Tag: Enzyme

Inflammatory responses fuel cardiovascular complications

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In a recent study published in the journal Circulation, researchers investigate the inflammatory response to acute respiratory distress syndrome (ARDS) within the heart.

Study: Virus-Induced Acute Respiratory Distress Syndrome Causes Cardiomyopathy Through Eliciting Inflammatory Responses in the Heart. Image Credit: Kateryna Kon / Shutterstock

The link between respiratory viral infections and CVD

Seasonal viral infections can range in severity from mild flu-like symptoms to potentially lethal ARDS. For example, despite being primarily a respiratory tract infection, coronavirus disease of 2019 (COVID-19) can lead to ARDS and other severe cardiovascular disease outcomes with high mortality rates.

Circulating immune cells may respond to COVID-19 by upregulating cytokine release, which can lead to myocardial injury. Cardiac macrophages, immune cells responsible for the myocardial inflammatory response, are increasingly being investigated for their role in ARDS. Recent evidence indicates that macrophage expansion, which can be accompanied by changes in the population size and relative abundances of various cardiac macrophages, is a characteristic feature of ARDS.

The main two types of cardiac macrophages include C-C chemokine receptor type 2 negative (CCR2^–) and CCR2⁺ macrophages. Further research is needed to determine the viral-induced contributions of these macrophages to adverse cardiac outcomes.

These data would allow clinicians to make informed intervention decisions and elucidate whether these outcomes are COVID-19-induced or if observed inflammation is a systemic immune response to viral infection. Furthermore, this information could support the development of future therapies to prevent cardiovascular disease (CVD) following recovery from COVID-19.

About the study

In the present study, researchers investigate the role of viral- and non-viral-induced ARDS-associated immune signals in altering cardiac macrophage populations, thereby impacting CVD parameters, including systemic inflammation.

This study was conducted at Massachusetts General Hospital and involved 33 control samples obtained from patients who died between September and December 2019, prior to the onset of COVID-19, as well as 21 samples obtained between May and July 2020 from patients who died from COVID-19-associated complications. Samples consisted of autopsy tissue excised from the left ventricular or septal region.

Simultaneously, in vivo studies involved a daily intratracheal administration of an ARDS cocktail of immunostimulatory agents to mice, which included resiquimod, imiquimod, lipopolysaccharide (LPS), and angiotensin-converting enzyme 2 (ACE2) inhibitor MLN-4760. This model allowed the researchers to reproduce clinical ARDS features in mice without the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).

Patient data included results obtained from electrocardiogram (ECG), echocardiography, lung computed tomography (CT) scan, blood gas analyses, body temperature evaluation, bronchoalveolar lavage fluid (BALF) characterization, blood pressure measurements, and flow cytometry. Both human and murine autopsy samples were processed using ribonucleic acid (RNA) isolation, real-time polymerase chain reaction (PCR) assay, and enzyme-linked immunosorbent assays (ELISAs) for protein and gene expression determinations.

Similar immune responses in non-viral- and SARS-CoV-2-associated ARDS

In the absence of viral infection, mice treated with the ARDS cocktail exhibited significant weight loss over the five-day cocktail treatment period. This was accompanied by hypothermia, a common feature of both ARDS and septic shock, as well as a mortality rate of over 40% by day five.

Mice with ARDS exhibited bilateral opacities and immune cell infiltrations within their lungs, as well as reduced blood oxygenation. Furthermore, increased D-dimer, neutrophil, and monocyte levels were observed, as well as reduced blood pressure and lower heart rates in ARDS mice. Other inflammatory pathways that were activated in ARDS mice included increased levels of interleukin 6 (IL-6), IL-1ß, tumor-necrosis factor α (TNF-α), and interferon y (IFN-y), all of which are also associated with SARS-CoV-2 infection.

In both non-infected ARDS and SARS-CoV-2-infected mice, an increased infiltration of interstitial macrophages and reduced levels of alveolar macrophages were observed. Although both mouse models exhibited increased levels of cardiac macrophages, this immune response was more pronounced in infected mice. Nevertheless, both models’ subsets of cardiac macrophages were altered to similar levels.

Upon comparison of control and COVID-19 patient myocardium samples, SARS-CoV-2 infection recruited a more significant number of CCR2+ CD68+ macrophages, thus indicating that a robust immune response is elicited after severe infection compared to other life-threatening diseases.

“Our findings indicate that systemic and myocardial inflammatory signals elicited by virally induced ARDS may contribute to the cardiovascular complications and high mortality rates of this condition. In addition, our study confirms previous reports that SARS-CoV-2 infection increases overall macrophage numbers in hearts.”

The cardiac benefits of TNF-α immune therapy

TNF-α neutralizing antibodies were also administered to mice to evaluate their effects on immune activation during ARDS. To this end, TNF-α immune therapy reduced weight loss, improved body temperature, increased blood oxygenation, and led to better survival rates. Histological analysis indicated that ARDS mice receiving anti-TNF-α therapy exhibited reduced macrophages, Cxcl2, IL-1ß, and IL-6 expression within the lungs.

