United States

Background
The ability to reduce ice growth and inhibit ice recrystallization has become a key factor in cryopreserving biological specimens, such as cells and tissues, for cell-based therapeutics like vaccines. However, current cryopreservation protocols can result in loss of cell viability due to cellular damage related to the freezing and thawing process. The use of cryoprotective agents has been effective in increasing post-thaw recoveries and functionality in appropriate models. Nevertheless, many cryoprotectant compounds demonstrate toxicity to cells, preventing their clinical application. Betaine is an organic molecule which has the potential to function as a cryoprotectant due to its physiological role in the body as an osmolyte. Therefore, the goal of this preliminary study was to observe the ability of betaine to inhibit ice recrystallization and to assess its cryopreservation properties. 

Methods
Ice recrystallization was assessed through a splat cooling assay. The betaine sample was dissolved in phosphate buffered saline at concentrations of 0.5, 2, 10, 15, 18, 20, 25, 50 mM. 10 𝜇 L of dissolved betaine was dropped from a height of 2m onto a -80 °C aluminum block forming an ice wafer which was transferred to a Peltier unit at -6.4°C for 5 minutes. The wafer was placed under a microscope and the ice crystals were photographed. Mean ice crystal size was obtained and compared to the negative control; phosphate buffered saline. A dose response curve representing the ice recrystallization inhibition activity was obtained. 

The cytotoxicity of betaine was assessed through a resazurin assay with HEPG2 liver cells. Cytotoxicity of Betaine was assessed at 0.5, 2, 10, 15, 18, 20, 25, and 50 mM. 1%- Triton-X was used as a negative control. 

Results
Dose response curves obtained from splat cooling assays indicated that betaine could reduce ice crystal growth at concentrations above 200mg/mL. However, it was unable to completely inhibit ice recrystallization. The half maximal inhibitory concentration (IC50) calculated from the dore response curves was found to be between 119.9mg/mL and 226.4mg/mL, which is not comparable to cryo-additives used in current cryoprotective protocols. In vitro cytotoxicity studies performed indicated biocompatibility up to 10mg/mL.

Conclusion
Our quantitative assessment suggests that the cryoprotective properties of betaine are not sufficient for its use as a cryoprotectant alone. Future directions include assessing the post-thaw viability of betaine and its cryoprotective abilities when combined with another cryoprotectant-like compound. 


AMA Research Challenge 2023

A quantitative assessment of the cryopreservation properties of betaine

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ATPase Inhibitory Factor 1 (IF1) is a nuclear-encoded ATP synthase-interacting mitochondrial protein. IF1 is known to be upregulated during periods of elevated stress, such as during myocardial infarctions or cardiac hypertrophy. Previously conducted lab research has demonstrated that mice with the absence of the protein IF1 were protected against pathological cardiac hypertrophy. To further investigate the extent of such findings, an experiment was designed to investigate the role of the IF1 protein in cardiotoxic conditions induced by Doxorubicin (DOX), a widely used anti-cancer drug. DOX is known to induce cardiotoxicity through the generation of reactive oxygen species, increased levels of lipid peroxidation, and mitochondrial dysfunction. Doxorubicin-induced cardiomyopathy is associated with a low prognosis and can serve to be fatal in certain populations. We hypothesized that mice with the absence of IF1 will demonstrate more preserved cardiac functions in the presence of DOX. 

