Manipulating Polyamines to Enhance Antibody Efficacy: A Novel Approach in Biotechnology

Consistent manufacturing and production of monoclonal antibodies (mAbs) is critical, and their functional profiles depend on cell culture conditions. Now, researchers from Japan have investigated the role of intracellular polyamines on N-glycan profiles of mAbs. They found that polyamine depletion led to an ER stress response in CHO cells, leading to an increase in galactosylation of mAbs. Supplementation of spermidine recovered N-glycan profiles. These findings will contribute to the stable production of antibody-based drugs.

Monoclonal antibodies (mAbs) are laboratory-designed proteins that mimic the immune system’s antibodies. To date, many therapeutic mAbs belonging to the immunoglobulin G (IgG) class of antibodies, have been approved for the treatment of cancer and autoimmune diseases. Cell lines such as the Chinese hamster ovary (CHO) cells are generally used to produce mAbs. Notably, the production and manufacture of mAbs are regulated by critical quality attributes (CQAs) to ensure their safety and efficacy in treatment.

An important CQA for mAbs is the N-linked glycosylation present at a specific position (Asn297). N-linked glycans consist of N-acetylglucosamine (GlcNAc), mannose (Man), fucose (Fuc), galactose (Gal), and sialic acid. The heterogeneity of the N-linked glycan profiles of mAbs can be attributed to the different numbers and linkages of additional saccharides. The composition of N-linked glycans affects the overall therapeutic efficacy, targeting ability, and immune-specificity of these antibodies. For example, antibody-dependent cellular cytotoxicity (ADCC) is influenced by the fucosylation and galactosylation of N-linked glycans. Complement-dependent cytotoxicity (CDC) is also affected by the galactosylation and sialylation of N-linked glycans. Hence, it’s crucial to meticulously regulate N-linked glycan profiles throughout the manufacturing process because the heterogeneity of the N-linked glycan profile of mAbs depends on the cell culture duration and changes in nucleotide sugars and glycosylation enzyme levels.

Recently, Dr. Kyohei Higashi, Associate Professor at Tokyo University of Science (TUS) in Japan, along with a team of researchers including Dr. Rin Miyajima and Dr. Masahiro Komeno, conducted a study to explore the effects of polyamines on N-linked glycan profiles of mAbs in CHO DP-12 cells. Their work was made available online on November 3, 2023 in the Journal of Biotechnology.

“Because the carbohydrate structure of mAbs changes depending on the state of the cells, we were interested in investigating the relationship between intracellular polyamines and the carbohydrate structure of mAbs from CHO cells.” explained Dr. Higashi, when asked about the motivation behind the research.

Polyamines (putrescine, PUT; spermidine, SPD; and spermine, SPM) are present in millimolar concentrations in all living organisms and play essential roles in normal cell growth and differentiation. PUT, SPD, and SPM contained two, three, and four amino groups, respectively. PUT is synthesized from ornithine (ORN) by ornithine decarboxylase (ODC), a rate-limiting enzyme in the polyamine biosynthesis pathway. SPD is synthesized from putrescine by spermidine synthase, and spermine is synthesized from spermidine by spermine synthase. Intracellular polyamine levels are regulated at various steps, including synthesis, degradation, and transport, and are affected by external stimuli, aging, and diseases. Because CHO cells lack arginase activity to produce ORN from arginine, they cannot produce polyamines in serum-free media, resulting in a decrease in intracellular polyamine levels, which causes a low growth rate and cell viability during long-term cultivation.

Intracellular polyamine levels can also be decreased by treatment with α-difluoromethylornithine (DFMO), an inhibitor of ODC. The depletion of intracellular polyamines by DFMO can be reversed by the addition of SPD to the growth media of CHO cells.

Upon introducing DFMO to the CHO cells, the team observed that IgG antibody galactosylation surged, along with an increase in the levels of β1,4-galactosyl transferase 1 (B4GALT1) mRNA. This mRNA is pivotal in governing the IgG galactosylation mechanism within CHO cells. What’s more, IgG production decreased by approximately 30% in DFMO-treated cells.

Dr. Higashi hypothesized that the decrease in IgG production was a result of endoplasmic stress (ER) stress response caused by polyamine depletion. During ER stress response, protein folding ceases, resulting in the arrest of the normal function of cells. Chaperone proteins assist in the correct folding of other protein classes and play a crucial role under both normal and stress conditions. The results of the ER stress response study confirmed the increased expression levels of chaperones for glycoprotein folding, in polyamine-depleted cells.

The team further observed that upon using tunicamycin, an ER stress inducer inhibiting N-glycosylation, ER stress from polyamine depletion triggered B4GALT1 mRNA expression, increasing IgG galactosylation in CHO cells.

The ability to maintain antibody glycosylation profiles via polyamine modulation has numerous implications. Controlled glycosylation is crucial for optimizing therapeutic proteins, such as antibodies, ensuring the stable production of antibodies in a uniform manner of biological activities, and potentially decreasing the manufacturing cost. Supplementation of polyamine could be accomplished by the addition only of SPD to serum-free medium, offer an easy and costless method to maintain the glycan structure of mAbs produced by CHO cells cultured in the serum-free medium. This insight might influence cell line development and bioproduction, facilitating the creation of biosimilars.

“Introducing polyamines, particularly SPD, to serum-free culture medium for CHO cells may contribute to consistent manufacturing and quality control of antibody production. We hope that this research will contribute to the stable production of antibody drugs and lead to lower drug prices” concludes Dr. Higashi.

