Home » Biology (Page 10)

Category Archives: Biology

Want to Get Involved In Research?

[su_heading size="15" margin="0"]The BioInnovation Group is an undergraduate-run research organization aimed at increasing undergraduate access to research opportunities. We have many programs ranging from research project teams to skills training (BIG-RT) and Journal Club.

If you are an undergraduate interested in gaining research experience and skills training, check out our website (https://bigucd.com/) to see what programs and opportunities we have to offer. In order to stay up to date on our events and offerings, you can sign up for our newsletter. We look forward to having you join us![/su_heading]

Newest Posts

Novel Pathway Elucidates Potential for Nitric-Oxide Produced by Tumor-Associated Macrophages to Confer Resistance to Chemotherapy Drug Cisplatin

January 24, 2020

By Reshma Kolala, Biochemistry & Molecular Biology ‘22

Authors Note: This past summer I was given the incredible opportunity to work in the Thurmond Lab at the City of Hope where I investigated a point mutant of the Syntaxin 4 protein on -cell function and apoptosis. The following piece reviews a publication that was fundamental to both the understanding and methodology of my project. 

 

Introduction

Cisplatin (CDDP) is a widely used chemotherapy drug that induces apoptosis in solid tumor cells, which are cells that lack cysts or liquid areas such as carcinomas, sarcomas, and lymphomas. The platinum-based chemotherapeutic agent was popularized in the late 1970s as the antitumoral toxicity of platinum compounds became known for their clinical efficacy against solid tumors (1). Although initially promising, many patients suffer a relapse due to the development of cisplatin resistance, largely as a result of their ability to overcome the apoptogenic effects of the drug. To elucidate the underlying mechanisms behind the propagation of cancer progression and chemotherapy resistance, an understanding of the tumor microenvironment is crucial. The tumor microenvironment is comprised of a complex and dynamic milieu that surrounds stromal cells. Among these cells, tumor-associated macrophages (TAMs) represent the largest population of infiltrating inflammatory cells in malignant tumors. TAMs have been suggested to possess a tumor-promoting phenotype that drives multiple mechanisms, most notably tumor cell proliferation and drug resistance (2). Initially, TAMs are in the classically-activated M1 state, in which their proinflammatory characteristics disables tumor growth. As tumors mature, however, they switch to an alternatively-activated M2 state, promoting tumor development and immunosuppression. As M2-like TAMs are major contributors to chemotherapeutic resistance, they are frequently targeted for cancer immunotherapies. 

M2-like TAMs are capable of producing nitric oxide (NO) via expression of inducible NO synthase (iNOS). NO is an important cell signaling molecule that is critical for many physiological processes such as neurogenesis and angiogenesis (3). At low levels, NO displays cytoprotective properties, promoting tumor growth, but can be cytotoxic to tumor cells when produced at high levels (4). The cytoprotective tendency of NO has been linked to the inhibition of the sphingomyelin-metabolizing enzyme acid sphingomyelinase (A-SMase). Traditionally, the activation of A-SMase (most commonly by chemotherapeutic drugs such as CDDP) drives the hydrolysis of sphingomyelin to generate ceramide. Ceramide, in coalition with other molecules, forms a cluster that drives transmembrane signaling of apoptotic death to effectively kill tumor cells (5). By contrast, it has been found that at relatively low concentrations NO hinders the beneficial apoptotic effect of A-SMase, resulting in resistance to the chemotherapeutic drug CDDP. The elucidation of this mechanism is the focus of research conducted by Perrotta et al. in 2018. 

A study led by Perrotta et al. investigated the potential for NO, a byproduct of TAMs, to be responsible for the mechanism conferring resistance to CDDP (6). An increased concentration of intracellular NO leads to the activation of the membrane-bound protein Syntaxin 4 (STX4) via a pathway that involves the production of cGMP and activation of protein kinase G (PKG). As STX4 aids in the translocation of A-SMase, an enzyme involved in apoptosis, to the plasma membrane, a decrease in the STX4 protein would result in resistance to the intended apoptotic effect of CDDP (Figure 1). However, it was found that a point mutant of the STX4 protein, namely the STX4-S78A mutant, is unable to be phosphorylated by PKG due to the chemical nature of the alanine side chain. This prevents proteasomal degradation, thus leading to successful tumoral apoptosis.

Figure 1: Nitric Oxide (NO)-Mediated Resistance to Apoptotic Effect of Cisplatin

A schematic of the Nitric Oxide-mediated resistance to chemotherapeutic drug CDDP. The introduction of CDDP (1) leads to an increase in the intracellular concentration of NO in TAMs (tumor associated macrophages). (2). This leads to the generation of cGMP via the cGMP pathway (4). This leads to PKG activation (5) and results in STX4-WT phosphorylation at Ser-78 residue (6a) to ultimately allow degradation of STX4 via the proteasome. The STX4-S78A mutant however, cannot be phosphorylated (6b), preventing STX4 degradation by proteasomes. If left intact, the STX4 protein mediates the binding of A-SMase to the plasma membrane (7), resulting in tumor cell death (8).

 

Methodology & Results

The presence of M2 polarized TAMs in U373 human glioma cells were confirmed through immunostaining of the M2 subtype marker CD206 and iNOS. The presence of double positive cells illustrated the ability for M2-TAMS in glioma cells to produce NO. To investigate the effect of CDDP-induced apoptosis, human glioma cells were cocultured with M2-TAMs and then treated with CDDP in the presence of the iNOS inhibitor L-NAME. Annexin V apoptosis staining data illustrated a three-fold decrease in tumor cell death when CDDP-treated U373 glioma cells were cocultured with M2-TAMs. However, the addition of L-NAME resulted in a roughly two-fold increase in the abundance of dead tumor cells. Similar results were observed in the GL261 murine cells. This demonstrates that NO induces resistance to the apoptotic effect of CDDP as the inhibition of the NO precursor iNOS resulted in increased efficacy of the CDDP treatment. 

The NO pathway operates via activation of the cGMP pathway. This was confirmed by administration of ODQ, a guanylate cyclase inhibitor that prevents NO-dependent cGMP generation, and DETA-NO (an NO donor) to U373 cells treated with CDDP. Results indicated a roughly two-fold increase in the percentage of apoptotic cells when treated with cGMP inhibitor ODQ, illustrating that the cGMP pathway is a significant contributor to CDDP. The generation of cGMP is correlated with the inhibition of CDDP-induced apoptosis, therefore, the presence of a cGMP inhibitor (ODQ) should increase levels of apoptosis, which is reflected in the data. 

It has been previously demonstrated that acid sphingomyelinase (A-SMase) is activated by CDDP. A-SMase activation often occurs via translocation to the plasma membrane, therefore a cell surface biotinylation assay was used in U373 to confirm increased expression of A-SMase at the plasma membrane 30 minutes post-CDDP treatment. As expected, western blotting data indicated heightened expression of the enzyme when compared to A-SMase expression in U373 cells treated either with DETA-NO or 8Br-cGMP (an activator of cGMP-dependent kinases).

The final step of the pathway conferring resistance to CDDP involves the phosphorylation of Syntaxin 4 (STX4). STX4 is a membrane-bound SNARE protein. SNARE proteins form a SNARE core complex that orchestrate vesicle fusion to the plasma membrane. In tumor cells, STX4 is known to control the trafficking of A-SMase from intracellular compartments to the plasma membrane, allowing the A-SMase to carry out the intended apoptotic effect of CDDP. However, data shows that the phosphorylation of STX4 at the Ser-78 residue promotes its subsequent proteasomal degradation. 

 

Conclusion 

It has been found that NO released by M2-polarized TAMS has led to resistance against a widely used chemotherapy drug CDDP. This is achieved via the generation of cGMP and the activation of PKG in response to increased intracellular concentrations of NO. This leads to phosphorylation of the STX4 protein at Ser-78, resulting in its degradation. The decrease of the STX4 protein immobilizes A-SMase, preventing the enzyme from reaching the plasma membrane to initiate tumoral apoptosis.

As previously mentioned, the effect of NO in large quantities yields cytotoxic properties. In smaller concentrations however, NO has exhibited protective effects. The dichotomous behavior of NO on tumor biology could be a result of a myriad of factors, including the conditions of the tumor microenvironment and its origin. The generation of NO by TAMs protects tumor cells from apoptosis through the indirect inhibition of A-SMase activity. It is important to note that this action is dependent on the ability of NO to generate cGMP in tumoral cells and block the CDDP-induced, STX4-dependent translocation of A-SMase to the plasma membrane.  

The elucidation of a chemotherapeutic resistance mechanism provides an understanding of Cisplatin’s efficiency and the origin of its drug-resistant tendencies. The observed proteasome-dependent degradation of STX4 may also be relevant to cancer therapies based on proteasome inhibitors. Prevention of the proteasomal degradation mechanism would increase efficacy of many chemotherapeutic treatments. This is due to the preservation of pro-apoptotic factors which would permit the programmed cell death of various proteins, preventing the accumulation of deleterious proteins. Currently, proteasome inhibitors are approved for treating multiple myeloma, a cancer of plasma cells or cells that produce antibodies. As NO is a major signaling molecule in the immune system, the elucidation of the CDDP resistance pathway yields further insight into how NO operates and proliferates. This renders research put forth by Perrotta et al. applicable to various fields of research beyond cancer. 

