What gives us our sense of taste?

Written By: Abel B. Cortinas

Original Article: Schroer et al. Chemical Senses 2018
The Gist of It:
Our sense of taste has a profound effect on how we experience the world around us, whether we know it consciously or not. But how do we convert chemical stimuli into “taste”? In mammals, there are 5 traditional “tastes” that scientists agree upon: (1) sweet, (2) sour, (3) bitter, (4) salty, and the newest (5) umami (this word has its roots in the Japanese language and generally describes foods that are savory). The complex pathway of recognizing chemical signals as the tastes spicy (like the chemical capsaicin in chili peppers or allyl isothiocyanate in wasabi), salty (like sodium chloride, a.k.a. table salt), or sweet (such as sucrose, a.k.a. table sugar) begins with our taste buds. Our taste buds are a cluster of cells that have unique protein receptors responsible for turning these signals into taste, and these receptors are grouped into the Type I and Type II Taste Receptor families, or T1R and T2R families. Scientists at the West Virginia School of Medicine studied the role of a protein called RGS21, a member of the T2R family. RGS21 is responsible for making bitter-tasting chemicals more tolerable (basically it helped diminish taste signals in cells), but no one knew how this protein affected all 5 of the traditional tastes in living organisms. To help fill this knowledge gap, these researchers specifically eliminated RGS21 in mice. The expectation was that by removing RGS21, taste signaling would be amplified on all accounts. Oddly, what they found was the opposite of what was expected. Mice without the RGS21 protein continued to have a tolerance (that is, a diminished response) to bitter-tasting chemicals (which was not expected), and they did not have an increased preference for sweet-, savory-, or salty-tasting chemicals (also not expected). Despite these originally confusing results, this study ultimately revealed that taste bud sensor interactions during taste signaling are complex and that more work needs to be done to fully understand how these proteins work together to help us taste.
KM_364e-20190725164204

THE INTERACTION BETWEEN TASTE BUD SENSORS AND CHEMICAL STIMULI IS COMPLEX, REQUIRING ADDITIONAL WORK AND EXPERIMENTATION

The Nitty Gritty:
In a mouse model of Regulator of G-protein Signaling (RGS) protein deficiency, Schroer et al. evaluated the contribution of RGS21 to taste-profiling between G protein-coupled receptors (GPCRs) and GTPase-accelerating proteins (GAPs). A conditional “floxed” Rgs21 knockout mouse strain was utilized to study these interactions. Rgs21-null mice did not have altered body mass, tongue morphology, or taste bud number, and were phenotypically “normal” compared with wild type mice. In a 2-bottle choice test, Rgs21-null mice interestingly demonstrated a lack of aversion to quinine sulfate and a reduced aversion to denatonium benzoate, tastants representing the bitter taste profiles mediated by the T2R family of GPCRs. To determine whether RGS21 loss affected the acute phase of taste responses, chorda tympani whole-nerve recordings were performed while stimulating the tongue with various taste stimuli. Overall, reduced organismal tastant responses were consistent with observations of reduced nerve recordings in Rgs21-null mice. While no significant differences were observed in the amplitude of responses for sour tastants HCl and citric acid, Rgs21-null mice had significantly depressed responses after exposure to bitter tastants. Also, in contrast to the behavioral responses, there was no significant difference found for Rgs21-null mice in response to NaCl exposure. Because loss of RGS21 in taste receptor cells is expected to result in prolonged signaling of taste GPCRs, the counterintuitive experimental findings are hypothesized to be due to desensitization of taste receptors and/or downstream signaling components. Additionally, bidirectional effects of GPCR signaling upon RGS protein loss have been seen with siRNA-mediated knock-down of RGS8 and RGS9, whereby a decrease in maximal response / agonist potency and efficacy were observed at some GPCRs; this may occur in Rgs21-null mice with respect to proximal umami and bitter signaling. Lastly, parallels exist between the findings here and the sensory-specific distribution of RGS3 in C. elegans. It is hypothesized that RGS21, similar to RGS3 in C. elegans, may play a role in dampening tastant signaling so that the mammalian gustatory system is not overwhelmed when tastants become too abundant on the lingual epithelium. When RGS21 is under-expressed or absent, normal exposure of GPCR-mediated tastants may lead to compensatory desensitization or down-regulation of tastant response machinery, a hypothesis currently being pursued by the authors.
Original Research Article: Schroer, A.B., et al. “Development of Full Sweet, Umami, and Bitter Taste Responsiveness Requires Regulator of G protein Signaling-21 (RGS21).” Chem Senses 43.5 (2018): 367-378.