New Theoretical Framework Unlocks Mysteries of Synchronization in Turbulent Dynamics

Data Assimilation (DA) is an important mathematical method for predicting turbulent flows for weather forecasting. However, the origins of the critical length scale, a crucial parameter in this method, and its dependence on the Reynolds number are not well understood. Now, researchers have developed a novel theoretical framework that treats DA as a stability problem to explain this parameter. This framework can contribute significantly to turbulence research and inspire novel data-driven methods to predict turbulence.

Weather forecasting is important for various sectors, including agriculture, military operations, and aviation, as well as for predicting natural disasters like tornados and cyclones. It relies on predicting the movement of air in the atmosphere, which is characterized by turbulent flows resulting in chaotic eddies of air. However, accurately predicting this turbulence has remained significantly challenging owing to the lack of data on small-scale turbulent flows, which leads to the introduction of small initial errors. These errors can, in turn, lead to drastic changes in the flow states later, a phenomenon known as the chaotic butterfly effect.

To address the challenge of limited data on small-scale turbulent flows, a data-driven method known as Data Assimilation (DA) has been employed for forecasting. By integrating various sources of information, this approach enables the inference of details about small-scale turbulent eddies from their larger counterparts. Notably, within the framework of DA methods, a crucial parameter known as the critical length scale has been identified. This critical length scale represents the point below which all relevant information about small-scale eddies can be extrapolated from the larger ones. Reynold’s number, an indicator of the turbulence level in fluid flow, plays a pivotal role in this context, with higher values suggesting increased turbulence. However, despite the consensus generated by numerous studies regarding a common value for the critical scale, an explanation of its origin and its relationship with Reynold’s number remains elusive.

To address this issue, a team of researchers, led by Associate Professor Masanobu Inubushi from the Tokyo University of Science, Japan, has recently proposed a theoretical framework. They treated the process of DA as a stability problem. “By considering this turbulence phenomenon as ‘synchronization of a small vortex by a large vortex’ and by mathematically attributing it to the ‘stability problem of synchronized manifolds,’ we have succeeded in explaining this critical scale theoretically for the first time,” explains Dr. Inubushi. The letter, published in Physical Review Letters on December 18, 2023, is co-authored by Professor Yoshitaka Saiki from Hitotsubashi University, Associate Professor Miki U. Kobayashi from Rissho University, and Professor Susumo Goto from Osaka University.

To this end, the research team employed a cross-disciplinary approach by combining chaos theory and synchronization theory. They focused on an invariant manifold, termed as the DA manifold, and conducted a stability analysis. Their findings revealed that the critical length scale is a key condition for DA; and is characterized by transverse Lyapunov exponents (TLEs), which ultimately dictate the success or failure of the DA process. Additionally, based on a recent discovery showing Reynolds number dependence of maximal Lyapunov exponent (LE) and the relation of TLEs with maximal LE, they concluded that the critical length scale increases with the Reynolds number, clarifying the Reynolds number dependence of the critical length scale.

Emphasizing the importance of these findings, Dr. Inubushi says, “This new theoretical framework has the potential to significantly advance turbulence research in critical problems such as unpredictability, energy cascade, and singularity, addressing a field that physicist Richard P. Feynman once described as ‘one of the remaining difficulties in classical physics.’”

In summary, the proposed theoretical framework not only enhances our understanding of turbulence, but also paves the way for novel data-driven methods that can enhance the accuracy and reliability of weather forecasting.

Let us hope for more accurate weather predictions soon!

Survival of the Fittest? New Study Shows How Cancer Cells Use Cell Competition to Evade Body’s Defenses

Cell competition, a defense system orchestrated by epithelial cells to suppress cancer formation, is altered in epithelial cells with sequential mutations. Activated Ras mutant epithelial cells, which would normally be eliminated into the lumen, instead infiltrate into the tissue to form invasive tumors. The underlying mechanisms were found to be increased MMP21 expression, via activation of NF-κB signaling. Analysis using human samples suggests that the NF-κB-MMP21 pathway contributes to early colorectal cancer progression.

Living cells compete with each other and try to adapt to the local environment. Cells that are unable to do so are eliminated eventually. This cellular competition is crucial as the surrounding normal epithelial cells use it to identify and eliminate mutant cancer cells. Studies have reported that when activating mutants of “Ras” proteins are expressed in mammalian epithelial cells, they are pushed toward the lumen, excreted along with other bodily waste, and eliminated by competition. Epithelial cells containing Ras mutants have been reported to be removed in this manner in several organs, including the small intestine, stomach, pancreas, and lungs. This suggests that cell competition is an innate defense system orchestrated by epithelial cells to prevent the accumulation of incidentally produced cancerous cells and thereby suppress cancer formation.

In general, mutations in multiple genes accumulate in a stepwise manner when normal cells become cancerous. However, it is not known how cell competition is affected by this process.

For instance, human colorectal cancer develops when the adenomatous polyposis coli (APC) gene becomes dysfunctional and activates “Wnt signaling,” followed by the activation of Ras signaling.

In a recent study, a team of researchers from Japan, led by Associate Professor Shunsuke Kon of the Department of Cancer Biology, Institute of Biomedical Research and Innovation, Tokyo University of Science (TUS), examined the effects of the accumulation of stepwise gene mutations on cell competition and investigated the role of cell competition in the actual cancer formation process. Their study was published in Nature Communications on November 3, 2023 with Mr. Kazuki Nakai, a third year PhD student at the Graduate School of Life Sciences in TUS, as the lead author.