 

References

  1. Dasari, S., & Tchounwou, P. B. (2014, October 5). Cisplatin in cancer therapy: molecular mechanisms of action. Retrieved from https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4146684/.
  2. Lin1, Y., & Jianxin. (2019, July 12). Tumor-associated macrophages in tumor metastasis: biological roles and clinical therapeutic applications. Retrieved from https://jhoonline.biomedcentral.com/articles/10.1186/s13045-019-0760-3.
  3. Nitric Oxide and Cell Stress – Cell Signaling and Neuroscience: Sigma-Aldrich. (n.d.). Retrieved from https://www.sigmaaldrich.com/life-science/cell-biology/cell-biology-products.html?TablePage=9552558.
  4. XU, W., LIU, L. Z., LOIZIDOU, M., AHMED, M., & CHARLES, I. G. (n.d.). The role of nitric oxide in cancer. Retrieved from https://www.nature.com/articles/7290133.
  5. Gorelik, A., Illes, K., Heinz, L. X., Superti-Furga, G., & Nagar, B. (2016, July 20). Crystal structure of mammalian acid sphingomyelinase. Retrieved from https://www.nature.com/articles/ncomms12196.
  6. Perrotta, C., Cervia, D., Di Renzo, I., Moscheni, C., Bassi, M. T., Campana, L., … Clementi, E. (2018, May 29). Nitric Oxide Generated by Tumor-Associated Macrophages Is Responsible for Cancer Resistance to Cisplatin and Correlated With Syntaxin 4 and Acid Sphingomyelinase Inhibition. Retrieved from https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5987706/.

How Expectations Shape Perception

January 17, 2020

By Neha Madugala, Cognitive Science, ‘21

Author’s Note: Previous studies in neuroscience have suggested that our expectations and prior experiences impact how we perceive reality and current tasks. This idea is embedded in Bayesian integration, also referred to as multisensory integration, which essentially studies how the brain combines information obtained from sensory neurons to affect perception and create a distinct outlook on an organism’s surroundings. It defines how we view and think about our environment. 

This topic was particularly interesting to me because expectations can serve to aid our further understanding of incoming information, but can also inhibit our understanding if the new information contradicts our predictions.

 

Scientists classify expectations as essentially creating a placebo effect. The idea is that an individual’s expectations can influence the outcome of a certain treatment, their personal performance, or their feelings towards a person, place, or object. These “prior beliefs” can shape our reality and influence our perception of things we encounter in our daily life. The source of this placebo is argued to either be due to a conditioned response, where a situation has occurred multiple times with the same outcome (behaviorist approach) or that the placebo is a result of expectancy (expectation approach) (4). 

In a recent study by MIT, researchers argued for the latter hypothesis suggesting that expectations are the suspect for shaping our interpretation of our surrounding environment. MIT neuroscientists were inspired by the idea of Bayesian integration, which is the process of incorporating prior knowledge with new and uncertain information (6). Jazayeri et al. trained monkeys in a task called “ready-set-go.” The monkeys are shown a starting signal and ending signal. After seeing both signals, they are expected to press a button after the same time interval between the starting and ending code has passed (6). The neuroscientists had short intervals, which spanned from 480 to 800 milliseconds and long intervals, which were 800 to 1,200 milliseconds. The monkeys were given a visual cue at the start of each trial signalling whether the trial would be a “short” or “long” scenario (6). 

Monkeys were trained in either the short or long intervals. Each prior condition consisted of four blocks and then was followed by the 800 msec block. When all the monkeys were given an interval of 800 msec, those trained with a shorter interval gave an average response that was a little less than 800 msec and those trained with a longer interval gave an average response that was a little longer than 800 msec (6). These results reveal that monkeys trained under longer time intervals had an expectation for the 800 msec time interval to be longer, and those trained under a shorter time interval had an expectation that the 800 msec time interval would be shorter. This experiment was reproduced in humans and they found similar results (6). 

The MIT researchers wanted to further determine which areas of the brain are responsible for the influence of expectations from the short and long trials affected the perception of the length of time for the 800 msce interval. Jazayeri et al, found that prior experiences strengthen a pattern of synaptic connections in a region located in the frontal cortex, which has previously been determined to be involved in temporal resolution (6). These patterns of synaptic connections were further computationally modeled. These models were found to perform the tasks in the same manner as the monkeys used in the previous experiments. 

In another study at the University of Wisconsin, researchers conducted a similar study in humans testing how prior expectations of taste can influence an individual’s perception of the taste. Sarinopoulos et al. tested how subjects responded to an aversive taste, which was a diluted solution of quinine hydrochloride, with deceiving and accurate cues to test how placebo altered the participants’ responses. One group was informed that they were about to receive a highly aversive taste, while the other group was deceived and told they would receive a less aversive taste. The group told that they would receive a less aversive taste acted as the experimental group and were used to test whether placebo is altered by expectations or further stated if expectations affect our perception. Sarinopoulos et al. compared the expectancy effect from the highly aversive to less aversive groups by measuring changes in the insula and the amygdala, which are activated by aversive tastes (5). 

The insula is associated with pain processing, which explains the presence of many sensory receptors for visceroceptive, referring to signals received from the heart, lungs, stomach, bladder, and other internal organs near the trunk region, and interoceptive inputs, including regions of the brain such as the thalamus, brainstem, insula, somatosensory, and anterior cingulate cortex (2). The amygdala, located near the insula, specifically the medial temporal lobe forms a part of the limbic system and plays a fundamental role in emotion processing, specifically fear and pleasure (1). 

The study was conducted using rapid event-related fMRI design. In order to create a basis of comparison, the study included various conditions with different levels of aversion and manipulation – totaling to seven experimental conditions. The wide array of conditions allows Sarinopoulos et al. to eliminate differences if the perception involves a more aversive or less aversive taste by including both aversive and pleasant taste with varying perceptions. 

They found that subjects demonstrated a consistent pattern of rostral anterior cingulate cortex (rACC), which plays an important role in decision-making and attention, and orbitofrontal cortex (OFC), which is also involved in decision-making, activation when presented with the misleading cue, followed by a decrease in the bilateral insula responses to the highly aversive taste. It was also associated with a smaller amygdala response. These findings suggest that the rACC and OFC are both correlated with the placebo effect and are associated with the aversive and pleasant perception of taste. 

 

References

  1. “Amygdala.” ScienceDaily, ScienceDaily, www.sciencedaily.com/terms/amygdala.htm.
  2. D.P.Papoiu, Alexandru. “Functional MRI Advances to Reveal the Hidden Networks Behind the Cerebral Processing of Itch.” ScienceDirect, Academic Press, 5 Aug. 2016, www.sciencedirect.com/science/article/pii/B9780128028384000285.
  3. “Figure 2f from: Irimia R, Gottschling M (2016) Taxonomic Revision of Rochefortia Sw. (Ehretiaceae, Boraginales). Biodiversity Data Journal 4: e7720. Https://Doi.org/10.3897/BDJ.4.e7720.” doi:10.3897/bdj.4.e7720.figure2f.
  4. Haour, France. “Mechanisms of the Placebo Effect and of Conditioning.” Neuroimmunomodulation, vol. 12, no. 4, 2005, pp. 195–200., doi:10.1159/000085651.
  5. Sarinopoulos, Issidoros, et al. “Brain Mechanisms of Expectation Associated with Insula and Amygdala Response to Aversive Taste: Implications for Placebo.” Brain, Behavior, and Immunity, U.S. National Library of Medicine, Mar. 2006, www.ncbi.nlm.nih.gov/pubmed/16472720.
  6. Trafton, Anne, and MIT News Office. “How Expectation Influences Perception.” MIT News, 15 July 2019, news.mit.edu/2019/how-expectation-influences-perception-0715.
    panelC.A.HanlonL.T.DowdleJ.L.Jones, Author links open overlay, et al. “Biomarkers for Success: Using Neuroimaging to Predict Relapse and Develop Brain Stimulation Treatments for Cocaine-Dependent Individuals.” ScienceDirect, Academic Press, 25 July 2016, www.sciencedirect.com/science/article/pii/S0074774216301131.

Reading into the Future: Development of Long-read DNA Sequencing

December 14, 2019

By Aditi Goyal, Genetics and Genomics, ‘22

At this moment, the next revolution in the field of biology is currently underway: third-generation sequencing, or Long-Read sequencing. Instead of relying on cluster-based short read technology (1), third-generation sequencing builds a DNA sequence on a nucleotide basis, therefore eliminating the extensive process of read alignment.

Until now, scientists across the world have been heavily relying on Next Generation Sequencing (NGS) for getting DNA sequences. This technology creates clusters of short DNA sequences, which range anywhere from 50 to 150 base pairs in length, by using fluorescent nucleotides (2). It is often referred to as sequencing by synthesis because a DNA sequence is created by tracking which nucleotides are being used to build the parallel strand. NGS has served the scientific community well, providing extremely high coverage and high accuracy reads, as well as slashing the cost and time to sequence an entire genome (2). However, the drawbacks are just as serious. While NGS is a fantastic candidate for bacterial or archaeal genomes, it fails to capture the complexity of eukaryotic genomes. About half of the human genome is comprised of repeated sequences (2). Currently, the function of these repeated regions remains unclear, partially due to the fact that it is not possible to get an accurate DNA sequence of these areas using short-read sequences. With a maximum read size of 150 base pairs for NGS, there are too many potential matches for a read that small for scientists to accurately assign that read to a region in the genome. Another major problem is the quality of each read. While the technology itself is very accurate, there are several sources of error that quickly cause the quality of each read to deteriorate, such as biases during the PCR of mixtures, polymerase errors, base misincorporation, cluster amplification errors, sequencing cycle errors, and incorrect image analysis (3). All these errors result in about 1% of bases being read incorrectly, which, when applied to a 3 billion base pair genome, can be incredibly damaging.

