Participants
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Osteoporosis impacts more than 18 million people every year and is characterized by low bone mineral density (BMD), which is linked to an increased risk of fracture. BMD is a measure of both the microstructure (bone volume fraction, BV/TV) and mineralization (tissue mineral density, TMD). The PPP6R3 gene, a regulatory subunit for PP6C (protein phosphatase six catalase subunit), is associated with a decrease of BMD in the lumbar spine. However, the mechanical implications have yet to be reported partly due to the complexity of obtaining measurements experimentally. To determine how the knockout of PPP6R3 impacts the mechanical properties of bone, we predicted the stiffness of the trabecular centrum in 49 L5 mouse vertebrae in compression. Three groups of mice were examined: wild-type (no gene knockouts), heterozygous (one gene knockout), and mutant (two gene knockouts). We considered two models for analysis, a finite element (FE) model based on micro-CT scans of the vertebrae, and an idealized model based on the dimensions and apparent modulus of the vertebrae. The mechanical response of bone is influenced by both the microstructure and the mineralization of the bone tissue. When the apparent modulus (as determined by BMD) is varied while the overall microstructure is held constant, the idealized model results showed a significant decrease in vertebral stiffness from wild-type to mutant vertebrae. When the tissue modulus (influenced by mineralization) was held constant and the microstructure was varied, the results of the FE models indicated no significant difference in stiffness between the groups. These findings suggest that the altered mechanical response in mouse vertebrae due to PPP6R3, is largely due to a difference in mineralization (TMD) as opposed to differences in microstructure (BV/TV) alone.
Hybrid exons– exons that can be used as either first or internal exons– are abundant, yet the mechanisms behind their usage are not well understood. Previous research has led to the creation of the hybrid-internal-terminal (HIT) pipeline, a tool that uses RNA sequencing junction reads to correctly classify exons as first, last, or hybrid (1). Of particular interest is the connection between splicing in hybrid exons and corresponding gene transcription levels. Preliminary data from the Fiszbein lab has found that directly altering hybrid exons at the DNA level (using CRISPR/Cas9 to remove splice sites) is known to regulate the usage of hybrid exons as well. We suspect this is due to the coupling of splicing with transcription (2). To confirm if this mechanism is linked to splicing, we aim to silence splice sites at the mRNA level using morpholinos. To explore this, hybrid exons were first validated in four genes. We then selected the hybrid exon for morpholino design based on two criteria: ensuring the hybrid exon and the desired splice sites were both present. We also evaluated these hybrid exons in both Human Embryonic Kidney (HEK293T) and cervical cancer (HeLa) cells to ascertain the best cell line for this experiment. Based on these criteria, we found one of these genes as the ideal hybrid exon for our experiment. Morpholinos were then electroporated into HEK239T cells to silence the splice sites in the desired gene. Currently, using qPCR, we are working on quantifying transcription levels. Further, using the HIT index pipeline to analyze RNA-Seq data sourced from the Genotype-Tissue Expression (GTEx) Database, we found a significant positive correlation between the usage of hybrid exons and splice site strength. Overall, our results confirm the linkage between splicing and gene transcription. Understanding this connection provides key insight into the functions of our genome, which can ultimately be used to help predict and treat several human diseases and cancers.