The study results showed that when Wnt signals were activated in epithelial cells, cell competition function was altered. Activated Ras mutant epithelial cells, which would normally be eliminated into the lumen, instead infiltrated diffusely into the tissue to form highly invasive cancerous tumors.

As senior author Dr. Kon explains, “We discovered that in epithelial tissues where Wnt and Ras signals, which commonly occur in human colorectal cancer, are activated in stages, the function of cell competition is altered. It was revealed that the production of cancer cells that diffusely infiltrate into the interstitium is promoted.”

Further, the research team identified an increased expression of matrix metalloproteinase 21 (MMP21) as one of the mechanisms underlying the production of diffusely invasive cancer cells in early colorectal cancer due to abnormal cell competition. This, in turn, was shown to be directly caused by activation of nuclear factor kappa B (NF-κB) signals via the innate immune system. Blocking NF-κB signaling restored the luminal elimination of Ras mutant epithelial cells. These findings raise some intriguing questions, such as “How do transformed cells sense the cellular content that leads to the NF-B-MMP21 pathway?” and “How do surrounding cells recognize transformed cells and prepare them for cellular extrusion?” These questions will almost certainly need to be addressed in the future.

The results of this research show that cancer cells with accumulated, sequential genetic mutations, alter the function of cell competition and use it to enhance their own invasive ability. Instead of being eliminated to the lumen, they infiltrate into the tissue, producing high-grade cancer cells. While the research team did note that the cancer histopathology of the mice used in this study resembled diffuse-type cancer in humans, future research is needed to determine whether the NF-κB-MMP21 pathway is relevant to other cancers. For instance, investigating scirrhous gastric cancer, a typical diffuse-type cancer, would be particularly interesting.

Overall, these findings demonstrate that Wnt activation disrupts cell competition, and confers invasive properties on transformed cells to escape primary epithelial sites. Understanding how the molecular landscape is re-modeled to change the fate of cancer cells with high mutational burdens could be used for therapeutic purposes. This could be of interest to researchers focused on Wnt signaling or cancer research, such as those at the Koch Institute for Integrative Cancer Research at MIT and Cancer Research UK, who are working towards common goals.

Dr. Kon concludes by saying, “This study further brings forth the prospect that cell competition constrains the order of appearance of mutations during tumor development, highlighting a link between cell competition and carcinogenesis. We hope that this will pave the way for the development of new cancer treatments from the standpoint of cell competition and infiltration for the benefit of our society.”

A Novel Lightweight Wearable Device to Perform Balance Exercises at Home

Falls are a serious risk for older individuals and people with compromised balance. However, there are no convenient devices to train one’s reactive posture control against unexpected perturbations outside of clinical settings. To tackle this issue, researchers from Japan have developed a lightweight wearable device to perform balance exercises at home. The experimental results showcase the potential of this device to improve postural control, thus helping prevent falls and fall-related injuries.

Maintaining balance and posture is quite a complex skill, even though it comes naturally to most people. However, postural control tends to worsen with age due to various reasons, such as muscle weakness coupled with changes in vision and sensory input. This explains why older people are much more prone to falling and suffering fall-related injuries than younger individuals. Approximately 40% of older individuals have been reported to fall at least once a year.

In this regard, over the past few decades, scientists have found that postural control can be improved through various exercises, which in turn helps prevent falls. It is possible to train and cultivate the ability to perform compensatory postural adjustments (CPAs) to counteract the effects of unexpected external perturbations. Although scientists have come up with specialized devices to perform balance exercises involving unexpected perturbations, these machines are generally bulky, expensive, and complex to use, rendering them suitable for clinical settings only.

But could there be a more practical way to perform these exercises comfortably at home? In a recent study published in IEEE Journal of Translational Engineering in Health and Medicine on 31 August 2023, a research team led by Assistant Professor Masataka Yamamoto from Tokyo University of Science (TUS), Japan, and including Professor Hiroshi Takemura, Mr. Daiki Yoshikawa, and Mr. Taku Washida from TUS, as well as Professor Koji Shimatani from the Prefectural University of Hiroshima, explore this question. For their research, the researchers developed an innovative wearable balance exercise device (WBED) and investigated its effects on CPAs and reactive postural control.

The proposed wearable device uses two pneumatic artificial muscles (PAMs) to generate unexpected perturbations. These PAMs, which resemble a pair of hollow shoulder straps or suspenders, can be forced to extend or contract by regulating the air pressure inside them. For this purpose, the WBED includes a set of electronically controlled valves connected to a can of compressed gas. This enables a computer program or smartphone application to control the valves and quickly fill or empty either PAM with gas, producing a force that pulls the user sideways in a specific direction.

To test whether WBED can truly improve reactive postural control, the researchers recruited 18 healthy adult males and divided them randomly into two groups: WBED and sham. All participants first underwent an evaluation of reactive balance. They had to hold a tandem stance for one minute while air cylinders on both sides of the hips pushed them laterally at unpredictable moments. The participants in the WBED group then performed a few rounds of balance training using the proposed device, while the sham group underwent the same exercises without unexpected perturbation. Lastly, a second evaluation was performed to check for improvements in postural control.

The researchers measured several variables as outcomes during the evaluations, including peak displacement, time at peak displacement, peak velocity, and root mean square of the soles’ center of pressure. Notably, participants in the WBED group exhibited lower displacement and peak velocity after exercising with the device. “Our results prove that perturbation-based balance exercises using WBED immediately improve the subjects’ reactive postural control,” remarks Dr. Yamamoto, satisfied with their findings. “Wearable exercise devices, such as the proposed WBED, could contribute to the prevention of falls and fall-related injuries.”

