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Efficient Sabre-Sheath Hyperpolarization Of Pyruvate And Other Alpha-Ketocarboxylates And Ultra-High-Qualityfactor Wireless Masing Magnetic Resonance Sensing Of Hyperpolarized Contrast Media
No full textOver the years, there has been research geared towards mitigating disease conditions such as cancers and tumors by molecular sensing of their aberrant metabolism in humans and animal models of diseases. Some of these biomolecules of interest are pyruvate, α-ketoglutarate, and ketoisocaproate, that play a central role in eukaryotic and human metabolic pathways. Nuclear magnetic resonance (NMR) spectroscopy and magnetic resonance imaging (MRI) have become predominant in investigating and imaging cancers and tumors for early detection and treatment. Although conventional NMR spectroscopy is limited by low sensitivity, we overcame this barrier through the use of hyperpolarization techniques. NMR hyperpolarization improves NMR sensitivity by 4-6 orders of magnitude by increasing the nuclear spin-order relative to the thermal equilibrium polarization level.
We used Parahydrogen-Induced Polarization (PHIP) and Signal Amplification by Reversible Exchange (SABRE) hyperpolarization techniques suitable for transferring parahydrogen-derived polarization to these biomolecules. SABRE is accomplished by simultaneous reversible exchange of parahydrogen (p-H2) and to-be-hyperpolarized contrast agent molecule on a hexacoordinate Iridium catalyst. Specially, we used a variant of SABRE called SABRE-SHEATH (Signal Amplification by Reversible Exchange in Shield Enables Alignment Transfer to Heteronuclei) where polarization is transferred from p-H2 to heteronucleus of the contrast media in the microtesla magnetic field. Chapters 1 and 2 are dedicated to creating an understanding of this study through careful explanation of the principles of NMR, MRI, hyperpolarization techniques and RASER effect.
Chapter 3 focuses on SABRE-SHEATH technique as it is explored to rapidly hyperpolarize 13C spins of [1-13C]pyruvate using parahydrogen as the source of spin order. The pyruvate interacts with an iridium-based polarization transfer catalyst with temperature and co-ligation of dimethyl sulfoxide and H2O playing key roles in controlling the exchange. By reducing the temperature, [1-13C]pyruvate exchange slows, achieving over 50% polarization in under 30 seconds. Using a 1.4 T NMR spectrometer, 39% polarization is measured and extrapolated to above 50% at saturation. The highest polarization of a 30 mM pyruvate sample, considering both free and bound forms, reaches 13%. Fast polarization buildup, outpacing spin-relaxation rates, and the ability to release hyperpolarized pyruvate through temperature cycling offer promise for future biomedical applications.
Chapter 4 explores the use of the SABRE-SHEATH technique to achieve fast hyperpolarization of 13C-labeled α-ketoglutarate. α-Ketoglutarate, a key metabolite in the tricarboxylic acid cycle, plays a vital role in metabolic pathways and is linked to conditions such as cancer. In this study, the SABRE-SHEATH technique generated 9.7% 13C polarization within just 1 minute. This method uses parahydrogen as a nuclear spin order source, and the polarization transfer was optimized at a field strength of 0.4 μT. The efficient hyperpolarization was driven by favorable relaxation dynamics, with a polarization buildup time of 11 seconds and a decay constant of 18.5 seconds. A higher ¹³C polarization value (17.3%) was achieved using natural-abundance α-ketoglutarate. Interestingly, deuteration had minimal effects on relaxation rates. This research highlights the potential of α-ketoglutarate as a hyperpolarized contrast agent, providing new opportunities for metabolic imaging, particularly in cancer diagnostics and brain function studies. The findings pave the way for developing hyperpolarized agents for real-time metabolic imaging applications.
Chapter 5 highlights the use of SABRE-SHEATH to rapidly hyperpolarize [1-13C]ketoisocaproate, a metabolic probe involved in branched-chain amino acid pathways. This technique achieved up to 18% polarization within one minute, a significant improvement compared to previous efforts. The optimization of temperature, pH, and p-H2 pressure played a key role in enhancing the hyperpolarization levels. We also explored the hyperpolarization of natural-abundance ketoisocaproate, which enabled the independent analysis of C-1 and C-2 carbons. Additionally, hyperpolarized [1-13C]ketoisocaproate was successfully detected in a post-mortem mouse using a 0.35 T clinical MRI scanner. These findings highlight the potential of ketoisocaproate as a hyperpolarized contrast agent for metabolic imaging, with applications in cancer and neurodegenerative disease research.
