Digital Commons Network^™

Integrating Interferon Gamma Receptor Pathways, Antigenicity, And Immune Contexture As Predictors Of Immunotherapeutic Strategies For Mucosal Melanomas, Matilde Monti, Jinglun Li, Obi Griffith, Malachi Griffith, William Vermi, Et Al.

2020-Current year OA Pubs

BACKGROUND: Mucosal melanomas (MM) arise from mucosal melanocytes at various anatomical sites. These tumors are rare, highly aggressive, and often associated with poor outcomes. Current treatments, including immune checkpoint inhibitors, show limited efficacy in advanced disease. Compared with cutaneous melanomas, there is a lack of data on the immunogenicity and interferon (IFN)-γ sensitivity of MM. In this study, we examined these features in sino-nasal melanomas (SN-MM) cell lines and clinical samples using microscopy and functional genomics.

METHODS: The immune contexture of SN-MM was analyzed by immunohistochemistry on 48 tumor biopsies. RNA sequencing and mass spectrometry-based proteomic approaches were used to …

A New Chromosome-Level Genome Assembly For Western Painted Turtle Chrysemys Picta Bellii, A Model For Extreme Physiological Adaptations, Junhao Chen, John R Bermingham Jr, Patrick Minx, Milinn Kremitzky, Zhenguo Lin, Daniel E Warren

2020-Current year OA Pubs

The western painted turtle, Chrysemys picta bellii, has the greatest tolerance to anoxia of any tetrapod studied to date. These turtles reside in the northern United States and southern Canada, and survive months of anoxia while submerged in ice-locked ponds and bogs. Reference genomes provide an important resource for elucidating the molecular bases for such unique physiological traits. The initial reference genome for this species, published in 2013, is highly fragmented, thereby limiting downstream analyses and biological interpretation. We created a new and improved assembly by combining PacBio HiFi, 10 × Genomics Chromium, Hi-C sequence data and Bionano optical mapping …

Development Of A Sustainable Blended Food Aid Product Using Local Ingredients For Nutritionally Vulnerable Populations In Haiti, Sherlie Jean Louis Dulience, Rachel Zimmerman, Michelle Dorce, Ilana Seff, Andrea Spray Bulungu, Patricia Kohl, Carolyn Lesorogol, Bazelais Dulience, Lora Iannotti

2020-Current year OA Pubs

OBJECTIVES: This study aimed to: (1) identify optimal blended food recipe options using local flours; (2) assess behaviors, attitudes, and practices around the use of blended foods among nutritionally vulnerable groups in Haiti; and (3) evaluate the nutrient composition of prototype blended food product relative to nutrient requirements for vulnerable populations.

METHODS: Blended food recipes made from local flours were identified through matrix scoring and stakeholder consensus. Focus groups (n = 7) assessed behaviors and attitudes toward blended foods. Prototype recipes were selected based on matrix scoring, program participant feedback, and feasibility of bringing to scale. Nutrient composition of the …

Single-Center Experience Of Voclosporin Use For Pediatric Lupus Nephritis: A Case Series, Ran Hazan, Nazlican Tang Civilibal, Kevin Baszis, Tarin M Bigley

2020-Current year OA Pubs

No abstract provided.

Brd4-Mediated Er Membrane Contact Creates Functionally Distinct Mitochondrial Subtypes, Brandon Chen, Rachel M Guerra, David J Pagliarini, Et Al.

2020-Current year OA Pubs

Inter-organellar communication is critical for cellular metabolism. One of the most abundant inter-organellar interactions occurs at the endoplasmic reticulum and mitochondria contact sites (ERMCSs). However, an understanding of the mechanisms governing ERMCS regulation and their roles in cellular metabolism is limited by a lack of tools that permit temporal induction and reversal. Through screening approaches, we identified fedratinib, an FDA-approved drug that dramatically increases ERMCS abundance by inhibiting the epigenetic modifier BRD4. Fedratinib rapidly and reversibly modulates mitochondrial and ER morphology, induces a distinct ER-mitochondria envelopment structure, and alters metabolic homeostasis. Moreover, ERMCS modulation depends on mitochondrial electron transport chain …

Longitudinal Profiling Of Tumor And Immune Compartments Uncovers Patterns Of Dysregulation And Associations With Response In Multiple Myeloma, Denis J Ohlstrom, Reyka G Jayasinghe, Yizhe Song, Ravi Vij, Li Ding, Et Al.