In the near future, the proposed device could revolutionize how people with a high tendency to fall perform balance training, especially in countries with a steadily aging population like Japan. “We designed WBED to be lightweight, portable, and easy to use both at home and in clinical settings. It weighs only 0.9 kg and takes less than three minutes to put on,” highlights Dr. Yamamoto. By training regularly with WBED, older individuals and people undergoing physical therapy can efficiently improve postural control and responsiveness, which in turn would prevent falls and improve their overall health. Notably, WBED could also be useful for athletes who want to improve their balance.

Let us hope that wearable devices become a mainstay in balance training and health care monitoring, providing a boost to the Internet of Things technology!

Cultivating Euglena in Tomato Juice

Euglena (Euglena gracilis) is a microalga containing chloroplasts and producing organic matter through photosynthesis in a well-lit environment, while taking in organic matter from outside in an unlit environment. It is known to be rich in nutrients like vitamins, minerals, amino acids, and essential fatty acids, such as DHA and EPA. Owing to the lack of cell walls, Euglena has a high digestion and absorption rate, making it attractive as a new source of nutritious and health enhancing food.

Moreover, Euglena protein is rich in methionine, a characteristic of animal protein, and its nutritional value is comparable to casein found in milk. Therefore, it is expected to be one of the solutions to the shortage of animal protein due to the effects of climate change and population growth, as well as one of the production technologies for space exploration, which is flourishing these days. In addition, Euglena also contains a high percentage of a special type of beta-1,3-glucan called paramylon, known for its immunomodulatory and hepatoprotective effects. Paramylon may also be effective in reducing atopic dermatitis, influenza, and arthritis symptoms, as well as in preventing colon cancer. However, the existing methods for food-grade manufacturing of Euglena are quite complicated.

Currently, Euglena can be propagated using both autotrophic as well as heterotrophic culture mediums. Conventionally, the Koren–Hutner (KH) medium, a higher yielding heterotrophic medium, is used for its culture. But it requires measuring and mixing 26 different chemicals. Moreover, after the microalgae has reproduced to high densities in large pools, it must be extracted, washed, concentrated, and dried to foods or nutritional supplements. The energy required for these processes accounts for about 30% of the total production cost, and other costs such as cultivation land and transportation costs are also incurred in the production of Euglena as a food ingredient.

Aimed at improving the efficiency of existing production processes, a team of researchers from Japan conducted experiments to find a promising method to grow Euglena in large quantities. As explained in their latest paper, the team examined several beverages to find a suitable growing medium for Euglena. This paper was made available online on August 14, 2023 and was published in Issue 5 of the journal Sustainable Food Technology on September 1st, 2023. The study was led by Assistant Professor Kyohei Yamashita from Tokyo University of Science (TUS) and co-authored by Dr. Kengo Suzuki and Dr. Koji Yamada from Euglena Co., Ltd. and Professor Eiji Tokunaga from TUS.

Interestingly, this study is a part of follow-up research for which a patent was filed by Dr. Yamashita during his doctoral course in 2017. Dr. Yamashita explains, “We had previously confirmed that E. gracilis can grow even when foods such as seaweed, dried sardines, and boiled rice are used as a source of essential vitamins.”

The researchers first cultured Euglena with initial cell density of 4.2 x 103 cells/mL statically under aerobic conditions for about 10 days. For this, they used either Cramers–Myers (CM) medium, an independent nutrient medium, or KH medium, a heterotrophic medium. The cell density increased to 106 cells/mL and 107 cells/mL, respectively. Next, they incubated Euglena with initial cell density of 1.6 x 104 cells/mL in 13 different beverages, including diluted grape juice (with juice-to-water ratio of 3:7 or 7:3), pineapple juice, apple juice, sweet wine, diluted carrot juice (with juice-to-water ratio of 3:7 or 7:3), tomato juice, orange juice, grapefruit juice, prune juice, coconut water, and maple water, and culture medium supplemented with essential vitamins B1 and B12 under aerobic conditions. The cells were cultured under ‘light’ (26 °C, white light irradiation) or ‘dark’ (23 °C, no light irradiation) conditions.

Interestingly, the researchers found that the cell density of Euglena cells reached a maximum when cultured in tomato juice, especially under light conditions, and increased to 107 cells/mL, the same level as in KH medium. This also resulted in a change in the appearance of the culture medium from red to green after incubation (as shown in Image 1). The bright green chloroplasts in Euglena cultured in tomato juice were observed to be tightly packed inside the cells. On the other hand, in the non-tomato juice, the number of chloroplasts was low, and the green color was lighter. These findings suggest that tomato juice is more suitable for the growth of Euglena than other beverages.

Furthermore, on culturing Euglena under aerobic conditions using tomato juice diluted with water (in a ratio of 3:7, 4:6, or 5:5) and without essential vitamins, it grew to approximately 100 times of its initial cell density to 106 cells/mL under all dilution conditions. This revealed that the nutrient composition of tomato juice itself is suitable for Euglena growth.

