Projects and Papers
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Chulso Moon 1,2,3, Maxie Gordon 2,3, David Moon 2 and Thomas Reynolds * 4  Citation: 

Int. J. Mol. Sci. 2021, 22, 12864. https://doi.org/ 10.3390/ijms222312864 Published: 28 November 2021 

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Abstract: Microsatellite instability (MSI), the spontaneous loss or gain of nucleotides from repetitive DNA tracts, is a diagnostic phenotype for gastrointestinal, endometrial, colorectal, and bladder cancers; yet a landscape of instability events across a wider variety of cancer types is beginning to be discovered. The epigenetic inactivation of the MLH1 gene is often associated with sporadic MSI cancers. Recent next-generation sequencing (NGS)-based analyses have comprehensively characterized MSI-positive (MSI+) cancers, and several approaches to the detection of the MSI phenotype of tumors using NGS have been developed. Bladder cancer (here we refer to transitional carcinoma of the bladder) is a major cause of morbidity and mortality in the Western world. Cystoscopy, a gold standard for the detection of bladder cancer, is invasive and sometimes carries unwanted complications, while its cost is relatively high. Urine cytology is of limited value due to its low sensitivity, particularly to low-grade tumors. Therefore, over the last two decades, several new “molecular assays” for the diagnosis of urothelial cancer have been developed. Here, we provide an update on the development of a microsatellite instability assay (MSA) and the development of MSA associated with bladder cancers, focusing on findings obtained from urine analysis from bladder cancer patients as compared with individuals without bladder cancer. In our review, based on over 18 publications with approximately 900 sample cohorts, we provide the sensitivity (87% to 90%) and specificity (94% to 98%) of MSA. We also provide a comparative analysis between MSA and other assays, as well as discussing the details of four different FDA-approved assays. We conclude that MSA is a potentially powerful test for bladder cancer detection and may improve the quality of life of bladder cancer patients.

Abstract: Several studies have shown that microsatellite changes can be profiled in urine for the detection of bladder cancer. The use of microsatellite analysis (MSA) for bladder cancer detection requires a comprehensive analysis of as many as 15 to 20 markers, based on the amplification and interpretations of many individual MSA markers, and it can be technically challenging. Here, to develop fast, more efficient, standardized, and less costly MSA for the detection of bladder cancer, we developed three multiplex-polymerase-chain-reaction-(PCR)-based MSA assays, all of which were analyzed via a genetic analyzer. First, we selected 16 MSA markers based on 9 selected publications. Based on samples from Johns Hopkins University (the JHU sample, the first set sample), we developed an MSA based on triplet, three-tube-based multiplex PCR (a Triplet MSA assay). The discovery, validation, and translation of biomarkers for the early detection of cancer are the primary focuses of the Early Detection Research Network (EDRN), an initiative of the National Cancer Institute (NCI). A prospective study sponsored by the EDRN was undertaken to determine the efficacy of a novel set of MSA markers for the early detection of bladder cancer. This work and data analysis were performed through a collaboration between academics and industry partners. In the current study, we undertook a re-analysis of the primary data from the Compass study to enhance the predictive power of the dataset in bladder cancer diagnosis. Using a four-stage pipeline of modern machine learning techniques, including outlier removal with a nonlinear model, correcting for majority/minority class imbalance, feature engineering, and the use of a model-derived variable importance measure to select predictors, we were able to increase the utility of the original dataset to predict the occurrence of bladder cancer. The results of this analysis showed an increase in accuracy (85%), sensitivity (82%), and specificity (83%) compared to the original analysis. The re-analysis of the EDRN study results using machine learning statistical analysis proved to achieve an appropriate level of accuracy, sensitivity, and specificity to support the use of the MSA for bladder cancer detection and monitoring. This assay can be a significant addition to the tools urologists use to both detect primary bladder cancers and monitor recurrent bladder cancer.

Abstract: The purpose of this study is to determine whether pharmacogenetic testing could be used to effectively customize postoperative pain medicine following total joint replacement. Methods: Buccal swabs were collected preoperatively from 107 patients. Pharmacogenetic testing was performed for genetic variants on a panel of 16 genes, including CYP2D6, CYP2C9, OPRM1, and CYP1A2, which affect the pharmacodynamics and pharmacokinetics of non-steroidal anti-inflammatory drugs and many opioids. Patients were randomized to a control group or custom group and blinded to their group. The control group was prescribed oxycodone, tramadol, and celecoxib for postoperative pain management. If any of those were not normally metabolized, they were not prescribed to the patients in the custom group, who were given an alternative drug (hydromorphone for narcotics, meloxicam for non-steroidal anti-inflammatory drugs). Patients recorded their pain level (0-10 numeric scale) and all medications taken daily for the first 10 days following surgery. Medication was converted to milligram morphine equivalents (MMEs). Results: Genetic variations to medications in our standard postoperative pain management protocol occurred in 24 of the 107 patients (22.4%). The 10-day MME consumed by patients in the control group with genetic variants was 162.6 mg. Patients with variants who had custom postoperative medication consumed only 86.7 MME in the same timeframe (P ¼ .126). The control group demonstrated a higher 10- day average pain level of 4.2 vs the custom group pain level of only 3.1 (P < .05). Conclusion: With custom postoperative pain prescriptions based on pharmacogenetic testing, patients were able to achieve lower pain levels while reducing the consumption of pain medication.