This is why long-read sequencing is such a breakthrough. By analyzing a DNA sequence from nucleotide to nucleotide, scientists can build considerably longer reads with a much higher confidence level as compared to NGS. Ideally, with this technology, scientists will be able to produce de novo whole genome sequences for patients with genetic disorders, allowing them to understand the root of their disease at an unmatched resolution. This could pave the way to accurately diagnosing and curing complex genetic diseases. In the last few years alone, several papers have been published on the impacts of long-read sequencing investigating diseases such as Parkinson’s disease, fragile X syndrome, Alzheimers, and ALS (11). Other applications include improving our understanding of human genetic diversity.  Recent studies show that the reference human genomes available today do not accurately represent humanity at a global level, but rather significantly overrepresent people of european descent (12). With the rise of long-read sequencing, it will be easier and cheaper to fully sequence a human genome, allowing us to expand the resources available and accurately reflect the human population.

 

There are currently several companies researching long-read sequencing, however, the most promising company appears to be Pacific Biosciences (Pac Bio) due to their development of single molecule real time sequencing (SMRT) (4, 5).

There are 2 key inventions that allow for the success of SMRT. The first is the fluorescent tagging.

Like with NGS, each nucleotide is modified to fluoresce a certain color, indicating which nucleotide it is, however with SMRT, the fluorescence is linked to the terminal phosphate of a nucleotide, instead of the base itself (8). Also similar to the NGS, the complementary strand continues to build. Now, when the DNA polymerase cleaves off the terminal phosphate, it releases the fluorescent group, which allows us to track which nucleotide was incorporated based on the color of the fluorescent.

The second innovation is the zero-mode waveguide (ZMW). The ZMW is a small nano chamber that contains the DNA sample during the sequencing process. It passes refracted light through so that the fluorescence of the nucleotides can be seen. This technology essentially acts as a microscope, allowing us to gain a powerful resolution of the DNA structure. Each ZMW can recognize over 10 base pairs per second with extreme accuracy. Additionally, given the ability for these ZMWs to be run in parallel, thousands of chambers can be sequenced at the same time, allowing for a fast cycle and long reads. 

The advantages of SMRT are clear: it allows for long reads to be built. This means that scientists will have the ability to understand the overall complexity of large eukaryotic genomes. Another advantage is the speed and portability of the technology. Once it is completely developed, SMRT will be able to sequence an entire human genome in under 3 minutes for less than $100 in a device the size of a flash drive, a stark difference from today’s estimate (9).

Like any novel technology, there are some challenges that must be overcome before SMRT can be used commercially. The most pressing is concerns over accuracy. Individual reads can contain 11-14% errors on average, dragging the quality score of the read down. However, developers have noticed that these errors occur at random across the genome. By using a 10x coverage method, 9 out of 10 times, SMRT will provide the correct sequence for that point, which allows the accuracy to rise to approximately 99.99%. 

Overall, SMRT is a revolutionary development that will soon change the way we understand biology. It will allow us to gain a holistic understanding of complex eukaryotic genome and will provide a higher resolution of the genome that we can use for further analysis.

 

References

  1. “Illumina Sequencing Technology.” Illumina, October 11, 2010. https://www.illumina.com/documents/products/techspotlights/techspotlight_sequencing.pdf.
  2. Treangen, Todd J, and Steven L Salzberg. “Repetitive DNA and next-Generation Sequencing: Computational Challenges and Solutions.” Nature reviews. Genetics. U.S. National Library of Medicine, November 29, 2011. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3324860/.
  3. Fox, Edward J, Kate S Reid-Bayliss, Mary J Emond, and Lawrence A Loeb. “Accuracy of Next Generation Sequencing Platforms.” Next generation, sequencing & applications. U.S. National Library of Medicine, 2014. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4331009/.
  4. Check Hayden, Erika. “Genome Sequencing: the Third Generation.” Nature News. Nature Publishing Group, February 6, 2009. https://www.nature.com/news/2009/090206/full/news.2009.86.html.
  5. Check Hayden, Erika. “Genome Sequencing: the Third Generation.” Nature News. Nature Publishing Group, February 6, 2009. https://www.nature.com/news/2009/090206/full/news.2009.86.html.
  6. Eid, John, Adrian Fehr, Jeremy Gray, Khai Luong, John Lyle, Geoff Otto, Paul Peluso, et al. “Real-Time DNA Sequencing from Single Polymerase Molecules.” Science. American Association for the Advancement of Science, January 2, 2009. https://science.sciencemag.org/content/323/5910/133.
  7. “Video: Introduction to SMRT Sequencing.” PacBio. Accessed November 7, 2019. https://www.pacb.com/videos/video-introduction-to-smrt-sequencing/.
  8. “Single Molecule Real Time Sequencing – Pacific Biosciences.” YouTube. YouTube. Accessed November 7, 2019. https://www.youtube.com/watch?v=v8p4ph2MAvI.
  9. Schadt, Eric E., Steve, Andrew, and Turner. “Window into Third-Generation Sequencing.” OUP Academic. Oxford University Press, September 21, 2010. https://academic.oup.com/hmg/article/19/R2/R227/641295.
  10. Roberts1, Richard J, Mauricio, and Michael C Schatz3. “The Advantages of SMRT Sequencing.” Genome Biology. BioMed Central, July 3, 2013. https://genomebiology.biomedcentral.com/articles/10.1186/gb-2013-14-7-405.
  11. Martin O Pollard, Deepti Gurdasani, Alexander J Mentzer, Tarryn Porter, Manjinder S Sandhu, Long reads: their purpose and place, Human Molecular Genetics, Volume 27, Issue R2, 01 August 2018, Pages R234–R241, https://doi.org/10.1093/hmg/ddy177

CD47-SIRPα Pathway as a Target for Cancer Therapeutics

December 6, 2019

By: Nicholas Garaffo, Biochemistry and Molecular Biology, 20’

Authors’ Note: I originally wrote this piece for my UWP 104E class Writing in the Science’s, but I have since expanded my topic and complicated my original analysis. Ultimately, I submitted this piece to the Norman J. Lang Prize, was awarded second place, and presented my research to the UC Davis college deans. I chose to focus my literary review on cell signaling pathways because I hope to study such topics in my PhD. This topic has impacted my life personally because my grandmother was diagnosed with non-hodgkin’s lymphoma my freshman year of college. In fact, during this review the drugs she was treated with were mentioned, and the CD47-SIRPa pathway may actually be used to treat such a disease. 

 

Abstract

According to the American Cancer Institute, in 2018, cancer had an estimated 1,735,350 new cases and 609,640 people died in the United States alone1. Like many deadly diseases, cancer has found ways to evade the immune system. Many cancers overexpress CD47—a widely expressed “don’t eat me” signal—which interacts with the immune cells’ signal receptor protein alpha (SIRPα), to prevent programmed cell removal (PrCR) 2. ‘Don’t eat me’ signals are a class of cell surface proteins that tell the immune system the corresponding cell is healthy and performing properly. Recent advances have been made to target the CD47-SIRPα pathway to prevent the antiphagocytic activity seen in many cancers. The scope of this review is limited to two new methods used to inhibit the CD47-SIRPα pathway: anti-CD47 and SIRPα antibodies, and small peptide inhibitors. The antibodies for CD47 have shown effectiveness in clinical trials. Antibody inhibition for CD47 and SIRPα were compared, and SIRPα produced better cell type specific inhibition, but similar on-target healthy cell phagocytosis caused anemia in both trials. Several factors, including degradation and inability to penetrate dense tumors, hinder antibody treatment in all cancer patients; therefore, small peptide inhibitors offer an alternate route for inhibition to occur. 

 

Introduction 

PrCR is an efficient and accurate process that clears dead, dying, or infectious cells. Phagocytic macrophages—neutrophils, dendritic cells and monocyte derivatives—perform PrCR, and acts independently of apoptosis—programmed cell rupture. Without such processes apoptosis would release cellular contents, such as proinflammatory signals, into the extracellular space3, 4. Such signals can activate inflammatory responses leading to organ and tissue damage. Cells that are under oxidative stress release chemotactic factors that attract immune cells4. Once the macrophage locates the infected cell, it recognizes the cell through “don’t eat me” or “eat me” ligands to prevent or induce cell engulfment, respectively. The scope of this review is limited to a single signal—receptor interaction between CD47—a widely expressed transmembrane protein5—and signal receptor protein alpha (SIRPα)—a receptor expressed on phagocytic immune cells. CD47 links to SIRPα and acts as a “don’t eat me” signal to prevent cellular phagocytosis. 

Macrophages activate specific transcription factors in response to environmental cues. Notch signaling describes the macrophages’ internal protein cascade upon receptor-ligand interactions. The macrophage responds by adjusting its polarization into either phagocytic, categorized as the M1 polarization, or non-phagocytic (M2)6. This is important because a macrophages’ phenotype is environmentally dependent on surrounding cell signals, and plays a critical role in PrCR. Upon binding CD47, SIRPα initiates a signal transduction via src homology-2 domain recruitment, a large protein complex. This complex importantly contains two tyrosine phosphatases: SHP-1 and SHP-2, which both interact with various proteins for signaling. Once activated, SHP-1 propagates a downstream antiphagocytic signal (M2) through an unknown mechanism2, 7, 8. Naturally, this ensures macrophages do not engulf healthy cells. In fact, a single CD47-SIRPα interaction is capable of preventing phagocytosis9. 

