Comparative analysis of high-throughput RNA extraction kits in Naïve Non-Human Primate (NHP) tissues for downstream applications utilizing Xeno Internal Positive Control (IPC).

Ribonucleic acid (RNA) extraction and purification play pivotal roles in molecular biology and cell and gene therapy, where the quality and integrity of RNA are critical for downstream applications. Automated high-throughput systems have gained interest due to their potential for scalability and reduced labor requirements compared to manual methods. However, ensuring high-throughput capabilities, reproducibility, and reliability while maintaining RNA yield and purity remains challenging. This study evaluated and compared the performance of four commercially available high-throughput magnetic bead-based RNA extraction kits across six types of naïve non-human primate (NHP) tissue matrices: brain, heart, kidney, liver, lung, and spleen. The assessment focused on RNA purity, yield, and extraction efficiency (EE) using Xeno Internal Positive Control (IPC) spiking. Samples (∼50 mg) were homogenized via bead-beating and processed according to the manufacturer's protocol on the KingFisher Flex platform in eight replicates. RNA purity and yield were measured using a NanoDrop® spectrophotometer, while EE was evaluated via real-time reverse transcription-quantitative polymerase chain reaction (RT-qPCR). The findings indicate consistent high RNA purity across all tested extraction kits, yet substantial variation in RNA yield. Extraction efficiency exhibited variations across tissue types, with decreasing trends observed from brain to lung tissues. These results underscore the importance of careful kit selection and method optimization for achieving reliable downstream applications. The MagMAX™ mirVana™ Total RNA Isolation Kit stands out as the most accurate and reproducible, making it the preferred choice for applications requiring high RNA quality and consistency. Other kits, such as the Maxwell® HT simplyRNA Kit, offer a good balance between cost and performance, though with some trade-offs in precision. These findings highlight the importance of selecting the appropriate RNA isolation method based on the specific needs of the research, underscoring the critical role of accurate nucleic acid extraction in gene and cell therapy research. In conclusion, this study highlights the critical factors influencing RNA extraction performance, emphasizing the need for researchers and practitioners to consider both kit performance and tissue characteristics when designing experimental protocols. These insights contribute to the ongoing efforts to enhance the reproducibility and reliability of RNA extraction methods in molecular biology and cell/gene therapy applications.

CRISPR PERSIST-On enables heritable and fine-tunable human gene activation.

Current technologies for upregulation of endogenous genes use targeted artificial transcriptional activators but stable gene activation requires persistent expression of these synthetic factors. Although general "hit-and-run" strategies exist for inducing long-term silencing of endogenous genes using targeted artificial transcriptional repressors, to our knowledge no equivalent approach for gene activation has been described to date. Here we show stable gene activation can be achieved by harnessing endogenous transcription factors ( EndoTF s) that are normally expressed in human cells. Specifically, EndoTFs can be recruited to activate endogenous human genes of interest by using CRISPR-based gene editing to introduce EndoTF DNA binding motifs into a target gene promoter. This Precision Editing of Regulatory Sequences to Induce Stable Transcription-On ( PERSIST-On ) approach results in stable long-term gene activation, which we show is durable for at least five months. Using a high-throughput CRISPR prime editing pooled screening method, we also show that the magnitude of gene activation can be finely tuned either by using binding sites for different EndoTF or by introducing specific mutations within such sites. Our results delineate a generalizable framework for using PERSIST-On to induce heritable and fine-tunable gene activation in a hit-and-run fashion, thereby enabling a wide range of research and therapeutic applications that require long-term upregulation of a target gene.

Hospital characteristics and perioperative complications of Hispanic patients following reverse shoulder arthroplasty-a large database study.

