The efficacy of antimicrobial solutions against multispecies bacterial biofilm with or without negative pressure wound therapy in an in vitro wound model.

OBJECTIVES: Biofilm is the major challenge in chronic wound management. Instilling a wound cleansing solution aids in wound bed cleaning and infectious pathogen elimination. Negative pressure wound therapy (NPWT) improves the wound-healing process. This study investigated the efficacy of two antimicrobials (Vashe Wound Cleanser and Prontosan Wound Irrigation Solution) against a multispecies bacterial biofilm with or without NPWT in an in vitro wound model. METHODS: A mixed multispecies biofilm containing Staphylococcus aureus, Pseudomonas aeruginosa, Streptococcus pyogenes, and Acinetobacter baumannii was developed and verified by scanning electron microscopy and fluorescent in situ hybridization. The efficacy of Vashe and Prontosan against multispecies biofilm with or without NPWT was evaluated by colony-forming unit (cfu) of each species and total bacterial number, and visually confirmed by live/dead stain and confocal microscopy. RESULTS: Prontosan reduced biofilm cell numbers significantly: 6 instils over 24 h resulting in 3.86 ±â€Š0.14 cfu log10 reduction without NPWT and 4.75 ±â€Š0.13 cfu log10 reduction combined with NPWT (P < 0.01) and 12 instils over 48 h resulting in 5.24 ±â€Š0.11 cfu log10 reduction without NPWT and biofilm eradication with NPWT (P < 0.001). NPWT alone or combined with Vashe failed to reduce multispecies biofilm numbers significantly over 24 or 48 h. CONCLUSIONS: Prontosan significantly reduced biofilm cell numbers, with better efficacy over 48 than 24 h, emphasizing the necessity for persistent and robust treatment. NPWT enhanced the effectiveness of Prontosan instillation. However, NPWT alone or combined with Vashe showed limited efficacy and difficulty when combating the multispecies biofilm in vitro.

Emerin deficiency drives MCF7 cells to an invasive phenotype.

During metastasis, cancer cells traverse the vasculature by squeezing through very small gaps in the endothelium. Thus, nuclei in metastatic cancer cells must become more malleable to move through these gaps. Our lab showed invasive breast cancer cells have 50% less emerin protein resulting in smaller, misshapen nuclei, and higher metastasis rates than non-cancerous controls. Thus, emerin deficiency was predicted to cause increased nuclear compliance, cell migration, and metastasis. We tested this hypothesis by downregulating emerin in noninvasive MCF7 cells and found emerin knockdown causes smaller, dysmorphic nuclei, resulting in increased impeded cell migration. Emerin reduction in invasive breast cancer cells showed similar results. Supporting the clinical relevance of emerin reduction in cancer progression, our analysis of 192 breast cancer patient samples showed emerin expression inversely correlates with cancer invasiveness. We conclude emerin loss is an important driver of invasive transformation and has utility as a biomarker for tumor progression.

Structural transitions in kinesin minus-end directed microtubule motility.

Kinesin motor proteins hydrolyze ATP to produce force for spindle assembly and vesicle transport, performing essential functions in cell division and motility, but the structural changes required for force generation are uncertain. We now report high-resolution structures showing new transitions in the kinesin mechanochemical cycle, including power stroke fluctuations upon ATP binding and a post-hydrolysis state with bound ADP + free phosphate. We find that rate-limiting ADP release occurs upon microtubule binding, accompanied by central ß-sheet twisting, which triggers the power stroke - stalk rotation and neck mimic docking - upon ATP binding. Microtubule release occurs with ß-strand-to-loop transitions, implying that ß-strand refolding induces Pi release and the recovery stroke. The strained ß-sheet during the power stroke and strand-to-loop transitions identify the ß-sheet as the long-sought motor spring.

Emerin deficiency drives MCF7 cells to an invasive phenotype.

During metastasis, cancer cells traverse the vasculature by squeezing through very small gaps in the endothelium. Thus, nuclei in metastatic cancer cells must become more malleable to move through these gaps. Our lab showed invasive breast cancer cells have 50% less emerin protein resulting in smaller, misshapen nuclei, and higher metastasis rates than non-cancerous controls. Thus, emerin deficiency was predicted to cause increased nuclear compliance, cell migration, and metastasis. We tested this hypothesis by downregulating emerin in noninvasive MCF7 cells and found emerin knockdown causes smaller, dysmorphic nuclei, resulting in increased impeded cell migration. Emerin reduction in invasive breast cancer cells showed similar results. Supporting the clinical relevance of emerin reduction in cancer progression, our analysis of 192 breast cancer patient samples showed emerin expression inversely correlates with cancer invasiveness. We conclude emerin loss is an important driver of invasive transformation and has utility as a biomarker for tumor progression.