TNF-α therapy also improved systolic dysfunction, cardiomyocyte apoptosis, and monocyte infiltration in ARDS mice. Total cardiac macrophage counts and reduced expression of IL-1ß, IL-6, and TNF-α within the myocardium were also observed, thus demonstrating the anti-inflammatory benefits associated with TNF-α immune therapy in the lungs and hearts of mice with ARDS.

Conclusions

The study findings demonstrate that SARS-CoV-2 infection leads to significant alterations in cardiac macrophage subset levels, particularly increased levels of CCR2+ macrophages, in both mice and humans. Even in the absence of SARS-CoV-2 or another virus, the immune response to ARDS-like injury is capable of inducing significant alterations in heart macrophage levels, which may increase the risk of cardiovascular complications and mortality associated with ARDS.

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March 24, 2024
Study highlights causal associations between gut microbes and hypothyroidism

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In a recent study published in Frontiers in Nutrition, researchers explored the association between the microbial community of the gut and hypothyroidism.

Study: Cross-talk between the gut microbiota and hypothyroidism: a bidirectional two-sample Mendelian randomization study. Image Credit: sdecoret/Shutterstock.com

Background

Hypothyroidism is a hormonal imbalance characterized by diminished thyroid gland activity and insufficient thyroid hormone synthesis, which can lead to heart disease, infertility, and poor brain development in children.

It has a tremendous economic and social impact on the individuals impacted. Research has revealed that the gut microbiome might indirectly influence thyroid function, with studies indicating a drop in Prevotella in hypothyroid patients and an increase in Phascolarctobacterium, resulting in decreased bacterial diversity and richness.

Gut microorganisms create short-chain fatty acids (SCFAs), which control thyroid cell expression and keep the intestinal barrier intact. Inadequate iodine consumption is a major cause of hypothyroidism, as the gut flora influences mineral absorption and enzyme activity in thyroid hormone production.

However, the precise relationship between gut microbes and hypothyroidism is unknown due to historical case-control studies and confounding variables such as age, environment, nutrition, and lifestyle.

Understanding the association between the intestinal microbiome and hypothyroidism requires extensive research into the underlying reasons and the development of novel therapeutic options.

About the study

The present two-sample and bidirectional Mendelian randomization (MR) researchers investigated whether gut microbes causally affect hypothyroidism development.

The team analyzed summary statistical data from genome-wide association studies (GWAS) provided by the FinnGen [26,342 hypothyroidism cases of hypothyroidism with 59,827 controls; 16,378,441 single-nucleotide polymorphisms (SNPs)] and MiBioGen consortia (n = 18,430).

They selected instrumental variables (IVs) from the MiBioGen consortium dataset, targeting SNPs related to gut microbial composition and gauging IV heterogeneity using Cochran’s Q statistics.

The team used several techniques, including the weighted median, MR-Egger, simple model, weighted model, inverse variance weighted (IVW), and MR-PRESSO, to determine whether gut microbes are causally associated with hypothyroidism.

They also performed reverse MR assessments for microbes that showed causal associations with hypothyroidism development in forward MR evaluation. For sensitivity analysis, they assessed horizontal pleiotropy and performed a leave-one-out analysis.

The researchers analyzed the 16S ribosomal ribonucleic acid (rRNA) gene variable sites V1-V2, V3-V4, and V4 to assess gut microbial abundances and taxonomic classifications by direct-type taxonomic binning.

They mapped microbiome quantitative trait loci (mbQTL) to detect genetic variants related to specific loci associated with gut bacteria. The researchers analyzed 119 taxa at the genus level, using 1,231 single-nucleotide polymorphisms as instrumental variables for assessment.

Results and discussion

In the IVW analysis, Akkermansia species (odds ratio 0.8), Ruminococcaceae UCG-011 isolate (odds ratio 0.9), Butyrivibrio species (odds ratio 0.9), and Holdemania species (odds ratio 0.9) exhibited protective effects against hypothyroidism.

In contrast, Anaerostipes species (odds ratio 1.2), Intestinimonas species (odds ratio 1.1), and Ruminiclostridium species (odds ratio 1.2) were detrimental to hypothyroidism.

Reverse MR estimates indicated no significant effects of hypothyroidism on the gut microbiome. Cochran’s Q statistics showed no significant heterogeneity among instrumental variables. The sensitivity analyses demonstrated the non-significant horizontal pleiotropy, and no SNPs considerably impacted the relationship between gut microbes and hypothyroidism.

Akkermansia, a gut microbe that strengthens the intestinal lining, boosts the mucus layer and regulates the immune system, is a promising probiotic or live biotherapeutic product therapy. Its intestinal repair and immunomodulatory functions may provide new insights into hypothyroidism prevention and treatment.