How does breast cancer behave like the immune system?

Written by: Ken Estrellas

Original Article: Hu et al. Biology Open 2019
The Gist of It:
Breast cancer is one of the most common cancers, with numerous cases reported worldwide every year. One of the earliest, localized stages of this disease, known as DCIS (ductal carcinoma in situ), represents about 1/5 of new breast cancer cases annually. Although the prognosis is positive, with 99% of patients surviving after 5 years and 95% surviving after 20 years, there is always a fear of the disease spreading beyond local tissue and beginning to metastasize. Understanding what drives disease progression in cancer is a topic of persistent interest. With that in mind, Dr. Hu and colleagues have demonstrated an interesting similarity between breast cancer cells and healthy macrophages, an important cell in the immune system – both types of cells respond positively to a protein known as CCL2. CCL2 is a chemokine, a type of protein that attracts certain types of cells to the tissues where it is being made. Normally, CCL2 binds to the receptor CCR2 to help attract macrophages in response to infections, inflammation, or other scenarios such as wound healing. In breast cancer, the presence of more CCL2 has been associated with disease progression. In this study, breast cancer cells that were modified to be even more responsive to CCL2 showed more invasive growth patterns, invasive potential, and expression of genes associated with tumor growth, along with reduced expression of a tumor suppressor gene. Notably, when breast cancer cells had the receptor CCR2 edited out, the cells were no longer responsive to CCL2, and their growth and invasion were inhibited when treated with CCL2. Additionally, expression of the gene ALDH1A1, which is known to be a pro-invasion factor for tumor cells, was associated with more CCR2 in the cells, more responsiveness to CCL2, and thus increased potential for invasion and metastasis. On the other hand, expression of the gene HTRA2, which is known to play a role in cell death, was associated with the opposite effect. Taken together, these findings paint a clearer picture of the mechanisms behind breast cancer metastasis and could someday help physicians diagnose and detect potential metastasis earlier in the disease cycle.
Screen Shot 2019-07-22 at 2.00.30 PM

Both macrophages and breast cancer cells show increased growth and responsiveness to the chemokine CCL2.

The Nitty Gritty
In this study, Hu et al. elucidated the roles of various components of the CCL2/CCR2 signaling axis in the proliferation, invasion, and potential metastasis of breast cancer cells. The breast cancer cell line SUM225, which has reduced expression of CCR2, was less prone to asymmetric cell growth patterns in the presence of recombinant CCL2 compared with the DCIS.com cell line, which has greater expression of CCR2. SUM225 cells modified to overexpress CCR2 subsequently showed increased responsiveness to CCL2, indicated by greater spheroid size, increased mean inversion index, increased expression of PCNA and TWIST1, and decreased expression of E-cadherin. Conversely, CRISPR-Cas9–mediated ablation of CCR2 in DCIS.com cells eliminated this effect, rendering the cells less responsive to CCL2 as indicated by decreased spheroid formation, lower levels of invasion, and decreased PCNA expression. Expression of the metabolic enzyme ALDH1A1 was increased in SUM225 cells treated with CCL2 and decreased in DCIS.com cells in which CCR2 expression was reduced by shRNA knockdown or CRISPR knockout treated with CCL2, suggesting that CCR2 positively regulates ALDH1A1 expression in both cell lines. This was supported by the fact that ALDH1A1 overexpression and knockdown affected the growth of CCR2-overexpressing SUM225 cells and CCR2-knockout DCIS.com cells, but not their invasion. The converse effect was observed with expression of the pro-apoptotic mitochondrial serine protease HTRA2, suggesting that CCR2 negatively regulates HTRA2. Taken together, these results reinforce previous literature associating the CCL2/CCR2 signaling axis with breast cancer metastasis and suggest that ALDH1A1 may play a specific role in mediating this process.
Original Research Article: Hu Q., et al. “Role of ALDH1A1 and HTRA2 expression in CCL2/CCR2-mediated breast cancer cell growth and invasion.” Biol Open 8.7 (2019): pii: bio040873. doi: 10.1242/bio.040873.