BACKGROUND: Hispanic patients are the youngest and fastest-growing ethnic group in the USA. Many of these patients are increasingly met with orthopedic issues, often electing to undergo corrective procedures such as reverse shoulder arthroplasty (RSA). This patient population has unique medical needs and has been reported to have higher incidences of perioperative complications following major procedures. Unfortunately, there is a lack of information on the hospitalization data and perioperative complications in Hispanic patients following procedures such as RSA. This project aimed to query the Nationwide Inpatient Sample (NIS) database to assess patient hospitalization information, demographics, and the prevalence of perioperative complications among Hispanic patients who received RSA. METHODS: Information from 2016-2019 was queried from the NIS database. Demographic information, incidences of perioperative complications, length of stay, and costs of care among Hispanic patients undergoing RSA were compared to non-Hispanic patients undergoing RSA. A subsequent propensity matching was conducted to consider preoperative comorbidities. RESULTS: The query of NIS identified 59,916 patients who underwent RSA. Of this sample, 2,656 patients (4.4%) were identified to be Hispanic, while the remaining 57,260 patients (95.6%) were found to belong to other races (control). After propensity matching, Hispanic patients had a significantly longer LOS (median = 1.4 days) than the patients in the control group (median = 1.0, P < 0.001). The Hispanic patients (89,168.5 USD) had a significantly higher cost of care than those in the control group (67,396.1 USD, P < 0.001). In looking at postoperative complications, Hispanic patients had increased incidences of acute renal failure (Hispanics: 3.1%, control group: 1.1%, P = 0.03) and blood loss anemia (Hispanics: 12.7%, control group: 10.9%, P = 0.03). CONCLUSIONS: Hispanic patients had significantly longer lengths of stay, higher costs of care, and higher rates of perioperative complications compared to the control group. For patients who are Hispanic and undergoing RSA, this information will aid doctors in making comprehensive decisions regarding patient care and resource allocation.

Perioperative complications of legally blind patients undergoing total hip arthroplasty - A national in-patient sample database study.

Background: Legally blind patients are more prone to injury-related falls, which may lead to hip fractures, often necessitating total hip arthroplasty (THA), as a corrective procedure. Many of these patients have unique medical needs and have increased rates of perioperative complications following surgical procedures. However, there is limited information on the hospitalization data and perioperative complications in this population following guidelines such as THA. The purpose of this study was to evaluate the patient characteristics, demographics, and prevalence of perioperative issues among THA patients who were legally blind. Methods: Using 2016-2019 data from the Nationwide Inpatient Sample (NIS), the incidence of perioperative complications, length of stay (LOS), and the cost of care (COC) among patients undergoing THA who were categorized as legally blind patients, compared to those who were not were analyzed. Propensity matching was conducted to consider associated factors that may influence perioperative complications. Results: From 2016 to 2019, 367,856 patients underwent THA, according to the NIS. Of those, 322 (0.1%) patients were categorized as legally blind, and the remaining 367,534 (99.9%) patients were not identified as legally blind (control). Legally blind patients were significantly younger than the control group (65.4 years vs. 66.7 years, p < 0.001). After propensity matching, legally blind patients had longer LOS (3.9 days vs. 2.8, p = 0.04), increased discharges to another facility (45.9% vs. 29.3%, p < 0.001), and fewer discharges to home (21.4% vs. 32.2%, p = 0.02) than control patients. Conclusions: The legally blind group had significantly longer LOS, higher rates of discharge to another facility, and lower rates of discharge to home compared to the control group. This data will help providers make informed decisions about patient care and resource allocation for legally blind patients undergoing THA.

The effect of cocaine on patients undergoing total hip arthroplasty.

Background: Cocaine use has surged in the past decade, with 4.8 million Americans (1.7% of the population greater than 12) reporting use in 2021, leading to a healthcare burden of 1.3 billion dollars. Cocaine users experience prolonged hospital stays, higher costs, worse surgical outcomes, increased risk of medical conditions, and inflammation-related osteoarthritis. The study aims to identify factors influencing length of stay, costs, and perioperative complications in cocaine users undergoing total hip arthroplasty (THA) to reduce these risks. Methods: This study utilized the NIS database, providing comprehensive information on patient demographics, length of stay, hospital costs, and complications. Statistical analyses were conducted using SPSS software, including propensity matching and significance testing, to compare outcomes between cocaine users (CU) and non-cocaine users (NCU) undergoing total hip arthroplasty. Results: After propensity matching, cocaine users had a significantly longer LOS (4.8 days) in comparison to non-cocaine users (2.6 days) (p < 0.001). Similarly, the CU group had a larger of care (87984.9) than the NCU group (69149.2) (p < 0.001). Cocaine users had significantly higher rates of blood loss anemia (OR: 3.24, 95% CI: 2.21, 4.73), blood loss anemia (OR: 1.59, 95% CI: 1.12, 2.24), blood transfusion (OR: 2.23, 95% CI: 1.04, 4.78), periprosthetic dislocation (OR: 6.57, 95% CI: 1.47, 29.32), and periprosthetic infection (OR: 4.59, 95% CI: 1.54, 13.68) than patients in the non-cocaine user's group. Conclusion: Cocaine users had a significantly longer length of stay, higher costs of care, and an increased number of post-operative complications compared to non-cocaine users. These data contribute to understanding the potential ramifications of cocaine users undergoing THA.