Chapter 6 focuses on advancements in NMR sensing using wireless maser detectors. We demonstrated how hyperpolarized nuclear spins can generate stimulated emission signals, similar to masers, improving the detection of hyperpolarized contrast agents like [1-13C]pyruvate, which is under investigation for cancer imaging. By enhancing the quality factor of the NMR detector through parametric pumping, a 22-fold increase in quality factor (up to 1,670) was reported, lowering the threshold for inducing nuclear spin masing. This approach allows for better signal detection under challenging conditions, such as low magnetic field homogeneity. This technology has potential for future applications in preclinical MRI scanners.
My dissertation will strengthen our understanding of the physics and chemistry of polarization transfer governing hyperpolarization of [1-13C]pyruvate, α-ketoglutarate, and ketoisocaproate in microtesla magnetic fields as well as RASER effects. HP contrast media utility for real time monitoring of diseases is already in clinical trials having gained attention and approval from relevant authorities because it a safe protocol that boosts MRI signals by several orders of magnitude. This work has the potential to augment or replace the 2.5h-long-PET exam by a non-ionizing ~10-minute-long HP MRI procedure
Splicing-Dependent Regulation Of Transcription: A Novel Role Of The Splicing Factor Prp19 In Rnapii-Transcribed Genes
No full textGene regulation in eukaryotes relies on complex, multi-layered processes that govern the precise control of transcription, the first step in gene expression. A growing body of evidence suggests that introns—non-coding regions within genes—play an integral role in regulating transcriptional activity beyond their traditional roles in RNA splicing. However, the exact mechanisms by which introns influence transcription remain poorly understood. This dissertation addresses these questions by examining the role of introns in transcriptional activation and the underlying mechanism that may facilitate this process in budding yeast.In preliminary investigations within our lab, the extent to which an intron can activate transcription of yeast genes was examined. It was observed that in the presence of an intron there is an augmentation in transcript levels of the cognate gene, underscoring the enhancement potential of introns on transcription. To further test the enhancement potential of an intron, I introduced the ACT1 intron into the promoter-proximal regions of four intronless yeast genes (INO1, MET16, CHA1, and GAL10) and examined their transcription levels under the influence of an intron versus a transcription activator. Remarkably, insertion of the intron restored transcription to levels comparable to those seen with active transcriptional activators, even in the absence of such activators. Transcription run-on assays further revealed that this intronic insertion could directly enhance transcription of nascent mRNA, indicating that the intron itself might promote transcriptional activation independently by facilitating the assembly of the preinitiation complex (PIC) at the promoter. Building upon these findings, I next investigated the broader regulatory mechanism in which splicing-competent introns enhance transcription. Based on studies from our lab demonstrating the formation of a gene loop—transcriptional structures formed by the interaction between promoter and terminator regions of a gene—in the presence of an activator or intron, I focused on the protein-protein interactions that might underscore loop-formation and subsequent enhanced transcription. Gene looping, previously shown to involve interactions between transcription factors such as TFIIB and termination complexes (CF1, CPF, and Rat1), was examined. Through quantitative proteomics of affinity-purified termination complexes, I discovered that termination factors not only interact with general transcription factors (i.e. TFIIB and TFIID) but also with splicing factors (Prp19, Prp43, Sub2, Snu114, Brr2, and Smb1), suggesting a collaborative role between transcription and splicing machinery in gene regulation. Notably, Prp19 emerged as a critical interactor with both transcription initiation and termination factors, prompting further analysis of its role in transcription. To elucidate the function of Prp19 in intron-mediated enhancement of transcription, I employed an auxin-inducible degron system to deplete Prp19, which resulted in a significant decrease in nascent transcription levels across both intron-containing and intron-less genes. Chromatin immunoprecipitation (ChIP) assays revealed that Prp19 associates with the promoter-proximal regions of these genes, supporting a novel role for Prp19 in the initiation of transcription. This effect appears mediated through the recruitment of the TFIID subunit TBP, linking Prp19 directly to transcription initiation and suggesting a broader regulatory function for splicing factors within transcription. Overall, this work demonstrates that introns can act independently to promote transcriptional activation, potentially by facilitating PIC assembly, and identifies Prp19 as a novel regulatory component in transcription initiation. These findings advance our understanding of how introns and associated protein complexes coordinate to regulate gene expression, offering new insights into the noncanonical roles of splicing factors and introns in transcription
High-Throughput Screening Of Fda-Approved Drugs For Protecting Retinal Pigment Epithelial Cells Against Er Stress Induced Cell Death: Implications For Age-Related Macular Degeneration
No full textPurpose: Age-related macular degeneration (AMD) is a leading cause of blindness in the elderly population, characterized by the degeneration of the retinal pigment epithelium (RPE). The link between endoplasmic reticulum (ER) stress and AMD is well established, however FDA-approved medications for the treatment of AMD caused by unregulated ER stress are limited and have potential side effects. The purpose of this study was to conduct a comprehensive screening of the FDA-approved drug library in order to identify potential candidates that could be repurposed in order to protect against the degeneration and death of RPE cells due to ER stress.