Nitrogen (N) is an essential nutrient for all living organisms, including plants, microbes, and animals. Increases in atmospheric concentrations of carbon dioxide, rising temperatures, and lengthening growing seasons are leading to increased demand for nitrogen by plants, just as there is declining nitrogen availability in the soil, a process known as nitrogen oligotrophication. The ability of forests to act as a carbon sink depends on nutrient availability. Therefore, with limited nitrogen available, forest productivity may diminish, and carbon sequestration is hindered. Climate change also leads to warmer winter temperatures, which reduces the depth and duration of the snowpack, affecting microbial activity throughout winter and during the critical spring snowmelt period. Reduced snowpack is thought to be a factor in the decrease in rates of soil N mineralization and soil solution N, which are key indicators of N availability to plants. The objective of this study was to determine the relationship between changes in winter and nutrient cycling, and specifically how snowmelt timing influences soil moisture in the northern hardwood forest. At intensive experimental sites on an elevation gradient at Hubbard Brook Experimental Forest in New Hampshire, snow was added or removed to induce a delay or earlier onset of spring snowmelt. Sieved, fresh soil was taken from root ingrowth and exclusion cores incubated in these plots, then was sub sampled, dried, and weighed for soil moisture analysis. Results show that our snow-removal treatment led to both earlier snowmelt and slightly lower soil moisture content compared to snow addition treatments which led to delayed snowmelt. Additionally, soil moisture tends to be higher in root exclusion cores and at higher elevation where greater N availability has been shown. Rates of N mineralization depend on soil moisture, therefore observing the impacts of a changing snowpack on soil moisture is crucial in understanding how future N cycling may change. Along with soil moisture, studying root biomass, net N mineralization, and soil solution for N availability may provide a deeper understanding of how nitrogen’s limited availability impacts the way forests respond to climate change.
With increasing ocean temperature, coral reefs are facing extinction. This could be mitigated by increasing coral thermal tolerance, by cross-breeding genetically distinct corals from different locations. We investigated coral thermal tolerance by creating larvae by crossing moms and dads from different genetic lineages of Porites cf. lobata that inhabits the inner (hotter) and outer reef (cooler) regions of Palau. We hypothesized that thermal stress will cause variable mortality in larval crosses, with parental location influencing larval thermal tolerance and higher survival. Larval heat tolerance was determined by exposing them to 30°C (control) or 39°C for five days and counting the viable larvae every six hours. Larval thermal tolerance was tested using linear mixed effects models with an interaction between mom location, dad location, treatment, and time. To compare and characterize larval gene expression, larval DNA and RNA were extracted, DNA was amplified, and sent for sequencing. As expected, larval mortality was higher at 39°C compared to 30°C. Larval mortality was variable across different crosses. Larval thermal tolerance was influenced by both the mom’s and dad’s locations, with mom’s location independently shaping larval thermal tolerance. Differences in thermal tolerance between larval crosses could be due to gene expression heritability or vertical transmission of symbionts from moms to offspring. To understand this, we will characterize and compare the gene expression and symbionts between larval crosses. Three cryptic lineages of P. lobata live in Palau, and they may produce larvae with different thermal tolerances. To test this, we will characterize the lineages of the parents. If thermal tolerance is heritable, coral extinction could be prevented through “genetic rescue” by introducing heat-tolerant genotypes. As the ocean temperatures increase, understanding the mechanism of coral thermal tolerance and developing thermal tolerant corals could help mitigate coral extinction, and improve the fate of coral reef ecosystems.
Iron based superconductors and antiferromagnetic van der Waals materials provide an interesting platform to study complex interactions of electronic and magnetic orders. The Hu Lab investigates the optical properties of these quantum materials using second harmonic generation and frequency resolved pump-probe spectroscopy in low energy regions. In order to resolve temperature dependent properties, the sample must be kept very cold, at temperatures as low as 4 Kelvin for the duration of the measurements. However, this presents a problem, as keeping the sample at 4 Kelvin causes layers of ice to form on the sample over time due to water vapor in the cooling chamber. This results in limited measurement time as well as potential errors in measurements due to absorption and interference from the ice. To improve measurements, there must be an efficient vacuum to pump water and hydrogen molecules out of the vacuum chamber, creating a low-pressure environment largely free of atmosphere. My project was to optimize the vacuum chamber by designing parts to modify the cryostats to improve the vacuum insulating the sample and allow experiments to continue longer and with more accuracy. This was accomplished by designing and machining the metal parts using CAD software to allow the turbopump to pull atmosphere from the cryostat from a more effective position, so that more air can be pumped out than was previously possible. Overall, this lengthens the cooling period and ensures accurate results, improving low temperature measurements in all lab set ups.
