Adenosine Diphosphate Research Articles

Abstract PO4-08-02: Genetic testing among patients with triple-negative breast cancer at a single health system: a quality improvement project

Abstract Background: Breast cancer is the most common type and the second leading cause of cancer-related deaths in women. Approximately 15-20% of patients have triple-negative breast cancer (TNBC) at diagnosis. TNBC is aggressive, difficult to treat, prone to relapse, and associated with an increased mortality rate. The incidence of BRCA1/2 mutation is about 20% in patients with TNBC (Hartman et al. 2012). Despite the high prevalence of germline mutations in cancer susceptibility genes among TNBC patients, many do not received genetic counseling (GC) or genetic testing (GT). National Comprehensive Cancer Network (NCCN) guidelines now recommend GT for all patients with TNBC, regardless of age, especially after the approval of Poly Adenosine diphosphate Ribose Polymerase (PARP) inhibitors for targeted therapy against BRCA1/2 mutations. The aim of this quality improvement (QI) project was to identify patients with TNBC who did not receive GT and design an intervention to understand potential barriers to receiving GC and GT. Methods: We implemented a QI project to identify previously diagnosed adult patients with TNBC (18 years and older) of any stage (early or advanced/metastatic) without prior GT or family history of genetic disorders with active follow-up at the Henry Ford health breast cancer clinic. Eligible patients diagnosed between January 2015 and January 2020 from the Syapse Learning Health Network database were included. Baseline information as well as additional data including demographics, breast cancer treatment details, and whether GT and/or GC was offered or not was obtained through electronic health record review after institutional review board committee approval. Bi-monthly meetings were held amongst breast cancer providers, genetic counselors, and nurse navigators to develop an intervention to contact the patients who did not undergo GT and/or GC. After obtaining the list of patients who did not undergo GT and/or GC despite meeting eligibility criteria, they were contacted by their primary breast cancer providers via telephone regarding their willingness to obtain GT and/or GC at this time and participate in a questionnaire-based interview to understand barriers towards the same. Results: In the 5 years, a total of 123 patients were noted to have been diagnosed with triple-negative breast cancer. Of these, 93 patients were actively being followed at the Henry Ford breast clinic at the time of the QI project. 2 of the 9 patients who underwent GT and GC were positive for BRCA1/2 mutation. Of the remaining 83 patients that did not receive GC or GT, 65 (78.3%) had no clear reason explained in the chart, 27 patients (32.5%) chose not to get tested, 23 (27.7%) were not deemed to be eligible upon initial assessment and 4 (4.8%) had pending orders placed by breast surgeons upon initial diagnosis. Unfortunately, 29 of these 83 patients were noted to have been deceased when the intervention was being designed. Remaining 54 patients were contacted by their primary breast cancer oncologists to determine their willingness in participating in the questionnaire-based interview. Conclusion: Based on the results of our QI project, we have now developed a process to contact the identified patients and renew the offer for GT and GC as well as participation in a questionnaire-based interview to understand the potential barriers to testing. We also plan to improve physician education and awareness by conducting mini sessions during our annual symposium meetings as well as grand rounds and develop a protocol for ordering GT and GC in patients with TNBC at our institution. Citation Format: Manasi Godbole, Mary Nyhuis, Travis Washburn, Joann Hirth, Haythem Ali. Genetic testing among patients with triple-negative breast cancer at a single health system: a quality improvement project [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO4-08-02.

Abstract PO3-09-04: Identification of germline pathogenic variants to guide systemic therapy: Prevalence and spectrum of PVs among Mexican patients with metastatic breast cancer