The synthetic food dye, Red 40, causes DNA damage, causes colonic inflammation, and impacts the microbiome in mice.

The incidence of colorectal cancer (CRC) among young people has been on the rise for the past four decades and its underlying causes are only just starting to be uncovered. Recent studies suggest that consuming ultra-processed foods and pro-inflammatory diets may be contributing factors. The increase in the use of synthetic food colors in such foods over the past 40 years, including the common synthetic food dye Allura Red AC (Red 40), coincides with the rise of early-onset colorectal cancer (EOCRC). As these ultra-processed foods are particularly appealing to children, there is a growing concern about the impact of synthetic food dyes on the development of CRC. Our study aimed to investigate the effects of Red 40 on DNA damage, the microbiome, and colonic inflammation. Despite a lack of prior research, high levels of human exposure to pro-inflammatory foods containing Red 40 highlight the urgency of exploring this issue. Our results show that Red 40 damages DNA both in vitro and in vivo and that consumption of Red 40 in the presence of a high-fat diet for 10 months leads to dysbiosis and low-grade colonic inflammation in mice. This evidence supports the hypothesis that Red 40 is a dangerous compound that dysregulates key players involved in the development of EOCRC.

Nontuberculous Mycobacterial Pulmonary Disease: A Clinical and Radiologic Update.

Nontuberculous mycobacterial pulmonary disease (NTM-PD) is increasingly recognized in the U.S. and worldwide as a debilitating disease that is challenging to diagnose and manage. Beyond the principle task of recognizing the protean imaging manifestations of NTM-PD, radiologists will need to appropriately communicate with pulmonology and infectious disease colleagues in multidisciplinary management discussions. This update on nontuberculous mycobacteria (NTM) species, their clinical significance, and imaging features aims to support these roles. Terminology mirrors that laid out by the American Thoracic Society (ATS) and Infectious Disease Society of America (IDSA) published guidelines on NTM-PD.1.

Exploring beyond the limit: How comparative stochastic performance affects retesting outcomes in six commercial SARS CoV-2 nucleic acid amplification tests.

Objectives: To examine the comparative stochasticity profile of six commercial SARS-CoV-2 nucleic acid amplification tests (NAATs) and how this may affect retesting paradigms. Methods: Commercial quality control (QC) material was serially diluted in viral transport media to create a panel covering 10-10,000 copies/ml. The panel was tested across six commercial NAATs. A subset of high cycle threshold results was retested on a rapid PCR assay to simulate retesting protocols commonly used to discriminate false positives. Results: Performance beyond the LOD differed among assays, with three types of stochasticity profiles observed. The ability of the rapid PCR assay to reproduce a true weak positive specimen was restricted to its own stochastic performance at the corresponding viral concentration. Conclusion: Stochastic performance of various NAATs overlap across low viral concentrations and affect retesting outcomes. Relying on retesting alone to discriminate false positives risk missing true positives even when a more sensitive assay is deployed for confirmatory testing.

Hepatobiliary Injuries: A Pictoral Essay and Literature Review.

Injuries to the liver and biliary tree carry significant morbidity and mortality if not diagnosed and managed rapidly. As clinical evaluation of suspected hepatobiliary injury is often limited in traumatic or post-surgical settings, imaging plays a critical role in the diagnosis of injury, assessment of treatment response and detection of delayed complications. In this paper, we review acute traumatic and iatrogenic hepatobiliary injuries and subsequent complications through different imaging modalities.

Prevention of stroke in people living with HIV.