Butyrivibrio bacteria, which break down plant fibers and produce butyric acid, can generate SCFAs and promote intestinal well-being, which may be a significant factor in hypothyroidism.

Holdemania is associated with several illnesses, including Parkinson’s disease and delirium. Hypothyroidism, characterized by reduced thyroid hormone levels, can lead to neuropsychiatric symptoms.

Excessive alcohol consumption is associated with elevated levels of Holdemania in the gastrointestinal tract, reducing butyric acid concentration.

The results indicated that anaerostipes, specialized anaerobes producing acetic and butyric acids, may contribute to hypothyroidism.

The finding may be due to confounding factors like age, sex, ethnicity, dietary habits, and medications. Hypothyroidism can cause impaired gastrointestinal motility and overgrowth of intestinal flora, potentially altering Anaerostipes abundance during recovery.

The study showed causal relationships between Akkermansia species and hypothyroidism, with increased Akkermansia inhibiting incidence and progression.

The researchers identified probiotics like Akkermansia, Holdemania, Ruminococcaceae UCG-011, and Butyrivibrio that protect against hypothyroidism, while Intestinimonas, Anaerostipes, and Ruminiclostridium had contrasting effects. However, additional randomized clinical trials are required to elucidate precise mechanisms researchers can target for personalized therapies enhancing precision care.

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March 20, 2024
Study uncovers how APOBEC enzymes drive cancer mutations

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A research team led by the University of California, Irvine has discovered the key role that the APOBEC3A and APOBEC3B enzymes play in driving cancer mutations by modifying the DNA in tumor genomes, offering potential new targets for intervention strategies.

The study, published today online in the journal Nature Communications, describes how the researchers identified the process by which APOBEC3A and APOBEC3B detect specific DNA structures, resulting in mutations at distinct positions within the tumor genome.

It’s critical to understand how cancer cells accumulate mutations leading to hot spots that contribute to disease progression, drug resistance and metastasis. Both APOBEC3A and APOBEC3B were known to generate mutations in many kinds of tumors, but until now we did not know how to identify the specific type caused by each. This finding will allow us to develop novel therapies to suppress mutation formation by directly targeting each enzyme accordingly.”

Rémi Buisson, UCI assistant professor of biological chemistry, corresponding author

In this study, graduate student Ambrocio Sanchez and postdoctoral fellow Pedro Ortega, both in Buisson’s laboratory at the UCI School of Medicine, developed a new method to characterize the particular kind of DNA modified by APOBEC3A and APOBEC3B. It revealed that the two enzymes do not recognize the same DNA sequences and structures within the genomes of cancer cells. Based on this observation, an innovative approach utilizing these unique target preferences was employed to classify cancer patients who had accumulated mutations caused by each enzyme.

“The next steps are to investigate whether mutations caused by these enzymes lead to various types of therapy resistance. It’s also critical to identify molecules that inhibit APOBEC3A and APOBEC3B to prevent mutations from forming. Our findings could, in the future, help to assess patient risk before treatment and suppress tumor evolution using the appropriate drug therapy,” Buisson said.

Other team members included undergraduate and graduate students and postdoctoral fellows from UCI, Harvard Medical School, the University of Southern California, the University of Texas at San Antonio and the University of Minnesota.

This work was supported by the National Institutes of Health’s Research Supplements to Promote Diversity in Health-Related Research program under award R37-CA252081-S; California Institute for Regenerative Medicine stem cell biology training grant TG2-01152; European Molecular Biology Organization postdoctoral fellowship ALTF 213-2023; Cancer Prevention and Research Institute of Texas research training award RP 170345 and Recruitment of Established Investigators award CPRIT RR220053; the National Cancer Institute under awards R37-VA252081 and P01-CA234228; the National Institute of Allergy and Infectious Diseases under award R01 AI150524; and access to UCI’s Genomics Research and Technology Hub, affiliated with the Chao Family Comprehensive Cancer Center, under grant P30-CA062203.

Source:

University of California – Irvine

Journal reference:

Sanchez, A., et al. (2024). Mesoscale DNA features impact APOBEC3A and APOBEC3B deaminase activity and shape tumor mutational landscapes. Nature Communications. doi.org/10.1038/s41467-024-45909-5.

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March 19, 2024
Scientists aim to understand why T cells do not sustain energy in tumors

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T cells are often called “assassins” or “killers” because they can orchestrate and carry out missions to hunt down bacteria, viruses, and cancer cells throughout the body. Mighty as they may be, recent research has shown that once T cells infiltrate the environment of a solid tumor, they lose the energy needed to combat the cancer.