Abstract BACKGROUND Currently, international genetic cancer risk assessment (GCRA) guidelines recommend genetic testing for the identification of germline pathogenic variants (PVs) in patients with metastatic breast cancer (MBC) to guide systemic therapy. Patients with PVs in BRCA genes are candidates to receive poly(adenosine diphosphate–ribose) polymerase inhibitors (PARPi). The prevalence of PVs in patients with HER2-negative MBC has been reported to be 10%. The proportion of MBC in low- and middle-income countries (LMIC) is higher. However, access to genetic testing in LMIC is limited and contributes to the gap in genetic epidemiology knowledge. We aimed to describe the frequency of germline PVs in breast cancer associated genes and clinic-pathologic characteristics of Mexican women with MBC. METHODS This retrospective study included Mexican women from 7 centers in Mexico, who were diagnosed with MBC and had genetic testing. Pathogenic and likely pathogenic variants were reported. Demographic, histopathological characteristics, family history and access to treatment with PARPi, were obtained from chart review. RESULTS From August 2017 to May 2023, 114 Mexican women with MBC were included, median age at diagnosis was 47 years (range 24-80 y), 42% had de novo metastatic, and 58% recurrent disease. 86.8% had infiltrating ductal carcinoma, 51.3% had HR+HER2- (n=58), 4.4% HR+HER+ (n=5), 5.3% HR-HER2+ (n=6), and 38.6% triple negative tumors (n=44). Sites of metastatic disease were as follows: 61.4% non-regional nodes (n=70), 57% bone (n=65), 40.3% lung (n=46), 30.7% liver (n=35), 15.8% central nervous system (n=18) and 7% peritoneal carcinomatosis (n=8). The frequency of PVs was 22% (25/114); 84% of the PVs were heterozygous, mainly identified in BRCA genes (BRCA1 n=12, BRCA2 n=9), and in 4 patients in other genes (PALB2 n=2, PTEN n=1, MUTYH n=1). Among PARPi candidates, 40% (10/25) received Olaparib, the majority received it on the second or subsequent lines of treatment (7/10) with a median progression free survival of 6 months (3-13 m). BRCA carriers were younger at BC diagnosis (38.5 vs. 49 years, p=0.00694) and had a significant higher proportion of family history of ovarian cancer (p=0.00039), but not for breast (p=0.06), prostate (p=0.71) or pancreatic cancer (p=0.098), compared to non-carriers. CONCLUSION We found a high frequency of PVs in BRCA genes in Mexican women with MBC. Access to the genetic testing and targeted therapy should be improve in the Mexican population in order to decrease disparities in cancer care in the Mexican Population. In addition, genetic testing allows to identify other family members at risk with cascade testing and to recommend them risk reduction strategies. Citation Format: Jose Luis Rodriguez-Olivares, Maria Fernanda Esparza-Orozco, Jose Alfredo Ramirez-Gonzalez, Roberto Ivan Sanchez-Reyes, Enrique Isay Talamantes-Gamez, Gregorio Quintero-Beulo, Gilberto Lopez-Rosas, Karla Alicia Centelles-López, Brizio Moreno-Jaime, Daniela Vazquez-Juarez, Ana Maria Hernandez-Murillo, Ilana de la Puente Tawil, Jeffrey Weitzel, Cynthia Villareal Garza, Yanin Chavarri-Guerra. Identification of germline pathogenic variants to guide systemic therapy: Prevalence and spectrum of PVs among Mexican patients with metastatic breast cancer [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO3-09-04.

Abstract PS05-02: PET Imaging of PARP Expression as a Biomarker of Response to Chemotherapy in Breast Cancer: A Nonrandomized Clinical Trial

Abstract Introduction: Poly–(adenosine diphosphate–ribose) polymerase (PARP) proteins are involved in double-stranded break repair, mediated through homologous recombination (HR) and non-homologous end joining (NHEJ). The majority of PARP activity in this context is attributed to PARP-1 (85-90%). PARP-1 binding to damaged DNA, catalytic activity, and subsequent release to allow access for other DNA repair proteins are necessary for efficient DNA repair. The critical function of PARP-1 in these processes makes this protein a unique potential biomarker of DNA repair capability. [18F]FluorThanatrace ([18F]FTT) is an 18F-labeled PARP inhibitor analog that binds to the same site as approved PARPi drugs. We hypothesized that the biologically relevant form of PARP in untreated breast cancer might provide an indication of functional tumor DNA repair capabilities and therefore predict chemotherapy response. Thus, this study aimed to determine whether a non-invasive quantitative measure of in vivo PARP expression correlated with response to chemotherapy in breast cancer. Methods: A single-arm prospective trial enriched for subjects with triple negative breast cancer (TNBC) was conducted at the University of Pennsylvania from May 2017 to March 2022 (NCT03083288 and NCT03846167). Inclusion criteria were primary breast cancer with tumor diameter of at least 1 cm on conventional imaging and willingness to undergo an [18F]FTT-PET scan prior to neoadjuvant chemotherapy. Participants provided written informed consent. [18F]FTT uptake in breast cancer was measured pre-therapy and tested for association with pathologic complete response (pCR). Subjects were scanned on an Ingenuity TF PET/CT (Philips Healthcare) following injection of [18F]FTT using a 20-minute static acquisition. Peak 1 cm3 average uptake (SUVpeak) were recorded from a spherical region-of-interest (ROI) covering the primary malignancy, guided by CT and prior imaging studies. All SUVs were partial volume corrected and normalized using muscle (NM). A Mann-Whitney test was used to determine if [18F]FTT uptake differed by pCR. Receiver operating characteristic (ROC) curves were constructed to test the performance of [18F]FTT uptake in predicting pCR. Statistical analyses utilized STATA v15.1 with significance based on a two-sided alpha level of ≤0.05. Participants: Twenty-six women with stage I-III breast cancer met inclusion criteria with age range of 29-74 years and self-reported race as Asian (1, 4%), Black (9, 37%) or White (16, 60%). One subject had bilateral tumors (ER+ stage IIA and HER2+ stage I) that were evaluated separately (27 total tumors). Breast tumors were pathologically confirmed and included 6 (22%) ER+/HER2-, 5 (19%) HER2+, and 16 (59%) TNBC. Germline sequencing was available for 21 participants. Results: Mean tumor diameter ranged from 15 to 91 mm (median 30 mm). A considerable range of [18F]FTT uptake was seen across subjects (SUVpeak/NM 0.66-6.5). [18F]FTT uptake was independent of subtype (ER+, HER2+, TN) (P=0.35), stage (P=0.39), and between mutations in genes associated with homologous recombination deficiency (i.e., BRCA1/2, PALB2) and wild-type (P=0.73). In the TNBC cohort, pre-treatment [18F]FTT uptake was higher in subjects who went on to have a pCR (n=16, P=0.05), while a trend toward higher uptake was seen when including all tumor types (n=27, P=0.11). ROC analysis of the value of [18F]FTT uptake for predicting pCR revealed an AUC of 0.79 and showed that a threshold SUV ratio < 2.47 predicted pCR in TNBC patients with a 100% specificity and 64% sensitivity. Conclusions: These early clinical results suggest a relationship between [18F]FTT uptake, a measure of PARP expression, and chemotherapy sensitivity in TNBC, warranting future studies to elucidate underlying mechanistic processes and potential clinical implications. Citation Format: Sarah Gitto, Anthony Young, Ira Bleiweiss, Amy Clark, Elizabeth McDonald. PET Imaging of PARP Expression as a Biomarker of Response to Chemotherapy in Breast Cancer: A Nonrandomized Clinical Trial [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PS05-02.