The microbiome of a Pacific moon jellyfish Aurelia coerulea.

The impact of microbiome in animal physiology is well appreciated, but characterization of animal-microbe symbiosis in marine environments remains a growing need. This study characterizes the microbial communities associated with the moon jellyfish Aurelia coerulea, first isolated from the East Pacific Ocean and has since been utilized as an experimental system. We find that the microbiome of this Pacific Aurelia culture is dominated by two taxa, a Mollicutes and Rickettsiales. The microbiome is stable across life stages, although composition varies. Mining the host sequencing data, we assembled the bacterial metagenome-assembled genomes (MAGs). The bacterial MAGs are highly reduced, and predict a high metabolic dependence on the host. Analysis using multiple metrics suggest that both bacteria are likely new species. We therefore propose the names Ca. Mariplasma lunae (Mollicutes) and Ca. Marinirickettsia aquamalans (Rickettsiales). Finally, comparison with studies of Aurelia from other geographical populations suggests the association with Ca. Mariplasma lunae occurs in Aurelia from multiple geographical locations. The low-diversity microbiome of Aurelia provides a relatively simple system to study host-microbe interactions.

Training Medical Student Counselors for the Rochester Model, a Hospital Tobacco Treatment Program.

Purpose: Providing effective tobacco dependence treatments to hospitalized patients remains a challenge. Prior to 2021, the Rochester Model program used staff nurses for both bedside and post-discharge counseling necessary to maintain abstinence. When nurse shortages and elevated job stress occurred during the COVID Pandemic, we proposed that medical students learn to counsel patients at the bedside and after discharge. Patients and Methods: Due to COVID restrictions, first- and second-year medical students trained using remote Zoom sessions. The total training time was 2.5 hr without role-play or additional evaluations. A survey measured the students' satisfaction, confidence, and counseling barriers. A smoking patient on a participating hospital unit can enroll in the program. Students delivered bedside counseling, then provided follow-up treatment and outcome calls along with New York State Quitline counselors. Results: The survey demonstrated that 89% of the students were satisfied with the training. The bedside counseling confidence was greater than the phone counseling confidence. All students felt the program experience has value to them as future physicians. 124 smoking patients enrolled, and outcomes followed out to 6 months. The 7-day point prevalence quit rates using the as-treated (patients contacted) analysis were 57% at 4 weeks, 48% at 3 months, and 43% at 6 months. The 7-day point prevalence quit rates using the intent-to-treat (all patients) analysis were 31% at 4 weeks, 16% at 3 months and 14% at 6 months. Conclusion: Medical students given minimal training are effective tobacco cessation counselors at no cost to the hospital system. The Rochester Model program using student counseling benefits patients, the students, and the health-care system.

Treatment of Refractory Congenital Pseudoarthrosis of Tibia with Contralateral Vascularized Fibular Bone Graft and Anatomic Distal Tibial Locking Plate: A Case Series and Literature Review.

BACKGROUND: Congenital pseudoarthrosis of the tibia (CPT) remains a challenge for physicians. Several treatment options have been proposed, but the standard of care remains inconclusive. In this study, we present three patients for whom the failure of prior treatments was managed with a contralateral vascularized fibular bone graft (VFG) and an anatomic distal tibial locking plate. METHODS: Between 2017 and 2021, three patients were referred for failed treatment of CPT. All patients had undergone multiple prior surgeries, including tumor excision and fixation with ring external fixators, plates, and screws. We performed radical tumor resection and reconstruction of bone defects with a VFG. The construct was fixed with an anatomic locking plate, and the patients were followed up for a mean of 45.7 months. RESULTS: All three patients were able to obtain graft union at 19.3 weeks. At the final follow-up, all grafts achieved bony hypertrophy without evidence of bone resorption or local tumor recurrence. There was a mean leg length difference of 8.5 cm preoperatively, compared with 6.3 cm postoperatively. The average lower leg angulation was 7.4 degrees and the average ankle range of motion was 58.3 degrees. The mean VAS score was 0 and the mean AOFAS score was 88.3. No significant complications were noted. CONCLUSIONS: Implantation of a VFG and an anatomic distal tibia locking plate can be considered an option for treatment-refractory CPT. Patients can expect to achieve bone consolidation, ambulate as tolerated, and have a low complication rate.

