAL02” by LASCCO SA, a Geneva-based biomedical company specialized in innovative technologies for diagnostics and therapeutics. The first clinical study, conducted on patients suffering from severe streptococcal pneumonia is scheduled for 2015.

“These in vivo studies strongly support our decision to conduct a first-in-human study next year in severely-ill patients with pneumococcal pneumonia” commented Samareh Azeredo da Silveira Lajaunias, Managing Director at LASCCO. “This new drug meets crucial medical needs, since virulence factors such as toxins are responsible for serious infection-related complications. These complications concern 23% of individuals affected by community-acquired pneumonia, extend hospitalisation in intensive care units, and tremendously increase the cost of care.”

A Nov. 2, 2014 LASCCO press release covers much of the same detail as the news items but is included here for completeness (i.e., my completeness issues).

Here’s a link to and a citation for the study,

Engineered liposomes sequester bacterial exotoxins and protect from severe invasive infections in mice by Brian D Henry, Daniel R Neill, Katrin Anne Becker, Suzanna Gore, Laura Bricio-Moreno, Regan Ziobro, Michael J Edwards, Kathrin Mühlemann, Jörg Steinmann, Burkhard Kleuser, Lukasz Japtok, Miriam Luginbühl, Heidi Wolfmeier, André Scherag, Erich Gulbins, Aras Kadioglu, Annette Draeger, & Eduard B Babiychuk. Nature Biotechnology (2014) doi:10.1038/nbt.3037 Published online 02 November 2014

This paper is behind a paywall.

Tackling antibiotic resistance with inhalable nanotherapeutics

A June 25, 2014 news item on Nanowerk highlights PneumoNP a new European Union ‘theragnostic’ research project (Note: Links have been removed) ,

A new research project (PneumoNP) is aimed at tackling antibiotic resistance in respiratory tract infections via the use of inhalable nanotherapeutic compounds. Funded under the FP7 programme by the European Commission, the 4-year long PneumoNP project brings together top research institutes, universities, clinicians and enterprises from 6 EU member states. This novel collaboration will contribute to answer the call of the World Health Organization (WHO), who recently released an alarming report on the global threat of antibiotic resistance.

The project will develop an innovative solution to antibiotic resistance by coupling new antibiotics to inhalable carrier molecules, resulting in more efficient targeting of antibiotics to infection-causing bacteria present in the respiratory tract.

An April 30, 2014 WHO news release details the level of antibiotic resistance,

New WHO report provides the most comprehensive picture of antibiotic resistance to date, with data from 114 countries

A new report by WHO–its first to look at antimicrobial resistance, including antibiotic resistance, globally–reveals that this serious threat is no longer a prediction for the future, it is happening right now in every region of the world and has the potential to affect anyone, of any age, in any country. Antibiotic resistance–when bacteria change so antibiotics no longer work in people who need them to treat infections–is now a major threat to public health.

The report, “Antimicrobial resistance: global report on surveillance”, notes that resistance is occurring across many different infectious agents but the report focuses on antibiotic resistance in seven different bacteria responsible for common, serious diseases such as bloodstream infections (sepsis), diarrhoea, pneumonia, urinary tract infections and gonorrhoea. The results are cause for high concern, documenting resistance to antibiotics, especially “last resort” antibiotics, in all regions of the world.

Key findings from the report include:

Resistance to the treatment of last resort for life-threatening infections caused by a common intestinal bacteria, Klebsiella pneumoniae–carbapenem antibiotics–has spread to all regions of the world. K. pneumoniae is a major cause of hospital-acquired infections such as pneumonia, bloodstream infections, infections in newborns and intensive-care unit patients. In some countries, because of resistance, carbapenem antibiotics would not work in more than half of people treated for K. pneumoniae infections.

Resistance to one of the most widely used antibacterial medicines for the treatment of urinary tract infections caused by E. coli–fluoroquinolones–is very widespread. In the 1980s, when these drugs were first introduced, resistance was virtually zero. Today, there are countries in many parts of the world where this treatment is now ineffective in more than half of patients.

Treatment failure to the last resort of treatment for gonorrhoea–third generation cephalosporins–has been confirmed in Austria, Australia, Canada, France, Japan, Norway, Slovenia, South Africa, Sweden and the United Kingdom. More than 1 million people are infected with gonorrhoea around the world every day.

Antibiotic resistance causes people to be sick for longer and increases the risk of death. For example, people with MRSA (methicillin-resistant Staphylococcus aureus) are estimated to be 64% more likely to die than people with a non-resistant form of the infection. Resistance also increases the cost of health care with lengthier stays in hospital and more intensive care required.

The suggestions offered for tackling antibiotic resistance will be familiar to many (from the news release),

 People can help tackle resistance by:

  •  using antibiotics only when prescribed by a doctor;
  •  completing the full prescription, even if they feel better;
  •  never sharing antibiotics with others or using leftover prescriptions.