Abstract PO2-09-03: Twenty-one gene recurrence scores in individuals with breast cancer associated with PALB2 germline pathogenic variants

Abstract Background: The 21-gene recurrence score assay (Oncotype DX, Genomic Health Inc., CA) is prognostic, quantifies predicted benefit of adjuvant chemotherapy, and informs clinical decisions for patients with early-stage, hormone receptor-positive (HR+), human epidermal growth factor receptor 2-negative (HER2-) breast cancer (BC). Differences in Oncotype recurrence scores (RS) in individuals with and without BRCA1 and BRCA2 (BRCA1/2) pathogenic variants (PV) have been demonstrated, but the results of this assay in patients with BC and germline PALB2 PV have not previously been described. This is of particular relevance as PALB2 BC has demonstrated biologic features similar to BRCA1/2 BC such as poly(adenosine diphosphate ribose) polymerase inhibitor (PARPi) sensitivity. Methods: Patients with early-stage, HR+, HER2-, invasive breast cancer and a germline PALB2 PV were ascertained from academic cancer center databases and the Prospective Registry of MultiPlex Testing (PROMPT), an online research registry for individuals who have had multigene panel testing for inherited cancer susceptibility. Study participants (pts) were categorized by age < 50 and ≥50. Oncotype DX RS were retrospectively reviewed and categorized as those that would and would not warrant consideration of chemotherapy (≥16 and < 16, respectively, for pts < 50; ≥26 and < 26, respectively, for pts ≥50). Yates corrected Chi-square tests were used to compare RS distributions between carriers and a previously published genetically unselected reference population. Results: Oncotype DX reports were reviewed for 20 pts with BC and germline PALB2 PV. Median RS was 19 (range 6-41) overall. Four patients were diagnosed under the age of 50 (range 33-48); median RS for these individuals was 19 (range 12-41). Sixteen patients were diagnosed at age 50 or greater (median 59, range 50-70); median RS for these individuals was also 19 (range 6-31). Compared to the genetically unselected reference population, Oncotype DX RS in pts with PALB2-associated BC demonstrated a numerical but not statistically significant shift in distribution towards categories in which chemotherapy would be considered. Among pts under the age of 50, 75% had Oncotype DX RS for which chemotherapy would be considered and 25% had Oncotype DX RS for which endocrine therapy alone would likely be recommended (p=0.726 compared to reference). Among pts aged 50 and older, 25% had Oncotype DX RS for which chemotherapy would be considered and 75% had Oncotype DX RS for which endocrine therapy alone would likely be recommended (p=0.579 compared to reference). Conclusions: Compared to data from the published genetically unselected commercial database, BC associated with PALB2 PV may have higher Oncotype DX RS reflective of intrinsically less favorable biology and greater therapeutic sensitivity. Larger datasets are forthcoming to confirm presence of significant differences in Oncotype DX RS distributions between BC with and without PALB2 PV. Table 1. Oncotype DX RS distribution by age among pts with gPALB2 PV and genetically unselected reference *Jakubowski et al., J Surg Oncol 2020. PMID 3249731 Citation Format: Payal Shah, Siddhartha Yadav, Jamie Brower, Jada Hamilton, Erica Simmons, Judy Garber, Kenneth Offit, Mark Robson, Fergus Couch, Susan Domchek. Twenty-one gene recurrence scores in individuals with breast cancer associated with PALB2 germline pathogenic variants [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO2-09-03.

The Impact of ADP Inhibition on Traumatic Brain Injury Outcomes.

Platelet inhibition correlates with severity of traumatic brain injury and may be associated with mortality. Adenosine diphosphate participates in platelet aggregation via the activation of the ADP receptors, P2Y1 and P2Y12. Prior work suggests this ADP pathway is significant in managing patients with head injuries. This study aimed to measure the influence of ADP inhibition on outcomes after a traumatic brain injury (TBI), as measured by thromboelastography with platelet mapping (TEG-PM). Outcomes were defined as (a) hospital length of stay; (b) ICU length of stay, (c) mortality, and (d) progression of hemorrhage on CT. The resulting cohort was split into quartiles to compare the effect of increasingly inhibited ADP values on the identified outcomes. Comparisons of 2 groups of patients were also conducted, one defined by ADP inhibition less than or equal to 60% and the other group by ADP inhibition of greater than 60%. 98 patients were included in final analysis, with 72.4% having ADP inhibition less than 60%. These patients were significantly older and had lower global injury severity scores (ISSs), although their head-specific ISS was equivalent. Compared to the group with ADP inhibition over 60%, there was no significant difference in mortality, hospital or ICU length of stay, or progression of lesion on CT. Patients with ADP less than 60% inhibited had smaller ISS and higher GCS, indicating they were less injured than those with greater ADP inhibition, consistent with prior literature. The equivalent ICU and hospital length of stay and mortality suggests that ADP inhibition plays a smaller role in outcomes. Additional study with a larger sample size and guideline-based assessments is necessary to further define the impact of ADP inhibition and to determine the role of platelet transfusion in this population.