To test the hypothesis, DOX was intraperitoneally injected into two separate groups of mice: mice for which the IF1 protein was knocked out (IF1-KO) and wildtype mice normally expressing the IF1 protein (IF1-WT). The drug was dissolved in a solvent consisting of dimethyl sulfoxide (DMSO), PEG-300, Tween-80, and distilled water in pre-designated proportions. Injections were performed on three separate days with 6 mg/kg of DOX administered on each injection day. Injections were conducted on Day 0, Day 2, and Day 4. Control mice were injected simply with solvent. Subsequently, transthoracic echocardiograms were performed using a VEVO F2 system at one-week intervals for three weeks ie. Day 7, Day 14, and Day 21. Long-axis M-Mode images were obtained to analyze systolic parameters: ejection fraction, fractional shortening, left ventricular mass, as well as left ventricle anterior and posterior wall thickness. Body mass was also measured initially and after the three-week interval. Findings demonstrated that in the presence of DOX, ejection fraction and fractional shortening, the two parameters of cardiac contraction, significantly decreased in IF1-WT mice; whereas no significant decrease was seen in IF1-KO mice. Furthermore, left ventricular anterior wall thickness was found to be significantly reduced in both IF1-WT and IF1-KO mice, suggesting that DOX may lead to a decrease in heart size. These results demonstrate that the absence of IF1 may provide protection against the cardiotoxic effects of DOX, thus supporting the hypothesis. Further studies could investigate the role of increased doses of DOX on cardiac physiology as well as the effects of increased time intervals through the performance of transthoracic echocardiograms after five to seven weeks. Future experiments using other pathophysiological and biochemical methodologies will gain further insights into the mechanisms of the protective effects of eliminating IF1. Experimental findings may yield new insights into the development of interventions to mitigate adverse effects of this drug in cancer patients under chemotherapy treatments.


Analysis of the Role of ATPase Inhibitory Factor 1 (IF1) in Doxorubicin-Induced Cardiotoxicity

Background: The larynx is important in breathing, swallowing, and speech production. The closure and opening of the space between the vocal folds (“glottis”), allows the larynx to fulfill its functions. The larynx is a symmetric organ. To move all parts of the larynx appropriately and achieve glottal sufficiency, symmetry is beneficial. Previous studies have reported on various forms of shape asymmetry and their consequence for function. Asymmetry in how the larynx is positioned within the neck has not been investigated before but could play a critical role in the development of shape asymmetry and thereby influence larynx functions. 
Methods: We investigated a stratified sample of 210 human subjects (105 females, 105 males) for positional asymmetry. Positional asymmetry was defined as the angle between the mid-sagittal organ plane and the mid-sagittal body plane. The larger the angle between the two, the greater the positional asymmetry. Positional asymmetry was measured in all subjects. With the help of regression models, the relationships between positional asymmetry and body size, larynx organ size, and larynx shape were explored. 
Results: Positional asymmetry ranged between 0 and 18°. It was larger in males (4.71°±3.05; mean±stdev) than in females (3.49°±3.11) (t-test, T105,105 = -2.85; p < 0.01). Larynx midsagittal plane was deflected either to the left or the right side. In females, the distribution was almost even (51.43%) but it was biased towards the right side in males (62.86%). Positional asymmetry was associated with larynx overall shape (females: R2= 0.05, P= 0.017; males: R2= 0.09, P= 0.002), and shape asymmetry (female: R2 = 0.059; p< 0.01, male: R2= 0.21; p<0.001), with larynx size in females (females: R2= 0.042, P= 0.036; males: R2= 0.003, P= 0.609), with age in females (females: R2 = 0.467; p= 0.027; males: R2 = 0.0232; p= 0.12), but not with body height (males: R2= 0.0082; P= 0.357 ; females: R2= 0.0061; P= 0.43) or with BMI (Body Mass Index) (female: R2 = 0.018; p= 0.169, males: R2= 0; p= 0.97). 
Conclusions: Our data highlight the value and challenges of investigating human phenotypic variation. Positional asymmetry of the larynx was associated with the shape and size variation of the vocal organ as well as with a person’s age.