2020-Current year OA Pubs

UNLABELLED: Multiple myeloma is a malignancy of clonally expanded plasma cells shaped by complex interactions with the immune microenvironment (IME). To investigate immune correlates of treatment response and disease progression, we conducted multi-omics profiling including CD138neg single-cell RNA sequencing of 243 bone marrow samples from 102 patients (631,226 cells) and CD138pos bulk RNA and whole-genome sequencing from 209 samples. In longitudinal analyses, interferon-γ signaling associated with markers of impaired T-cell memory after autologous stem cell transplant, whereas naïve B-cell abundance and immunoglobulin diversity correlated with improved progression-free survival (HR = 0.48; P = 2.3e-4). At disease progression, multiple myeloma cells …

An Acetylation-Dependent Switch Underlies Host Disease Tolerance During Streptococcal Infection, Sumit Kumar Paudel, Sowmya Gannavaram, Michael G Caparon, Wei Xu

2020-Current year OA Pubs

Building on our finding that Streptococcus pyogenes pyruvate dehydrogenase (PDH) suppresses host disease tolerance (DT) via short-chain fatty acid (SCFA)-mediated modulation of host acetyl-CoA and IL-10 levels, we characterize the global transcriptomic and epigenetic mechanisms underlying this immunometabolic manipulation. Combining new histological and ultrastructural analyses with an in-depth re-analysis of single-cell and bulk RNA-seq datasets to more comprehensively characterize the DT response, we show that PDH deficiency is associated with broad immunologic rewiring, characterized by intracellular bacterial containment within phagocytes, expansion of pro-resolving myeloid cells, and altered cell-cell communication. Metabolic analysis of ΔPdh-infected tissues revealed a shift away from acetyl-CoA …

Decreased Metallothionein-3 Expression In The Human Spinal Cord Is A Common Feature Of Amyotrophic Lateral Sclerosis And Multiple Sclerosis, Adam P Gunn, James B W Hilton, Soumya Mukherjee, Jeffrey R Liddell, Stephen W Mercer, Catriona A Mclean, Colin L Masters, Peter J Crouch, Blaine R Roberts

2020-Current year OA Pubs

Amyotrophic lateral sclerosis (ALS) and multiple sclerosis (MS) are devastating adult-onset neurological diseases with distinct pathological manifestations. Although growing evidence implicates copper dysregulation in both diseases, our understanding of this connection remains incomplete. In this study, we investigated metallothionein-3 (MT3), which is a key regulator of copper metabolism in the central nervous system (CNS), and its relationship to copper in the ALS and MS spinal cord. Using combined quantitative mass spectrometry approaches and immunohistochemical observations, we found a significant decrease in ALS and MS spinal cord MT3 levels compared to controls. This was correlated with levels of copper and MT3-copper …

Glucagon-Like Peptide-1 Receptor Agonists And Risk Of Substance Use Disorders Among Us Veterans With Type 2 Diabetes: Cohort Study, Miao Cai, Taeyoung Choi, Yan Xie, Ziyad Al-Aly

2020-Current year OA Pubs

OBJECTIVES: To investigate whether initiation of glucagon-like peptide-1 (GLP-1) receptor agonists is associated with both reduced risks of incident alcohol, cannabis, cocaine, nicotine, opioid, and other substance use disorders (SUDs) in people with no history of SUDs (protocol 1) and with reduced risk of SUD related adverse clinical outcomes among people with a pre-existing SUD (protocol 2).

DESIGN: Emulation of eight parallel, new user, active comparator target trials using electronic health records: seven trials for each incident SUD outcome (protocol 1) and one trial for adverse outcomes in people with pre-existing SUD (protocol 2).