Improving platelet function following prophylactic platelet transfusion in patients with hematological malignancies.

Platelet transfusion is a standard treatment to prevent bleeding in patients with hematological malignancies. Although transfusions can improve platelet count, their impact on platelet function remains controversial. We conducted flow cytometry to assess platelet function before and after transfusion and performed subgroup analyses to examine differences based on blood type, corrected count increment (CCI), and platelet microparticles. Overall, 50 patients who received prophylactic platelet transfusion were enrolled. CD42b expression increased, whereas CD41 expression decreased after transfusion. Apheresis platelets exhibited the lowest expression of PAC-1 and P-selectin when exposed to agonist stimulations. PAC-1 expression increased under high adenosine diphosphate (ADP) stimulation, while P-selectin expression increased under both high ADP and thrombin receptor-activating peptide stimulation. In the subgroup analysis, patients with a CCI >4500 and those with the same blood types exhibited a more significant increase in PAC-1 and P-selectin expression under agonist stimulation. When comparing apheresis platelets collected on different days, only the percentage of platelet-derived microparticles showed a significantincrease. Prophylactic transfusion improved platelet function. Platelet function significantly improved in patients with a CCI >4500, those with the same blood types as that of apheresis platelets, or those with platelet-derived microparticle levels <4.7%. No significant improvement in platelet function was noted after the transfusion of different blood types with acceptable compatibility or the transfusion of incompatible blood types. Our results suggest that transfusing platelets with the same blood type remains the optimal choice.

Differential Impact In Vivo of Pf4-ΔCre-Mediated and Gp1ba-ΔCre-Mediated Depletion of Cyclooxygenase-1 in Platelets in Mice.

Low-dose aspirin is widely used for the secondary prevention of cardiovascular disease. The beneficial effects of low-dose aspirin are attributable to its inhibition of platelet Cox (cyclooxygenase)-1-derived thromboxane A2. Until recently, the use of the Pf4 (platelet factor 4) Cre has been the only genetic approach to generating megakaryocyte/platelet ablation of Cox-1 in mice. However, Pf4-ΔCre displays ectopic expression outside the megakaryocyte/platelet lineage, especially during inflammation. The use of the Gp1ba (glycoprotein 1bα) Cre promises a more specific, targeted approach. To evaluate the role of Cox-1 in platelets, we crossed Pf4-ΔCre or Gp1ba-ΔCre mice with Cox-1flox/flox mice to generate platelet Cox-1-/- mice on normolipidemic and hyperlipidemic (Ldlr-/-) backgrounds. Ex vivo platelet aggregation induced by arachidonic acid or adenosine diphosphate in platelet-rich plasma was inhibited to a similar extent in Pf4-ΔCre Cox-1-/-/Ldlr-/- and Gp1ba-ΔCre Cox-1-/-/Ldlr-/- mice. In a mouse model of tail injury, Pf4-ΔCre-mediated and Gp1ba-ΔCre-mediated deletions of Cox-1 were similarly efficient in suppressing platelet prostanoid biosynthesis. Experimental thrombogenesis and attendant blood loss were similar in both models. However, the impact on atherogenesis was divergent, being accelerated in the Pf4-ΔCre mice while restrained in the Gp1ba-ΔCres. In the former, accelerated atherogenesis was associated with greater suppression of PGI2 biosynthesis, a reduction in the lipopolysaccharide-evoked capacity to produce PGE2 and PGD2, activation of the inflammasome, elevated plasma levels of IL-1β, reduced plasma levels of HDL-C, and a reduction in the capacity for reverse cholesterol transport. By contrast, in the latter, plasma HDL-C and α-tocopherol were elevated, and MIP-1α (macrophage inflammatory protein-1α) and MCP-1 (monocyte chemoattractant protein 1) were reduced. Both approaches to Cox-1 deletion similarly restrain thrombogenesis, but a differential impact on Cox-1-dependent prostanoid formation by the vasculature may contribute to an inflammatory phenotype and accelerated atherogenesis in Pf4-ΔCre mice.

Cellular adenylate energy charge and adenine nucleotides in brain tissue during hypoglycemia in newly born BALB/c mice pups