Statins associate with lower risk of biliary tract cancers: A systematic review and meta-analysis.

BACKGROUND: Biliary tract cancers (BTCs), encompassing cholangiocarcinoma (CCA), gallbladder (GBC), and ampulla of Vater cancers (AVC), are common hepatobiliary cancer after hepatocellular carcinoma with a high mortality rate. As there is no effective chemopreventive agent to prevent BTCs, this study aimed to explore the role of statins on the risk of BTCs. METHODS: PubMed, Embase, and Cochrane Library from inception until 24 April 2020 were searched according to the Meta-Analyses of Observational Studies in Epidemiology (MOOSE) guidelines. The adjusted risk ratios (aRRs) of BTCs and individual cancer were pooled using a random-effects model. RESULTS: Eight observational studies (3 cohort and 5 case-control studies) were included with 10,485,231 patients. The median age was 68.0 years (IQR: 67.0-71.5) and 48.3% were male. Statins were associated with a lower risk of all BTCs (aRR: 0.67; 95% CI: 0.51-0.87). The pooled aRR for CCA was 0.60 (95% CI: 0.38-0.94) and GBC was 0.78 (95% CI: 0.68-0.90). There was only one study on AVC with aRR of 0.96 (95% CI: 0.66-1.41). The pooled aRR for lipophilic and hydrophilic statins was 0.78 (95% CI: 0.69-0.88) and 0.70 (95% CI: 0.61-0.80), respectively. The effects were attenuated in studies that adjusted for aspirin and/or non-steroidal anti-inflammatory drugs (aRR: 0.80, 95% CI: 0.72-0.89) and metformin (aRR: 0.80, 95% CI: 0.72-0.90). CONCLUSIONS: Statins, both lipophilic and hydrophobic, were associated with a lower risk of BTCs, particularly CCA and GBC.

Membrane curvature governs the distribution of Piezo1 in live cells.

Piezo1 is a bona fide mechanosensitive ion channel ubiquitously expressed in mammalian cells. The distribution of Piezo1 within a cell is essential for various biological processes including cytokinesis, cell migration, and wound healing. However, the underlying principles that guide the subcellular distribution of Piezo1 remain largely unexplored. Here, we demonstrate that membrane curvature serves as a key regulator of the spatial distribution of Piezo1 in the plasma membrane of living cells. Piezo1 depletes from highly curved membrane protrusions such as filopodia and enriches to nanoscale membrane invaginations. Quantification of the curvature-dependent sorting of Piezo1 directly reveals the in situ nano-geometry of the Piezo1-membrane complex. Piezo1 density on filopodia increases upon activation, independent of calcium, suggesting flattening of the channel upon opening. Consequently, the expression of Piezo1 inhibits filopodia formation, an effect that diminishes with channel activation.

Association of Left Ventricular Remodeling Assessment by Cardiac Magnetic Resonance With Outcomes in Patients With Chronic Aortic Regurgitation.