Assessing Positional Asymmetry of the Larynx

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**Abstract:**



The role of the triglyceride-glucose index (TyG) in obesity and its progression to Type 2 diabetes

Various growth factors have been shown to affect the proliferation, migration, and adhesion of keratinocytes in the face of wound healing. The expression of wound repair genes is suspected to be regulated by circadian rhythm. How wound repair functions over the course of a circadian rhythm has yet to be fully described. Moreover, much is unknown about the impact of bacteria on the circadian expression of wound healing genes. Here, we intend to use periodontal disease as a model to describe the effect of a septic environment on expression of wound healing genes by epithelial cells. Furthermore, we aim to demonstrate that stimulation of epithelial cells with pathogenic bacteria alters the circadian patterns of wound repair gene expression. In the experiment, OKF4 cells were seeded, and artificial wounds were created in each well. The cells were challenged with pathogenic oral bacteria, and cell lysates were collected at T=0, T=8, and T=22 hours. PCR quantified gene expression levels. Microscopically, increased cell spreading was observed after T=22 hours in the wounded cells. Impaired cell spreading was observed in the wells challenged with pathogenic bacteria. The expression of EGF and FGF-7 appeared to follow a circadian rhythm. The presence of pathogenic bacteria downregulated the expression of genes involved in keratinocyte migration. These findings support the hypothesis that genes involved in wound healing follow a circadian rhythm and are altered in the face of infection. Continued research into this topic can lead to the development of therapies targeted towards restoring the expression of genes that aid in wound repair complicated by a septic environment or overlying infection. Further elucidating the circadian pattern of expression of these genes can also result in optimization of the timing that therapies are administered to maximize their therapeutic effect thereby improving patient outcomes. 


Altered Wound Healing Response after Challenge with Pathogenic Bacterium

Background: Neuroinflammation has been implicated in the development and progression of numerous neurodegenerative disorders and studies suggest systemic inflammation may precede central inflammation. Chronic low-grade systemic inflammation is a hallmark of metabolic disorders like Type II diabetes mellitus (T2DM), which is a risk factor for cognitive dysfunction. In fact, researchers have proposed the term “Type III diabetes” for Alzheimer’s disease due to the shared molecular and cellular mechanisms among diabetes and cognitive decline. For both of these pathologies, diet has been recognized as a modifiable risk factor, yet the specific impact of diet on neuroinflammation remains poorly understood. Current literature suggests that a high intake of saturated fats and processed foods may promote neuroinflammation and worsen neurodegeneration, while a ketogenic diet has been linked to cognitive improvement in dementia via regulation of neuroinflammatory pathways. This ongoing work seeks to uncover how diet composition influences neuroinflammation, specifically how diet modulates the activity of the NLRP3-inflammasome.

Methods: To address this question, we utilized a novel biosensor mouse developed in our laboratory that allows us to measure caspase-1/NLRP3-inflammasome activity in vivo. Due to the strong association of dementia and T2DM, and the established abnormalities in blood glucose levels in T2DM, this study focuses on high glycemic index (HGD) and ketogenic (KD) diets. These diets will allow us to determine whether rapid spikes in blood sugar seen with the HGD increase NLRP3-inflammasome neuroinflammation, potentially contributing to neurodegeneration, and if the extremely low carbohydrate load in the KD has the opposite effect.

Results: Long term administration of a KD promotes glucose intolerance but does not increase neuroinflammation, which a HGD increases NLRP3-inflammasome-dependent neuroinflammation without altering glucose regulation in wild-type male mice.

Conclusion: We found that the ketogenic diet impairs glucose metabolism, but does not lead to increased neuroinflammation, while the HGD showed the opposite phenotype. Future experiments will assess inflammatory cytokines systemically and centrally, determine levels of pro-inflammatory transcripts and cleaved caspase-1 protein levels in the brain, and evaluate behavior deficits to probe at the mechanism behind these unexpected results.