SETTING: US Department of Veterans Affairs. …

Sodium Content Of Packaged Foods And Beverages In Nigeria: A Cross-Sectional Comparative Study With Who Global Sodium Benchmarks, Kenya, And South Africa, Adedayo E Ojo, Alexandra Jones, Kylie Howes, Vanessa Alfa, Anthony Orji, Oluwafemi A Stephen, Carla Pool, Alex Kibet, Fraser Taylor, Diederick E Grobbee, Bruce Neal, Mark D Huffman, Dike B Ojji, Sanne A E Peters

2020-Current year OA Pubs

High intake of processed foods, especially those with high sodium content, is a contributor to hypertension and cardiovascular disease. This study aimed to compare the sodium content of packaged foods and beverages in Nigeria to WHO Global Sodium Benchmarks and similar products in Kenya and South Africa. The study examined packaged foods from major retail stores in the capital cities of the Federal Capital Territory, Kano, and Ogun states in Nigeria from November 2020 to March 2021. Benchmark values were based on the 2021 WHO Global Sodium Benchmarks. We used secondary data from packaged food surveys conducted in South Africa …

A Neural Circuit Framework For Economic Choice: From Building Blocks Of Valuation To Compositionality In Multitasking, Aldo Battista, Camillo Padoa-Schioppa, Xiao-Jing Wang

2020-Current year OA Pubs

Value-guided decisions are a cornerstone of cognition, yet the underlying circuit-level mechanisms remain elusive. We used reinforcement learning to train recurrent neural network models endowed with Dale's law on a battery of economic choice tasks, which revealed a two-stage computational framework. First, value estimation occurs at the input level, where learned weights store subjective preferences and approximate the non-linear multiplication of reward magnitude and probability to yield expected values. This feedforward mechanism enables generalization to novel choice options. Second, option values are compared within the recurrent network, where specific connectivity patterns mediate robust winner-take-all decisions, with both excitatory and inhibitory …

Sex-Related Differences In Clinical Profile, Management, And Outcomes Of Patients With Type A And B Acute Aortic Dissection: Observations From Irad, Elizabeth Jackson, Andreina Carbone, Sherene Shalhub, Chih-Wen Pai, Marek Ehrlich, Toru Suzuki, G Chad Hughes, Anil Bhan, Rumi Faizer, Antonio Cittadini, Alan Braverman, Eric Isselbacher, Kim Eagle, Christoph Nienaber, Eduardo Bossone

2020-Current year OA Pubs

BACKGROUND: Available data about sex differences in patients with acute aortic dissection (AAD) are scant and conflicting. The aim of this study is to assess sex-related differences in clinical characteristics, presentation, diagnostic findings, management, and outcomes among patients with type A (TA-AAD) and type B AAD (TB-AAD).

METHODS: Data about 11 586 patients enrolled in the IRAD (International Registry of Acute Aortic Dissection) from 1996 to 2022, were analyzed; 7819 (67.5%) experienced TA-AAD and 3867 (33.0%) TB-AAD. Men and women were compared with regard to diagnosis, treatment, and in-hospital and long-term survival.

RESULTS: One third of patients were women (TA-AAD, …

Exploring The Role Of Social Networks In Youth Mental Health, Nicholas Szoko, Alexander Riley, Jacquelin Rankine, Alison J Culyba

2020-Current year OA Pubs

Social network analysis (SNA) offers an innovative approach to understand youths' complex social support systems. Growing work highlights links between youths' social networks and mental health. We used SNA to examine associations between social network characteristics and psychological empowerment, depression, anxiety, and post-traumatic stress among youth living in areas with concentrated disadvantage. We administered egocentric social network surveys in which youth (i.e., egos) enumerated individuals in their social network (i.e., alters), answered questions about each alter, and described how alters were connected. We measured psychological empowerment and mental health symptoms with validated instruments. There were 111 youth in the analytic …

The Relationship Between Apparent Potentiation And The Magnitude Of The Control Response, Joe Henry Steinbach, Gustav Akk