ARTICLE Functional Characterization of Undenatured Type II Collagen Supplements: Are They Interchangeable? 

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ABSTRACT Undenatured (native) type II collagen is a dietary supplement ingredient reported to support joint health in healthy individuals by providing relief from symptoms of stiffness and discomfort and improving mobility. This benefit is thought to occur through oral tolerance, a mechanism whereby the immune system distinguishes between innocuous material in the gut and potentially harmful foreign invaders. The presence of antigenic epitopes in undenatured type II collagen, but not in denatured (hydrolyzed) collagen, is thought to be the basis for the therapeutic benefits. The purpose of this study was to investigate the physicochemical and analytical characteristics of type II collagen supplements currently available on the market and to explore whether they might be sufficiently similar in their physical properties to yield similar benefits in promoting joint health. Collagen type II supplement powders (raw material) and capsules (products in the market) were examined for color, particle size, quality profiles, fatty acid profiles, electron microscopy, and were analyzed for amino acid content as well as antigenic potential via an ELISA assay. Powders labeled as undenatured type II collagen were found to have markedly different properties, including the size of collagen fibers as per electron microscopy and antigenic configuration as per the ELISA assay. As significant differences were found between products, it allows consumers and practitioners to not assume that products labeled as undenatured (native) type II collagen are interchangeable.

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In 2001, the Human Genome Project reached a momentous milestone: after working on it for 15 years, scientists published, for the very first time, the DNA sequence of one human genome. Today, scientists can sequence roughly 45 human genomes in a single day. The field of DNA sequencing has come a long way. As it gets better and faster, researchers are setting out to tackle some technical hurdles — namely, challenging samples and human errors — that curb their current pace. The exponential acceleration in our capacity to sequence 3.2 billion human base pairs has transformed our approach to therapeutics, particularly for cancer. SelectScience interviews Dr. Robert B. Harris, the chief scientific officer at NEXT Molecular Analytics, an experienced scientist who has embraced the fast-evolving DNA sequencing movement. The goals of NEXT Molecular Analytics, among others, include applying genomics to help patients and doctors make informed decisions about managing cancer risks. Cancer therapy no longer just “reacts” to the presence of disease, but has become more and more proactive. Using the tools of next-generation sequencing (NGS), we can now scrutinize the cancer genome and identify DNA aberrations. We can investigate tumor biology, aid clinicians with diagnosis and prognosis, and finally, look optimistically in the direction of precision oncology. But first, we must overcome the challenge of extracting DNA from tough samples and eliminating human errors — steps that affect NGS results. Some researchers are restrained by the poor DNA yield from a typical formalin-fixed paraffinembedded (FFPE) tissue sample that arrives from a pathology lab. And some others, like Dr. Harris and his team, face another type of constraint — no room for errors due to limited sample accessibility. An ongoing project at NEXT involves studying and predicting inheritable cancer risk, with a focus on 30–60 cancer-related genes. Dr. Harris and his team perform targeted sequencing on the DNA obtained from cheek swabs of thousands of subjects. The sample pool, spread across individuals from multiple countries, is often difficult to re-obtain. “In a clinical context one of the biggest challenges when working with these types of samples is generating sufficient amounts of target DNA for analysis,” notes Dr. Harris.  

Abstract: Bladder cancer is one of the most financially burdensome cancers globally, from its diagnostic to its terminal stages. The impact it imposes on patients and the medical community is substantial, exacerbated by the absence of disease-specific characteristics and limited disease-free spans. Frequent recurrences, impacting nearly half of the diagnosed population, require frequent and invasive monitoring. Given the advancing comprehension of its etiology and attributes, bladder cancer is an appealing candidate for screening strategies. Cystoscopy is the current gold standard for bladder cancer detection, but it is invasive and has the potential for undesired complications and elevated costs. Although urine cytology is a supplementary tool in select instances, its efficacy is limited due to its restricted sensitivity, mainly when targeting low-grade tumors. Although most of these assays exhibit higher sensitivity than urine cytology, clinical guidelines do not currently incorporate them. Consequently, it is necessary to explore novel screening assays to identify distinctive alterations exclusive to bladder cancer. Thus, integrating potential molecular assays requires further investigation through more extensive validation studies. Within this article, we offer a comprehensive overview of the critical features of bladder cancer while conducting a thorough analysis of the FDA-approved assays designed to diagnose and monitor its recurrences.