“During static incubation, tomato juice diluted with water separated into a solid sediment layer and an upper aqueous solution layer in the container, and Euglena proliferated actively near the boundary of these layers. Therefore, when cultured under aerobic conditions using ‘tomato (filtered) medium,’ in which solid components were removed from tomato juice, Euglena were distributed throughout the entire culture medium,” points out Dr. Yamashita. Notably, the cell density was greater than that in the unfiltered tomato juice medium. This indicates that the removal of solid components may mitigate the effects of density, including growth space, light and nutrient acquisition, and waste accumulation.

Finally, the team cultured Euglena in CM medium with glutamic acid, a nutrient characteristic of tomato juice. The cell density reached two to three times that of the CM medium, but only about half that of the tomato juice medium. These findings suggest that components other than glutamic acid contained in tomato juice also contribute to the good growth of Euglena.

“Euglena is rich in nutrients and functional ingredients, so it is possible to easily fortify foods by converting some of the nutrients in the food into Euglena. Being simple and economically feasible, we expect this method to be useful for carbon-neutral and sustainable food production. It could also contribute to the achievement of sustainable development goals related to food and hunger and has the potential to contribute as a food production technology in space exploration,” concludes Dr. Yamashita, expressing his hopes for the future development of this research.

Template for Success: Shaping Hard Carbon Electrodes for Next-Generation Batteries

Sodium- and potassium-ion batteries are promising next-generation alternatives to the ubiquitous lithium-ion batteries (LIBs). However, their energy density still lags behind that of LIBs. To tackle this issue, researchers from Japan explored an innovative strategy to turn hard carbon into an excellent negative electrode material. Using inorganic zinc-based compounds as a template during synthesis, they prepared nanostructured hard carbon, which exhibits excellent performance in both alternative batteries.

Lithium-ion batteries (LIBs) are, by far, the most widely used type of rechargeable batteries, spanning numerous applications. These include consumer electronics, electric vehicles (e.g., Tesla cars), renewable energy systems, and spacecrafts. Although LIBs deliver the best performance in many aspects when compared to other rechargeable batteries, they have their fair share of disadvantages. Lithium is a rather scarce resource, and its price will rise quickly with its availability going down in the future. Moreover, lithium extraction and improperly discarded LIBs pose huge environmental challenges as the liquid electrolytes commonly used in them are toxic and flammable.

The shortcomings of LIBs have motivated researchers worldwide to look for alternative energy storage technologies. Sodium (Na)-ion batteries (NIBs) and potassium-ion batteries (KIBs) are two rapidly emerging options that are cost-efficient as well as sustainable. Both NIBs and KIBs are projected to be billion-dollar industries by the end of the decade. Governments across the world, including that of the US, Austria, Hong Kong, Germany, and Australia, are promoting research and innovation in this field. Moreover, companies such as Faradion Limited, TIAMAT SAS, and HiNa Battery Technology Co. Ltd., are investing heavily in this technology. Both Contemporary Amperex Technology Co. Limited and Build Your Dreams are expected to introduce electric vehicle battery packs with NIBs soon.

Unfortunately, however, the capacity of the electrode materials used in NIBs and KIBs still lags behind that of LIBs. Against this backdrop, a research team led by Professor Shinichi Komaba from Tokyo University Science (TUS), Japan, has been working to develop groundbreaking high-capacity electrode materials for NIBs and KIBs. In their latest study, published in Advanced Energy Materials on November 9, 2023, they report a new synthesis strategy for nanostructured “hard carbon” (HC) electrodes that deliver unprecedented performance. The study was co-authored by Mr. Daisuke Igarashi, Ms. Yoko Tanaka, and Junior Associate Professor Ryoichi Tatara from TUS, and Dr. Kei Kubota from the National Institute for Materials Science (NIMS), Japan.

But what is HC and why is it useful for NIBs and KIBs? Unlike other forms of carbon, such as graphene or diamond, HC is amorphous; it lacks a well-defined crystalline structure. Additionally, it is strong and resistant. In an earlier 2021 study, Prof. Komaba and his colleagues had found a way to use magnesium oxide (MgO) as a template during the synthesis of HC electrodes for NIBs, altering their final nanostructure. The process had led to the formation of nanopores within the electrodes upon MgO removal, which, in turn, had vastly increased their capacity to store Na+ ions.

Motivated by their previous findings, the researchers explored whether compounds made from zinc (Zn) and calcium (Ca) could also be useful as nano-templates for HC electrodes. To this end, they systematically investigated different HC samples made using zinc oxide (ZnO) and calcium carbonate (CaCO3) and compared their performance with the ones synthesized using magnesium oxide (MgO).

Preliminary experiments showed that ZnO was particularly promising for the negative electrode of NIBs. Accordingly, the researchers optimized the concentration of ZnO embedded in the HC matrix during synthesis, demonstrating a reversible capacity of 464 mAh g–1 (corresponding to NaC4.8) with a high initial Coulombic efficiency of 91.7% and a low average potential of 0.18 V vs. Na+/Na.

The team achieved remarkable results by incorporating this powerful electrode material into an actual battery. “The NIB fabricated using the optimized ZnO-templated HC as the negative electrode exhibited an energy density of 312 Wh kg–1,” highlights Prof. Komaba. “This value is equivalent to the energy density of certain types of currently commercialized LIBs with LiFePO4 and graphite and is more than 1.6 times the energy density of the first NIBs (192 Wh kg–1), which our laboratory reported back in 2011.” Notably, the ZnO-templated HC also exhibited a significant capacity of 381 mAh g–1 when incorporated into a KIB, further showcasing its potential.

Taken together, the results of this study show that using inorganic nanoparticles as a template to control the pore structure may provide an effective guideline for the development of HC electrodes. “Our findings prove that HCs are promising candidates for negative electrodes as an alternative to graphite,” concludes Prof. Komaba.