One mechanism cancer uses to evade the immune system is through the CD47-SIRPα pathway. For cancer to propagate it must: prevent apoptosis, divide rapidly, and evade the immune system11. Many cancers overexpress CD47 and it is hypothesized that CD47 accumulation acts as a camouflage. Since CD47 is sufficient to prevent PrCR of healthy cells, when cancers overexpress this signal they can effectively prevent phagocytic clearance. Therefore, inhibiting the CD47-SIRPα pathway is a favorable route for therapeutics2. Efforts have been made to target CD47 and SIRPα individually through monoclonal antibodies (mAb) and high-affinity small peptides. These methods, coupled with known cancer therapeutics like Rituximab, have been shown to decrease tumor cell density in vitro, in vivo, and in clinical trials14. The main goal here is to assess the potential adverse effects presented in each therapeutic. Major hurdles include the potential for other phagocytic inhibitors, off-target effects, and the lack of long-term effects. 

 

Antibody targeting of CD47 and SIRPα shows inhibition of anti-phagocytic signaling 

Antibody targeting of CD47 is an effective therapeutic for specific cancers. Acute myelogenous leukemia (AML) is maintained by self-renewing leukemia stem cells (LSC) which evade phagocytosis through increased CD47 expression2, 4. By targeting CD47, researchers hope to activate a focused immune response against tumor cells. Both, in vitro and in vivo analysis of an anti-CD47 antibody (B6H12.2) in an AML LSC model reported a 3-5 fold increase in phagocytosis compared to macrophages and tumor cells alone12. In contrast, an anti-SIRPα antibody reported an increased phagocytosis only when coupled with trastuzumab—a known breast cancer therapeutic10. This contradiction is important, firstly, because it shows antibodies alone are insufficient to increase phagocytosis. Secondly, it hypothesizes other “don’t eat me” signals continue to inhibit phagocytosis after the CD47-SIRPα has been blocked. Lastly, it shows two alternate ways to inhibit the CD47-SIRPα pathway. The anti-SIRPα antibody is argued as a favored cancer therapeutic because CD47 is widely expressed across cell types. Targeting CD47 may cause unwanted on-target CD47 phagocytosis. Despite this possibility, an in vivo analysis of B6H12.2 reported no additional phagocytic activity even with equivalently coated cells4. However, therapeutic exposure only lasted 14 days and animal models were sacrificed afterwards; therefore, long term effects have not been assessed. 

 

Anti-CD47 antibody development towards human variant 

A limitation to antibody therapeutics is inter-species variation. B6H12.2s’ affinity decreased from mice to humans due to CD47 variation. Therefore, a human anti-CD47 antibody (5F9) was produced and grafted to immunoglobulin G4 scaffold (IgG4)13. The resulting antibody (Hu5F9-G4) was tested in vitro for its affinity towards human CD47 and 

revealed strong attraction, illustrated by the incredibly small amount of dissociation betweenCD47-SIRPa (KD=1×10-12). Hu5F9-G4 was further tested in cynomolgus monkeys to assess potential toxicity in a human-like model. No serious adverse events were characterized except dose dependent anemia which was expected due to the high CD47 expression on red blood cells and reverted naturally after antibody treatment2. However, using healthy monkeys was a limitation to this study; tumor cell phagocytosis was not assessed in vivo. Furthermore, the toxic effects were only tested in a three week period and no long-term effects were characterized. 

 

Clinical trials for the human CD47 antibody variant 

Clinical trials of Hu5F9-G4 antibody coupled with rituximab are currently being conducted. Toxicity and effectiveness were assessed in 22 patients with aggressive and indolent lymphoma (this can be thought of as metastatic and benign cancer, respectively)14. From this sample, 50% had an objective response and 36% had a complete response. Furthermore, by day 28, white and red blood cells had approximately 100% of their CD47 receptors occupied. This is important because blocking all CD47-SIRPα interactions is needed for effective results and, since all cells are not degraded, other signals must be preventing phagocytosis in healthy cells9. As seen in other animal models, dose-dependent anemia was the most common side-effect but normal levels of red blood cells reverted at lower dosages or after the treatment period2. This coupled treatment showed promising results for patients with aggressive and indolent lymphoma. 

 

High-affinity small peptides as an alternate CD47-SIRPα inhibitor 

Another issue with antibody therapeutics is their poor permeability into dense tumors15. Given this hurdle, an alternate route is small peptide inhibitors against the CD47-SIRPα pathway. By antagonizing CD47 or SIRPα, the small peptides should block any anti-phagocytic signaling and allow PrCR to occur. Small peptides are highly specific antagonists modeled after invariable regions of their target. By analyzing the human SIRPαs’ binding domain, a competitive antagonist for human CD47 was produced16. The high-affinity SIRPα monomer (CV1) was tested in vitro to assess its affinity towards human and mouse CD47. CV1 presented the same inhibition between human and mouse CD47 variants (50,000-fold affinity increase and KD=34.0 pm). Since small peptides are modeled after invariable regions, their affinities are similar between species. This is important because affinity testing for humans can now be estimated through animal models; thereby, eliminating toxic and costly human trials. Furthermore, ex vivo co-treatment of CV1 with anti-Her2/neu—a well studied breast cancer antibody—increased phagocytosis of human breast cancer cells compared to anti-Her2/neu alone. This coupled treatment was tested in vivo and revealed increased anti-tumor responses in a mouse breast cancer model. Co-treatment illustrates the possibility for more “don’t-eat-me” signals present on cancer cells. Despite CV1s’ efficacy, its high affinity caused on-target CD47 binding across all cell types. Although this high-affinity is wanted in therapeutics, unwanted red blood cell phagocytosis occurred and resulted in anemia. This side-effect, however, is common between all CD47 inhibitors and naturally reverted after treatment16,17. 

A solution to CD47 on-target side-effects is antagonizing SIRPα instead. CD47 is expressed widely across cell lines, while SIRPα is present on a subset of macrophages; therefore, SIRPα is arguably the favored target for cancer therapeutics10. One potential SIRPα antagonist, which showed similar potency as CV1, is Velcro-CD47– a high-affinity CD47 variant synthesized through a novel protein “velcro” technique9. Through in vitro analysis, Velcro-CD47 enhanced mAb-mediated phagocytosis by inhibiting anti-phagocytic signals. It is important to note that the small peptide inhibitors do not, by themselves, promote phagocytosis. While antibodies illicit a targeted immune response, small peptides rely on the immune systems’ natural clearance or other cancer therapeutics to clear cancer cells. 

Other small peptide therapeutics for CD47-SIRPα inhibition include 4N1K and its derivative PKHB1. There has been substantial evidence that 4N1K increases PrCR in vivo18,19,20,21. Several papers highlight a difference between CD47 +/+ and CD47 -/- tumor cells removal upon 4N1K treatment22. Unlike B6H12/Hu5F9, 4N1K is able to potentiate PrCR of chronic lymphocytic leukemia (CLL) in soluble conditions; however, in human serum, 4N1K is degraded by proteases faster than antibodies-more than 90% was degraded in an 1-hour incubation18. This therapy, therefore, requires more injections for an accurate response. Furthermore, 4N1K has conflicting evidence for its CD47 specificity, and may cause off-target effects23. In order to combat these issues, two terminal residues were replaced on 4N1K with their D analogues. This new therapeutic, PKHB1, lasted longer in human serum, maintained its solubility, and continued to bind CD47. PKHB1 was then tested in vivo and showed higher rates of CLL PrCR 18. PKHB1 is currently in pre-clinical trials for CLL treatment.

 

Conclusion

Cancer therapeutics continue to progress towards more accurate and less toxic forms. In turn, this eliminates the need for deleterious options like chemotherapy. CD47-SIRPα presents a target for future immunological therapeutics. Although anemia and off-target effects must be further assessed, CD47-SIRPα inhibitors present a feasible and effective option. 

Anti-CD47 antibodies increase tumor cell phagocytosis in a coupled therapy with Rituximab and show accurate responses in Phase I clinical trials14. In response to phagocytosis of healthy CD47-expressing cells, anti-SIRPα antibodies have been developed which illustrated similar phagocytic responses in vivo with higher cell type specificity10. Regardless of the target, blocking the CD47-SIRPα pathways still cause anemia in patients. This is an expected and treatable side-effect that naturally reverts after a short-term treatment. No long-term effects of antibody treatments have been assessed and remains a limitation to these studies. To combat the limitations seen in antibody treatments, small peptide inhibitors are being developed for the CD47-SIRPα pathway. Velcro-CD47 presented a novel protein manufacturing technique and provided a high-affinity peptide to prevent inhibitor signals16. 4N1K has been shown to increase tumor cell phagocytosis between CD47 +/+ and CD47-/- but small peptide inhibitors are hindered by their short half-life in blood serum due to protease activity. 

By blocking the CD47-SIRPα pathway and other inhibitor signals, researchers can trigger a natural immune clearance of cancer cells. Although differences between CD47 and SIRPα therapeutics, long-term effects, and 4N1K off-target effects must be further assessed, preliminary research indicates this pathway as a potential target for future therapeutics. 