Aim: Hypoglycemia occurs in the neonatal period but the exact pathophysiology of the resulting brain injury at the cellular level is not well known. Therefore, a neonatal murine model was developed with insulin-induced hypoglycemia, to analyze the in-vitro effects of hypoglycemia on brain nucleotides and adenylate energy charge (AEC) throughout the first ten days of life. Methods: Newly born BALB/c pups between one and ten days of age were used. In each age group, six pups were subjected to insulin-induced hypoglycemia and six others served as controls. In both groups, immediately after euthanasia, brain tissues were collected. The in-vitro effects of hypoglycemia on brain nucleotides [adenosine monophosphate (AMP), adenosine diphosphate (ADP), and adenosine triphosphate (ATP)] were analyzed using liquid chromatography with tandem mass spectrometry (LC-MS/MS) as well on AEC. Results: In the controls, the cellular AEC steadily decreased with age by at least 50% over the 10-day study period (P &lt; 0.05) except in the parietal tissue (P = 0.30) where it remained stable throughout that period. The most marked decrease was observed in the occipital tissue (P &lt; 0.001). In the hypoglycemic mice, AEC in both the parietal and occipital tissues decreased significantly more than in the controls, more rapidly and pronounced between day 2 and 5 in the occipital tissue, reaching very low levels from day 5 onward. Except in the occipital tissue, none of the adenine nucleotides on its own, including ATP, reflected the cellular AEC. Conclusions: Over the first ten days of life, hypoglycemia progressively depleted cellular AEC in the brain, unlike cellular ATP concentration which did not appropriately reflect cellular energy.

GS-441524-Diphosphate-Ribose Derivatives as Nanomolar Binders and Fluorescence Polarization Tracers for SARS-CoV-2 and Other Viral Macrodomains.

Viral macrodomains that can bind to or hydrolyze protein adenosine diphosphate ribosylation (ADP-ribosylation) have emerged as promising targets for antiviral drug development. Many inhibitor development efforts have been directed against the severe acute respiratory syndrome coronavirus 2 macrodomain 1 (SARS-CoV-2 Mac1). However, potent inhibitors for viral macrodomains are still lacking, with the best inhibitors still in the micromolar range. Based on GS-441524, a remdesivir precursor, and our previous studies, we have designed and synthesized potent binders of SARS-CoV-2 Mac1 and other viral macrodomains including those of Middle East respiratory syndrome coronavirus (MERS-CoV), Venezuelan equine encephalitis virus (VEEV), and Chikungunya virus (CHIKV). We show that the 1'-CN group of GS-441524 promotes binding to all four viral macrodomains tested while capping the 1″-OH of GS-441524-diphosphate-ribose with a simple phenyl ring further contributes to binding. Incorporating these two structural features, the best binders show 20- to 6000-fold increases in binding affinity over ADP-ribose for SARS-CoV-2, MERS-CoV, VEEV, and CHIKV macrodomains. Moreover, building on these potent binders, we have developed two highly sensitive fluorescence polarization tracers that only require nanomolar proteins and can effectively resolve the binding affinities of nanomolar inhibitors. Our findings and probes described here will facilitate future development of more potent viral macrodomain inhibitors.

Characterization of a novel mouse platelet transfusion model.

Platelet transfusions are increasing with medical advances. Based on FDA criteria, platelet units are assessed by in vitro measures; however, it is not known how platelet processing and storage duration affect function in vivo. Our study's aim was to develop a novel platelet transfusion model stored in mouse plasma that meets FDA criteria adapted to mice, and transfused fresh and stored platelets are detectable in clots in vivo. Platelet units stored in mouse plasma were prepared using a modified platelet-rich plasma (PRP) collection protocol. Characteristics of fresh and stored units, including pH, cell count, in vitro measures of activity, including activation and aggregation, and post-transfusion recovery (PTR), were determined. Lastly, a tail transection assay was conducted using mice transfused with fresh or stored units, and transfused platelets were identified by confocal imaging. Platelet units had acceptable platelet and white cell counts and were negative for bacterial contamination. Fresh and 1-day stored units had acceptable pH; the platelets were activatable by thrombin and adenosine diphosphate, agreeable with thrombin, had acceptable PTR, and were present in vivo in clots of recipients after tail transection. In contrast, 2-day stored units had clinically unacceptable quality. We developed mouse platelets for transfusion analogous to human platelet units using a modified PRP collection protocol with maximum storage of 1 day for an 'old' unit. This provides a powerful tool to test how process modifications and storage conditions affect transfused platelet function in vivo.

An energy-conserving reaction in amino acid metabolism catalyzed by arginine synthetase

All forms of life are presumed to synthesize arginine from citrulline via a two-step pathway consisting of argininosuccinate synthetase and argininosuccinate lyase using citrulline, adenosine 5'-triphosphate (ATP), and aspartate as substrates. Conversion of arginine to citrulline predominantly proceeds via hydrolysis. Here, from the hyperthermophilic archaeon Thermococcus kodakarensis, we identified an enzyme which we designate "arginine synthetase". In arginine synthesis, the enzyme converts citrulline, ATP, and free ammonia to arginine, adenosine 5'-diphosphate (ADP), and phosphate. In the reverse direction, arginine synthetase conserves the energy of arginine deimination and generates ATP from ADP and phosphate while releasing ammonia. The equilibrium constant of this reaction at pH 7.0 is [Cit][ATP][NH3]/[Arg][ADP][Pi] = 10.1 ± 0.7 at 80 °C, corresponding to a ΔG°' of -6.8 ± 0.2 kJ mol-1. Growth of the gene disruption strain was compared to the host strain in medium composed of amino acids. The results suggested that arginine synthetase is necessary in providing ornithine, the precursor for proline biosynthesis, as well as in generating ATP. Growth in medium supplemented with citrulline indicated that arginine synthetase can function in the direction of arginine synthesis. The enzyme is widespread in nature, including bacteria and eukaryotes, and catalyzes a long-overlooked energy-conserving reaction in microbial amino acid metabolism. Along with ornithine transcarbamoylase and carbamate kinase, the pathway identified here is designated the arginine synthetase pathway.