Since the exfoliation of graphene, 2-dimensional (2D) materials have been widely studied. However, a newer and less understood family of 2D materials are transition metal-phosphorus trichalcogenides (MPX3). These 2D semiconductors are commonly used in electronics, such as optoelectronics and nanoelectronics. The specific MPX3 material we focused on is nickel phosphorus trisulfide (NiPS3), an antiferromagnetic van der Waals material. The antiferromagnetic properties of thin layer NiPS3 have already been studied in depth, but here we report how these properties potentially change when layers are stacked on top of each other. We did this by mechanically exfoliating 3-layer samples of NiPS3 using the tape method, then stacking them to create a 6-layer heterostructure using the dry transfer method. We were then able to observe the differences between the initial 3-layer samples and the 6-layer heterostructure using Raman and Photoluminescence (PL) spectroscopy. They are both nondestructive tests used to study the vibrational modes and electron behaviors of crystals. The PL results showed a difference between spin orientation of the heterostructure sample and the 3-layer samples, meaning, as we hypothesized, the coupling between layers in the heterostructure causes a potential difference in the spin structure. Overall, we improved the technique of exfoliating NiPS3, successfully made a heterostructure sample that can be studied more in depth and got closer to understanding the coupling between layers of NiPS3. Further research on NiPS3 can lead to understanding this phenomenon and its potential applications in the electronic world.
As ocean temperatures and acidification levels rise due to anthropogenic change, our coral reefs, which home over a quarter of all marine species, are experiencing increasingly frequent events of mass coral bleaching. Coral bleaching is the result of disruption of the coral symbiotic relationship with their dinoflagellate algae and is related to a coral’s thermal tolerance. Recent advancements in sequencing technology have allowed for the discovery of cryptic lineages, which are genetically different yet morphologically similar, genetic lineages. These cryptic lineages live in different reef environments, with some living in naturally warmer habitats that experience temperatures expected under future climate change scenarios. These cryptic lineages are therefore of particular interest to study whether and how corals might adapt to future climate change conditions. Here, we observe three cryptic lineages of the coral species Porites cf. lobata and assess their survivorship after reciprocal transplantation between naturally warmer inner sites and naturally cooler outer sites, over the course of one year. We hypothesized that coral nubbins taken from lineages typically residing in the warmer, inner reef sites, would experience higher survival than coral nubbins from lineages that are typically found in the cooler, outer reef habitats. After conducting image analysis for all fifty-four nubbin samples, we found evidence supporting our hypothesis as coral nubbins from lineage 2 (L2) experienced higher survival when cross transplanted into an inner reef habitat, but interestingly also flourished in the outer reef habitats. In contrast, coral nubbins from lineage 1 (L1), which is typically found in the cooler, outer sites of the reef, experienced increased mortality when transplanted to inner reef sites. Interestingly, lineage 3 (L3) which is found throughout both inner and outer sites showcased initial signs of mortality during the first six months of the experiment and later of recovery during the latter half of the experiment. Such results suggest a correlation between coral lineage, their thermal tolerance and thus, they’re ability to survive under future environmental stressors. Such findings provide an important foundation of understanding coral responses to climate change and the potential variation in responses as a result of cryptic coral lineages.