Methods: The FDA Pharmakon compound library (containing 1760 medicines) was acquired from MicroSource Discovery Systems, Inc. (USA). These compounds were received in microplates at 10 mM, dissolved in DMSO, and stored at -80°C until needed. A human retinal pigment epithelium cell line (ARPE-19) was exposed to tunicamycin (TM) with or without various FDA-approved drugs to induce ER stress. Lactate dehydrogenase (LDH) release into the media was measured over 24, 48 and 72 hours to assess cellular toxicity. Statistical analysis was performed using a two-way ANOVA test followed by post hoc tests.
Results: Treatment of ARPE-19 cells with tunicamycin (TM) resulted in cell death, as evidenced by LDH leakage after 48 and 72 hours of treatment. Significantly, medications containing beta-lactam compounds, including penicillin and cephalosporin derivatives, emerged as the most effective drug category in mitigating TM-induced RPE cell death. Furthermore, a detailed exploration of the structure-activity relationship was undertaken to identify the specific chemical groups responsible for this observed protective effect. Based on these results, increasing the hydrophilicity of the benzene ring reduces the protective effect, similar to amoxicillin. As well, converting the primary amino group in ampicillin into a secondary amino group, such as those found in piperacillin and azlocillin, eliminates its activity. Replacing the primary amino group with a carboxylic group extends the protective effect of ampicillin as seen in carbenicillin. Deletion of the primary amino or carboxyl group at position 2 of the side chain, as in Penicillin G, resulted in partial protection of RPE cells against ER stress-induced cell death. The protective effect could be enhanced by adding chlorine atoms like in cloxacillin and dicloxacillin or methoxy groups like in methicillin. Similarly, the presence of a six-membered ring adjacent to the beta-lactam ring enhances the protective effect, as seen in cephalosporin. A number of moieties can be added to cephalosporin derivatives\u27 R1 side chains to enhance their protection against TM-induced RPE cell death. Specifically, incorporating methyl (CH₃), chlorine (Cl), ester groups, thiadiazole rings, and tetrazole rings resulted in complete (100%) protection, highlighting the importance of these structural modifications in improving therapeutic efficacy. As a result of these modifications, the drug is likely to interact more effectively with retinal cells. This will reduce the possibility of damage caused by ER stress and related degenerative processes in the retina.
Conclusion: The study concludes that beta-lactam medications, including penicillin and cephalosporin derivatives, can serve as potent protections against RPE cell degeneration and death as a result of ER stress. A number of these compounds, particularly those with specific structural modifications, are highly effective at preserving the viability of RPE cells. These findings offer promising avenues for developing therapeutic strategies to combat AMD associated with unregulated ER stress
Effects Of Gender, Ethnicity, And Drinking Level On Judgments Of College Students’ Personal Effectiveness: A Vignette Study
No full textObjective: Opinions about alcohol use differ across ethnicities, and in many Middle Eastern communities, alcohol use is stigmatized. Despite this, few studies have investigated how ethnicity influences alcohol-related stigma and perceptions of individuals who drink. Moreover, existing research on stigma and substance use has often relied on surveys, which can be affected by social desirability bias or limited insight. To address these issues, the current study used vignettes to examine the degree to which participant ethnicity and vignette character drinking level, gender, and ethnicity influenced ratings of vignette character conscientiousness, responsibility, and success.Method: Middle Eastern and non-Middle Eastern participants (N = 347) were randomly assigned to rate the perceived conscientiousness, responsibility, and success of 1 of 12 vignette characters who varied on ethnicity (Arab American vs. non-Arab American), gender (male vs. female), and drinking level (non, light, or heavy). A structural equation model was specified to examine main and interaction effects of these variables on participant ratings. Results: Vignette characters who drank heavily were perceived as significantly less conscientious, responsible, and successful than those who drank less or abstained, particularly by Middle Eastern participants. Additionally, male characters who drank heavily received lower ratings than female characters who drank heavily. Conclusions: Individuals who drink heavily may be perceived as low in conscientiousness, responsibility, and success, especially in Middle Eastern communities, where cultural and religious norms discourage use. Additionally, males who drink heavily may be perceived as less conscientious, responsible, and successful than females who drink heavily
Y-STR Haplotypic Polymorphisms for the Hakka Population in West China and Phylogenic Comparison with Other Chinese Populations