2020-Current year OA Pubs

The effect of a potentiating drug on ion channel function is typically evaluated by comparing current responses to the control agonist in the presence and absence of the potentiator. Differences in ratios of responses are then taken as proof of distinct potentiation properties when comparing modulation by different compounds. In these experiments, the concentration of the agonist is typically kept low to generate a small fractional control response. The precise relative magnitude of the control response is, however, not standardized among labs and can range from a concentration producing a response equal to just 2% of maximal (EC2) to over …

Cell Type-Specific Gene Regulatory Atlas Prioritizes Drug Targets And Repurposable Medicines In Alzheimer's Disease, Yunxiao Ren, Ming Hu, Yang E Li, Andrew A Pieper, Jeffrey Cummings, Feixiong Cheng

2020-Current year OA Pubs

Alzheimer's disease (AD) is a complex and poorly understood neurodegenerative disorder that lacks sufficiently effective treatments. Computational and integrative analyses that leverage multiomic data provide a promising strategy to uncover disease mechanisms and identify therapeutic opportunities. Here, we develop a cell type-specific regulatory atlas of the human middle temporal gyrus via leveraging single-nucleus RNA-seq (1,197,032 nuclei) and ATAC-seq (740,875 nuclei) data sets from 84 donors across four stages of AD neuropathological change (ADNC). We observe differential gene expression for six major cell types intensified at severe ADNC. Integrating peak-to-gene linkages and motif enrichment analyses, we reconstruct transcription factor (TF)-target gene …

Media Reporting Of The 2024 Us Preventive Services Task Force Mammography Guideline Update, Tamar Parmet, Yue Li, Joseph Cappella, Laura D Scherer, Erika A Waters, Ashley J Housten

2020-Current year OA Pubs

No abstract provided.

Building Capacity On Hypertension Management In Nigeria, Shivani Mishra, Daphne Lew, Et Al.

2020-Current year OA Pubs

No abstract provided.

Shiga Toxin-Producing Escherichia Coli Outbreak In Canadian Daycare Centers, Mohamed Eltorki, Oluwatimilehin O Ajayi, Jina Seok, Jianling Xie, Francesco A Rizzuti, Byron M Berenger, Phillip I Tarr, Andrew T Pavia, Kate Snedeker, Silviu Grisaru, Otto G Vanderkooi, Linda Chui, Gillian A M Tarr, Gemma Vomiero, Stephen B Freedman

2020-Current year OA Pubs

IMPORTANCE: Shiga toxin-producing Escherichia coli (STEC) infections have significant morbidity, with 15% to 20% of children developing hemolytic uremic syndrome (HUS), more than half of whom require kidney replacement therapy (KRT).

OBJECTIVE: To describe health care resource utilization, clinical outcomes, and outcomes associated with daily laboratory monitoring for thrombotic microangiopathy (TMA) in a large point-source STEC outbreak.

DESIGN, SETTING, AND PARTICIPANTS: This retrospective cohort study was conducted among a population-based cohort of individuals exposed to a contaminated food item served in 11 childcare centers in Calgary, Alberta, Canada, on August 29, 2023. Children and adults with confirmed STEC infection linked …

Trends In Mammography Use Among Women Aged 40 To 74 Years In The Us, 2002-2022, Syed Mahfuz Al Hasan, Debbie L Bennett, Adetunji T Toriola

2020-Current year OA Pubs

IMPORTANCE: Understanding how prior policies, systemic factors, and public health events have shaped mammography use among US women is essential for interpreting the 2024 US Preventive Services Task Force (USPSTF) recommendation update and identifying gaps to inform equitable public health strategies.

OBJECTIVE: To analyze mammography use trends in US women aged 40 to 49 and 50 to 74 years from 2002 to 2022 and assess variation by sociodemographic, health care access, behavioral, and geographic factors, including associations with the 2009 USPSTF recommendation and the COVID-19 pandemic.