Experimental Evaluation of a Novel Device to Quantify Canal Cleanliness: An In Vitro Study

Endodontic treatments are performed to avoid extractions and maintain the natural dentition. Root canal treatments are undertaken to eliminate or prevent an infection within the root canal system. Chemical and mechanical root canal debridement are the main methods used in endodontics to remove necrotic tissue, microorganisms, and microbial byproducts from the canal. However, to date there is no objective method to clinically determine the proper root canal disinfection level and thus proceed with the obturation. Clinicians just rely on their experience and habits or can trust in empirical methods such as the insertion of paper cones inside the canal and then check their appearance after the removal. Even in the in vitro and ex vivo scientific studies there is no objective method to analyze and compare the efficacy of different endodontic chemo-mechanical techniques and materials. The most frequently used method is to visually analyze some areas with a scanning electron microscope (SEM), even if the resulting images are hardly quantifiable and could greatly vary according to the analyzed area. A new device to clinically test the cleanliness of a root canal and display the result in an objective score was recently developed. The device analyzes the luminescence generated by an enzyme cycling method that process the adenosine triphosphate (ATP), adenosine diphosphate (ADP) and adenosine monophosphate (AMP) present in organic residues. The aim of the present in vitro study was to test the efficacy and reliability of this novel device (Endocator) in a controlled in vitro environment, before using it in clinical practice. The device sensitivity was tested on 5 single canal resin blocks. Three consecutive sampling were executed by one operator for each block to test the device repeatability. Results were recorded according to Endoscore (ES) and relative light unit (RLU) scales. Descriptive analysis and comparison between the 5 resin blocks and the 3 consecutive sampling were performed. Only the comparison between the first and third measurements both for ES (p = 0.00115999) and RLU (p = 0.00532749) resulted significant. Endocator was able to determine small variations of canal contamination in a controlled laboratory environment, showing high sensitivity and repeatability.

Early manifestation of hypophosphatemic rickets in goslings: a potential role of insufficient muscular adenosine triphosphate in motility impairment of early P-deficient geese

We aimed to determine the onset time of hypophosphatemic rickets and investigate the mechanism of motility impairment through adenosine triphosphate (ATP) production in goslings. Two hundred and sixteen 1-day-old male Jiangnan white geese were randomly divided into 3 groups, with 6 replicates and 12 geese per replicate. Birds were fed on 3 diets: a control diet (nonphytic phosphorus, NPP, 0.38%), a P-deficient diet (PD; NPP, 0.08%), and a high P diet (HP; NPP, 0.80%) for 14 d. Subsequently, all birds were shifted to the control diet for an additional 14 d. The cumulative incidence of lameness increased significantly (P < 0.01) starting on d 4, reaching over 80% on d 7 and 100% on d 12 in the PD group. Drinking and eating frequency decreased from d 4 and d 5, respectively, in the PD group compared to the other groups (most P < 0.01). The PD group exhibited shorter and narrower beaks, higher (worse) curvature scores of the beak and costochondral junctions, swelling caput costae, and dirtier feathers since d 4, in contrast to the control and HP groups (most P < 0.01). The HP had bigger (P < 0.05) beak and sternum sizes than the control groups on d 4 to 11. Leg muscle ATP levels were lower (P < 0.01 or 0.05) on d 4 to 11; in contrast, adenosine diphosphate (d 7-11) was higher in PD compared to the control (P < 0.05). Leg muscle ATP level had positive linear (R2 > 0.40) correlations (r > 0.60) with eating and drinking frequencies on d 7 and 11 (P < 0.01). Bone stiffness, feather cleanliness, and ATP levels recovered (P > 0.05) to the control level, whereas bone size did not recover (P < 0.05) in PD and HP after eating the control diet for 2 wk. The onset time of hypophosphatemic rickets was around 4 d in goslings, and insufficient leg muscle ATP was related to the impaired motility observed in early P-deficient geese.

Individualized antiplatelet therapy for non-cardiogenic ischemic stroke

ObjectiveThis research aims to investigate the impact of individualized antiplatelet therapy guided by thromboelastography with platelet mapping (TEG-PM) on the clinical outcomes of patients with non-cardiogenic ischemic stroke. MethodsAmong a total of 1264 patients, 684 individuals diagnosed with non-cardiogenic ischemic stroke underwent TEG-PM testing. Based on the adjustment of antiplatelet medication, these patients were divided into individual and control groups. Within the individual group, in accordance with the TEG-PM test results, a Maximum amplitude (MA) value greater than 47mm was defined as high residual platelet reactivity (HRPR), while an MA value less than 31mm was defined as low residual platelet reactivity (LRPR). Patients with arachidonic acid (AA) less than 50% and adenosine diphosphate (ADP) less than 30% were classified as aspirin-resistant or clopidogrel-resistant. Treatment strategies for antiplatelet medication were subsequently adjusted accordingly, encompassing increment, decrement, or replacement of drugs. Meanwhile, the control group maintained their original medication regimen without alterations. ResultsThe individual group included 487 patients, while the control group had 197. In the individual group, approximately 175 patients (35.9%) were treated with increased medication dosages, 89 patients (18.3%) with reduced dosages, and 223 patients (45.8%) switched medications. The results showed that the incidence rate of ischemic events in the individual group was lower than that of the control group (5.54% vs. 12.6%, P = 0.001), but no significant difference was observed in bleeding events. Cox regression analysis revealed age (hazard ratio, 1.043; 95% CI, 1.01-1.078; P = 0.011) and coronary heart disease (hazard ratio, 1.902; 95% CI, 1.147-3.153; P = 0.013) as significant risk factors for adverse events. ConclusionIndividualized antiplatelet therapy based on TEG-PM results can reduce the risk of ischemic events in patients with non-cardiogenic ischemic stroke without increasing the risk of bleeding events or mortality. Advanced age and coronary heart disease were identified as risk factors affecting the outcomes of individualized antiplatelet therapy.

