Novel C. diff structures are required for infection, offer new therapeutic targets

 Iron storage "spheres" inside the bacterium C. diff -- the leading cause of hospital-acquired infections -- could offer new targets for antibacterial drugs to combat the pathogen.

A team of Vanderbilt researchers discovered that C. diff (Clostridioides difficile) produces the spheres, called ferrosomes, and that these structures are important for infection in an animal model. The findings, reported Nov. 15 in the journal Nature, are also a rare demonstration of a membrane-bound structure inside a pathogenic bacterium.

Bacteria have long been thought not to contain organelles (such as a nucleus, mitochondria and other specialized structures) like eukaryotic cells, but that biological dogma appears to be incorrect.

"The emerging idea that bacteria do compartmentalize biochemical processes in a way similar to eukaryotic cells really flips the field of microbiology on its head," said Eric Skaar, PhD, MPH, the Ernest W. Goodpasture Professor of Pathology and director of the Vanderbilt Institute for Infection, Immunology, and Inflammation.

Skaar, co-corresponding author Qiangjun Zhou, PhD, assistant professor of Cell and Developmental Biology, and their colleagues were intrigued by findings reported several years ago that some environmental bacteria produce iron-containing ferrosomes.

They knew that the genes in these bacteria were conserved in C. diff and other anaerobic bacteria (bacteria that die in the presence of oxygen), and they set out to determine if C. diff produces ferrosomes to manage its need for iron. Like all living organisms, C. diff requires iron to survive and grow. Skaar and his team have focused on how pathogens like C. diff acquire iron and other metals, with a goal of finding new pathways that could be exploited to "starve" pathogens of essential nutrients.

C. diff causes about 500,000 infections and more than 29,000 deaths in the United States each year, according to the Centers for Disease Control and Prevention, and treatment options are limited. People taking antibiotics that disrupt the healthy microbes in the gut are at increased risk for C. diff infection, which causes diarrhea and colitis. New strategies for treating this urgent public health threat are needed, Skaar said.

To look for iron inside C. diff, the researchers first drew on expertise and resources in the Vanderbilt Institute of Nanoscale Science and Engineering (VINSE).

"The best way to look for the accumulation of elements in a small space like a cell is with a method called STEM-EDS, which has not commonly been used for biological samples," Skaar said. "We were fortunate to have access to a STEM-EDS instrument and collaborators at VINSE, and we quickly proved that there was an accumulation of iron 'dots' within the bacterium."

Co-first authors Hualiang Pi, PhD, and Rong Sun, PhD, led studies to show that those iron dots represented organelles that were important to C. diff infection.

Pi and Skaar's team found that two genes (fezA and fezB), which are similar to those in environmental bacteria, were required for ferrosome formation. Using C. diff bacteria missing these genes, they showed that ferrosomes are required for C. diff to fully colonize and cause disease in an animal model. They found that ferrosomes were even more important for C. diff infection in a model of inflammatory bowel disease, demonstrating that these iron-containing structures help the bacterium combat "nutritional immunity" -- the host response of producing proteins to bind iron and attempt to starve the pathogen.

Sun and Zhou's team used cryogenic electron microscopy (cryo-EM) and cryo-tomography to show that the ferrosome structures were encased in a membrane, classifying them as organelles.

Skaar noted that "Vanderbilt's unique geography" -- the proximity of experts in engineering, cell biology and the Medical Center -- and specialized tools for STEM-EDS and cryo-EM made the research possible.

The results "establish ferrosome formation and all the factors involved in ferrosome formation as potential targets for new antibacterial drugs against an important infectious disease," Skaar said. "Anytime we find new factors involved in host-pathogen interactions and show that they're important for infection, that opens entirely new opportunities to make classes of antibacterial drugs that have not existed before. That is especially important in the face of rising antimicrobial resistance that we're seeing globally."

In future studies, the researchers plan to explore how ferrosomes are formed, whether other gut pathogens produce ferrosomes, and whether these structures might be shared in the gut as a source of iron. Skaar is also particularly interested in pursuing the emerging area of bacterial organelles.

"We think our study is a rare demonstration of an organelle in a pathogenic bacterium," he said. "Now we want to know if there are other subcellular compartments in bacteria that we're interested in that could teach us about how these cells perform various physiologic processes."