 

Reference: 

  1. “Cancer Statistics”. National Cancer Institute, 2019, https://www.cancer.gov/about-cancer/understanding/statistics.
  2. Oldenborg PA, Zheleznyak A, Fang Y, Lagenaur CF, Gresham HD, Lindberg FP. “Role of CD47 as a Marker of Self on Red Blood Cells.” Science. 2001;288: 2051- 54 
  3. Lagasse E, and Weissman IL. “bcl-2 inhibits apoptosis of neutrophils but not their engulfment by macrophages.” J Exp Med. 1994 Mar 1;179(3):1047-52. 
  4. Chao MP, Alizadeh AA, Tang C, Jan M, Weissman- Tsukamoto R, Zhao F, Park CY, Weissman IL, Majeti R. “Therapeutic antibody targeting of CD47 eliminates human acute lymphoblastic leukemia.” Cancer Res. 2011 Feb 15; 71(4): 1374-84. 
  5. Brown EJ, and Frazier WA. “Integrin-associated protein (CD47) and its ligands.” Trends Cell Biol. 2001 Mar;11(3): 130-5. 
  6. Alvey C, and Discher DE. “Engineering macrophages to eat cancer: from “marker of self” CD47 and phagocytosis to differentiation.” J Leukoc Biol. 2017;102: 31–40. 
  7. Barclay AN, and Brown MH. “The SIRP family of receptors and immune regulation.” Nat Rev Immunol. 2006 Jun;6(6): 457-64 
  8. Lin Y, Zhao JL, Zheng QJ, Jiang X, Tian J, Liang SQ, Guo HW, Qin HY, Liang YM, Han H. “Notch Signaling Modulates Macrophage Polarization and Phagocytosis Through Direct Suppression of Signal Regulatory Protein α Expression.” Front Immunol. 2018 July 30. 
  9. Ho CC, Guo N, Sockolosky JT, Ring AM, Weiskopf K, Ozkan E, Mori Y, Weissman IL, Garcia KC. “‘Velcro’ engineering of high affinity CD47 ectodomain as signal regulatory protein α (SIRPα) antagonists that enhance antibody-dependent cellular phagocytosis.” J Biol Chem. 2015 May 15;290(20): 12650-63. 
  10. Zhao XW et al. “CD47-signal regulatory protein-α (SIRPα) interactions form a barrier for antibody-mediated tumor cell destruction.” Proc Natl Acad Sci USA. 2011 Nov 8;108(45): 18342-7. 
  11. Ottaviano M, De Placido S, Ascierto PA. “Recent success and limitations of immune checkpoint inhibitors for cancer: a lesson from melanoma.” Virchows Arch. 2019 Feb 12. 
  12. Majeti R, Chao MP, Alizadeh AA, Pang WW, Siddhartha J, Gibbs Jr. KD, Rooijen N, and Weissman IL. “CD47 is an adverse prognostic factor and therapeutic antibody target on human acute myeloid leukemia stem cells.” Cell. 2009 July 13;138(2): 286-299. 
  13. Liu J et al. “Pre-Clinical Development of a Humanized Anti-CD47 Antibody with Anti-Cancer Therapeutic Potential.” PLoS ONE. 2015. 
  14. Advani R et al. “CD47 Blockade by Hu5F9-G4 and Rituximab in Non-Hodgkin’s Lymphoma.” N Engl J Med. 2018 Nov 1;378(18):1711-21. 
  15. Chames P, Van Regenmortel M, Weiss E, Baty D. “Therapeutic antibodies: successes, limitations and hopes for the future.” Br J Pharmacol. 2009 May;157(2): 220-33. 
  16. Weiskopf K et al. “Engineered SIRPα variants as immunotherapeutic adjuvants to anti-cancer antibodies.” Science. 2013 July 5;341(6141). 
  17. Willingham SB, et al. “The CD47-signal regulatory protein alpha (SIRPα) interaction is a therapeutic target for human solid tumors.” Proc Natl Acad Sci. 2012;109:6662. 
  18. Martinez-Torres AC, Quiney C, Attout T, et al. “CD47 agonist peptides induce programmed cell death in refractory chronic lymphocytic leukemia B cells via PLCγ1 activation: evidence from mice and humans.” PLoS Med. 2015;12(3): e1001796. 
  19. Soto-Pantoja DR, et al. “Therapeutic opportunities for targeting the ubiquitous cell surface receptor CD47.” Expert Opin Ther Targets. 2013 Jan;17(1):89-103. 
  20. Kanda S, Shono T, Tomasini-Johansson B, Klint P, Saito Y. “Role of Thrombospondin-1-Derived Peptide, 4N1K, in FGF-2-Induced Angiogenesis.” Exp Cell Res. 1999;252, 262–272. 
  21. Kalas W et al. “Thrombospondin-1 receptor mediates autophagy of RAS-expressing cancer cells and triggers tumour growth inhibition.” Anticancer Res. 2013;33(4):1429-38. 
  22. Fujimoto T.-T., Katsutani S., Shimomura T., Fujimura K. “Thrombospondin-bound integrin-associated protein (CD47) physically and functionally modifies integrin alphaIIbbeta3 by its extracellular domain.” J Biol Chem. 2013;278 26655–26665 
  23. Jeanne A, Schneider C, Martiny L, Dedieu S. “Original insights on thrombospondin-1-related antireceptor strategies in cancer.” Front Pharmacol. 2015;6: 252. 

CRISPR/HDR Platform Allows for the Production of Monoclonal Antibodies with the Constant Region of Choice

November 22, 2019

By Sharon Yang, Cell Biology, ‘20

Author’s Note: I first came across an article talking about this new innovation on Science X. Having worked with hybridomas and antibodies through various internships, I was deeply intrigued by this discovery and secured an original paper to learn more about its potential applications. Because of the revolutionizing usage of antibodies in the medical field, it is vital to understand how this finding will facilitate antibody-based therapies in clinical research.  

 

Introduction

Since the discovery of antibodies and their applications in therapeutics, many diseases once deemed incurable now have a treatment, if not a cure. Antibodies are proteins that recognize and bind to specific antigens (proteins that are considered “foreign” to the body). The immune system recognizes this antibody-antigen complex and removes the foreign substance from the body. Monoclonal antibodies (mAbs) are specific for one type of antigen and are produced using hybridomas, immortal cell lines that secrete only one type of antibody. The specificity of a mAb is determined by its antigen binding variable region. Though the variable region is of critical importance, the constant region (also known as the Fc region) is also essential to the therapeutic efficacy of mAbs. The Fc region has many different variants, called isotypes. Each isotype has its own unique function in making the immune system respond in different ways. After an antibody binds to an antigen by its variable region, the Fc region of the antibody elicits a response from the immune system, which serves as the basis for antibody-based therapeutics.

A recent study conducted in the summer of 2019 by Schoot and colleagues demonstrates how the use of genetic engineering on hybridomas can modify the Fc region of mAbs to that of a different species, isotype, or format. This new versatile platform grants ease of production of monoclonal antibodies that have different constant regions but retain the same variable regions. 

The research team utilized a one-step clustered regularly interspaced short palindromic repeat (CRISPR)/homology-directed repair (HDR) technique to create a recombinant hybridoma that secretes a mAb in the Fc format of choice — a highly attractive alternative to the conventional recombinant production methods that were often time-consuming, challenging, and expensive.

As the team emphasizes, “[CRISPR/HDR] is a simple alternative approach requiring a single electroporation step to obtain an unlimited source of target antibody in the isotype format of choice” (1). Through using CRISPR/HDR, the team was able to seamlessly generate monovalent Fab’ fragments and a panel of different isotypes for the same monoclonal antibody.

 

CRISPR/Cas9 and Homology-Directed Repair

In their genetic engineering method, the researchers took advantage of an ancient bacterial immunity mechanism: the CRISPR/Cas9 system. When a bacteria is invaded by a virus, the bacteria stores snippets of viral DNA and creates segments of DNA called CRISPR arrays. When a virus with the same DNA segment attacks again, the bacteria creates RNA from the CRISPR arrays to target the virus; the RNA is called the guide RNA. The nuclease protein Cas9 is used to cut the DNA apart at a very specific site determined by the guide RNA, disabling the virus. CRISPR/Cas9 works in a similar fashion in the lab. Scientists create a guide RNA that binds to Cas9, which then targets a specific site on the DNA to be cut (2).

When CRISPR/Cas9 cuts DNA, it induces a double-strand break (DSB). Homology-directed repair (HDR) occurs when the intact donor strand contains high sequence homology to the damaged DNA strand. Through HDR, scientists can integrate a sequence or gene of their liking into the genome, which Schoot and colleagues perform in their study (3).  

 

The Generation of Fab’ Fragments

The fragment antigen-binding (Fab’) is a region on the antibody that binds to the antigen. It consists of a single heavy chain and light chain. To create a Fab’ fragment-secreting hybridoma using CRISPR/HDR, the team selected NLDC-145, a hybridoma clone that secretes mAbs of rat IgG2a (rIgG2a) isotype. The antigen of rIgG2a is DEC205, an endocytic receptor found on immune cells. The team electroporated NLDC-145 cells with Cas9 and an appropriate guide RNA to induce double-strand breaks at the hinge region; to repair the double-strand break, they designed an HDR Fab’ donor construct for homology-directed repair. The HDR Fab’ donor construct also inserts specific tags onto the protein, allowing for easy purification of the Fab’ fragment. 