In turn, this could make NIBs viable for practical applications, such as the development of sustainable consumer electronics and electric vehicles as well as low carbon footprint energy storage systems for storing energy from solar and wind farms.

Novel Enzyme Family Could Provide Insights into Bacterial Pathogenicity

Gram-negative bacteria like E. coli and Salmonella are a global cause of concern as they can cause disease outbreaks. They release osmo-regulated periplasmic glucans (OPGs)—a diverse group of long-chain carbohydrates—that have a role in infection. Researchers from Japan have investigated two OPG-related genes, OpgG and OpgD, in E. coli. Their discovery of a novel family of β-1,2-glucanases could provide insights into bacterial pathogenicity.

Gram-negative bacteria cause a variety of infectious diseases in plants and animals alike. Outbreaks of Salmonella and E. coli infections often make headlines due to their severity, and people have to resort to allopathic as well as natural remedies, increasing the burden on the healthcare system. While antibiotics offer an effective solution against bacterial infections, the increasing incidence of antibiotic-resistant bacteria have prompted researchers to identify other possible treatments against these infections. With technological advances and modern medicine, researchers are looking into the possibility of disrupting the pathogenicity of the bacteria at a molecular level by interfering with molecular processes at the gene as well as protein level.

Gram-negative bacteria, notorious for their infection capability, produce osmo-regulated periplasmic glucans (OPGs)—long-chain carbohydrates made of multiple glucose units—in the extracellular and/or periplasmic space. Initially, it was believed that OPGs were by-products produced under low solute concentrations, but recent reports confirm that they are crucial for pathogenicity, symbiosis, cell adhesion, and signaling.

However, the enzymes involved in the synthesis, regulation, and degradation of OPGs are not fully known. Genetic analysis revealed that the removal of opgH and/or opgG genes, partially responsible for OPG synthesis, causes bacteria to lose their infection capability, suggesting strong potential links of these genes with bacterial pathogenicity.

Although the structure of OpgG from E. coli (EcOpgG) has been elucidated, the mechanism of action of OpgG and OpgD from E. coli (EcOpgG and EcOpgD, respectively) remains unclear. Understanding the enzymes involved in OPG synthesis and the mechanisms underlying their function could provide us vital insights into the pathogenicity of Gram-negative bacteria, allowing us to develop more effective ways to deal with bacterial infections.

To bridge this gap in knowledge, Mr. Sei Motouchi from Tokyo University of Science, Dr. Kaito Kobayashi from the National Institute of Advanced Industrial Science and Technology (AIST), Associate, Associate Professor Hiroyuki Nakai from Niigata University and Professor Masahiro Nakajima from the Tokyo University of Science conducted structural and functional analyses of EcOpgD and EcOpgG. The study was published in Communications Biology on September 21, 2023.

Sharing the motivation behind this study, Professor Nakajima tells us, “Glycans are important biological macromolecules that play a variety of roles in living organisms, including pathogenicity and symbiosis. Their structure is very diverse and complex, and thus there are many types of enzymes that may synthesize and degrade them. However, we humans know only a small fraction of them”.

The researchers investigated the functions of OPG-related genes in the model organism E. coli. Functional analyses revealed that E. coli OpgD (EcOpgD) was an endo-β-1,2-glucanase, which specifically broke down β-1,2-glucans. It also had similar kinetic properties as those of general glycoside hydrolases (GH), further confirming its identity as a β-1,2-glucanase.

Structural analysis using crystallography revealed a high degree of similarity between the structures of EcOpgG and EcOpgD. However, the two enzymes had remarkably different activity. Upon further investigation, the researchers found that a few amino acids forming the reaction pathway, termed ‘Loop A’, were critical for enzyme activity and regulated the rate of reaction. EcOpgG and EcOpgD differed in their catalytic functions, possibly due to the difference in the amino acids in the Loop A region. The LoopA region diversifies among this group of enzymes, which may lead to functional diversity. Nevertheless, the basis of the catalytic center is shared in this group of enzymes. This common point will help scientists develop therapies that could potentially disrupt OPG synthesis and hinder the infection capability of bacteria.

Further, while the two enzymes belonged to the same family of GHs, their structure did not match with any of the existing GH enzymes. Thus, the authors confirmed that they belonged to a novel GH family, namely GH186. This information opens avenues for research into therapies that can target GH186 proteins to stop the progression of bacterial infections.

Professor Masahiro concludes by explaining the long-term applications of the study, “Although it was known that some Gram-negative plant pathogens synthesize OPGs for pathogenicity, most of the key enzymes for their synthesis had not been identified, preventing the development of agrochemicals targeting OPGs. We have identified a family of enzymes (GH186) involved in the direct synthesis of OPGs and elucidated their detailed functions, which has presented us with new targets (GH186) to inhibit pathogens and provides a solid foundation for ‘structure-based pesticide discovery’”.

The findings of this study lay down a strong foundation for further investigation of OPGs and related genes and may usher in a new era of disease management.