Passing dangerous cargo: How cells can transfer the mutant protein that causes Huntington’s disease

Written By: Kaitlyn Sadtler

Original Article: Sharma & Subramaniam. Journal of Cell Biology 2019
The Gist of It:
Huntington’s disease is a heritable degenerative brain disorder that is caused by a mutant protein – called Huntingtin (HTT) – which has something called a “triplet repeat expansion.” The diseased, mutant version has a CAG triplet repeat that is expanded, which means that the DNA sequence “CAG” is mistakenly replicated many more times than it should be (40–50+ of these CAG repeats); this causes clumping of the proteins and the disease. The effects of this disease are both mental and physical, with symptoms ranging from involuntary movements and impaired balance to depression and anxiety. There is no cure, and patients will die from their illness within 15–20 years of the initial onset of symptoms, which usually occurs from ages 30–50. Recently, researchers have discovered that the diseased protein can spread to neighboring brain cells through tunnel-like passages that form between a “donor cell” and an “acceptor cell”. This process depends upon the action of another protein called Rhes. In a set of experiments, the researchers were able to show that the protein Rhes creates small tunnels between cells that can transport mutant HTT, but not normal HTT. Also, the protein Rhes can tag things with a small protein called “SUMO” (think of this like putting a post-it note on a page of a book). If Rhes is genetically modified so it can no longer put its SUMO post-it note on mutant HTT, then the transport of mutant HTT from one cell to another is decreased. Figuring out how these mutant proteins spread in the human body can help identify new targets for treating diseases such as Huntington’s by stopping further progression or even ameliorating existing symptoms.
KM_364e-20190710104007

A protein called Rhes helps make connections between neighboring cells and transport mutant Huntingtin protein (the cause of Huntington’s disease) between brain cells.

The Nitty Gritty:
Previous research has identified the presence of tunneling nanotubes (TNTs) that provide cytoplasmic connections between neighboring cells. In this study, Sharma and Subramaniam describe TNT-like structures that are capable of transporting mutant Huntingtin protein (mHTT) but not wild-type HTT from a donor cell to an acceptor cell. This transfer is dependent upon the GTPase/SUMO E3-like protein Rhes, which is found mainly in brain tissue. When transfected into striatal cells (STHdhQ7/Q7), GFP-linked Rhes induced the formation of filipodia-like protrusions, which were not present in cells transfected with GFP-RhoA or GFP alone. These protrusions connected with other cells transiently in a “kiss-and-run” fashion wherein membrane fusion was detected via scanning and transmission electron microscopy. This behavior was prevented with the addition of the actin polymerization inhibitor cytochalasin D. The transition of Rhes between cells with TNT-like connections was shown by tagging Rhes with GFP and acceptor cell membranes with mCherry and measuring double-positive cells via flow cytometry. TNT-like Rhes tunnels are capable of transiting mHTT between neighboring cells but not wild type HTT or other proteins such as mTOR or wild type Tau. If the SUMOylation activity of Rhes was inhibited (Rhes C263S), mHTT SUMOylation was inhibited, or SUMO was knocked out using CRISPR/Cas9, transport of mHTT was significantly inhibited. These findings describe a form of transfer of mHTT from neighboring cells through direct connection of the cytoplasms via Rhes-dependent TNT-like connections.
Original Research Article: Sharma, M. & Subramaniam, S. “Rhes travels from cell to cell and transports Huntington disease protein via TNT-like protrusion.” J Cell Biol 218.6 (2019): 1972.