Importance: Chronic aortic regurgitation (AR) causes left ventricular (LV) volume overload, which results in progressive LV remodeling negatively affecting outcomes. Whether cardiac magnetic resonance (CMR) volumetric quantification can provide incremental risk stratification over standard clinical and echocardiographic evaluation in patients with chronic moderate or severe AR is unknown. Objective: To compare LV remodeling measurements by CMR and echocardiography between patients with and without heart failure symptoms and to verify the association of remodeling measurements of patients with chronic moderate or severe AR but no or minimal symptoms with clinical outcomes receiving medical management. Design, Setting, and Participants: This multicenter retrospective cohort study included consecutive patients with at least moderate chronic native AR evaluated by 2-dimensional transthoracic echocardiography and CMR examination within 90 days from each other between January 2012 and February 2020 at Allina Health System. Data were analyzed from June 2021 to January 2022. Exposures: Clinical evaluation and risk stratification by CMR. Main Outcomes and Measures: The end point was a composite of death, heart failure hospitalization, or progression of New York Heart Association functional class while receiving medical management, censoring patients at the time of aortic valve replacement (when performed) or at the end of follow-up. Results: Of the 178 included patients, 119 (66.9%) were male, 158 (88.8%) presented with no or minimal symptoms (New York Heart Association class I or II), and the median (IQR) age was 58 (44-69) years. Compared with patients with no or minimal symptoms, symptomatic patients had greater LV end-systolic volume index (LVESVi) by CMR (median [IQR], 66 [46-85] mL/m2 vs 42 [30-58] mL/m2; P < .001), while there were no significant differences by echocardiography (LVESVi: median [IQR], 38 [30-58] mL/m2 vs 27 [20-42] mL/m2; P = .07; LV end-systolic diameter index: median [IQR], 21 [17-25] mm/m2 vs 18 [15-22] mm/m2; P = .17). During the median (IQR) follow-up of 3.3 (1.6-5.8) years, 50 patients with no or minimal symptoms receiving medical management developed the composite end point, which, in multivariate analysis adjusted for age and EuroSCORE II, was independently associated with LVESVi of 45 mL/m2 or greater and aortic regurgitant fraction of 32% or greater, the latter adding incremental prognostic value to CMR volumetric assessment. Conclusions and Relevance: In patients with chronic moderate or severe AR, patients presenting with heart failure symptoms have greater LVESVi by CMR than those with no or minimal symptoms. In patients with no or minimal symptoms, CMR quantification of LVESVi and AR severity may identify those at risk of death or incident heart failure and therefore should be considered in the clinical evaluation and decision-making of these patients.

Decoding accelerometry for classification and prediction of critically ill patients with severe brain injury.

Our goal is to explore quantitative motor features in critically ill patients with severe brain injury (SBI). We hypothesized that computational decoding of these features would yield information on underlying neurological states and outcomes. Using wearable microsensors placed on all extremities, we recorded a median 24.1 (IQR: 22.8-25.1) hours of high-frequency accelerometry data per patient from a prospective cohort (n = 69) admitted to the ICU with SBI. Models were trained using time-, frequency-, and wavelet-domain features and levels of responsiveness and outcome as labels. The two primary tasks were detection of levels of responsiveness, assessed by motor sub-score of the Glasgow Coma Scale (GCSm), and prediction of functional outcome at discharge, measured with the Glasgow Outcome Scale-Extended (GOSE). Detection models achieved significant (AUC: 0.70 [95% CI: 0.53-0.85]) and consistent (observation windows: 12 min-9 h) discrimination of SBI patients capable of purposeful movement (GCSm > 4). Prediction models accurately discriminated patients of upper moderate disability or better (GOSE > 5) with 2-6 h of observation (AUC: 0.82 [95% CI: 0.75-0.90]). Results suggest that time series analysis of motor activity yields clinically relevant insights on underlying functional states and short-term outcomes in patients with SBI.

Preparation and Characterization of Targeted Microbubbles.

Targeting of microbubbles (ultrasound contrast agents for molecular imaging) has been researched for more than two decades. However, methods of microbubble preparation and targeting ligand attachment are cumbersome, complicated, and lengthy. Therefore, there is a need to simplify the targeted microbubble preparation procedure to bring it closer to clinical translation. The purpose of this publication is to provide a detailed description and explanation of the steps necessary for targeted microbubble preparation, functional characterization and testing. A sequence of the optimized and simplified procedures is presented for two systems: a biotin-streptavidin targeting pair model, and a cyclic RGD peptide targeting the recombinant αvß3 protein, which is overexpressed on the endothelial lining of the tumor neovasculature. Here, we show the following: covalent coupling of the targeting ligand to a lipid anchor, assessment of the reagent quality, and tests that confirm the successful completion of the reaction; preparation of the aqueous precursor medium containing microbubble shell components, followed by microbubble preparation via amalgamation; assessment of the efficacy of lipid transfer onto the microbubble stabilizer shell; adjustment of microbubble size distribution by flotation at normal gravity to remove larger microbubbles that might be detrimental for in vivo use; assessment of microbubble size distribution by electrozone sensing; evaluation of targeted binding of the microbubbles to receptor-coated surface in a static binding assay test (in an inverted dish); and evaluation of targeted binding of the microbubbles to receptor-coated surface in a parallel plate flow chamber test.