A research team led by Jessica Thaxton, PhD, MsCR, associate professor of cell biology and physiology and co-leader of the Cancer Cell Biology Program at the UNC Lineberger Comprehensive Cancer Center, aimed to understand why T cells do not sustain energy in tumors. Using their expertise in tumor immunity and metabolism, the Thaxton Lab, led by the Katie Hurst, MPH, and 4^th year graduate student Ellie Hunt, found that a metabolic enzyme called Acetyl-CoA Carboxylase (ACC) causes T cells to store fat rather than burning fat for energy.

Our discovery fills a long-standing gap in knowledge regarding why T cells in solid tumors don’t appropriately generate energy. We inhibited the expression of ACC in mouse cancer models, and we observed that T cells were able to persist much better in solid tumors.”

Jessica Thaxton, PhD, MsCR, associate professor of cell biology and physiology and co-leader of the Cancer Cell Biology Program at the UNC Lineberger Comprehensive Cancer Center

The new findings and immunotherapeutic strategies, which were published in Cell Metabolism, could be used to make multiple types of T-cell therapies more effective for patients, possibly encompassing both checkpoint and chimeric antigen receptor (CAR) T-cell therapies.

In the field of cancer immunotherapy, it has long been known that T cells are not able to create their cellular energy, called adenosine triphosphate or ATP, when they are inside of a solid tumor.

In 2019, Thaxton’s lab studied a T cell with optimal antitumor function. In a publication in Cancer Immunology Research, Hurst and Thaxton used a proteomics screen to identify enzymes associated with the optimal antitumor metabolism of these T cells. Through this screen, the two discovered that ACC expression may limit the ability of T cells to make ATP in tumors. ACC, a key molecule that is involved in many metabolic pathways, blocks cells from breaking down fat and using it as fuel for energy in mitochondria.

“Acetyl-CoA carboxylase can drive the balance between storing lipids versus breaking down those lipids and feeding them into the citric acid cycle for energy,” said Thaxton. “If ACC is flipped ‘on’, cells generally store lipid. If ACC is ‘off’, cells tend to use the lipid in their mitochondria to make ATP.”

Using Hunt’s expertise in confocal imaging, the research team was able to observe lipid stores in T cells isolated from multiple types of cancers. The observation, as well as other experiments, confirmed the team’s hypothesis that T cells were storing lipids instead of breaking them down.

Thaxton’s team then used CRISPR Cas9-mediated gene deletion to see what would happen if they “deleted” ACC from the picture. There was a rapid reduction in the amount of lipid storage in T cells, and the team was able to visualize fat relocating to the mitochondria to be used to generate energy.

Thaxton now hypothesizes that T cells may need a “delicate balance” of lipids to persist in solid tumors with a certain amount of lipid dedicated to cancer cell assassination and low levels of fats being maintained in stores.

The latest findings could prove to be useful in enhancing chimeric antigen receptor (CAR) T-cell therapies. This cutting-edge technology takes T cells out of cancer patients, modifies them in the lab to hunt down tumor cells, and then re-infuses the cells to fight the patient’s cancer. Preliminary data from Thaxton’s lab demonstrates that even the manufactured T cells contain excess lipid stores.

The lab is starting to look in patient samples to understand how researchers can possibly flip the ACC metabolic switch directly in patient tumors, negating the need to take out and reinfuse cells back into the body. But researchers must first determine how this could affect other immune cell populations in the body, such as macrophages.

Source:

University of North Carolina Health Care

Journal reference:

Hunt, E. G., et al. (2024). Acetyl-CoA carboxylase obstructs CD8+ T cell lipid utilization in the tumor microenvironment. Cell Metabolism. doi.org/10.1016/j.cmet.2024.02.009

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March 15, 2024
New England Biolabs® Launches NEBNext® Enzymatic 5hmC-seq Kit, for enzyme-based 5hmC detection at single-base resolution

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New England Biolabs (NEB^®) today announced the launch of the NEBNext Enzymatic 5hmC-seq Kit (E5hmC-seq^™), a novel enzyme-based method for the specific detection of 5hmC sites. The gentle, enzyme-based approach enables high yields and high-quality data, with an input range of 100 pg to 200 ng.

While the biological importance of 5hmC modification is less clear than that of 5mC, the abundance of 5hmC varies significantly between tissues, suggesting that it may play a critical role in gene regulation and other biological processes.

“So far, the study of 5hmC has been hampered by the lack of precise detection methods,” said Fiona Stewart, Associate Director, NEBNext Portfolio Management. “While NEBNext Enzymatic Methyl-seq (EM-seq^™), our gold standard for methylation detection, detects both 5mC and 5hmC, it does not distinguish between them. Additionally, bisulfite-based methods suffer from reduced data quality due to the sample fragmentation and loss of DNA that results from the damaging bisulfite treatment, thereby limiting their practical utility.