No full textThe Hakka, who have undergone a series of great migrations, are usually identified with people who speak the Hakka language or share at least same Hakka ancestry. As the largest Hakka dialect island in West China, the Dongshan region was closely linked with the great migration wave of Hakka. However, the paternal genetic profiles of Dongshan Hakka have never been revealed. In the present study, 41 Y-chromosomal short tandem repeat (Y-STR) loci included in the SureID PathFinder Plus Kit were analyzed in 353 unrelated male individuals (171 Hakka and 182 Han) of Sichuan Province, China. By analyzing 166 different haplotypes among Dongshan Hakka and 176 different haplotypes among Sichuan Han males, haplotype diversity was calculated as 0.9997, with a discrimination capacity of 0.9708, for the Hakka population, and 0.9996 and 0.9670 for the Sichuan Han population, respectively. Most of the Y-STR loci were highly informative in both populations except DYS645. The genetic relationships were evaluated by comparing the Hakka population with 11 other groups that are relevant to Hakka migration routes. The results of the multidimensional scaling plot and phylogenetic tree indicate that the Dongshan Hakka population was closely related to Han nationalities from Anhui, Jiangxi, and Fujian Provinces
Autism Spectrum Disorder: An Insider’s Perspective in Public and Academic Libraries
Get PDFThe purpose of this paper is to bring to light the social issue of the Autism Spectrum Disorder (ASD) and how public and academic libraries are making strides to better serve this branch of the population. Definitions will be provided to better explain what challenges ASD patrons face. In turn, librarians across America, specifically in Illinois and Ohio in recent years, have come together to discuss in both literature and national forums what they are continually doing to better serve, educate, and support their patrons and train their librarians. ASD is a complex social issue faced by millions; one widely covered and not easily solved alone, but through strategic planning and partnerships, libraries are formulating some creative solutions to meet these challenges. Also touched on will be the perspectives of librarians with ASD; the challenges they face each day, and where they see the future for people with ASD working in a library setting. Let us discover how libraries are rising above these challenges and becoming better informed to equip one another to serve all their patrons
Sex-Ratio, Health, and Social Status: A Biographical Description of Middle and Late Period Bay Area Children
No full textThis article presents new information pertaining to the demographic profile of the juvenile burial assemblage (n = 39) from a Late Holocene site located on the eastern shore of the San Francisco Bay. CA-ALA-329 is commonly referred to as Ryan Mound and now bears the Muwekma Ohlone name Mánni Muwékma Kúksú Hóowok Yatiš Túnnešte-tka, which means “place where the people of the kúksú (bighead) pendants are buried.” This site has been extensively studied and has contributed significantly to our understanding of life on the bay during the Middle and Late Periods. However, most of the previous studies have focused on adults. The goal of the present study was to identify patterns in the profiles of those who died prematurely: their sex, the degree of stress they experienced based on skeletal indicators of disease/malnutrition, and their social status based on associated grave goods. Results show high incidence of skeletal indicators in the sample consistent with nutritional deficiency, disease/infection, and/or metabolic disorders. This suggests that this population was experiencing stress. Individual circumstances, such as age and sex, may also have contributed to poor health because infants have the highest prevalence of cribra orbitalia and periostitis. The distribution of wealth, as evidenced by burial goods associated with the sample, shows some correlation with age at death and types of artifacts. Distribution of wealth also differs temporally. Inequality seems to have been highest in the Middle Period, whereas inequality decreased but overall wealth increased into the Late Period
The Celebrity Effect: How Social Media Changed Ozempic Utilization by Medicaid Patients
Get PDFBackground: Recently, glucagon-like peptide-1 receptor agonists (GLP-1 agonists), a drug class used to treat Type 2 Diabetes, has gained popularity on social media for cosmetic weight loss. Celebrity endorsement of Ozempic, brand name of GLP-1 agonist semaglutide, has increased public demand and caused supply shortages. However, effects on Medicaid patients, who use Ozempic for diabetes, have yet to be investigated.
Methods: We sourced publicly available drug utilization datasets from Medicaid.gov. Nationwide Medicaid reimbursement data for Ozempic, Wegovy (another brand name of semaglutide), and Jardiance (different diabetes medication drug class) were extracted for 2021, 2022, and 2023. Rates of change per quarter per calendar year were calculated, and two-tailed student’s paired T-tests were conducted.
Results: Social media promotions for Ozempic largely began 2022Q4 (Oct 1-Dec 31). Medicaid Ozempic utilization prior to 2022Q4 were significantly different from Jardiance regarding reimbursed units, number of prescriptions, total amount, and Medicaid amount (p0.05), potentially correlating with a difference in publicity for weight loss use compared to Ozempic.
Conclusion: This study strongly suggests that social media has impacted Ozempic usage spanning different socioeconomic classes nationwide. Plastic surgeons with aesthetic services should be conscientious of the downstream effects of prescribing cosmetic weight loss drugs and manage patient expectations accordingly as social media continues to drive public demand