Secondary Structural Model of MALAT1 Becomes Unstructured in Chronic Myeloid Leukemia and Undergoes Structural Rearrangement in Cervical Cancer.

Long noncoding RNAs (lncRNAs) influence cellular function through binding events that often depend on the lncRNA secondary structure. One such lncRNA, metastasis-associated lung adenocarcinoma transcript 1 (MALAT1), is upregulated in many cancer types and has a myriad of protein- and miRNA-binding sites. Recently, a secondary structural model of MALAT1 in noncancerous cells was proposed to form 194 hairpins and 13 pseudoknots. That study postulated that, in cancer cells, the MALAT1 structure likely varies, thereby influencing cancer progression. This work analyzes how that structural model is expected to change in K562 cells, which originated from a patient with chronic myeloid leukemia (CML), and in HeLa cells, which originated from a patient with cervical cancer. Dimethyl sulfate-sequencing (DMS-Seq) data from K562 cells and psoralen analysis of RNA interactions and structure (PARIS) data from HeLa cells were compared to the working structural model of MALAT1 in noncancerous cells to identify sites that likely undergo structural alterations. MALAT1 in K562 cells is predicted to become more unstructured, with almost 60% of examined hairpins in noncancerous cells losing at least half of their base pairings. Conversely, MALAT1 in HeLa cells is predicted to largely maintain its structure, undergoing 18 novel structural rearrangements. Moreover, 50 validated miRNA-binding sites are affected by putative secondary structural changes in both cancer types, such as miR-217 in K562 cells and miR-20a in HeLa cells. Structural changes unique to K562 cells and HeLa cells provide new mechanistic leads into how the structure of MALAT1 may mediate cancer in a cell-type specific manner.

Autonomic and Cardiac Repolarization Lability in Long QT Syndrome Patients.

OBJECTIVE: Long QT-Syndrome (LQTS) patients are at risk of arrhythmias and seizures. We investigated whether autonomic and cardiac repolarization measures differed based on LQTS genotypes, and in LQTS patients with vs. without arrhythmias and seizures. METHODS: We used 24-h ECGs from LQTS1 (n = 87), LQTS2 (n = 50), and LQTS genotype negative patients (LQTS(-), n = 16). Patients were stratified by LQTS genotype, and arrhythmias/seizures. Heart rate variability (HRV) and QT variability index (QTVI) measures were compared between groups during specific physiological states (minimum, middle, & maximum sympathovagal balance, LF/HF). Results were further tested using logistic regression for each ECG measure, and all HRV measures in a single multivariate model. RESULTS: Across multiple physiological states, total autonomic (SDNN) and vagal (RMSSD, pNN50) function were lower and repolarization dynamics (QTVI) were elevated in LQTS(+), LQTS1, and LQTS2, compared to LQTS(-). Many measures remained significant in the regression models. Multivariate modeling demonstrated that SDNN, RMSSD, and pNN50 were independent markers of LQTS(+) vs. LQTS(-), and SDNN and pNN50 were markers for LQTS1 vs. LQTS(-). During sympathovagal balance (middle LF/HF), RMSSD and pNN50 distinguished LQTS1 vs. LQTS2. LQTS1 patients with arrhythmias had lower total (SDNN) and vagal (RMSSD and pNN50) autonomic function, and SDNN remained significant in the models. In contrast, ECG measures did not differ in LQTS2 patients with vs. without arrhythmias, and LQTS1 and LQTS2 with vs. without seizures. CONCLUSION: Autonomic (HRV) and cardiac repolarization (QTVI) ECG measures differ based on LQTS genotype and history of arrhythmias in LQTS1. SDNN, RMSSD, and pNN50 were each independent markers for LQTS genotype.

Naturally occurring modified ribonucleosides.