Tendons, the critical connectors between muscles and bones, serve an indispensable role in the musculoskeletal system, enabling seamless movement and mechanical support. Understanding the full range of factors that regulate tendon health and repair is critical in developing effective treatments for both acute injuries and degenerative disorders. Although it is known that estrogen plays a significant role in maintenance and health functions of these connective tissues, the impact of estrogen on tendons is an area of scientific interest that remains incompletely understood. (1) Higher estrogen levels have been correlated with an increase of collagen synthesis in tendons and ligaments. (2) Some studies also suggest estrogen revealed a positive impact on tendon strength and elasticity. However, the exact effect of these varying levels is not completely understood. We aim to illuminate this relationship and uncover how estrogen affects tissue composition and metabolism. Our study involves the extraction and sterile culture of tendons from ten mice, comprising equal representation of both sexes. To emulate diverse hormonal environments, we subject these tendons to varying estrogen concentration: Cycling Levels (100 pM), mirroring the physiological fluctuations observed in cycling female mice, and Therapeutic Levels (10nM), simulating estrogen supplementation in therapeutic scenarios. We also compare our findings with previously collected data from tendons treated with no hormone and ovulation levels (1nM) to get a more holistic understanding of dose-dependent effects. Through a series of biochemistry experiments we assessed two pivotal proteins in the tendon structure, (1) collagen, which is essential for giving tendons their strength, and (2) glycosaminoglycans (GAGs), which play a crucial role in providing elasticity to the tendon’s structure. The results suggested a dose-despondent response to estrogen, but it surprisingly wasn't linear. The balance of ECM remodeling might have been disrupted, thus causing the nonlinear dose-dependent response. The precise reason for these fluctuations remains unknown, further research is needed to fully uncover the outcome. Our research marks a significant step towards advancing the understanding of tendon biology and brings us closer to a future with improved treatment options for tendon-related afflictions. This understanding may offer valuable insights into the interplay of sex hormones and tissue health.
Citations: 1. Hansen M, Kjaer M. Sex Hormones and Tendon. Adv Exp Med Biol. 2016;920:139-149. doi:10.1007/978-3-319-33943-6_13
2. Leblanc DR, Schneider M, Angele P, Vollmer G, Docheva D. The effect of estrogen on tendon and ligament metabolism and function. J Steroid Biochem Mol Biol. 2017;172:106-116. doi:10.1016/j.jsbmb.2017.06.008
3. Chidi-Ogbolu N, Baar K. Effect of Estrogen on Musculoskeletal Performance and Injury Risk. Front Physiol. 2019;9:1834. doi:10.3389/fphys.2018.01834
Microplastics - fragments of plastic less than 5 millimeters long - are pervasive in coastal marine environments. Microplastics may harm marine life not only via ingestion, but also by releasing reactive oxygen species (ROS), e.g., hydrogen peroxide (H₂O₂), when exposed to ultraviolet (UV) light. As a pervasive marine pollutant, microplastics may challenge the resilience of marine organisms and impact their evolution, but few studies have investigated variability in organismal response to microplastics. As a salt marsh inhabitant exposed to varying levels of microplastics and known to harbor variation in resistance to ROS, we hypothesize that populations of the starlet sea anemone, Nematostella vectensis, will exhibit variation in susceptibility to microplastic pollution. Understanding the basis of such variation will be critical to predicting how microplastics impact coastal marine communities. We exposed three clonal lines of N. vectensis from Belle Isle Marsh (Boston, MA) to H₂O₂, as ROS tolerance may predict microplastic tolerance in N. vectensis. Additionally, we sampled sediment and collected N. vectensis from six sites in Squantum Marsh (Quincy, MA), part of an effort to characterize variability in microplastic prevalence in Massachusetts salt marshes, while sourcing N. vectensis from a range of geographic locations and microplastic exposure levels for subsequent experiments. As in a prior study, we found that extended peroxide exposure (14 days) caused a reduction in tentacle number/extension. However, unlike this prior study, we observed a partial acclimation to H₂O₂ exposure in all three lines of N. vectensis, who extended significantly more tentacles on days 3-4 of exposure than on days 1-2. We were unable to accurately quantify microplastic particles within the body cavity of N. vectensis using a fluorescence microscopy approach, because the microplastics and N. vectensis fluoresced at the same wavelength. N. vectensis is a tractable laboratory and field model for studying organismal impacts of microplastic pollution. Future studies will expose N. vectensis clones known to vary in ROS tolerance to microplastics, in the presence and absence of UV light, and characterize variability in microplastic contamination in Massachusetts salt marshes, attempting to relate this to variability in organismal resistance to microplastic exposure. Going forward, it will be important to optimize methods for quantifying microplastics inside the anemone’s body.