To test secretion of the Fab’ fragment, they stained JAWSII, a DEC205-expressing cell line, with the supernatants of NLDC-145 clones that had undergone CRISPR/HDR. Flow cytometry assays showed that a large portion of Fab’-secreting hybridomas were successfully created. Further assays showed that the secreted Fab’ fragments retained their binding capabilities. It is worth noting that the researchers also used the same strategy to convert other hybridoma lines to become recombinant, Fab’-producing lines, with similar success; this demonstrates that this engineering technique is flexible and not just limited to one cell line (1).

 

The Generation of Isotype Panels

In a similar manner to creating monoclonal Fab’-generating hybridomas, the team also used the one-step CRISPR/HDR technique to create hybridomas capable of producing a wide array of isotype variants for the same mAb. This time, the cell line subject was hybridoma MIH5, which secretes monoclonal rIgG2a that targets mouse PD-L1, an immune checkpoint protein. The goal was to make clones of MIH5 to each produce one isotype of the chimeric (having both rat and mouse-related parts) monoclonal antibodies: mIgG1, mIgG2a, mIgG2b, mIgG3, mIgA, and a mutant form of mIgG2a (mIgG2asilent).

MIH5 cells were cotransfected (introduced with DNA) with a Cas9 vector containing the appropriate guide RNA and a construct from a panel of isotype HDR donor constructs (each isotype had its own unique HDR donor construct). Following knock-in integration, flow cytometry analysis showed that the engineered chimeric mAbs were successfully secreted. Thus, the creation of recombinant hybridomas for a panel of isotypes was successfully engineered (1). This invention allows for the creation of monoclonal antibodies with different Fc regions, providing researchers an easy way to “customize” their antibodies to elicit a specific response from the immune system. Researchers may choose which isotype variant they want on their antibody, which is fully dependent on their target (antigen) of interest and how the immune system behaves towards it. This has vast potential in antibody-based therapeutics, in that this system can be used for the optimization of potential drugs to become more potent and dynamic.

 

Biochemical Applications

To test the functional capability of isotype-switched mAbs, Schoot and colleagues tested the antibodies’ capability to induce an important immune mechanism: antibody-dependent cellular cytotoxicity (ADCC). In order to test ADCC in vitro, mouse colon adenocarcinoma cells were labeled with chromium-51, and then taken in by MIH5 Fc isotype variants. After adding whole blood, they measured chromium-51 release. On the other hand, B cell depletion by MIH5 Fc variants was used to measure ADCC in in vivo experiments. Analyses of these studies show that chimeric mAbs created by CRISPR/HDR hybridomas have the same biochemical and immune effector characteristics as their recombinant and naturally occurring counterparts (1). Something to highlight is that instead of treading through the laborious process of producing  recombinant antibodies in the conventional way (often consisting of multiple rounds of optimized sequencing, cloning, transfection), this one-step mechanism grants smooth and rapid generation of recombinant antibodies that perform their expected functions (1).

 

Conclusion

The ability to create monoclonal antibodies with the freedom to choose what goes on their constant regions possess many applications in the vast field of medicine and engineering. Being able to construct a very specific monoclonal antibody (the engineering element) that stimulates the immune system in a certain, beneficial way (the medical component) intertwines the two fields together to propel us closer towards treating diseases more efficiently and effectively. This system also represents an optimized version of recombinant engineering, which saves valuable time and funds that can be used towards conducting further studies. A simple, yet powerful and flexible approach, this versatile CRISPR/HDR platform aims to facilitate antibody engineering and research for the scientific community, and is accelerating the rate at which new clinical trials can be performed.

 

References

  1. Schoot, J. M. V. D. et al. Functional diversification of hybridoma produced antibodies by CRISPR/HDR genomic engineering. Science Advances 5, (2019).
  2. Ran, F Ann et al. “Genome engineering using the CRISPR-Cas9 system.” Nature protocols vol. 8,11 (2013): 2281-2308. doi:10.1038/nprot.2013.143
  3. Cortez, Chari. “CRISPR 101: Homology Directed Repair.” Addgene Blog, Addgene, 12 Mar. 2015, blog.addgene.org/crispr-101-homology-directed-repair.

Finding a Solution in the Source: Exploring the Potential for Early Beta Cell Proliferation to Disrupt Autoreactive Tendencies in a Type 1 Diabetes Model

September 6, 2019

By Reshma Kolala, Biochemistry & Molecular Biology ‘22

Residing in the pancreas are clusters of specialized cells, namely alpha, beta (), and delta cells. cells, more specifically, are insulin-secreting cells that are instrumental in the body’s glucose regulation mechanism. An elevation of the extracellular glucose concentrations allows glucose to enter cells via GLUT2 transporters, where it is subsequently metabolized. The resultant increase in ATP catalyzes the opening of voltage-gated Ca2+ channels, triggering the depolarization of the plasma membrane which in turn stimulates insulin release by cells (1). In individuals with Type 1 Diabetes (T1D), however, pancreatic islet beta cells are damaged by pro-inflammatory cytokines that are released by the body’s own immune cells. The loss of functional beta cell mass induces a dangerous dysregulation of glucose levels, resulting in hyperglycemia along with other harmful side effects. The absence of a regulatory factor in the bloodstream forces those with T1D to take insulin intravenously to remedy the consequences.  

A new study led by Dr. Ercument Dirice, a Harvard Medical School (HMS) instructor and research associate at the Joslin Diabetes Center. has suggested that an increase in cell mass early in life diminishes the autoreactive behavior of immune cells towards cells, therefore halting the development of T1D (2). In a typical T1D model, the secretion of antigens from cells induces a response from the body’s immune cells. These immune cells bind to the epitopes (the recognizable portion of an antigen) that are displayed on the surface of professional antigen presenting cells (APC’s) which are littered throughout the pancreatic islets (3). This binding action induces a destructive autoimmune response to antigens secreted by cells, resulting in loss of functional beta-cell mass. It was found however that by increasing cell mass at an early age where the organs of the immune system are still developing, the immune cells stopped attacking cells. 

The novel approach presented by Dirice et al. departs from the traditional method of targeting various other components involved in the destructive autoimmune response, namely APC’s or the pro-inflammatory cytokines associated with T1D progression. This method instead focuses on the source of the autoimmune marked “pathogenic” antigens, the cells themselves. 

The studies were completed using two models of female non-obese diabetic (NOD) mice. One was a genetically engineered model of female mice (NOD-LIRKO) that showed increased cell growth soon after birth while the second model was done using a live mouse that was injected at an early age with an agent known to increase cell proliferation. While maintaining more than 99.5% isogenicity (4), it was found that the mice with increased cell mass had a significantly lower predisposition to develop diabetes when compared against the NOD control mice, which developed severe diabetes between 20-35 weeks of age. The study also observed the interaction between the modified cells and immune cells by monitoring the concentration of these immune cells in the spleen. In doing this, researchers were able to conclude which mice had a greater risk of developing T1D based on if mice had an abnormal increase in the concentration of these cells. 

At first glance, this method appears counterintuitive as an increase in cell mass may lead one to naively assume that this would result in increased autoantigen production. This precise hypothesis illustrates the beauty of this approach. Although the specific details of this mechanism have yet to be made clear, it is believed that the rapid turnover of cells “confuses” the autoimmune reaction. The proliferated   cells present unusual autoantibodies that are not observed in typical T1D progression. Dr. Rohit Kulkarni, a fellow HMS professor and researcher at Joslin noted that there is thought to be some alteration in the new cells where the autoantigens typically produced are reduced or dilated (2). As a result of the slow presentation of antigens, there is a lower proportion of autoreactive immune cells. This essentially results in a “reshapen immune profile that specifically protects   cells from being targeted.” Some degree of autoimmunity would continue to exist in the body, so further immunosuppressive treatment would be required. 

Early cell proliferation has been previously speculated to have a protective effect in those with reduced functional cell mass as in a Type 1 Diabetes model. Once this preventative quality is better understood, applications of this research may be further explored. Despite still being in the beginning stages, this novel approach holds tremendous potential for application to T1D if this method is able to be translated to a human model. The massive prevalence of a T1D diagnosis is illustrated by 2014 census data that states that T1D affects roughly 4.7% of the world’s adult population. Although extensive research continues to be done on several aspects of the disease, the introduction of new data by Dirice et al. may push us a small step closer to solving one of the body’s greatest metabolic mysteries. 

References

  1. Komatsu, M., Takei, M., Ishii, H., & Sato, Y. (2013, November 27). Glucose-stimulated insulin secretion: A newer perspective. Retrieved from https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4020243/
  2. Yoon, J., & Jun, H. (2005). Autoimmune destruction of pancreatic beta cells. Retrieved from https://www.ncbi.nlm.nih.gov/pubmed/16280652
  3. Pushing early beta-cell proliferation can halt autoimmune attack in type 1 diabetes model. (2019, May 06). Retrieved from https://www.sciencedaily.com/releases/2019/05/190506124102.htm
  4. Burrack, A. L., Martinov, T., & Fife, B. T. (2017, December 05). T Cell-Mediated Beta Cell Destruction: Autoimmunity and Alloimmunity in the Context of Type 1 Diabetes. Retrieved from https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5723426/
  5. Dirice, E., Kahraman, S., Jesus, D. F., Ouaamari, A. E., Basile, G., Baker, R. L., . . . Kulkarni, R. N. (2019, May 06). Increased β-cell proliferation before immune cell invasion prevents progression of type 1 diabetes. Retrieved from https://www.nature.com/articles/s42255-019-0061-8?_ga=2.76180373.1669397493.1557550910-1092251988.1557550910

A Chemical Report on Heptachlor (Heptachlor Epoxide)

August 16, 2019

By Kaiming Tan

Author’s Note: This report analyzes and explains the biological, chemical, and environmental importance of heptachlor. More and more in today’s society, we are utilizing synthetic compounds as agricultural insecticides, which makes understanding what these chemicals do to our bodies and the environment of utmost importance. Farming strategies may seem far-removed from our daily lives, but these chemicals do not stay on the farm. They travel to our cities, to our grocery stores and markets, then make their way onto our dinner plates and into our children’s stomachs. I am constantly amazed at the power of scientific research to transform and demystify the detrimental nature of environmental and biological toxicants; this, combined with my passion in toxicology, has inspired me to research this topic and write this manuscript.