The chemical identity of RNA molecules beyond the four standard ribonucleosides has fascinated scientists since pseudouridine was characterized as the "fifth" ribonucleotide in 1951. Since then, the ever-increasing number and complexity of modified ribonucleosides have been found in viruses and throughout all three domains of life. Such modifications can be as simple as methylations, hydroxylations, or thiolations, complex as ring closures, glycosylations, acylations, or aminoacylations, or unusual as the incorporation of selenium. While initially found in transfer and ribosomal RNAs, modifications also exist in messenger RNAs and noncoding RNAs. Modifications have profound cellular outcomes at various levels, such as altering RNA structure or being essential for cell survival or organism viability. The aberrant presence or absence of RNA modifications can lead to human disease, ranging from cancer to various metabolic and developmental illnesses such as Hoyeraal-Hreidarsson syndrome, Bowen-Conradi syndrome, or Williams-Beuren syndrome. In this review article, we summarize the characterization of all 143 currently known modified ribonucleosides by describing their taxonomic distributions, the enzymes that generate the modifications, and any implications in cellular processes, RNA structure, and disease. We also highlight areas of active research, such as specific RNAs that contain a particular type of modification as well as methodologies used to identify novel RNA modifications. This article is categorized under: RNA Processing > RNA Editing and Modification.

Real-Time Indoor Geolocation Tracking for Assisted Healthcare Facilities.

A leading cause of physical injury sustained by elderly persons is the event of unintentionally falling. A delay between the time of fall and the time of medical attention can exacerbate injury if the fall resulted in a concussion, traumatic brain injury, or bone fracture. The authors present a solution capable of finding and tracking, in real-time, the location of an elderly person within an indoor facility, using only existing Wi-Fi infrastructure. This paper discusses the development of an open source software framework capable of finding the location of an individual within 3m accuracy using 802.11 Wi-Fi in good coverage areas. This framework is comprised of an embedded software layer, a Web Services layer, and a mobile application for monitoring the location of individuals, calculated using trilateration, with Kalman filtering employed to reduce the effect of multipath interference. The solution provides a real-time, low cost, extendible solution to the problem of indoor geolocation to mitigate potential harm to elderly persons who have fallen and require immediate medical help.

Secondary Structural Model of Human MALAT1 Reveals Multiple Structure-Function Relationships.

Human metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) is an abundant nuclear-localized long noncoding RNA (lncRNA) that has significant roles in cancer. While the interacting partners and evolutionary sequence conservation of MALAT1 have been examined, much of the structure of MALAT1 is unknown. Here, we propose a hypothetical secondary structural model for 8425 nucleotides of human MALAT1 using three experimental datasets that probed RNA structures in vitro and in various human cell lines. Our model indicates that approximately half of human MALAT1 is structured, forming 194 helices, 13 pseudoknots, five structured tetraloops, nine structured internal loops, and 13 intramolecular long-range interactions that give rise to several multiway junctions. Evolutionary conservation and covariation analyses support 153 of 194 helices in 51 mammalian MALAT1 homologs and 42 of 194 helices in 53 vertebrate MALAT1 homologs, thereby identifying an evolutionarily conserved core that likely has important functional roles in mammals and vertebrates. Data mining revealed that RNA modifications, somatic cancer-associated mutations, and single-nucleotide polymorphisms may induce structural rearrangements that sequester or expose binding sites for several cancer-associated microRNAs. Our findings reveal new mechanistic leads into the roles of MALAT1 by identifying several intriguing structure-function relationships in which the dynamic structure of MALAT1 underlies its biological functions.

Stability of an RNA•DNA-DNA triple helix depends on base triplet composition and length of the RNA third strand.

Recent studies suggest noncoding RNAs interact with genomic DNA, forming an RNA•DNA-DNA triple helix that regulates gene expression. However, base triplet composition of pyrimidine motif RNA•DNA-DNA triple helices is not well understood beyond the canonical U•A-T and C•G-C base triplets. Using native gel-shift assays, the relative stability of 16 different base triplets at a single position, Z•X-Y (where Z = C, U, A, G and X-Y = A-T, G-C, T-A, C-G), in an RNA•DNA-DNA triple helix was determined. The canonical U•A-T and C•G-C base triplets were the most stable, while three non-canonical base triplets completely disrupted triple-helix formation. We further show that our RNA•DNA-DNA triple helix can tolerate up to two consecutive non-canonical A•G-C base triplets. Additionally, the RNA third strand must be at least 19 nucleotides to form an RNA•DNA-DNA triple helix but increasing the length to 27 nucleotides does not increase stability. The relative stability of 16 different base triplets in DNA•DNA-DNA and RNA•RNA-RNA triple helices was distinctly different from those in RNA•DNA-DNA triple helices, showing that base triplet stability depends on strand composition being DNA and/or RNA. Multiple factors influence the stability of triple helices, emphasizing the importance of experimentally validating formation of computationally predicted triple helices.