The Impact of Diet on Neuroinflammation

Background
On March 11, 2020, the WHO declared the COVID-19 outbreak a pandemic. Multiple aspects of healthcare were interrupted, as individuals were instructed to stay home, protect other citizens, and avoid potential exposure. Individuals with HIV heavily rely on healthcare providers for essential medical treatment and critical testing. They are particularly susceptible to the upheaval in healthcare services and access. We undertook this study to evaluate the impact of the COVID-19 pandemic on diverse aspects of HIV patient care. 
Methods
Using the Cascades analysis template provided by the New York State Department of Health populated with our patient data, we compared the effects of the COVID-19 pandemic on established clinic patients on Antiretroviral Therapy (ARV) and whether they received viral load testing (VL) or kept viral load suppression (VS). Established clinic patients had two or more visits to our Infectious Disease clinic for HIV management before COVID-19. We also highlighted differences between viral load testing and suppression status within groups when comparing 2019 and 2021 against 2020. Data were stratified by demographics, including; age 20 to 60 years or older, race (Black, White, Asian), and Ethnicity (Hispanic or Non-Hispanic). The relationship between these outcomes was analyzed via the Chi-square test of independence and logistic regression. A p-value < 0.05 indicated statistical significance. 1,498 patients were seen in our Infectious disease clinic and on antiretroviral therapy during 2019-2021.
Results
A significantly higher percentage of patients did not receive testing in 2020 compared to 2019 & 2021 (OR 0.427, CI [0.237- 0.75], p-value = 0.0035, Chi-square p-value = 0.004). During 2019 & 2021, we observed a higher percentage of detectable patients; however, the year was unlikely to be related to decreased suppression rates (p-value= 0.79). Age is a significant predictor of the outcome for viral testing and suppression (F-value < 0.049, F-value < 0.0001) with an average of 55-56 years. Interestingly, we observed no significant difference in the odds or probability between patient race or ethnicity in obtaining VL testing or achieving suppression.
Conclusion
The observation during our analysis that stood out most prominently was the impact of the COVID-19 pandemic in 2020, likely influenced by nationwide medical facility closures and patient reluctance to leave their homes. Telemedicine appointments were a significant response to COVID-19 and were a tool to combat interruption of care. There is further emphasis on the advantages of home delivery of ARV provided by specialty pharmacies, especially under extreme circumstances. Home medication deliveries, telemedicine visits, and considering age-related factors in HIV-care planning during the earliest part of the pandemic may have prevented deterioration in monitoring these at-risk populations. These findings underscore the imperative of adaptive healthcare delivery methods to navigate the consequences of unforeseen medical emergencies effectively.

Disruptions in the HIV Continuum of Care During COVID-19: Lessons from a Clinic-Based Study

Objective: Since the COVID-19 pandemic began, the number of individuals suffering from mental health disorders has increased substantially without an equal increase in the number of mental health workforce members. This has created difficulty in creating safe discharge plans for hospitalized patients, discouraged patients from seeking necessary help when suffering and can deter patients from completing treatment due to scheduling and financial barriers. The goal of this project is to evaluate availability and cost of outpatient psychiatric and psychological services in the Phoenix, Arizona area. 

Methods: Psychiatry and psychology practices were identified via an online search in the Phoenix, AZ area and contacted by phone or email to enquire about the services they provide. Ten psychiatric and ten psychological practices were contacted. Information was obtained including type of services (psychiatry, psychology, neuropsychiatric testing), type of provider (MD/DO, NP, PA, PhD/PsyD, master’s level counselor, social worker), soonest available appointment and cost of appointment (cash pay vs accepted insurance). Wait times to reach a representative were also recorded. 

Results: For psychiatric services, the average wait time to speak to a representative over the phone was 5 minutes. The average soonest available appointment was 12 days ranging from same day to 5 weeks from date of call. The most common provider available for the soonest available appointment was a physician psychiatrist or NP. Neuropsychiatric testing was offered at 3/10 practices. Insurance was accepted at 8/10 contacted locations, with cash pay costs ranging from $225-450 for intake and $100-300 for each follow up appointment. For psychological services, the average wait time to speak to a representative was 48.5 minutes. The average soonest available appointment was 18 days from date of call ranging from same day to 2 months. The most common provider available for the soonest available appointment was a Psychologist (PsyD/PhD) or master’s level counselor. Neuropsychiatric testing was offered at 4/10 practices. Four practices accepted insurance, all practices accepted cash pay with costs ranging from $75-350 for an intake appointment and $75-265 for each follow up appointment.