Scientists 3D-print hair follicles in lab-grown skin

 A team led by scientists at Rensselaer Polytechnic Institute has 3D-printed hair follicles in human skin tissue cultured in the lab. This marks the first time researchers have used the technology to generate hair follicles, which play an important role in skin healing and function.

The finding, published in the journal Science Advances, has potential applications in regenerative medicine and drug testing, though engineering skin grafts that grow hair are still several years away.

"Our work is a proof-of-concept that hair follicle structures can be created in a highly precise, reproducible way using 3D-bioprinting. This kind of automated process is needed to make future biomanufacturing of skin possible," said Pankaj Karande, Ph.D., an associate professor of chemical and biological engineering and a member of Rensselaer's Shirley Ann Jackson, Ph.D. Center for Biotechnology and Interdisciplinary Studies, who led the study.

"The reconstruction of hair follicles using human-derived cells has historically been a challenge. Some studies have shown that if these cells are cultured in a three-dimensional environment, they can potentially originate new hair follicles or hair shafts, and our study builds on this work," Karande said.

When it comes to engineering human skin, hair may at first seem superfluous. However, hair follicles are quite important: They produce sweat, helping regulate body temperature, and they contain stem cells that help skin heal.

Hair follicles are also an entry point for topical drugs and cosmetics, making them an important part of dermatological testing. But today, initial safety testing is done on engineered skin tissues that lack hair follicles.

"Right now, contemporary skin models -- the engineered structures that mimic human skin -- are quite simple. Increasing their complexity by adding hair follicles would give us even more information about how skin interacts with topical products," said Carolina Catarino, Ph.D., first author of the study, who earned her doctorate at Rensselaer and is now a researcher developing new skin testing methods at Grupo Boticário, a cosmetics company in her home country of Brazil.

"Dr. Karande's lab is at the forefront of skin tissue engineering. This team has already successfully printed skin with working blood vessels, and this latest research is an exciting next step in developing and testing better treatments for burns and other skin conditions," said Deepak Vashishth, Ph.D., director of the Shirley Ann Jackson, Ph.D. Center for Biotechnology and Interdisciplinary Studies.

"Dr. Karande's work is a great example of advances being made by RPI researchers at the interface of engineering and life sciences with impact on human health," said Shekhar Garde, Ph.D., dean of Rensselaer's School of Engineering. "Bringing multichannel 3-D printing to biological realm is opening exciting opportunities that would have been hard to imagine in the past."

The researchers created their follicle-bearing skin with 3D-printing techniques adapted for printing at the cellular level.

The scientists begin by allowing samples of skin and follicle cells to divide and multiply in the lab until there are enough printable cells. Next, the researchers mix each type of cell with proteins and other materials to create the "bio-ink" used by the printer. Using an extremely thin needle to deposit the bio-ink, the printer builds the skin layer by layer, while also creating channels for depositing the hair cells. Over time, the skin cells migrate to these channels surrounding the hair cells, mirroring the follicle structures present in real skin.

Right now, these tissues have a lifespan of two to three weeks, which is not enough time for hair shafts to develop. The research team's future work aims to extend that period, allowing the hair follicle to mature further and paving the way for their use in drug testing and skin grafts.

Realistic talking faces created from only an audio clip and a person's photo

 A team of researchers from Nanyang Technological University, Singapore (NTU Singapore) has developed a computer program that creates realistic videos that reflect the facial expressions and head movements of the person speaking, only requiring an audio clip and a face photo.

DIverse yet Realistic Facial Animations, or DIRFA, is an artificial intelligence-based program that takes audio and a photo and produces a 3D video showing the person demonstrating realistic and consistent facial animations synchronised with the spoken audio (see videos).

The NTU-developed program improves on existing approaches, which struggle with pose variations and emotional control.

To accomplish this, the team trained DIRFA on over one million audiovisual clips from over 6,000 people derived from an open-source database called The VoxCeleb2 Dataset to predict cues from speech and associate them with facial expressions and head movements.

The researchers said DIRFA could lead to new applications across various industries and domains, including healthcare, as it could enable more sophisticated and realistic virtual assistants and chatbots, improving user experiences. It could also serve as a powerful tool for individuals with speech or facial disabilities, helping them to convey their thoughts and emotions through expressive avatars or digital representations, enhancing their ability to communicate.