Restoring the Function of a Human Cell Surface Protein in Yeast Cells

G protein-coupled receptors (GPCRs) are the largest and most diverse group of cell surface proteins in humans. These receptors, which can be seen as ‘traffic directors,’ transmit signals from the outside to the inside of cells and are involved in many physiological processes. Given their prominent roles in cellular communication, cell growth, immune responses, and sensory perception, many drugs have been developed to target GPCRs, for the treatment of conditions such as asthma, allergies, depression, hypertension, and heart disease. In fact, more than 300 GPCR-related drugs are currently in clinical trials, 36% of which target over 60 novel GPCR targets without an already-approved drug. Moreover, drugs that target GPCRs account for as much as 27% of the global market share of therapeutic drugs, with aggregated sales close to US$890 billion between 2011 and 2015. Thus, any technique that could accelerate research on GPCRs is likely to trigger a large ripple effect, ultimately bringing more effective treatments to millions of people.

Today, approaches such as cryo-electron microscopy, optogenetics, computational approaches and artificial intelligence, biosensors and label-free technologies, and single-cell technologies are being explored for GPCR drug discovery and development. Among them, the single-cell approach based on yeast is one of the most useful platforms to study GPCRs. Besides its widespread application in beer and bread making, the yeast species Saccharomyces cerevisiae has a long history of being used as a host to research human derived GPCRs. Although some GPCRs can be engineered to enhance their stability and function to facilitate experiments, most GPCRs do not function well in yeast cells. This long-standing problem has greatly slowed progress in our understanding of GPCRs and the development of new drugs that target them.

Against this backdrop, a research team from Tokyo University of Science (TUS), Japan, recently came up with an innovative strategy to restore the activity of human derived GPCR human histamine 3 (H3R) in S. cerevisiae. Their study, published in Volume 13 of Scientific Reports on September 26, 2023, was led by Associate Professor Mitsunori Shiroishi and co-authored by Ms. Ayami Watanabe and Ms. Ami Nakajima, all from TUS.

“H3R is mainly expressed in the nervous system. It is involved in cognitive function, and its inhibition is associated with the therapeutic outcomes of various conditions, such as ADHD, schizophrenia, Alzheimer’s disease, and narcolepsy,” explains Dr. Shiroishi. Through preliminary experiments, the team showed that H3R becomes non-functional when expressed in yeast.

To restore its function, the research team utilized a technique called error-prone polymerase chain reaction to introduce random mutations in the H3R gene. After producing a random mutant library of H3R, they introduced modified DNA segments into yeast cells and cultivated them in the presence of an H3R agonist—a compound that binds to H3R and sets off a measurable response. By screening through multiple cultures, the researchers obtained four mutants in which the normal activity of H3R was restored. These mutants responded exclusively to a type of yeast strain that harbors certain G-chimera proteins. The mutations responsible for the restored activity were located near the amino acid sequence motifs important for GPCR activation.

This innovative approach to study GPCRs could have profound implications, particularly in the fields of medicine and cell biology. “Our research could help elucidate the function of GPCRs and may even lead to the development of drugs with fewer side effects, as well as bolster drug discovery for diseases for which there is currently no treatment,” remarks Dr. Shiroishi. There are many therapeutic areas where GPCR-targeting drugs are being actively developed, including neurological disorders like Alzheimer’s and schizophrenia, cardiovascular diseases such as hypertension and heart failure, various types of cancer, and metabolic disorders.

A deeper understanding of GPCR variations and how they impact individuals differently could also lead to new approaches to personalized medicine. Tailoring GPCR-targeted drugs to an individual’s genetic makeup and their specific disease profile may greatly improve treatment outcomes. Furthermore, generic GPCR treatments reaching a vast number of people worldwide might also become a reality, which would reduce the burden on healthcare systems.

We are certain that the findings of this study will pave the way to a healthier future for everyone.

Evaluating the Shear Viscosity of Different Water Models

The 4-point Optimal Point Charge (OPC) and 3-point OPC (OPC3) models are highly accurate water models, used extensively in molecular simulations to reproduce the properties of bulk water. However, there are no reports on whether these models can accurately reproduce the viscosity of water. Recently, a researcher from Japan tested the performance of the OPC and OPC3 models by evaluated their shear viscosities and comparing them to experimental results.

Water is one of the most abundant substances on Earth and partakes in countless biological, chemical, and ecological processes. Thus, understanding its behavior and properties is essential in a wide variety of scientific and applied fields. To do so, researchers have developed various water models to reproduce the behavior of bulk water in molecular simulations. While these simulations can provide valuable insights into the specific properties of water, selecting an appropriate model for the system under study is crucial. Today, two water models have become very popular among biomolecular researchers: the 4-point Optimal Point Charge (OPC) and 3-point OPC (OPC3) models. These models are known for their ability to reproduce several properties of water with high accuracy, including density, heat of vaporization, and dielectric constant. However, there is limited information on whether OPC and OPC3 water models can accurately predict the shear viscosity of water.

The viscosity of water greatly affects how water molecules interact with other substances and surfaces, dictating critical phenomena such as diffusion and absorption. This affects the texture and taste of foods and beverages, as well as how oils and liquids interact with food during cooking. More importantly, the viscosity of water needs to be considered when designing and manufacturing pharmaceutical products, as well as many types of lubricants and polymeric materials. In addition, it influences how water and water-based solutions flow through small tubes, such as those in our circulatory system and in microfluidic devices.

Recently, Associate Professor Tadashi Ando from Tokyo University of Science conducted a study to test the performance of the OPC and OPC3 models, by evaluating their shear viscosities and comparing the values to the experimental calculations. These findings were published in Volume 159, Issue 10 of The Journal of Chemical Physics on September 14, 2023.