Conclusion: In the Phoenix area, access to psychological services including talk therapy and neuropsychiatric testing is more difficult to obtain than psychiatric services. Barriers to these services include longer wait times to speak to representatives for scheduling and information, which can discourage patients from making appointments, longer wait times to the soonest available appointment which can be detrimental to those who are struggling and need help quickly and lack of services covered by insurance. A comprehensive mental health treatment plan often involves both therapy and medication management. This information suggests a focus of increasing both the volume of psychological and psychiatric providers and providers willing to accept insurance to meet the collaborative needs of appropriate mental health care in this location.

Accessibility of Outpatient Mental Health Resources in the GreaterPhoenix, Arizona Area

Background: 
Metabolic syndrome (MetS) is characterized by interrelated metabolic risk factors that increase the risk of cardiovascular disease. With a global prevalence of about 25% in the adult population, prevalence differs across different racial/ethnic populations and is higher in non-Hispanic White (NHW) than non-Hispanic Black (NHB) Americans. Epidemiologic studies have indicated that poor diet quality is associated with fluctuations in blood glucose and dyslipidemia, two essential components of MetS. The study examined the associations between metabolic syndrome (MetS), self-rated dietary quality (SRDQ), and self-rated health (SRH) in non-Hispanic White (NHW) and non-Hispanic Black (NHB) American adults. 


Methods: 
Data (n=3008) from the 2017-2018 US National Health and Nutritional Health Survey were used for this study. Race/ethnic-specific prevalence odds ratio (POR) from multivariable logistic regression analysis was used to evaluate the cross-sectional associations between MetS, SRPDQ, and SRPH. Statistical adjustments were made for age, sex, education, annual household income, alcohol use, smoking, and lack of exercise. 


Results: 
Approximately 39% and 31.2% of NHW study participants with MetS rated their dietary quality and health status as poor compared to 44.7% and 40.8% of NHB with MetS, respectively. SRH was positively correlated with SRDQ in NHW (r=0.281, p <.001) and NHB (r=0.495, p<.001) study participants with MetS. SRPDQ was associated with increased odds of MetS [POR=1.78;95% CI:1.16-2.66] and SRPH [POR=3.73;95% CI: 2.87-4.84] in NHW individuals, and with MetS [POR=1.45;95% CI:1.07-2.83] and SRPH [POR=4.35;95% CI:3.17-5.98] in NHB individuals, controlling for covariates. MetS was associated with increased odds of SRPH in NHW [POR=2.28;95% CI:1.04-3.68] and NHB [POR=2.56;95% CI:1.28-5.12] individuals. SRPDQ was found to mediate the association between MetS and SRPH and increased the association by 25.9% and 46.1% in NHW and NHB individuals, respectively. 


Conclusions: 
This study shows a positive association between SRPDQ and MetS, and that MetS is associated with SRPH in NHW and NHB persons. Longitudinal studies are needed to investigate whether MetS accelerates declines in overall health in NHW and NHB adults and inform clinical and public health interventions to promote optimal dietary quality. Race/ethnic-specific dietary intervention may help prevent and control MetS and its subsequent sequelae, including poor health outcomes.

Self-rated Dietary Quality, Metabolic Syndrome, and Self-rated Health in non-Hispanic ​Black and non-Hispanic White Americans.

Purpose: To explore the effects of recalibration on a cardiac surgery risk of mortality (STS-ROM) model in a majority-minority population (25% white). 

Methods: 3,721 patients from 2009-20 were identified in a cardiac surgery registry at a tertiary care center in Hawai'i. Recalibration was performed by training a new model built on only this local data, with in-hospital mortality as a dependent variable, and the following three independent variable strategies: (1) only STS-ROM score (add a flat value), (2) the same independent variables as the original STS-ROM (same model, new coefficients), and (3) entirely new variable selection (local-only model). In keeping with STS methodology, logistic regression models were created with a 70:30 test:train split and optimism-adjusted LASSO variable selection when warranted. Models were evaluated via observed/expected (O/E) ratios and area under the curve (AUC). Clinical impact was evaluated in the test set by comparing classifications of the new models compared to the original STS-ROM risk classification (Low/Medium/High). During this timeframe, STS-ROM was a primary determinant of receiving surgical aortic valve replacement vs transthoracic aortic valve replacement. Thus we also calculated the percentage of aortic valve patients who would have qualified for a different procedure had we used the local model. 