Corresponding author Associate Professor Lu Shijian, from the School of Computer Science and Engineering (SCSE) at NTU Singapore, who led the study, said: "The impact of our study could be profound and far-reaching, as it revolutionises the realm of multimedia communication by enabling the creation of highly realistic videos of individuals speaking, combining techniques such as AI and machine learning. Our program also builds on previous studies and represents an advancement in the technology, as videos created with our program are complete with accurate lip movements, vivid facial expressions and natural head poses, using only their audio recordings and static images."

First author Dr Wu Rongliang, a PhD graduate from NTU's SCSE, said: "Speech exhibits a multitude of variations. Individuals pronounce the same words differently in diverse contexts, encompassing variations in duration, amplitude, tone, and more. Furthermore, beyond its linguistic content, speech conveys rich information about the speaker's emotional state and identity factors such as gender, age, ethnicity, and even personality traits. Our approach represents a pioneering effort in enhancing performance from the perspective of audio representation learning in AI and machine learning." Dr Wu is a Research Scientist at the Institute for Infocomm Research, Agency for Science, Technology and Research (A*STAR), Singapore.

The findings were published in the scientific journal Pattern Recognition in August.

Speaking volumes: Turning audio into action with animated accuracy

The researchers say that creating lifelike facial expressions driven by audio poses a complex challenge. For a given audio signal, there can be numerous possible facial expressions that would make sense, and these possibilities can multiply when dealing with a sequence of audio signals over time.

Since audio typically has strong associations with lip movements but weaker connections with facial expressions and head positions, the team aimed to create talking faces that exhibit precise lip synchronisation, rich facial expressions, and natural head movements corresponding to the provided audio.

To address this, the team first designed their AI model, DIRFA, to capture the intricate relationships between audio signals and facial animations. The team trained their model on more than one million audio and video clips of over 6,000 people, derived from a publicly available database.

Assoc Prof Lu added: "Specifically, DIRFA modelled the likelihood of a facial animation, such as a raised eyebrow or wrinkled nose, based on the input audio. This modelling enabled the program to transform the audio input into diverse yet highly lifelike sequences of facial animations to guide the generation of talking faces."

Dr Wu added: "Extensive experiments show that DIRFA can generate talking faces with accurate lip movements, vivid facial expressions and natural head poses. However, we are working to improve the program's interface, allowing certain outputs to be controlled. For example, DIRFA does not allow users to adjust a certain expression, such as changing a frown to a smile."

Besides adding more options and improvements to DIRFA's interface, the NTU researchers will be finetuning its facial expressions with a wider range of datasets that include more varied facial expressions and voice audio clips.

New compound outperforms pain drug by indirectly targeting calcium channels

 A compound -- one of 27 million screened in a library of potential new drugs -- reversed four types of chronic pain in animal studies, according to new research led by NYU College of Dentistry's Pain Research Center and published in the Proceedings of the National Academy of Sciences (PNAS).

The small molecule, which binds to an inner region of a calcium channel to indirectly regulate it, outperformed gabapentin without troublesome side effects, providing a promising candidate for treating pain.

Calcium channels play a central role in pain signaling, in part through the release of neurotransmitters such as glutamate and GABA -- "the currency of the pain signal," according to Rajesh Khanna, director of the NYU Pain Research Center and professor of molecular pathobiology at NYU Dentistry. The Cav2.2 (or N-type) calcium channel is the target for three clinically available drugs, including gabapentin (sold under brand names including Neurontin) and pregabalin (Lyrica), which are widely used to treat nerve pain and epilepsy.

Gabapentin mitigates pain by binding to the outside of the Cav2.2 calcium channel, affecting the channel's activity. However, like many pain medications, gabapentin use often comes with side effects.

"Developing effective pain management with minimal side effects is crucial, but creating new therapies has been challenging," said Khanna, the senior author of the PNAS study. "Rather than directly going after known targets for pain relief, our lab is focused on indirectly targeting proteins that are involved in pain."

Inside the channel

Khanna has long been interested in a protein called CRMP2, a key regulator of the Cav2.2 calcium channel that binds to the channel from the inside. He and his colleagues previously discovered a peptide (a small region of amino acids) derived from CRMP2 that could uncouple CRMP2 from the calcium channel. When this peptide -- dubbed the calcium channel-binding domain 3, or CBD3 -- was delivered to cells, it acted as a decoy, blocking CRMP2 from binding to the inside of the calcium channel. This resulted in less calcium entering the calcium channel and less neurotransmitter release, which translated to less pain in animal studies.