 

Keywords

Heptachlor, heptachlor epoxide, insecticide, environmental toxicology, biological toxin

 

Introduction

Heptachlor was introduced into the United States in the 1940s and 1950s, among the other chlorinated hydrocarbon insecticides, such as DDT (4). In 1971, 500,000 kilograms of heptachlor were applied to agricultural fields in soils and seeds to prevent termite infection in woods (4). However, after it became evident that heptachlor’s toxicity was a serious health concern, the Environmental Protection Agency (EPA) banned all registered use of heptachlor because of its carcinogenic properties and bioaccumulation in food and water. With the exception of treatment of fire ants in underground power transformers, there is no use of heptachlor in the United States now (2). In contrast, heptachlor is still used as an insecticide in some areas in Asia, Africa, and Eastern Europe (5). The form of greater toxicological concern is heptachlor epoxide, which is a metabolite of heptachlor in soil and the human body. Heptachlor epoxide is more toxic because it degrades slower than heptachlor, thus, is more persistent in the environment. Both heptachlor and heptachlor epoxide can be found in freshwater, estuarine, and marine systems (6). This review will cover the current literature on heptachlor toxicity and investigate the impact of heptachlor on biological systems.

 

Biological Fate (Absorption, Distribution, Metabolism, Elimination) 

Humans and animals can be exposed to heptachlor and heptachlor epoxide through soil, air and water; however, the main route of exposure is oral, through consuming contaminated food or drinking contaminated water. Children and infants are especially vulnerable to heptachlor exposure if they consume large amounts of breast milk from their mother. For instance, if the mother’s breast milk is contaminated by heptachlor, heptachlor can be easily consumed by the infant through breast milk feeding. In human breast milk, heptachlor epoxide was detected ranging from 0.13 to 128 ppb (parts per billion) (8). Another route of exposure is through inhalation. When heptachlor is deposited into the soil, it becomes heptachlor epoxide, which then spreads into the air. As a result, workers who use heptachlor to kill termites are potentially susceptible to inhaling heptachlor epoxide from the air (2). In addition, touching contaminated soils causes exposure to heptachlor dermally (1). 

Heptachlor persists in the environment after agricultural application. Even though there was no information on the background levels of heptachlor and heptachlor epoxide in the air or soil, there are 20 to 800 ppt (parts per trillion) heptachlor in drinking water and groundwater in the United States (2). Contaminated fish and shellfish were detected with levels of 2 to 750 ppb of heptachlor and 0.1 to 480 ppb of heptachlor epoxide. To date, there is no available data regarding the biological fate of heptachlor on humans. However, in rats, at least 50% of the orally-administered dose of heptachlor is absorbed in the gastrointestinal tract.The absorption is indicated by the presence of heptachlor and/or heptachlor epoxide in the rats’ liver (9). When heptachlor epoxide enters the bloodstream after absorption, it becomes the predominant metabolite in the body of laboratory female rats. Heptachlor epoxide in the bloodstream is positively correlated to the heptachlor dose administered (10). In other words, the higher heptachlor dose one consumed, the more heptachlor epoxide would appear in the bloodstream. 

Because of heptachlor epoxide’s high lipophilicity (likelihood in dissolving fats), its residue is found highest in adipose tissue in human infants (0.32±0.10ppm), while undetectable in the brain. These data also suggest that heptachlor epoxide can be transferred between pregnant women and their babies through the placenta (11). Furthermore, heptachlor and heptachlor epoxide can be stored in human breast milk. Jonsson et al.’s study in 1977 sampled the breast milk of 51 women who had no previously known exposure to heptachlor and detected an average concentration of heptachlor epoxide between 0.0027ppm and 0.019ppm (12). To the researchers, the results proved worrisome as the women’s breast milk contained high levels of heptachlor, which could damage the infant. The results were even more of a concern because there was no known interaction between the women studied and heptachlor.

After heptachlor is absorbed in the body, the primary metabolite of heptachlor in humans and rats is heptachlor epoxide. Heptachlor and heptachlor epoxide are usually metabolized by Cytochrome P450 (CYP450) enzymes. CYP450 enzymes are the body’s major class of enzymes responsible for detoxification. Moreover, heptachlor alters liver function by increasing gluconeogenic enzymes’ activities, which upregulates glucose synthesis from glycogen. Since most of heptachlor is metabolized in the liver, humans with liver diseases may have increased bioaccumulation of heptachlor epoxide because of decreased CYP450 enzyme activity (13). Despite the metabolism of heptachlor in the liver, most heptachlor epoxide can be stored in the adipose tissue because of its high lipophilicity and long half-life (1). 

In terms of excretion, orally-administered heptachlor in rats was excreted in the form of heptachlor epoxide and also as heptachlor. Radiolabeled heptachlor epoxide is excreted ten times more in the feces compared to urine after ten days of oral administration in rats, due to the high lipophilicity of heptachlor metabolites (8).

 

Mechanism of Action

The primary organ and organ systems that heptachlor targets are the liver, central nervous system and reproductive system, while secondary target organs include kidneys and lungs. Heptachlor is primarily metabolized in the liver into heptachlor epoxide, which has the same toxic potential. The reproductive system is a sensitive target for heptachlor toxicity because this system lacks a comprehensive detoxification system like the CYP450 enzymes in liver. CYP450 enzymes facilitate chemical reactions that chemically convert the toxicant into a more hydrophilic metabolite, thereby enhancing toxicant elimination by urine. Oral exposure of 1.8 mg/kg/day to female rats over 14 days caused decreased fertility due to decreased estradiol and progesterone levels. Estradiol and progesterone are important hormones produced by the ovaries, placenta, and adrenal glands; decreased levels may result in endocrine disruption and delayed sexual development. Oral exposure of 0.65 mg/kg/day over 70 days caused decreased sperm count in male rats. The mechanism of heptachlor’s reproductive toxicity remains unknown (1-2).

GABA, the molecule that normally binds the GABA receptor, is an amino acid which can also serve as an inhibitory neurotransmitter in the mammalian brain. Heptachlor primarily acts as a noncompetitive antagonist of the chloride channels of the GABAA receptors in mammals.  In other words, heptachlor blocks the Chlorine channel in the cells thus preventing GABAA activation (1-2, 14). By inhibiting the inhibitory neuron, heptachlor causes hyperexcitability of the cells. Although the heptachlor toxicity mechanism in liver is unknown, it is observed that in rats that presence of heptachlor elevates intracellular calcium levels and induces protein kinase C (PKC), which in turn activates the activator protein-1 DNA binding thereby causing tumorigenesis (2, 14). 

When animals are exposed to heptachlor above the toxic threshold, symptoms occur including tremors, convulsions, ataxia, and changes in EEG patterns (1). Also, rats who were orally exposed to >320 ppm of heptachlor had consistently lower body weight than rats that were unexposed (15). In humans, clinical symptoms of heptachlor toxicity include seizure, vomiting, and convulsions (2). 

Although heptachlor epoxide is theoretically an effective mutagen because of its high reactivity as an electrophile, research studies on heptachlor mutagenicity yielded mostly negative results. For example, heptachlor was not mutagenic in the auxotrophic strains of E. coli by reversion bioassays (14,15). In addition, heptachlor is proven to increase liver tumor incidence as an epigenetic promoter in previously initiated B6C3F1 mice. Heptachlor is a carcinogen in mice by inhibiting intercellular communications to promote tumor growth (14,16). On the other hand, research studies that investigate heptachlor’s teratogenicity, or tendency to disturb embryonic development, yielded mostly negative results. For instance, although feeding rats with diets containing heptachlor produces pups with higher mortality, no congenital malformation was found (2, 14, 17). 

In male rats, oral LD50 (lethal dose for 50% of the study population) of heptachlor is 40 to 100 mg/kg body weight based on data from two studies. In contrast, the oral LD50 of heptachlor in male chickens is 62 mg/kg body weight (1, 17). Symptoms displayed by heptachlor acute toxicity in animal subjects include hyperexcitability, tremors, convulsions and paralysis (18). 

Long-term exposure to heptachlor can cause liver damage in animals. In rats, prolonged exposure of sub-lethal doses of heptachlor is associated with increased CYP450 enzyme induction and other hepatic microsomal enzymes, in addition to liver hypertrophy (18). In humans, chronic exposure to heptachlor results in storage in adipose tissue and breast milk, because heptachlor is very lipophilic. Infants are at risk of being exposed to large doses of heptachlor if they consume contaminated breast milk (17). Despite the lack of human studies on long-term exposure, a multi-generation study conducted on rats concluded that oral exposure of 6 mg/kg/day of heptachlor is associated with decreased litter size, increased mortality and lens cataract (2). Because of its high lipophilicity, heptachlor residues can remain in the body over time. Heptachlor’s long-term toxicity damages the body gradually, further underlying the need for strict regulations of heptachlor use worldwide.  