Patching together cures: A new method to deliver antibiotics to patients

Written By: Rebecca Tweedell

Original Article: Rodgers et al. Antimicrobial Agents and Chemotherapy 2019
The Gist of It:
Bacterial infections are a major threat to newborn babies. In developed countries, improved sanitation, monitoring, and treatment have greatly reduced the risk of infection, but in low- and middle-income countries, bacterial infections can still be a death sentence for a baby. One of the critical antibiotics used to combat these bacterial infections is gentamicin because it is cheap and effective. Unfortunately, it is normally given as a shot into the muscle (like the flu vaccine is), and this requires trained medical workers with access to safe needle disposal, which is not always available in the areas where the drug is needed the most. To avoid the drawbacks of gentamicin, researchers have been working on finding new ways to get the drug into people’s bodies. Researchers at Queens University Belfast in the United Kingdom recently invented a microarray patch that can deliver gentamicin without the need for shots. The microarray patch looks similar to the nicotine patches that you may have seen, and it uses short, pain-free, microscopic needles that dissolve to release the gentamicin. This allows the microarray patch to be used by healthcare workers without the need for special training and needle disposal. The researchers put these patches to the test in a mouse model of Klebsiella pneumoniae infection; K. pneumoniae is a bacterium that can cause pneumonia, bloodstream infections, and intra-abdominal infections in humans. Mice were infected with K. pneumoniae and then treated with a traditional shot of gentamicin or the microarray patch. The researchers found that both treatments led to significant decreases in the number of bacteria in the lungs and lymphoid tissue in the mice. This means that microarray patches could potentially be used in place of gentamicin shots, reducing the need for needles, needle disposal, and trained staff. Similar methods could also be used to improve the delivery of other treatments, vastly improving access to medical care for those in remote and poor areas and saving the lives of thousands of infants.
iceberg_rt

Mice infected with K. pneumoniae were successfully treated with a microarray patch that delivers gentamicin.

The Nitty Gritty:
Microarray patches containing gentamicin were fabricated in two parts, the baseplate and the needles. Baseplates were made from 15% polyvinylpyrrolidone (PVP) cast into a mold to create a thin rectangular cuboid shape. The microscopic needles were made by adding a solution containing 3.4% sodium hyaluronate, 1% PVP, and 10% gentamicin sulfate to a sawtooth-shaped mold and applying 3–4 bar pressure for 15 minutes to push the mix into the grooves of the mold. The baseplate was then applied to the base of the needles, and the entire assembly was dried for 48 hours before the full microarray plate was removed from the mold. Each resulting needle was approximately 500 μm long and 300 μm wide at the base. To test the efficacy of the microarray plate in vivo, C57BL6 mice were inoculated with K. pneumoniae intranasally. Eight hours after infection, mice were treated with an intramuscular injection of gentamicin (7.5 mg/kg body weight), the gentamicin microarray plate (placed on the ear and secured with micropore tape, then replaced with a fresh microarray plate at 24 hours post-infection), or no treatment. Bacterial levels in the nasal-associated lymphoid tissue (NALT), lungs, and spleens were quantified 48 hours post-infection. Mice treated with the microarray plate had statistically significant reductions in bacterial levels in the NALT and lungs compared with bacterial levels in untreated mice. Treatment with the intramuscular injection of gentamicin produced similar results. These results showed in vivo evidence that the microarray plate could be used as an alternative delivery method for life-saving gentamicin treatment.
Original Research Article: Rodgers, A.M., et al. “Control of Klebsiella pneumoniae infection in mice by using dissolving microarray patches containing gentamicin.” Antimicrob Agents Chemother 63 (2019): e02612-18.