“To address these challenges, we developed the NEBNext Enzymatic 5hmC-seq Kit, which allows for the specific detection of 5hmC sites using a two-step enzymatic conversion workflow,” said Stewart. “The enzymatic method minimizes DNA damage and allows for the discrimination of 5hmC from unmodified cytosine and 5mC after Illumina^® sequencing. Additionally, E5hmC-seq data can be subtracted from EM-seq data, allowing for precise determination of individual 5mC and 5hmC sites.”

The kit includes the reagents required for E5hmC-seq conversion and library preparation compatible with Illumina sequencing; index primers for multiplexing are available separately. A conversion module is also available, for applications beyond library prep.

For more information, visit www.NEBNext.com.

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March 8, 2024
Revolutionary tool for tracking physical interactions between different cells

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One of the fundamental goals of basic biology is understanding how diverse cell types work in concert to form tissues, organs, and organ systems. Recent efforts to catalog the different cell types in every tissue in our bodies are a step in the right direction, but only one piece of the puzzle. The great mystery of how those cells communicate with one another remains unsolved.

Now, a new paper in Nature describes uLIPSTIC, a tool capable of laying the groundwork for a dynamic map tracking the physical interactions between different cells-;the elusive cellular interactome. The authors have been perfecting the technology since 2018 and the latest iteration can in principle allow researchers to directly observe any cell-to-cell interaction in vivo.

With uLIPSTIC we can ask how cells work together, how they communicate, and what messages they transfer. That’s where biology resides.”

Gabriel D. Victora, Rockefeller University

Kiss-and-run

Ever since single-cell mRNA sequencing came into its own, researchers have been scrambling to connect the dots and explain how diverse cells unite to form tissue. Several methods of cataloging cell-to-cell interactions have already emerged, but all have considerable shortcomings. Early efforts that involved direct observation under a microscope failed to retrieve interacting cells for further analysis; subsequent attempts leaned on advanced imaging techniques that intuit how cells might interact based on their structure and proximity to other cells. No approach captured true physical interactions and signal exchange between cell membranes.

Enter LIPSTIC, an innovative approach from the Victora lab that involved labeling cellular structures that touch when two cells make fleeting, “kiss-and-run” contact before parting ways. The labels ensured that, if one cell “kissed” another, it would leave a mark akin to a lipstick, enabling easy identification and quantification of physical interactions between cells.

Originally, the platform had narrow applications. Victora and colleagues designed LIPSTIC to record a very specific kind of cell-to-cell interaction between T cells and B cells, a major focus of their lab. Other researchers, however, began clamoring for a version of LIPSTIC that would work on other cellular interactions too. “We could have tailored a LIPSTIC for every type of interaction,” Victora says. “But why not try to make a universal version, instead?”

Mapping every interaction

In the original version of LIPSTIC, a “donor” cell uses an enzyme borrowed from bacteria to place a labeled peptide tag onto the surface of an “acceptor” cell upon contact-;the biochemical equivalent of applying lipstick to one cell and looking for a kiss print on another. That method required knowing exactly how the “kiss” would occur, identifying molecules the donor cell uses to interact with recipient cells and painstakingly forcing the tags onto those molecules. But over time the team discovered that dousing the cells with a high volume of enzyme and its target would ensure that any interaction that one cell had with another cell would be tracked just as efficiently.

“If you cram partner cells with enough enzyme and target, you can make any any cell pair capable of LISPTIC labeling without needing to know in advance what molecules these cells will use for their interaction,” Victora says.

The result was a uLIPSTIC, a universal platform not bound by foreknowledge of molecules, ligands, or receptors. Scientists can now theoretically smear uLIPSTIC on any cell, without preconceived notions of how it would interact with its environment, and observe physical cell-to-cell interactions. To demonstrate the power of the platform, the team showed that uLIPSTIC could expand beyond LIPSTIC’s narrow repertoire of B cells and T cells to track how dendritic cells kickstart the body’s immune response against tumors and food allergens.

“The reception to uLIPSTIC has been great,” says Sandra Nakandakari-Higa, a PhD student in the Victora lab and lead author on the paper. “We’re already getting a lot of inquiries from other labs about how they can adapt our system to their models.”

The team hopes to eventually use uLIPSTIC to discover the receptor-ligand pairs key to cellular interactions, in an effort to better understand how cells unite into tissue at the molecular level. Eventually, the team envisions uLIPSTIC as a key tool in the effort to generate comprehensive atlases describing how cells interact to form tissue-;a key to the long-awaited interactome.

Source:

Journal reference:

Nakandakari-Higa, S., et al. (2024). Universal recording of immune cell interactions in vivo. Nature. doi.org/10.1038/s41586-024-07134-4.

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March 6, 2024
SolasCure publishes phase IIa clinical trial report in leading wound care journal

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SOLASCURE Ltd (SolasCure), a biotechnology company developing a novel treatment to transform chronic wound care, has today announced the publication of its CLEANVLU Phase IIa clinical trial report in the International Wound Journal, a leading wound care journal.