 

Overview of Latest Research

Recent research on heptachlor focuses on its toxicity, specifically on the oxidative stress caused by heptachlor and its metabolism in aquatic animals. For example, Vineela et al.’s study investigates the impact of sub-lethal concentrations of heptachlor on carp fish Catla catla by measuring enzymatic activities of mostly Phase II enzymes (19). Phase II enzymes, a part of the CYP450 enzyme class, primarily perform conjugation reactions to convert the chemical into more water-soluble form to enhance urine elimination. Oral exposure of heptachlor at 1.46mg/L (20% of LC50 concentration) for 45 days causes a significant increase in lipid peroxidation, superoxide dismutase, glutathione-S-transferase and catalase activity in Catla catla. The results suggest that carp fish have a sensitive biological defense system against heptachlor, because low concentration activates the detoxification by increased biomarkers of primarily Phase II activity to prevent heptachlor toxicity and organ damage (19). 

In addition to heptachlor toxicity, current research also focuses on how to remove existing heptachlor from the environment via microbial degradation, as this method is more environmentally mindful and cost-effective than current physicochemical methods. Qiu et al.’s study discovered a novel strain of bacteria, named strain H, that can metabolize heptachlor efficiently. Strain H is a Gram-negative, short rod-shaped, single-cell bacterial strain that can degrade heptachlor at a rate of 88.2% degradation in 130 hours when exposed to 300 μg/L of heptachlor at 30oC. The main metabolites of heptachlor by strain H include heptachlor epoxide, chlordane epoxide, and 1-hydroxychorodene. This innovation allows possible bioremediation by microorganisms like strain H in heptachlor-contaminated soil and water to reduce heptachlor toxicity and threat to the environment and animals (20). 

Recent studies on heptachlor took a creative approach in gaining a deeper understanding in reducing heptachlor toxicity by enzymatic activity in aquatic model organisms and inventing novel microorganisms to metabolize heptachlor into less toxic metabolites. These new techniques will benefit public health by developing cost-effective ways for toxicant removal and controlling the environmental/biological fate of toxicants without causing additional harm.   

 

Conclusions

When initially used as an insecticide, heptachlor’s toxicity became a threatening health concern. The ban of commercial heptachlor use in the United States was a step in the right direction, as misuse of heptachlor can cause severe environmental consequences such as prolonged residue in soil and water habitats along with toxicities in humans and animals. Based on this research, other countries should pursue a substitute pesticide that has less potential for environmental and biological damage than heptachlor. 

Most studies conducted regarding heptachlor toxicity were conducted between the 1950s and the late 1980s. Future research can focus on the dose-response relationship of heptachlor exposure within large populations and whether genetic polymorphism contributes to the metabolism of heptachlor. Environmental toxicologists can also study the synergistic toxicity of heptachlor on environmental damage with other pesticides, since common pesticides often contain more than one chemical (1,2).

 

Acknowledgment

The author would like to thank Dr. Matthew Wood and Mr. Thomas Sears for providing feedback on early versions of this manuscript. 

 

Works Cited (in order of appearance) 

  1. Reed, N.R., & Koshlukova, S. (2014). Heptachlor. In Encyclopedia of Toxicology (pp. 840-844).
  2. United States. Agency for Toxic Substances Disease Registry, & Syracuse Research Corporation. (2007). Toxicological Profile for Heptachlor and Heptachlor Epoxide.
  3. Ivie, G., Knox, W., Khalifa, J., Yamamoto, R., & Casida, S. (1972). Novel photoproducts of heptachlor epoxide, Trans -chlordane, and Trans -nonachlor. Bulletin of Environmental Contamination and Toxicology, 7(6), 376-382.
  4. Hodgson, E. (2004). A textbook of modern toxicology (3rd ed.). Hoboken, N.J.: Wiley-Interscience.
  5. United States. Environmental Protection Agency. Office of Research Development. (2002). The Foundation for Global Action on Persistent Organic Pollutants a United States Perspective.
  6. Schimmel, S., Patrick, J., & Forester, J. (1976). Heptachlor: Toxicity to and uptake by several estuarine organisms. Journal of Toxicology and Environmental Health, 1(6), 955-965.
  7. Mnif, W., Hassine, A. I. H., Bouaziz, A., Bartegi, A., Thomas, O., & Roig, B. (2011). Effect of Endocrine Disruptor Pesticides: A Review. International Journal of Environmental Research and Public Health, 8(6), 2265–2303. http://doi.org/10.3390/ijerph8062265
  8. Savage, E., Keefe, T., Tessari, J., Wheeler, H., Applehans, F., Goes, E., & Ford, S. (1981). National study of chlorinated hydrocarbon insecticide residues in human milk, USA. I. Geographic distribution of dieldrin, heptachlor, heptachlor epoxide, chlordane, oxychlordane, and mirex. American Journal of Epidemiology, 113(4), 413-22.
  9. Tashiro, S., & Matsumura, F. (1978). Metabolism of trans -nonachlor and related chlordane components in rat and man. Archives of Environmental Contamination and Toxicology, 7(1), 113-127.
  10. Radomski, J., & Davidow, B. (1953). The metabolite of heptachlor, its estimation storage, and toxicity. The Journal of Pharmacology and Experimental Therapeutics, 107(3), 266-72.
  11. Curley, A., Copeland, M., & Kimbrough, R. (1969). Chlorinated Hydrocarbon Insecticides in Organs of Stillborn and Blood of Newborn Babies. Archives of Environmental Health: An International Journal, 19(5), 628-632.
  12. Adeshina, F., & Todd, E. (1990). Organochlorine compounds in human adipose tissue from north Texas. Journal of Toxicology and Environmental Health, 29(2), 147-156.
  13. Komori, Nishio, Kitada, Shiramatsu, Muroya, Soma, . . . Kamataki. (1990). Fetus-specific expression of a form of cytochrome P-450 in human livers. Biochemistry, 29(18), 4430-3.
  14. Whitacre, David M. (2008). Reviews of Environmental Contamination and Toxicology(Vol. 234). New York, NY: Springer New York.
  15. Moriya, Ohta, Watanabe, Miyazawa, Kato, & Shirasu. (1983). Further mutagenicity studies on pesticides in bacterial reversion assay systems. Mutation Research, 116(3-4), 185-216.
  16. Williams, G., & Numoto, S. (1984). Promotion of mouse liver neoplasms by the organochlorine pesticides chlordane and heptachlor in comparison to dichlorodiphenyltrichloroethane. Carcinogenesis, 5(12), 1689-96.
  17. United Nations Environment Programme, World Health Organization, International Labour Organisation, & Commission of the European Communities. (1976). Environmental health criteria. Geneva: World Health Organization.
  18. World Health Organization, & International Program on Chemical Safety. (1988). Heptachlor health and safety guide. Geneva : Albany, NY: World Health Organization ; WHO Publications Center USA [distributor].
  19. Vineela, D., Janardana Reddy, S., & Kiran Kumar, B. (2017). Impact of Heptachlor on Antioxidant Enzyme Markers of Fish Catla Catla. World Journal of Pharmaceutical Research, 6(12), 759-771. doi:10.20959/wjpr201712-9679
  20. Qiu, Liping, Wang, Hu, & Wang, Xuntao. (2018). Conversion mechanism of heptachlor by a novel bacterial strain. RSC Advances, 8(11), 5828-5839.

The Effect of Trastuzumab on HER2-Signaling in Breast Cancers to Induce Cardiotoxicity

July 5, 2019

By Karissa Cruz, B.S. Biochemistry and Molecular Biology, Spring ‘19

Author’s Note: I wrote this piece as part of my UWP 104F assignments and ended up becoming really interested in what I wrote about. I specifically chose this topic because I think breast cancer is a smart, complex disease, and the treatment can change day-to-day. I wanted to shed light on a widely accepted breast cancer treatment that is now under review after discovering that it can cause cardiac dysfunction.

(more…)

Lethal and Perplexing: An Overview of Metal Phosphides and Their International Impact

June 28, 2019

By Taylor Riedley, Biology, ‘20

Author’s Note: I wrote this paper for my UWP 102B class with Dr. Brenda Rinard in Winter Quarter 2019. For the preliminary stage of an assignment, I reviewed primary research on metal phosphide poisoning and wrote a formal paper geared towards a medical audience. For the final stage, I translated my formal paper into this one, intending it for a curious though not necessarily scientific audience. I chose this topic because I am considering an occupation in forensic pathology, and while I searched for articles about pathological findings, I came across an autopsy of a victim of metal phosphide poisoning. I was struck by the discrepancy between the general outward symptoms and the lethal organ damage of phosphide patients. I was further appalled when I learned that metal phosphides are within ready reach of children. I viewed this paper as an opportunity to draw attention to the need for more research on metal phosphides, and especially in this more accessible incarnation, to encourage readers to advocate for those who are not given the information necessary to stay safe.

(more…)

Why Your Dog is Itchy: A Review of Common Skin Allergies

June 21, 2019

By Jeffrey Nguyen, Animal Biology, ‘19

Author’s Note:  I originally wrote this piece for UWP 104E: Writing in the Sciences. The assignment called for an explanation of any scientific topic to the general public and I thought to write on a topic that would be both useful and relatable to pet owners. Skin allergies affect dogs of all breeds and can bear severe consequences if left untreated. I hope that this paper increases awareness on animal health and convinces pet owners to consider taking a second glance in those moments they observe something out of the ordinary.

(more…)

Post navigation

← Older posts Newer posts →