A June 25, 2014 PneumoNP press release describes both the European Union’s response to massive, global antibiotic resistance and the specifics of the new programme (PneumoNP),

In this context, the European Commission launched 15 projects under its7 Framework Programme to fight antimicrobial resistance, with PneumoNP being one of these projects. Started in 2014, the aim of this 4-year project is to develop novel therapeutic and diagnostic tools for bacterial respiratory tract infections, focusing on infections caused by Klebsiella pneumoniae. PneumoNP will pioneer the development of a therapeutic treatment based on a combination of nanocarriers coupled to new antibiotics. This novel combination is expected to enhance the efficiency of antibiotic delivery to the patient. The project is expected to generate:

  • a new inhalable drug system made of a new nanotherapeutic system (an antimicrobial peptide or an active pharmaceutical ingredient and a nanocarrier);
  • a new aerosol technology that will allow direct access to the main focus of infection;
  • an innovative efficiency-efficacy test to follow-up the treatment;
  • a new diagnostic test for faster detection and identification of antibiotic resistance in bacteria causing respiratory infections.

European funding allows PneumoNP to combine scientific research capacities with the expert healthcare capabilities of European enterprises. The result is an interdisciplinary collaboration between 11 teams from 6 EU member states – Spain, Italy, France, Germany, The Netherlands, and Denmark. Each partner has a distinct yet collaborative role according to its own expertise involving a total of 8 work packages.

There is a figure in the news release which illustrates the PneumoNP concept,

Figure 2: PneumoNP concept

Figure 2: PneumoNP concept

There is more information about PneumoNP on its website. I wasn’t able to glean much in the way of technical details (are they using silver nanoparticles, what kind of nanocarriers are they considering, etc.) but I imagine those will emerge with time. There is this from the homepage which features the relatively new (to me) word, theragnostic,

Development of a theragnostic system for the treatment of lung Gram-negative bacterial infections

I assume they are conflating two processes, therapeutics and diagnostics for theragnostics.

DARPA, innovation, passwords, people, and nanotherapeutics

There have been a few articles recently about (US) DARPA (Defense Advance Research Projects Agency) that have roused my interest in how they view innovation and business. The first piece I’m mentioning is a request for a proposal (RFP) on nanotherapeutics in a Nov. 22, 2011 news item on Nanowerk,

Through the U.S. Department of Defense’s Small Business Innovation Research (SBIR) program, DARPA is currently soliciting research proposals to develop a platform capable of rapidly synthesizing therapeutic nanoparticles targeted against evolving and engineered pathogens (SB121-003: Rapidly Adaptable Nanotherapeutics pdf).

Here’s part of the problem they’re trying to solve,

Acquired resistance compromises our ability to fight emergent bacterial threats in injured warfighters and our military treatment facilities. For burn patients in particular, multidrug-resistant Acinetobacter calcoaceticus-baumannii complex (ABC) is a common cause of nosocomial infection, causing severe morbidity as well as longer hospital stays. Typically, antimicrobial resistant infections require a hospital stay three times as long and are in excess of four times as expensive. Therefore, new and innovative methods to control bacterial infection in the military health system are of critical importance.

Here’s what they want,

Recent advances in nanomaterials, genome sequencing, nucleotide synthesis, and bioinformatics could converge in nanotherapeutics with tailored sequence, specificity, and function that can overcome earlier challenges. Collectively, these core technologies could permit the development of an innovative pharmaceutical platform composed of nanoparticles with tethered small interfering RNA (siRNA) oligonucelotides whose sequence and objective can be reprogrammed “on-the-fly” to inhibit multiple targets within multiple classes of pathogens.

This topic is focused on the development of a revolutionary rapidly adaptable nanotherapeutic platform effective against evolving and engineered pathogens. The biocompatible materials used to fabricate the nanoparticle should optimize cellular targeting, intracellular concentration, target sequence affinity, resistance to nuclease, and knockdown of target genes. The platform should leverage state-of-the-art genomic sequencing and oligonucleotide synthesis technologies to permit rapid programmability against evolving biologic threats.

I have taken a look at the RFP and, predictably, there’s a militaristic element to the introduction,

DARPA’s mission is to prevent technological surprise for the United States and to create technological surprise for its adversaries. The DARPA SBIR [Small Business Innovation Research] and STTR [Small Business Technology Transfer] Programs are designed to provide small, high-tech businesses and academic institutions the opportunity to propose radical, innovative, high-risk approaches to address existing and emerging national security threats; thereby supporting DARPA’s overall strategy to bridge the gap between fundamental discoveries and the provision of new military capabilities. (p. 1)

In short, we should never be caught with our pants down but we would like to catch our enemies in that position.

I was surprised to find that the responders are expected to create a business plan that includes information about markets, customers, and sales (from the RFP),

5. Market/Customer Sets/Value Proposition – Describe the market and customer sets you propose to target, their size, and their key reasons they would consider procuring the technology.

• What is the current size of the broad market you plan to enter and the “niche” market opportunity you are addressing?

• What are the growth trends for the market and the key trends in the industry that you are planning to target?

• What features of your technology will allow you to provide a compelling value proposition?