Peptides are difficult to synthesize as drugs because they are short-acting and easily degrade in the stomach, so the researchers sought to create a small molecule drug based on CBD3. Starting with the 15 amino acids that make up the CBD3 peptide, they honed in on two amino acids that studies showed were responsible for inhibiting calcium influx and mitigating pain.

"At that point, we realized that these two amino acids could be the building blocks for designing a small molecule," said Khanna.

From 27 million to one

In collaboration with colleagues at the University of Pittsburgh, the researchers ran a computer simulation that screened a library of 27 million compounds to look for a small molecule that would "match" the CBD3 amino acids.

The simulation narrowed the library down to 77 compounds, which the researchers experimentally tested to see if they lessened the amount of calcium influx. This further pared the pool down to nine compounds, which were assessed using electrophysiology to measure decreases in electrical currents through the calcium channels.

One compound, which the researchers named CBD3063, emerged as the most promising candidate for treating pain. Biochemical tests revealed that CBD3063 disrupted the interaction between the CaV2.2 calcium channel and CRMP2 protein, reduced calcium entering the channel, and lessened the release of neurotransmitters.

"Many scientists have screened the same library of compounds, but have been trying to block the calcium channel from the outside. Our target, these two amino acids from CRMP2, is on the inside of the cell, and this indirect approach may be the key to our success," said Khanna.

Four labs, four types of pain

Khanna's lab then tested CBD3063 with mouse models for pain related to injury. The compound was effective in alleviating pain in both male and female mice -- and notably, in a head-to-head test with the drug gabapentin, the researchers needed to use far less CBD3063 (1 to 10 mg) than gabapentin (30 mg) to reduce pain.

To explore whether CBD3063 helped with different types of chronic pain, Khanna partnered with researchers at Virginia Commonwealth University, Michigan State University, and Rutgers University. Collaborators ran similar studies administering CBD3063 to treat animal models of chemotherapy-induced neuropathy, inflammatory pain, and trigeminal nerve pain -- all successfully reversing pain, similar to gabapentin.

But unlike gabapentin, the use of CBD3063 did not come with side effects, including sedation, changes to cognition such as memory and learning, or changes to heart rate and breathing.

What's next

The researchers are continuing to study CBD3063, refining its chemical composition and running additional tests to study the compound's safety and assess if tolerance develops.

Long-term, they hope to bring a CBD3063-derived drug to clinical trials in an effort to offer new options for safe and effective pain relief.

"Identifying this first-in-class small molecule has been the culmination of more than 15 years of research. Though our research journey continues, we aspire to present a superior successor to gabapentin for the effective management of chronic pain," said Khanna.

Additional authors include Kimberly Gomez, Tyler Nelson, Heather Allen, Aida Calderon-Rivera, Sara Hestehave, Erick Rodríguez Palma, Paz Duran, Santiago Loya-Lopez, Samantha Perez-Miller, and May Khanna of NYU Dentistry's Pain Research Center; Elaine Zhu and Jing Wang of NYU Grossman School of Medicine; Handoko and Paramjit Arora of NYU's Department of Chemistry; Ulises Santiago and Carlos Camacho of the University of Pittsburgh; Yuan Zhou, Angie Dorame, and Aude Chefdeville of the University of Arizona; Upasana Kumar, Rory Shields, Wanhong Zuo, Huijuan Hu, and Olga Korczeniewska of Rutgers University; Eda Koseli, Bryan McKiver, and M. Imad Damaj of Virginia Commonwealth University; Denise Giuvelis and Tamara King of the University of New England; Kufreobong Inyang and Geoffroy Laumet of Michigan State University; Dongzhi Ran, Yi Lu, and Xia Liu of Chongqing Medical University; Marcel Patek of Bright Rock Path LLC; and Aubin Moutal of St. Louis University.

This 3D printer can watch itself fabricate objects

 With 3D inkjet printing systems, engineers can fabricate hybrid structures that have soft and rigid components, like robotic grippers that are strong enough to grasp heavy objects but soft enough to interact safely with humans.

These multimaterial 3D printing systems utilize thousands of nozzles to deposit tiny droplets of resin, which are smoothed with a scraper or roller and cured with UV light. But the smoothing process could squish or smear resins that cure slowly, limiting the types of materials that can be used.