First, Dr. Ando set up molecular dynamics simulations of up to 2,000 water molecules using popular water models, including OPC, OPC3, and variants of the Transferable Intermolecular Potential 3-point (TIP3P) and 4-point (TIP4P) models. Next, he used an approach known as the Green-Kubo formalism—a commonly used method from statistical mechanics to study viscosity and heat conduction in various materials— to calculate the viscosity of the models.

The calculated viscosities for both OPC and OPC3 water models were very close to each other for temperatures ranging from 273 K to 373 K. Notably, for temperatures above 310 K, the viscosity predicted by these models was very close to that predicted by previous experimental findings. However, this was not the case at lower temperatures. Dr. Ando explains, “Compared to other water models, the performance of the OPC and OPC3 models in terms of predicting the shear viscosity was lower than that of TIP4P and TIP3P variants, but only for temperatures below 293 K.” Notably, at 273 K and 293 K, the shear viscosities of the two models were around 10% and 20% lower, respectively, as compared to those derived experimentally.

In addition to viscosity, Dr. Ando also assessed the performance of the OPC and OPC3 models for predicting other important water properties, such as surface tension and self-diffusion. The performance of OPC and OPC3 for these properties was remarkably accurate. “Based on the results of this study, along with those from previous reports, we can conclude that the OPC and OPC3 are among the best nonpolarizable water models at present, accounting for the various static and dynamic properties of water,” highlights Dr. Ando.

Overall, this study provides a thorough understanding of the advantages and limitations of water models. With any luck, this will help scientists polish these models to make them even more useful across various technological fields!

Researchers Offer Insights into Solid-Electrolyte Interphases in Next-Gen Aqueous Potassium-Ion Batteries

Aqueous potassium-ion batteries are a promising alternative to lithium-ion batteries owing to their safety and low cost. However, not much is known about the properties of the solid-electrolyte interphases (SEI) that form between the electrode and the aqueous electrolyte. To address this knowledge gap, researchers from Japan have now conducted a study using advanced scanning electrochemical microscopy and operando electrochemical mass spectrometry. Their findings provide a deeper understanding of SEI in next-generation batteries.

Lithium-ion batteries (LIBs) have become immensely popular as the go-to power source for a wide variety of electronic devices and vehicles over the past two decades. Although it is hard to overstate the transformative effects that LIBs have had on modern societies, this technology has a fair share of disadvantages that cannot be ignored any further. These include the limited availability of lithium as well as safety and environmental concerns. These drawbacks have motivated scientists around the world to look for alternative battery technologies, such as aqueous batteries. Potassium-ion batteries (KIBs) are a prominent example; these batteries are made from abundantly available materials and are much safer than LIBs. Moreover, KIBs can utilize a water-in-salt electrolyte (WISE), which makes them more stable thermally and chemically.

However, the prevention of hydrogen evolution at the negative electrode for its stabilization is a major challenge in high-voltage aqueous batteries. While solid-electrolyte interphases (SEI) that form between these electrodes and the electrolyte solution help stabilize the electrodes in LIBs (by preventing electrolyte decomposition and self-discharge of the batteries), they have been scarcely researched in the context of KIBs.

To address this major knowledge gap, a research team from Tokyo University of Science– (TUS), Japan, has recently conducted a pioneering study to gain insights into SEI formation and their properties in WISE-based KIBs. Their findings were published online in the journal Angewandte Chemie International Edition on August 18, 2023. The study, led by TUS Professor Shinichi Komaba, is co-authored by Junior Associate Professor Ryoichi Tatara, Dr. Zachary T. Gossage, and Ms. Nanako Ito, all from TUS.

The researchers mainly employed two advanced analytical techniques—scanning electrochemical microscopy (SECM) and operando electrochemical mass spectrometry (OEMS)—to observe how SEI forms and reacts in real time during the operation of a KIB with a 3,4,9,10-perylenetetracarboxylic diimide negative electrode and 55 mol/kg K(FSA)0.6(OTf)0.4∙1H2O, a WISE developed by the team in a previous study.

The experiments revealed that SEI forms a passivating layer in WISE akin to that seen in LIBs, with slow apparent electron transfer rates, helping suppress hydrogen evolution. This can ensure stable performance and higher durability of KIBs. However, the researchers observed that the coverage of the SEI layer was incomplete at higher operating voltages, leading to hydrogen evolution.

Taken together, the results reveal the need to explore potential avenues to enhance SEI formation in future aqueous batteries. “While our results reveal interesting details on the properties and stability of SEI found in one particular WISE, we should also focus on reinforcing the SEI network to achieve improved functionality,” comments Prof. Komaba. “SEI could perhaps be improved by the development of other electrolytes that produce unique SEIs, but also through the incorporation of electrolyte additives or electrode surface pretreatment.”

This study also highlights the power of SECM and OEMS for gaining a solid understanding of electrode–electrolyte interactions in next-generation batteries. “These techniques provide a powerful means for tracking the development, coverage, ion transfer, and stability of SEI and can easily be adapted for a variety of electrolytes and electrodes,” explains Prof. Komaba. “We hope that this work encourages other researchers to further explore SECM and OEMS as advanced characterization methods that can be incorporated with traditional battery measurements to gain deeper insights.”

The development of aqueous batteries such as KIBs will be instrumental for sustainable societies in the future, since they could replace the expensive and hazardous LIBs currently used in electric vehicles, smart grids, renewable energy systems, and marine applications. By making energy storage more accessible, aqueous batteries will aid the transition toward carbon-neutral energy generation, paving the way for a greener future.

With any luck, further studies will lead us to promising LIB alternatives soon!