SolasCure’s first investigational product, Aurase Wound Gel, is a hydrogel releasing tarumase (provisional INN), a recombinant enzyme derived from medical maggots, which aims to promote wound healing through debridement and wound bed preparation.

The Phase IIa data, which demonstrates proof-of-concept and safety of Aurase Wound Gel in humans, has now been peer-reviewed and published, providing strong validation as SolasCure progresses into further clinical studies, and marks a significant milestone for the Company.

Chronic wounds are a major healthcare challenge, with around 100 million people suffering from these wounds globally. This represents a significant unmet need as patients and healthcare systems lack safe, pain-free, and effective treatment solutions.

Recent clinical data suggest that, after 20 weeks of the current standard of care treatment, complete wound closure is achieved in as little as 25%–50% of chronic or hard-to-heal wounds.2 Aurase Wound Gel aims to address this global challenge by being the first treatment to target all elements of wound bed preparation: debridement, moisture provision, infection control and overall promotion of healing.

SolasCure’s CLEANVLU Phase IIa trial was performed in venous leg ulcer (VLU) patients across centers in the US, UK, and Hungary. The trial compared five escalating dose concentrations to baseline the use of tarumase for enzymatic debridement and wound bed preparation. Patients were treated three times per week, for four weeks.

The study established proof-of-concept that tarumase successfully debrides wounds, with faster and more complete debridement and improved healing observed at increased enzyme concentrations. The trial also demonstrated a strong safety profile, with no indications of systemic absorption, antibody generation, or systemic effects on coagulation.

Significantly, application of Aurase Wound Gel was shown to be pain-free, did not add to the patients’ existing pain burden, and had no evidence of local tolerability issues.

Further Phase II studies plan to use randomized controlled groups over a longer period, with stratification for factors that may affect debridement and wound healing, to explore the efficacy of tarumase at higher concentrations.

“The opportunity for Aurase Wound Gel to truly transform chronic wound care is very exciting, as no other treatment to date aims to target all elements of wound care management in a single product. The peer-review publication of our Phase IIa data not only provides important validation to enable further Phase II studies, but also highlights the clinical potential of Aurase Wound Gel to treat millions of patients globally safely and effectively, addressing an urgent and unmet medical need. With this excellent data we are now fundraising to support the next phase of SolasCure’s clinical and product development.”

Andy Weymann MD, MBA, Chairman of the Board, SolasCure

“Debridement is a key first step of successful wound bed preparation, itself a prerequisite for wound healing. Achieving timely complete and pain-free debridement which is agnostic of the patient setting is an urgent unmet medical need. SolasCure’s Aurase Wound Gel has shown in this publication positive safety and proof-of-concept results, which bring this product a significant step closer to providing relief to those suffering from chronic wounds worldwide.”

Rob Kirsner, MD, Ph.D, Head of Medical Advisory Board at SolasCure, Chairman and Harvey Blank Professor of Dermatology at the University of Miami

For more information about SolasCure, please visit: https://solascure.com/.

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March 4, 2024
Rare gene mutations in hereditary Alzheimer’s disease disrupt amyloid production, study shows

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A University of Kansas study of rare gene mutations that cause hereditary Alzheimer’s disease shows these mutations disrupt production of a small sticky protein called amyloid.

Plaques composed of amyloid are notoriously found in the brain in Alzheimer’s disease and have long been considered responsible for the inexorable loss of neurons and cognitive decline. Using a model species of worm called C. elegans that’s often used in labs to study diseases at the molecular level, the research team came to the surprising conclusion that the stalled process of amyloid production -; not the amyloid itself -; can trigger loss of critical connections between nerve cells.

The research, appearing in the journal Cell Reports, was headed by Michael Wolfe, Mathias P. Mertes Professor of Medicinal Chemistry at KU.

The research team focused on the rare inherited mutations because these mutations are found in genes that encode proteins that produce amyloid.

If we can understand what’s happening in this inherited form of the disease where a single mutation can trigger it. that might be a clue to what’s going on in all the other cases.”

Michael Wolfe, Mathias P. Mertes Professor of Medicinal Chemistry at KU

The rare mutations are particularly devastating, as they fate the mutation carrier to Alzheimer’s disease in middle age, and children of a mutation carrier have a 50% chance of inheriting the disease-causing mutation.

Wolfe said hereditary Alzheimer’s disease shows the same pathology, the same presentation clinically and the same progression of symptoms as the “common, garden-variety” of Alzheimer’s related to old age.

“You see the same amyloid plaques in the hereditary disease,” he said. “We think that these inherited mutations, though rare, are key to what’s going on with all Alzheimer’s disease.”

Wolfe, who earned his doctorate at KU and returned to the university seven years ago for collaborative research opportunities, joined forces with Brian Ackley, associate professor of molecular biology at KU, whose lab specializes in research with the C. elegans model worm. The research team also included other KU collaborators as well as investigators in Beijing, China, and at Harvard Medical School.