In the era of effective antiretroviral therapy (ART), HIV has become a manageable disease marked by an elevated risk of non-AIDS-related comorbidities, including stroke. Rates of stroke are higher in people living with HIV (PLWH) compared with the general population. Elevated stroke risk may be attributable to traditional risk factors, HIV-associated chronic inflammation and immune dysregulation, and possible adverse effects of long-standing ART use. Tailoring stroke prevention strategies for PLWH requires knowledge of how stroke pathogenesis may differ from non-HIV-associated stroke, knowledge of long-term stroke outcomes in HIV, and accurate stroke risk assessment tools. As a result, the approach to primary and secondary stroke prevention in PLWH relies heavily on guidelines developed for the general population, with an emphasis on optimization of traditional vascular risk factors and early initiation of ART. This review summarizes existing evidence on HIV-associated stroke mechanisms and considerations for stroke prevention for PLWH.

Intracranial CNS Infections: A Literature Review and Radiology Case Studies.

Acute infections of the intracranial central nervous system (CNS) often present as neurological emergencies, where missed or delayed diagnosis and treatment can be catastrophic to the patient. Accurate and timely identification of the underlying etiologies, which are critical in directing life-saving therapies, can be achieved through neuroimaging. This article will provide a thorough review of radiologic findings in common infections of the brain, from primarily compartmentalized infections of multimicrobial etiologies, to CNS manifestations of specific immunocompromised-selective pathogens, of herpes simplex virus, and of tuberculosis. We also briefly discuss the epidemiology, etiology, clinical features, treatment guidelines, complications, and long-term sequelae of these infections.

Incidence and predictors of positive intraoperative cultures in primary shoulder arthroplasty following prior ipsilateral shoulder surgery.

BACKGROUND: To our knowledge, the rate of positive intraoperative cultures in patients undergoing primary shoulder arthroplasty with prior ipsilateral nonarthroplasty shoulder surgery is unknown. The aim of this study was to determine the incidence and predictors of positive cultures in these patients. METHODS: We performed a retrospective review of patients with prior ipsilateral shoulder surgery with intraoperative cultures taken at the time of primary shoulder arthroplasty. We evaluated culture results, demographics, and number of prior surgeries. Regression analysis was used to determine patient-related risk factors that predict positive cultures. RESULTS: A total of 682 patients underwent primary shoulder arthroplasty, 83 had at least 1 prior ipsilateral shoulder surgery: 65.1% male, mean age 64.2 ± 10.9 years. For the cohort of 83 patients, an average of 3.2 ± 1.2 tissue samples were obtained for each patient, with a mean of 0.84 ± 1.14 tissue cultures being positive (range 0-5). Thirty-seven of the 83 patients (44.5%) had at least 1 positive culture, with Cutibacterium acnes the most frequent organism (31/37; 83.4%). An average of 1.9 ± 0.96 tissue cultures resulted positive (range 1-5) for the 37 patients who had positive cultures, 40.5% (15/37) had only 1 positive tissue culture (12/15 C acnes, 2/15 Staphylococcus epidermidis, and 1/15 vancomycin-resistant enterococcus). Male sex and history of prior shoulder infection were predictive of culture positivity (odds ratios: 2.5 and 20.9, respectively). Age, race, medical comorbidities, number of prior shoulder surgeries, and time from index shoulder surgery were not predictive of culture positivity. CONCLUSION: About 45% of patients with no clinical signs of infection and a history of prior ipsilateral shoulder surgery undergoing primary shoulder arthroplasty grew positive intraoperative cultures. The significance of these findings remains unclear with regard to risk of periprosthetic infection and how these patients should be managed.

Panaxynol, a bioactive component of American ginseng, targets macrophages and suppresses colitis in mice.

Ulcerative colitis has a significant impact on the quality of life for the patients, and can substantially increase the risk of colon cancer in patients suffering long-term. Conventional treatments provide only modest relief paired with a high risk of side effects, while complementary and alternative medicines can offer safe and effective options. Over the past decade, we have shown that both American ginseng and its hexane fraction (HAG) have anti-oxidant and anti-inflammatory properties that can suppress mouse colitis and prevent colitis-associated colon cancer. With the goal of isolating a single active compound, we further fractionated HAG, and found the most abundant molecule in this fraction was the polyacetylene, panaxynol (PA). After isolating and characterizing PA, we tested the efficacy of PA in the treatment and prevention of colitis in mice and studied the mechanism of action. We demonstrate here that PA effectively treats colitis in a Dextran Sulfate Sodium mouse model by targeting macrophages for DNA damage and apoptosis. This study provides additional mechanistic evidence that American ginseng can be used for conventional treatment of colitis and other diseases associated with macrophage dysfunction.