Factors that modulate platelet reactivity as measured by 5 assay platforms in 3429 individuals

BackgroundAssessment of platelet function is key in diagnosing bleeding disorders and evaluating antiplatelet drug efficacy. However, there is a prevailing “one-size-fits-all” approach in the interpretation of measures of platelet reactivity, with arbitrary cutoffs often derived from healthy volunteer responses. ObjectivesOur aim was to compare well-used platelet reactivity assays. MethodsBlood and platelet-rich plasma obtained from the Framingham Heart Study (N = 3429) were assayed using a range of agonists in 5 platelet assays: light transmission aggregometry, Optimul aggregometry, Multiplate impedance aggregometry, Total Thrombus-Formation Analysis System, and flow cytometry. Using linear mixed-effect models, we determined the contribution of preanalytical and technical factors that modulated platelet reactivity traits. ResultsA strong intra-assay correlation of platelet traits was seen in all assays, particularly Multiplate velocity (r = 0.740; ristocetin vs arachidonic acid). In contrast, only moderate interassay correlations were observed (r = 0.375; adenosine diphosphate Optimul Emax vs light transmission aggregometry high area under the curve). As expected, antiplatelet drugs strongly reduced platelet responses, with aspirin use primarily targeting arachidonic acid–induced aggregation, and explained substantial variance (β = −1.735; P = 4.59 × 10−780; variance proportion = 46.2%) and P2Y12 antagonists blocking adenosine diphosphate responses (β = −1.612; P = 6.75 × 10−27; variance proportion = 2.1%). Notably, female sex and older age were associated with enhanced platelet reactivity. Fasting status and deviations from standard venipuncture practices did not alter platelet reactivity significantly. Finally, the agonist batch, phlebotomist, and assay technician (more so for assays that require additional sample manipulation) had a moderate to large effect on measured platelet reactivity. ConclusionCaution must be exercised when extrapolating findings between assays, and the use of standard ranges must be medication-specific and sex-specific at a minimum. Researchers should also consider preanalytical and technical variables when designing experiments and interpreting platelet reactivity measures.

Evolutionary trajectory of TRPM2 channel activation by adenosine diphosphate ribose and calcium

Ion channel activation upon ligand gating triggers a myriad of biological events and, therefore, evolution of ligand gating mechanism is of fundamental importance. TRPM2, a typical ancient ion channel, is activated by adenosine diphosphate ribose (ADPR) and calcium and its activation has evolved from a simple mode in invertebrates to a more complex one in vertebrates, but the evolutionary process is still unknown. Molecular evolutionary analysis of TRPM2s from more than 280 different animal species has revealed that, the C-terminal NUDT9-H domain has evolved from an enzyme to a ligand binding site for activation, while the N-terminal MHR domain maintains a conserved ligand binding site. Calcium gating pattern has also evolved, from one Ca2+-binding site as in sea anemones to three sites as in human. Importantly, we identified a new group represented by olTRPM2, which has a novel gating mode and fills the missing link of the channel gating evolution. We conclude that the TRPM2 ligand binding or activation mode evolved through at least three identifiable stages in the past billion years from simple to complicated and coordinated. Such findings benefit the evolutionary investigations of other channels and proteins.

Thrombosis inhibited by Corydalis decumbens through regulating PI3K-Akt pathway

Ethnopharmacological relevanceCorydalis decumbens (Thunb.) Pers. was used as stasis-eliminating medicine traditionally to treat cardiovascular disease potentially attributed to its antithrombotic effect, but lack of pharmacological research on it. Aim of the studyTo investigate the antithrombotic effect of C. decumbens and its preliminary mechanism. Materials and methodsA carrageenan-induced mouse thrombus model and adenosine diphosphate stimulated platelet aggregation of rabbits were used to confirm the inhibitory effect of C. decumbens extract and compounds on thrombosis in vivo. Then, H2O2-induced human umbilical vein endothelial cells (HUVECs) injury model was further adopted to verify the effects of bioactive compounds in vitro. Moreover, in silico network pharmacology analyses and molecular docking were performed to predict the underlying mechanisms, targets, and pathways, and which were further confirmed through western blotting assay. ResultsThe administration of total extract (TE), total alkaloids (TA) and TET (tetrahydropalmatine) resulted in a significant reduction in black tail thrombus and congestion, along with a decreasing in platelet aggregation of rabbits. A superior antithrombotic effect indicated the bioactive fraction, and then the isolated bioactive compounds, TET and protopine (PRO) increased cell survival, and decreased reactive oxygen species (ROS) and lactate dehydrogenase (LDH) release in H2O2-induced HUVECs injury model. Moreover, the two alkaloids targeted 33 major proteins and influenced 153 pathways in network pharmacology prediction. Among these, HSP90AA1, COX-2, NF-κB/p65, MMP1 and HIF-1α were the key proteins and PI3K-Akt emerged as the major signaling pathway. Further western blotting results supported that five key proteins were downregulated by the two bioactive compounds in H2O2-stimulated HUVECs model. ConclusionC. decumbens exerted protective effect on thrombosis through inhibiting PI3K-Akt pathway and related key proteins, which supported the traditional use and presented potential antithrombotic alkaloids for further investigation.