Co-authors with KU’s Department of Medicinal Chemistry were Sujan Devkota, Vaishnavi Nagarajan, Arshad Noorani and Sanjay Bhattarai; co-authors at KU’s Department of Molecular Biosciences were Ackley and Yinglong Miao; and co-authors from KU’s Center for Computational Biology were Hung Do and Anita Saraf. Other KU co-authors were Caitlin Overmeyer of the Graduate Program in Neurosciences and Justin Douglas of KU’s Nuclear Magnetic Resonance Core Lab. The KU personnel collaborated with Rui Zhou of Tsinghua University in Beijing and Masato Maesako of Harvard Medical School.

Wolfe said the discovery could point the way toward new approaches to Alzheimer’s therapies, and he hoped fellow researchers and developers of drug therapies would pay close attention to his team’s results.

“Our findings suggest what’s needed is a stimulator of the amyloid-producing enzyme, to restart stalled processes and address both problems: eliminating stalled protein complexes that lead to degeneration of nerve cell connections and producing more soluble forms of amyloid. This approach could address both contributing factors simultaneously.”

Source:

Journal reference:

Devkota, S., et al (2024). Familial Alzheimer mutations stabilize synaptotoxic γ-secretase-substrate complexes. Cell Reports. doi.org/10.1016/j.celrep.2024.113761.

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February 29, 2024
Scientists uncover a new doorway for SARS-CoV-2 into human cells

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In a recent study published in the journal Proceedings of the National Academy of Sciences, researchers demonstrated that human transferrin receptor (TfR) mediates severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection.

Coronavirus disease 2019 (COVID-19), caused by SARS-CoV-2, presents influenza-like manifestations, including mild-to-severe pneumonia, acute respiratory distress syndrome, multiorgan failure, and fatal lung injury. Further, the etiology and pathogenesis of COVID-19 are not entirely understood and targeted therapies remain inadequate.

The viral spike protein binds to the host receptor, angiotensin-converting enzyme 2 (ACE2), for cellular entry. Although SARS-CoV-2 preferentially infects cells in the respiratory tract, the virus has been detected in virtually all organs. Studies have revealed the presence of SARS-CoV-2 RNA in diverse cells lacking ACE2, suggesting that other receptors or co-receptors may mediate viral entry.

Study: Human transferrin receptor can mediate SARS-CoV-2 infection. Image Credit: Kateryna Kon / Shutterstock

The study and findings

In the present study, researchers identified TfR as an alternative receptor mediating the cellular entry of SARS-CoV-2. First, they used co-immunoprecipitation (Co-IP) to identify host proteins interacting with the viral spike in Calu-3 cells. This revealed 293 proteins, including 42 transmembrane proteins; two proteins were associated with entry (ACE2 and TfR). Next, the team evaluated TfR expression in the respiratory tract and liver in mice.

TfR expression, both transcript and protein levels, was substantially higher in the lungs and trachea than in other tissues. Using immunohistochemical analysis, the researchers investigated the effects of SARS-CoV-2 on TfR expression in the lungs of humanized ACE2 (hACE2) mice and monkeys. This revealed a 1.5- and 1.8-fold increase in TfR expression in mice and monkeys, respectively.

In addition, surface plasmon resonance revealed direct interactions between the viral spike and human TfR. Notably, the spike protein lacked interactions with Syrian hamster or mouse TfR. Docking analysis predicted two peptide sequences (QK8: QDSNWASK and SL8 SKVEKLTL) in TfR to be involved at the interface of TfR-spike interactions.

Mutagenesis and Co-IP revealed that the A529 residue in TfR was essential for interactions with the spike. Further analysis indicated that physiological interactions between spike and TfR occurred at the cellular surface and during endocytosis. This was confirmed by electron microscopy using SARS-CoV-2 pseudoviral spike and HEK293/hACE2 and BHK-21/TfR cells.

Next, the team evaluated the effects of soluble TfR, anti-TfR antibody, and SL8 and QK8 peptides on SARS-CoV-2 infection using reverse-transcription polymerase chain reaction (RT-PCR) and plaque assays. Results showed their inhibitory effects on SARS-CoV-2 in Vero E6 and Calu-3 cells. Cytotoxicity was not observed even at 1,000 nM.

Confocal microscopy revealed that TfR was widespread on the surface of Calu-3 and Vero E6 cells, with the colocalization of TfR and SARS-CoV-2 at the surface and during endocytosis. Notably, treatment with the anti-TfR antibody inhibited the colocalization. Further, electron microscopy showed that viral particles were present in the cytosol and clathrin-coated pits in Vero E6 cells; likewise, treatment with anti-TfR antibody inhibited viral internalization.