The Polymer ANalysis and Discovery Array (PANDA) is an automated robotic system that uses electrochemistry to characterize how different films conduct electricity. One method of characterization is using an electroluminescent panel and shining it through the thin polymer film. During the analysis of the experiment the system needs to turn on the light and turn it off again, instead of having a person control it and risk human error a controller is used. The controller was designed to work with the system and turn on the light at a certain time and turn it off again. Although the controller could not be used alone because it only supplied 5 volts at a time, we were able to connect the controller to a voltage-controlled power strip. Another part of the project was creating a digital twin of the full system, and other parts used in the system, like the vials. This digital twin can be used to create animations of the full system that demonstrates how the system moves. The digital twin will also make it easier to create future accessories or tools for the system. The last part of my project was designing a mount for a camera lens that would then be 3D printed and mounted onto the PANDA, so that the camera could take pictures during the experiment. All parts of my project will be used in the experiments, the microcontroller will control the electroluminescent lights, the lens mount will hold the camera and the digital twin will be used to create additional tools and accessories for the PANDA. This will help facilitate the characterization of different polymers and how they conduct electricity so that this information can be used to create a new generation of lithium-ion batteries.
Gold (1) complexes are an area of interest for catalysis due to aurophilic interactions which could be leveraged for catalysis and luminescent and phosphorescent properties which could be used in LED type applications. In order to research aurophilicity, a gold catalyst must be first created. N-heterocyclic carbenes ligands were attached to gold to help stabilize gold(I) complexes; this synthetic route enables other ligands to be attached to the other side of the linear complex. Two different carbene ligands were used with mesityl or isopropyl substituents at nitrogen. Upon successful synthesis of the known gold carbene complexes, pyrenyl isonitrile was attached to the gold center. Furthermore, the gold carbene complexes were used in attempts to synthesize the bimetallic complex [(L)AuPt(PPh3)(CNC)], L = carbene ligand. These gold complexes will continue to be studied for applications in catalysis and for their luminescent and phosphorescent properties.
Digitonthophagus gazella is a species of dung beetle that exhibits nutrition-based polyphenism. In the more researched horn-dimorphic beetle Onthophagus taurus, high-nutrition males grow larger and develop large horns. Comparatively, low-nutrition males grow short horns or no horns at all. Different phenotypes display different mating tactics. Large males guard females and fight other males, whereas smaller males use a sneaker tactic to bypass large males. Understudied D. gazella beetles have a more moderate horn dimorphism than O. taurus. This study seeks to characterize whether the fighter behavior seen in large O. taurus males is conserved in D. gazella. Additionally, we examined if aggressive behavior instances and duration correlate to body size. We recorded and observed beetle behaviors using previously published methods, in which we performed behavioral assays that placed two size-matched male beetles in clear tubes. We found that similarly to O. taurus, large D. gazella males fight with other male beetles, suggesting that this behavior is conserved across species. We also found that body size does not tightly correlate to aggression in D. gazella. Other traits such as horn sizes or smaller body size should also be studied. Our research will serve as a basis for future studies on D. gazella and other beetle species, especially for those that investigate the molecular mechanisms that regulate alternative mating tactics. Exploring the interaction between polyphenism and breeding behaviors across species gives us a better understanding of the evolution of behavior and its relationship with morphology.