Ex Vivo Antiplatelet Effects of Oral Anticoagulants.

The impact of non-vitamin K antagonist oral anticoagulants (NOACs) on platelet function is still unclear. We conducted a comprehensive ex vivo study aimed at assessing the effect of the four currently marketed NOACs on platelet function. We incubated blood samples from healthy donors with concentrations of NOACs (50, 150 and 250 ng/mL), in the range of those achieved in the plasma of patients during therapy. We evaluated generation of thrombin; light transmittance platelet aggregation (LTA) in response to adenosine diphosphate (ADP), thrombin receptor-activating peptide (TRAP), human γ-thrombin (THR) and tissue factor (TF); generation of thromboxane (TX)B2; and expression of protease-activated receptor (PAR)-1 and P-selectin on the platelet surface. All NOACs concentration-dependently reduced thrombin generation compared with control. THR-induced LTA was suppressed by the addition of dabigatran at any concentration, while TF-induced LTA was reduced by factor-Xa inhibitors. ADP- and TRAP-induced LTA was not modified by NOACs. TXB2 generation was reduced by all NOACs, particularly at the highest concentrations. We found a concentration-dependent increase in PAR-1 expression after incubation with dabigatran, mainly at the highest concentrations, but not with FXa inhibitors; P-selectin expression was not changed by any drugs. Treatment with the NOACs is associated with measurable ex vivo changes in platelet function, arguing for antiplatelet effects beyond the well-known anticoagulant activities of these drugs. There are differences, however, among the NOACs, especially between dabigatran and the FXa inhibitors.

Differential effects of physiological agonists on the proteome of platelet-derived extracellular vesicles.

Arterial thrombosis manifesting as heart attack and stroke is the leading cause of death worldwide. Platelets are central mediators of thrombosis that can be activated through multiple activation pathways. Platelet-derived extracellular vesicles (pEVs), also known as platelet-derived microparticles, are granular mixtures of membrane structures produced by platelets in response to various activating stimuli. Initial studies have attracted interest on how platelet agonists influence the composition of the pEV proteome. In the current study, we used physiological platelet agonists of varying potencies which reflect the microenvironments that platelets experience during thrombus formation: adenosine diphosphate, collagen, thrombin as well as a combination of thrombin/collagen to induce platelet activation and pEV generation. Proteomic profiling revealed that pEVs have an agonist-dependent altered proteome in comparison to their cells of origin, activated platelets. Furthermore, we found that various protein classes including those related to coagulation and complement (prothrombin, antithrombin, and plasminogen) and platelet activation (fibrinogen) are attributed to platelet EVs following agonist stimulation. This agonist-dependent altered proteome suggests that protein packaging is an active process that appears to occur without de novo protein synthesis. This study provides new information on the influence of physiological agonist stimuli on the biogenesis and proteome landscape of pEVs.

Multidimensional engineering of Rhodobacter sphaeroides for enhanced photo-fermentative hydrogen production

The photo-fermentative hydrogen production by purple non-sulfur photosynthetic bacteria (PNSB) is regarded as a promising approach for sustainable hydrogen production. In PNSB, biohydrogen synthesis is catalyzed by nitrogenase which requires two essential cofactors of adenosine triphosphate (ATP) and electron. To avoid the insufficient ATP supply that limits the nitrogenase activity for hydrogen production, we systematically engineered Rhodobacter sphaeroides to improve the hydrogen production by manipulating the light harvesting (LH) complexes, the electron transfer chain (ETC), the adenosine diphosphate (ADP) synthetic pathway, the F0F1-ATPase expression, and the nitrogenase expression. To improve the photosynthetic efficiency, we engineered the LH complexes by eliminating pucBA and overexpressing pufQ and spbA. Then, the ETC was boosted by deleting qxt-type oxidase and integrating the electron carrier cytochrome cy at the site of cyt cbb3 oxidase. In addition, we increased the supply of precursor ADP by overexpressing adk, and the abundance of F0F1-ATPase was efficiently enhanced through overexpressing atpXF. Further transposon engineering with the integration of nifHDK and atpXF resulted in a mutant YH27 with a substantial improvement of performance over the base strain, and its nitrogenase activity and hydrogen production were enhanced by 404 % and 93 %, respectively. Taken together, the multidimensional strategies enabled the engineered R. sphaeroides with a hydrogen productivity of 61.2 mL/(Lh) and a yield of 3.0 mol/mol acetate under the fed-batch condition, which represents one of the most efficient biohydrogen-producing strains.