Next, ACE2 was knocked out (KO) from Calu-3 and Vero E6 cells and the cells were infected with SARS-CoV-2. This inhibited infection by 40% to 50%, suggesting that ACE2 might not be the only receptor mediating infection. In addition, TfR knockdown (KD) inhibited infection by 30%, whereas its overexpression (OE) promoted infection. TfR KO was not performed as it is lethal. TfR OE or KD did not impact ACE2 expression.

Further, the team transfected C57 mice with adenovirus vector (Ad5) expressing hACE2 or humanized TfR (hTfR) and infected them with SARS-CoV-2. Viral load in the lungs in Ad5-hTfR and Ad5-hACE2 mice was significantly higher than in Ad5-empty mice. Finally, the researchers evaluated the effects of the anti-TfR antibody on infection in rhesus macaques. Anti-TfR antibody inhibited viral replication and reduced pneumonia.

Viral load in the respiratory epithelium was also significantly lower between 3- and 7 days post-infection (dpi) compared to controls. Radiographs taken at 0 and 5 dpi revealed significantly less severe pulmonary infiltration in antibody-treated macaques relative to controls. Antibody-treated animals had no significant pulmonary lesions, while controls showed lung lesions of varying degrees.

Conclusions

Taken together, the study described the human TfR as a receptor for SARS-CoV-2. TfR can directly bind to the viral spike at an affinity comparable to that of ACE2. Notably, mouse TfR and the viral spike lacked interactions. Soluble TfR, SL8, and QK8 peptides and anti-TfR antibodies can inhibit infection. The team also illustrated the antiviral effects of the anti-TfR antibody in rhesus macaques. Overall, TfR could serve as an alternative infection pathway, facilitating viral entry through endocytosis.

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February 28, 2024
Innovative Subak tool offers affordable solution for detecting nuclease digestion

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A new tool could reduce costs for diagnosing infectious diseases.

Biomedical researchers from The University of Texas at Austin have developed a new, less expensive way to detect nuclease digestion – one of the critical steps in many nucleic acid sensing applications, such as those used to identify COVID-19 and other infectious diseases.

A new study published in the journal Nature Nanotechnology shows that this low-cost tool, called Subak, is effective at telling when nucleic acid cleavage occurs, which happens when an enzyme called nuclease breaks down nucleic acids, such as DNA or RNA, into smaller fragments.

The traditional way of identifying nuclease activity, Fluorescence Resonance Energy Transfer (FRET) probe, costs 62 times more to produce than the Subak reporter.

“To make diagnostics more accessible to the public, we have to reduce costs,” said Soonwoo Hong, a Ph.D. student in the lab of Tim Yeh, associate professor in the Cockrell School of Engineering’s Department of Biomedical Engineering, who led the work. “Any improvements in nucleic acid detection will strengthen our testing infrastructure and make it easier to widely detect diseases like COVID-19.”

The research team – which also included Jennifer Brodbelt, professor of chemistry at UT Austin’s College of Natural Sciences, and MinJun Kim, professor of mechanical engineering in Southern Methodist University’s Lyle School of Engineering – replaced the traditional FRET probe with Subak reporter in a test called DETECTR (DNA endonuclease-targeted CRISPR trans reporter).

Subak reporters are based on a special class of fluorescent nanomaterials known as silver nanoclusters. They are made up of 13 silver atoms wrapped inside a short DNA strand. This organic/inorganic composite nanomaterial is too small to be visible to the naked eye and ranging from 1 to 3 nanometers (one billionth of a meter) in size.

Nanomaterials at this length scale, such as semiconductor quantum dots, can be highly luminescent and exhibit different colors. Fluorescent nanomaterials have found applications in TV displays and biosensing, such as the Subak reporters.

We have very clear evidence from mass spectrometry that transformation from Ag₁₃ to Ag₁₀ underlines the green to red color conversion observed in the sample, after DNA template digestion.”

Jennifer Brodbelt, professor of chemistry at UT Austin’s College of Natural Sciences

Subak reporters, which can be synthesized at room temperature in a single-pot reaction, cost just $1 per nanomole to make. In contrast, FRET probe – which employs complex steps to label a donor dye and a quencher – costs $62 per nanomole to produce.

“These highly luminescent silver nanoclusters can be called quantum dots as they show strong size-tunable fluorescence emission due to quantum confinement effect,” Yeh said. “No one can precisely tune the cluster size (and the corresponding emission color) until our demonstration of Subak,” which highlights the innovation of this research.

In addition to further testing the Subak reporter for nuclease digestion, the team also wants to investigate whether it can be a probe for other biological targets.

The work is supported by a National Science Foundation grant to Yeh and Brodbelt.

Source:

University of Texas at Austin

Journal reference:

Hong, S., et al. (2024). A non-FRET DNA reporter that changes fluorescence colour upon nuclease digestion. Nature Nanotechnology. doi.org/10.1038/s41565-024-01612-6.

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February 27, 2024