Results: The STS-ROM model underestimated risk overall (O/E: 1.14). Adding a flat value (model 1) performed marginally better (O/E: 1.07) but building a new model (2) using local data and STS-ROM variables performed the best (O/E: 0.99). Model 3, which used only local data, significantly overestimated risk (O/E: 0.82). All models aside from Model 3 had similar AUC (0.82-0.83). If the best model (2) was used instead of the STS-ROM, 3.72% of all patients and 4.1% of non-white patients would have moved up in risk category. Of those already at intermediate risk, 15.6% would have been elevated to high risk. In the aortic valvular population, where STS-ROM was an especially important variable in clinical decision making, 16.7% of surgical replacements may have qualified for less-invasive procedures instead. 

Conclusion: Recalibrating national risk models using local data from our majority-minority population was superior in all models, but entirely new models without national input studies performed worse. Given that these risk models are major factors in surgical decision making, recalibration can have a significant impact on patient care. 


Surgical Risk Models in a Majority-Minority Population: The Clinical Effect of Local Recalibration

Background: The long-term sequelae of undiagnosed and untreated obstructive sleep apnea (OSA) are well-established. Current research supports the existence of racial disparities in prevalence, risk factors, and treatment utilization in OSA. However, most studies focus on adult patients or sample from limited cohorts. The aim of this study is to investigate possible racial disparities in the diagnosis and treatment of OSA in patients aged 0-20 years.
Methods: The TriNetX national database was utilized to identify patients under the age of 22 who were diagnosed with OSA in this retrospective cohort study. The incidence of surgical treatment (tonsillectomy, adenoidectomy, or adenotonsillectomy) and Positive Airway Pressure (PAP) treatment, as well as polysomnography (PSG) rates were examined across multiple races with sub-analyses performed on demographics including age, ethnicity, sex, BMI class, and comorbidities.
Results: We identified 143,111 patients with a diagnosis of OSA using ICD-10 codes across 71 healthcare organizations. 96,181 patients were Caucasian, 41,463 were African American (AA), 4,002 were Asian, 1,127 were American Indians or Alaska Natives (AI/AN), and 338 were Pacific Islanders/Hawaiian Natives (PI/HN). Rates of surgical treatment differed by race (p<0.001), with AA (37.4%), Caucasian (39.5%), AI/AN (37.5%), and PI/HN (34.9%) compared to Asian patients (27.2%). AA (26.3%) and Caucasian (26.4%) children were more likely to obtain a PSG compared to AI/AN (23.8%), Asian (20.2%), and PI/HN (16.9%), p<0.001. In addition, more Caucasian patients (36%) received surgical treatment before 12 years of age compared to AA (34.1%), Asian (24.6%), AI/AN (32.7%), and PI/HN (31.4%) groups (p<0.001) despite AA patients receiving PSG in young children. The adjusted odds of CPAP use were 1.6 times greater for AI/AN vs. Caucasian (95%CI 1.06-2.4) and 1.2 times greater for AA vs. Caucasian (95%CI 1.1-1.3). Significant differences remained after controlling for demographics, BMI, and comorbidities.
Conclusion: There are differential rates of PSG and OSA treatment utilization when analyzed by race in this national sample of pediatric OSA patients. Given the importance of diagnosing and treating OSA early to prevent undesirable outcomes, more studies should be done to confirm these differences and investigate the factors contributing to them.

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A quantitative assessment of the cryopreservation properties of betaine

Next from AMA Research Challenge 2023

Analysis of the Role of ATPase Inhibitory Factor 1 (IF1) in Doxorubicin-Induced Cardiotoxicity