Sex-based differences in myocardial gene expression in recently deceased organ donors with no prior cardiovascular disease.

Sex differences in the development of the normal heart and the prevalence of cardiomyopathies have been reported. The molecular basis of these differences remains unclear. Sex differences in the human heart might be related to patterns of gene expression. Recent studies have shown that sex specific differences in gene expression in tissues including the brain, kidney, skeletal muscle, and liver. Similar data is limited for the heart. Herein we address this issue by analyzing donor and post-mortem adult human heart samples originating from 46 control individuals to study whole-genome gene expression in the human left ventricle. Using data from the genotype tissue expression (GTEx) project, we compared the transcriptome expression profiles of male and female hearts. We found that genes located on sex chromosomes were the most abundant ones among the sexually dimorphic genes. The majority of differentially expressed autosomal genes were those involved in the regulation of inflammation, which has been found to be an important contributor to left ventricular remodeling. Specifically, genes on autosomal chromosomes encoding chemokines with inflammatory functions (e.g. CCL4, CX3CL1, TNFAIP3) and a gene that regulates adhesion of immune cells to the endothelium (e.g., VCAM1) were identified with sex-specific expression levels. This study underlines the relevance of sex as an important modifier of cardiac gene expression. These results have important implications in the understanding of the differences in the physiology of the male and female heart transcriptome and how they may lead to different sex specific difference in human cardiac health and its control.

Assessment of the Radiation Effects of Cardiac CT Angiography Using Protein and Genetic Biomarkers.

OBJECTIVES: The purpose of this study was to evaluate whether radiation exposure from cardiac computed tomographic angiography (CTA) is associated with deoxyribonucleic acid (DNA) damage and whether damage leads to programmed cell death and activation of genes involved in apoptosis and DNA repair. BACKGROUND: Exposure to radiation from medical imaging has become a public health concern, but whether it causes significant cell damage remains unclear. METHODS: We conducted a prospective cohort study in 67 patients undergoing cardiac CTA between January 2012 and December 2013 in 2 U.S. medical centers. Median blood radiation exposure was estimated using phantom dosimetry. Biomarkers of DNA damage and apoptosis were measured by flow cytometry, whole genome sequencing, and single cell polymerase chain reaction. RESULTS: The median dose length product was 1,535.3 mGy·cm (969.7 to 2,674.0 mGy·cm). The median radiation dose to the blood was 29.8 mSv (18.8 to 48.8 mSv). Median DNA damage increased 3.39% (1.29% to 8.04%, p < 0.0001) and median apoptosis increased 3.1-fold (interquartile range [IQR]: 1.4- to 5.1-fold, p < 0.0001) post-radiation. Whole genome sequencing revealed changes in the expression of 39 transcription factors involved in the regulation of apoptosis, cell cycle, and DNA repair. Genes involved in mediating apoptosis and DNA repair were significantly changed post-radiation, including DDB2 (1.9-fold [IQR: 1.5- to 3.0-fold], p < 0.001), XRCC4 (3.0-fold [IQR: 1.1- to 5.4-fold], p = 0.005), and BAX (1.6-fold [IQR: 0.9- to 2.6-fold], p < 0.001). Exposure to radiation was associated with DNA damage (odds ratio [OR]: 1.8 [1.2 to 2.6], p = 0.003). DNA damage was associated with apoptosis (OR: 1.9 [1.2 to 5.1], p < 0.0001) and gene activation (OR: 2.8 [1.2 to 6.2], p = 0.002). CONCLUSIONS: Patients exposed to >7.5 mSv of radiation from cardiac CTA had evidence of DNA damage, which was associated with programmed cell death and activation of genes involved in apoptosis and DNA repair.

Diagnostic yield of electroencephalography in a general inpatient population.