Researchers from MIT, the MIT spinout Inkbit, and ETH Zurich have developed a new 3D inkjet printing system that works with a much wider range of materials. Their printer utilizes computer vision to automatically scan the 3D printing surface and adjust the amount of resin each nozzle deposits in real time to ensure no areas have too much or too little material.

Since it does not require mechanical parts to smooth the resin, this contactless system works with materials that cure more slowly than the acrylates which are traditionally used in 3D printing. Some slower-curing material chemistries can offer improved performance over acrylates, such as greater elasticity, durability, or longevity.

In addition, the automatic system makes adjustments without stopping or slowing the printing process, making this production-grade printer about 660 times faster than a comparable 3D inkjet printing system.

The researchers used this printer to create complex, robotic devices that combine soft and rigid materials. For example, they made a completely 3D-printed robotic gripper shaped like a human hand and controlled by a set of reinforced, yet flexible, tendons.

"Our key insight here was to develop a machine vision system and completely active feedback loop. This is almost like endowing a printer with a set of eyes and a brain, where the eyes observe what is being printed, and then the brain of the machine directs it as to what should be printed next," says co-corresponding author Wojciech Matusik, a professor of electrical engineering and computer science at MIT who leads the Computational Design and Fabrication Group within the MIT Computer Science and Artificial Intelligence Laboratory (CSAIL).

He is joined on the paper by lead author Thomas Buchner, a doctoral student at ETH Zurich, co-corresponding author Robert Katzschmann, PhD '18, assistant professor of robotics who leads the Soft Robotics Laboratory at ETH Zurich; as well as others at ETH Zurich and Inkbit. The research will appear in Nature.

Contact free

This paper builds off a low-cost, multimaterial 3D printer known as MultiFab that the researchers introduced in 2015. By utilizing thousands of nozzles to deposit tiny droplets of resin that are UV-cured, MultiFab enabled high-resolution 3D printing with up to 10 materials at once.

With this new project, the researchers sought a contactless process that would expand the range of materials they could use to fabricate more complex devices.

They developed a technique, known as vision-controlled jetting, which utilizes four high-frame-rate cameras and two lasers that rapidly and continuously scan the print surface. The cameras capture images as thousands of nozzles deposit tiny droplets of resin.

The computer vision system converts the image into a high-resolution depth map, a computation that takes less than a second to perform. It compares the depth map to the CAD (computer-aided design) model of the part being fabricated, and adjusts the amount of resin being deposited to keep the object on target with the final structure.

The automated system can make adjustments to any individual nozzle. Since the printer has 16,000 nozzles, the system can control fine details of the device being fabricated.

"Geometrically, it can print almost anything you want made of multiple materials. There are almost no limitations in terms of what you can send to the printer, and what you get is truly functional and long-lasting," says Katzschmann.

The level of control afforded by the system enables it to print very precisely with wax, which is used as a support material to create cavities or intricate networks of channels inside an object. The wax is printed below the structure as the device is fabricated. After it is complete, the object is heated so the wax melts and drains out, leaving open channels throughout the object.

Because it can automatically and rapidly adjust the amount of material being deposited by each of the nozzles in real time, the system doesn't need to drag a mechanical part across the print surface to keep it level. This enables the printer to use materials that cure more gradually, and would be smeared by a scraper.

Superior materials

The researchers used the system to print with thiol-based materials, which are slower-curing than the traditional acrylic materials used in 3D printing. However, thiol-based materials are more elastic and don't break as easily as acrylates. They also tend to be more stable over a wider range of temperatures and don't degrade as quickly when exposed to sunlight.

"These are very important properties when you want to fabricate robots or systems that need to interact with a real-world environment," says Katzschmann.

The researchers used thiol-based materials and wax to fabricate several complex devices that would otherwise be nearly impossible to make with existing 3D printing systems. For one, they produced a functional, tendon-driven robotic hand that has 19 independently actuatable tendons, soft fingers with sensor pads, and rigid, load-bearing bones.

"We also produced a six-legged walking robot that can sense objects and grasp them, which was possible due to the system's ability to create airtight interfaces of soft and rigid materials, as well as complex channels inside the structure," says Buchner.

The team also showcased the technology through a heart-like pump with integrated ventricles and artificial heart valves, as well as metamaterials that can be programmed to have non-linear material properties.

"This is just the start. There is an amazing number of new types of materials you can add to this technology. This allows us to bring in whole new material families that couldn't be used in 3D printing before," Matusik says.