Our body’s soldiers are also architects: macrophages and skin healing

Written By: Kaitlyn Sadtler

Original Article: Shook et al. Journal of Investigative Dermatology 2016
The Gist of It:
When you get a cut on your skin, lots of things happen. One of the biggest responsibilities of our skin is to protect us from the outside world so that the bad bugs on the outside can’t get inside and wreak havoc in the form of infections. Our immune system can help fight these infections after an injury and also help rebuild the damaged tissue. Researchers from Yale looked at a specific type of cell – the macrophage – and explored what types of macrophages are important for healing and regeneration. Macrophages can have different personalities depending upon the challenge they are facing; in immunology these personalities are called “polarization states”. Two of these states are helpfully named M1 and M2 – not too much information in those names but bear with me. M1 macrophages are important for what you think of immediately when you think of our immune system; they fight off dangerous bacteria and viruses and protect our body from infection. M2 macrophages on the other hand can help fight other dangers like parasites in your gut, but they are also very important in healing wounds. These M2 macrophages are known for the presence of a protein called CD206 on their surface, and they start appearing in larger numbers about 3 days after an injury to the skin. The researchers at Yale identified a new sub-population of these macrophages that have both CD206 and another protein called CD301b, and they found that these macrophages are critical for skin healing and regeneration. While most macrophages in an injury have CD206, and the majority of these also have CD301b, there are some that don’t have CD301b. When they took mice that didn’t have any macrophages and transplanted in just the macrophages without CD301b, they didn’t get proper healing. These studies help us moving forward by identifying specific cells that we can target to promote healing and regeneration.
47_doodle

M2-Macrophages that express CD206 have been associated with wound healing. Here, researchers show that a subset of CD301b+CD206+ macrophages are the main mediators of their pro-regenerative response.

The Nitty Gritty:
Using a mouse model of full-thickness skin injury, Shook et al. identified a population of CD301b+CD206+ pro-regenerative macrophages that were necessary for skin repair and regeneration. Using flow cytometry, researchers evaluated the expression of Ly6C and CD206 on F4/80+ macrophages at days 1–7 post-injury. Ly6Chi macrophage populations peaked at 1.5 days post-injury, corresponding with increased expression of Il17, Il1b, Il1a, and Ifng mRNA at the same timepoint; these were defined as “early-stage” macrophages. CD206+ macrophages were most prevalent at 5 days post-injury and corresponded with increases in Il10 and Tgfb1 mRNA expression and were termed “mid-stage” macrophages. Using LysMcre-iDTR–based depletion of macrophages continuously or at the early or mid-stages of healing, they showed that there was significantly lower re-epithelialization, cell proliferation, fibroblast population numbers, and vascularization when macrophages were either continuously depleted or depleted during mid-stage healing. Depletion of early stage macrophages had no to minimal effect on skin healing and regeneration. To further characterize macrophage sub-classes, they analyzed the expression of CD301b (protein product from the gene Mgl2), which had previously been described as a marker of M2 macrophages (Raes et al. J Leuk Bio 2004). Populations of CD301b+ macrophages peaked in skin wounds at 5–7 days post-injury. Depletion of CD301b+ macrophages using Mgl2DTR mice resulted in similar deficits in healing as those seen in the continuous and mid-stage macrophage depletions described previously. Furthermore, addition of sorted CD206+CD301b+ macrophages to the wound bed of injured mice induced higher levels of re-epithelization, cell proliferation, and fibroblast migration when compared to vehicle control or transplantation of CD206+CD301 macrophages. These data show a critical role for CD301b+CD206+ macrophages in tissue regeneration.
Shook, B., et al. “CD301b+ macrophages are essential for effective skin wound healing.” J Invest Dermatol 136.9 (2016): 1885-1891.