OBJECTIVE: To determine the frequency and clinical predictors of seizures and markers of epileptiform activity in a noncritically ill general inpatient population. PATIENTS AND METHODS: We performed a retrospective cohort study of patients 18 years and older who underwent inpatient electroencephalography (EEG) between January 1, 2005, and December 31, 2010, for an indication of spells or altered mental status. The EEGs and reports were reviewed for ictal activity, interictal epileptiform abnormalities, and nonepileptiform abnormalities. Demographic and clinical data were gathered from the electronic medical record to determine seizure predictors. RESULTS: Of 2235 patients screened, 1048 met the inclusion criteria, of which 825 (78.7%) had an abnormal EEG finding. Seizures occurred in 78 of 1048 patients (7.4%), and interictal epileptiform discharges were noted in 194 of 1048 patients (18.5%). An intracranial mass and spells as the indication for the EEG were independently associated with the group of patients experiencing seizures in a multivariate logistic regression model (adjusted for age, sex, EEG indication, intracranial mass, stroke, and history of epilepsy). Ninety-seven percent of patients (69 of 71) experienced their first seizure within 24 hours of monitoring, and the presence of seizures was associated with a lower likelihood of being discharged (odds ratio, 0.45; 95% CI, 0.27-0.76). CONCLUSION: Seizures occurred at a high frequency in hospitalized patients with spells and altered mental status. The EEG may be an underused investigative tool in the hospital with the potential to identify treatable causes of these common disorders.

Diagnostic yield of electroencephalography in a general inpatient population.

OBJECTIVE: To determine the frequency and clinical predictors of seizures and markers of epileptiform activity in a non-critically ill general inpatient population. PATIENTS AND METHODS: We performed a retrospective cohort study of patients 18 years and older who underwent inpatient electroencephalography (EEG) between January 1, 2005, and December 31, 2010, for an indication of spells or altered mental status. The EEGs and reports were reviewed for ictal activity, interictal epileptiform abnormalities, and nonepileptiform abnormalities. Demographic and clinical data were gathered from the electronic medical record to determine seizure predictors. RESULTS: Of 2235 patients screened, 1048 met the inclusion criteria, of which 825 (78.7%) had an abnormal EEG finding. Seizures occurred in 78 of 1048 patients (7.4%), and interictal epileptiform discharges were noted in 194 of 1048 patients (18.5%). An intracranial mass and spells as the indication for the EEG were independently associated with the group of patients experiencing seizures in a multivariate logistic regression model (adjusted for age, sex, EEG indication, intracranial mass, stroke, and history of epilepsy). Ninety-seven percent of patients (69 of 71) experienced their first seizure within 24 hours of monitoring, and the presence of seizures was associated with a lower likelihood of being discharged (odds ratio, 0.45; 95% CI, 0.27-0.76). CONCLUSION: Seizures occurred at a high frequency in hospitalized patients with spells and altered mental status. The EEG may be an underused investigative tool in the hospital with the potential to identify treatable causes of these common disorders.

Neurotransmitter signaling in postnatal neurogenesis: The first leg.

Like the liver or other peripheral organs, two regions of the adult brain possess the ability of self-renewal through a process called neurogenesis. This raises tremendous hope for repairing the damaged brain, and it has stimulated research on identifying signals controlling neurogenesis. Neurogenesis involves several stages from fate determination to synaptic integration via proliferation, migration, and maturation. While fate determination primarily depends on a genetic signature, other stages are controlled by the interplay between genes and microenvironmental signals. Here, we propose that neurotransmitters are master regulators of the different stages of neurogenesis. In favor of this idea, a description of selective neurotransmitter signaling and their functions in the largest neurogenic zone, the subventricular zone (SVZ), is provided. In particular, we emphasize the interactions between neuroblasts and astrocyte-like cells that release gamma-aminobutyric acid (GABA) and glutamate, respectively. However, we also raise several limitations to our knowledge on neurotransmitters in neurogenesis. The function of neurotransmitters in vivo remains largely unexplored. Neurotransmitter signaling has been viewed as uniform, which dramatically contrasts with the cellular and molecular mosaic nature of the SVZ. How neurotransmitters are integrated with other well-conserved molecules, such as sonic hedgehog, is poorly understood. In an effort to reconcile these differences, we discuss how specificity of neurotransmitter functions can be provided through their multitude of receptors and intracellular pathways in different cell types and their possible interactions with sonic hedgehog.