The locus coeruleus (LC), a brain region in the brainstem, is responsible for releasing the neurotransmitter norepinephrine to the central nervous system and plays a role in modulating various independent behaviors including stress response, wakefulness, and attention and memory in cognitive tasks. We used designer receptors exclusively activated by designer drugs (DREADDs), a common chemogenetic technique used to modulate activity in specific areas of the brain by either exciting or inhibiting neuron firing temporarily when activated by the DREADDs ligand. First, an excitatory DREADDs was introduced to the LC neurons of the rats. Then they were injected with either Deschloroclozapine (DCZ), the DREADDs ligand, or saline, used as a control, prior to training in a task. We analyzed confocal imaging brain slices of the rat’s LC stained for the proteins: cFOS, which is used as an indicator of recent neural activity, and mCherry, which indicates DREADDs expression. We hypothesized that we would see higher numbers of cFOS+ cells in the DCZ group compared to the saline group because it would mean the DREADDs are working as anticipated. We found a higher number of cFOS+ cells and a higher percentage of mCherry+ cells that coexpressed cFOS in the DCZ compared to the saline group, supporting our hypothesis. Future researchers can use this data to better understand the LC which plays a role in many psychiatric disorders including anxiety, depression, post-traumatic stress syndrome, and attention deficit/hyperactivity disorder, as well as in many neurodegenerative disorders including Alzheimer’s disease and Parkinson’s disease.
Method development is crucial in the field of organic chemistry. Novel methods are able to be utilized when synthesizing complex natural products. Such products can have potential biological activity and can subsequently serve as lead compounds in drug development. Multicomponent reactions (MCRs) are especially important in method development as they can greatly increase the efficiency in making target molecules. Herein, we perform three-component coupling reactions that are able to occur in one-pot, using ester enolates. We are investigating the role of Michael acceptors in this MCR and how changing coupling partners can affect reactivity. Analytical methods were used to confirm the structure and mass of compounds. Through performing these multicomponent reactions, we observed competition which resulted in both conjugate and carbonyl addition during the Michael addition step. Further reaction optimization is needed; however, this work was able to give insight into the reaction limitations and expanded the substrate scope to make an eventual chemical library of bioactive molecules.
How does the environment (abiotic, biotic, and social) influence animal behavior in the wild? In the clown anemonefish (Amphiprion percula), parental care is performed by a dominant female and a subordinate male, with behaviors varying between individual mates and their surroundings. The environmental factor studied here, egg predation and cannibalization, is challenging to research in-field. Instead, we record the behaviors of multiple laboratory breeding pairs before and after the experimental removal of half their eggs. The data we collected for male and female tending time has high repeatability and shows no significant increase or decrease post-experimental treatment. Thus, the amount of time breeding pairs spend caring for their eggs is uninfluenced by egg removal and may depend on other factors. These results clarify the effects of egg predation and cannibalization in A. percula, enforcing that these events do not influence tending time.
The development of a fruit fly, Drosophila melanogaster, and other organisms is regulated by shadow enhancers, seemingly redundant enhancers that control a common gene. These shadow enhancers help balance transcriptional noise and maintain embryo stability. They also regulate gap genes like Kruppel, which intersect to form the gap gene network. The gap gene network is responsible for the formation of certain body parts of the fly. It is expected that when the embryo undergoes environmental stresses like heat shock or hypoxia, transcriptional noise will increase. With an increase in transcriptional noise, there will be adverse effects upon the embryo’s development. However, when only the proximal enhancer is present in the Kruppel gap gene shadow enhancer system, transcriptional noise stays constant after the application of heat shock. I tested whether this uniquely stable response is related to the transcription factor binding site STAT92E, which is on the proximal enhancer. I used site-directed mutagenesis and cloning processes to introduce a mutation to our F23 plasmid. With gel electrophoresis, I verified the plasmid DNA samples and sequenced them to confirm the mutation was present. The results of this experiment will not only contribute to a deeper understanding of the Kruppel gap gene and shadow enhancers present in humans but may also provide insight into developmental defects and the impact of environmental stressors on development.