DARPA – 3

• Have you validated the significance of these features and if not, how do you plan to validate?

6. Competition Assessment – Describe the competition in these markets/customer sets and your anticipated advantage (e.g., function, performance, price, quality, etc.)

7. Funding Requirements – List your targeted funding sources (e.g., federal, state and local, private (internal, loan, angel, venture capital, etc.) and your proposed plan and schedule to secure this funding.

Provide anticipated funding requirements both during and after Phase II required to:

• mature the technology

• as required, mature the manufacturing processes

• test and evaluate the technology

• receive required certifications

• secure patents, or other protections of intellectual property

• manufacture the technology to bring the technology to market for use in operational environments

• market/sell technology to targeted customers

8. Sales Projections – Provide a schedule that outlines your anticipated sales projections and indicate when you anticipate breaking even. (pp. 2-3)

I do understand that the US has a military-industrial complex which fuels much of the country’s economic growth; I just hadn’t expected that the military would care as much as they do (as per this RFP) about  their suppliers’ business plans and financial health. It makes sense. After all, you want your suppliers to stay in business as it’s expensive and time-consuming to find new ones.

I don’t know if this is a new philosophy for the agency but it does seem to fit nicely with the current director’s Regina Dugan’s approach. From a Q & A between Dugan and Adam L. Penenberg for an Oct. 19, 2011 article in Fast Company,

That seems a key part of your mission since you got here–that it’s not enough to be doing cutting-edge research.
When deputy director Kaigham Gabriel and I got here, we understood that DARPA is one of the gems of the nation. We had been asked to take good care of her. For me, part of that meant really understanding why DARPA has this half-century of success in innovation. And the first element in DARPA’s success is the power that lies at the intersection of basic science and application, in the so-called Pasteur’s Quadrant. Do you know Stokes’s theory of innovation?

Absolutely not.
Donald E. Stokes wrote a theory of innovation in the late 1990s. Till then, most people thought of innovation as a linear process. You do basic science; then you do more advanced science; then you do the application work; then you commercialize it. What Stokes suggested is that it doesn’t happen that way at all. He preferred to think of it in a quadrant fashion, defining one row as very deep science and the other as light science; the two columns were a low-application drive and a high-application drive. Pasteur’s Quadrant happens at the deep-science-, high-application-drive quadrant. That’s DARPA’s absolute power lane. It’s called Pasteur’s Quadrant because serious concerns about food safety drove his research.

A very recent example of how it works for us is the blast-gauge work that we do. Here’s a big problem: TBI, traumatic brain injuries. So the way we approach it at DARPA is to say, “Okay, let’s understand the basic science, the phenomenology. How is it that an encounter with a blast injures the brain? What levels of blasts cause what levels of injury? Is it the overpressure? Is it the acceleration? What is it?” A medical person from DARPA researched this and discovered it was the overpressure. And the DARPA physicist says, “We know how to measure that.” Together, they devise this little blast gauge that’s the size of a couple stacks of quarters [the gauge helps doctors measure a soldier’s blast-exposure level, enabling better assessment of injuries]. They develop it in one year, going through four iterations of the electronics. That’s fast.

All of this leads back to the idea of shipping products. The defense world is like a mini-society. It has to deploy to anyplace in the world on a moment’s notice, and it has to work in a life-or-death situation. That kind of focus, that kind of drive to ship an application, really does inspire greater genius. And the constancy of funding that comes with that–in good times or bad, whether this party or that party is in power–also helps inspire innovation.

Dugan later goes on to describe her first weeks at DARPA (she was sworn in July 2009) where she and the deputy director made it their mission to meet every single person on staff, all 217 of them.

Still on the theme of innovation and DARPA, there’s a Nov. 16, 2011 article, DARPA Is After Your Password, by Neal Ungerleider in Fast Company which has to be of huge interest to anyone who has passwords,

According to DARPA press materials, the agency is focusing on creating cutting-edge biometric identification products that can identify an individual user through their individual typing style. In the future, DARPA hopes smart computers will be able to verify account-holders’ identities through their typing speed, finger motions and quirks of movement.

Materials published by DARPA seem to indicate that researchers at the agency believe most contemporary account passwords–at least those adhering to best practices–are clunky, hard to remember, and ultimately insecure. According to program manager Richard Guidorizzi, “My house key will get you into my house, but the dog in my living room knows you’re not me. No amount of holding up my key and saying you’re me is going to convince my dog you’re who you say you are. My dog knows you don’t look like me, smell like me or act like me. What we want out of this program is to find those things that are unique to you, and not some single aspect of computer security that an adversary can use to compromise your system.”

Nobody likes entering passwords. Nobody likes remembering passwords. Nobody likes forgetting passwords. Creating a painless, easy, and secure password-replacement system will be a major cash cow for any firm that can effectively bring it to market. [emphasis mine]

My enthusiasm for a world without passwords aside, I do note the interest in having the technology come to market. I wonder if DARPA will accrue some financial benefit, i.e. a licensing agreement. I did quickly skim the RFP but was unable to confirm or disprove this notion.