DESIGN, SETTING, AND PARTICIPANTS: This cross-sectional study used data from the Behavioral Risk Factor …

Single Zonular Fiber Extraction And Characterization: A Platform For Biomechanical, Biochemical And Structural Analyses, Riley Forinash, Sean Staggs, Steven Bassnett, Juan Rodriguez

2020-Current year OA Pubs

Zonular fibers are essential for suspending the lens within the eye and, in primates, for transmitting forces generated by the ciliary body to drive ocular accommodation. Despite their biomechanical importance, the response of these fibers to sudden strains, and the molecular basis of their exceptional durability remain poorly understood. This is due, in part, to the absence of tools for studying them in isolation. Here, we present a custom-built, cost-effective apparatus designed to isolate, manipulate, and characterize individual zonular fibers under controlled physical and chemical conditions. The apparatus enables quantification of elastic moduli, stress-relaxation behavior, Poisson's ratio, and response to …

Parkinson's Disease As A Somato-Cognitive Action Network Disorder, Jianxun Ren, Evan M Gordon, Nico U F Dosenbach, Et Al.

2020-Current year OA Pubs

Parkinson's disease (PD) is an incurable neurological disorder that often begins insidiously with sleep disturbances and somatic symptoms, progressing to whole-body motor and cognitive symptoms

Accuracy And Reliability Of Artery-Vein Differentiation In Small-Field Macular Oct Angiography, Haneen Alfauri, Tugce Ilayda Turer, Cyriac Manjaly, Aditya Santoki, Senyue Hao, Marin Woronets, Chao Zhou, Rithwick Rajagopal

2020-Current year OA Pubs

PURPOSE: Accurate artery-vein (AV) differentiation in small-field macular optical coherence tomography angiography (OCTA) remains challenging due to a lack of standardized guidelines. We propose and validate criteria for

APPROACH: Small field-of-view (FOV) OCTA scans were analyzed using established AV criteria for large-field (

RESULTS: A total of 465 vessels from 20 healthy eyes were evaluated across 3 pseudo-branching orders using the criteria for OCTA. Annotators achieved high accuracy (95.1%, 92.3%) and strong intra/inter-rater reliability (

CONCLUSIONS: Accurate and reproducible AV labeling is feasible in

Cdk4/6 Inhibition Mitigates Chemotherapy-Induced Expansion Of Tp53-Mutant Clonal Hematopoiesis, Irenaeus C C Chan, Giulia Petrone, J Scott Beeler, Duc Tran, Griffen Mustion, Catrina Fronick, Carlos Cruchaga, Kelly L Bolton, Et Al.

2020-Current year OA Pubs

Therapy-related myeloid neoplasm (tMN) is a fatal consequence of exposure to cytotoxic therapy administered in the treatment of cancer. Individuals with pre-existing TP53 clonal hematopoiesis (CH) are at high risk of tMN, with avoidance of therapy being the only strategy to reduce tMN risk. Here, in four randomized clinical trials, we show that the CDK4/6 inhibitor trilaciclib, given in conjunction with a variety of chemotherapeutic regimens and across diverse populations of patients with cancer, mitigates chemotherapy-related expansion of CH clones with mutations in DNA damage response genes, including TP53. This finding was also observed in a syngeneic mouse model of …

Cholecalciferol (Vitamin D3) Is An Agonist Of The Alzheimer's Disease-Associated Immune Receptor Trem2, Hunter B Dean, Ryan A Tuckey, Rory A Greer, Jessica A Greven, Shan-Zhong Yang, Daniel S Elston, Gunnar N Eastep, Yuwei Song, Thomas J Brett, Yuhua Song, Erik D Roberson

2020-Current year OA Pubs

Triggering Receptor Expressed on Myeloid Cells 2 (TREM2) is one of the strongest genetic risk factors for late-onset Alzheimer's disease (AD). Several TREM2 ligands are known, including charged lipids and AD-associated proteins like apolipoprotein E and amyloid-β, but the full range of endogenous ligands for TREM2 remains unknown. Here we combined virtual screening of the Human Metabolome Database with molecular dynamics simulations, binding free energy estimation, and biolayer interferometry to identify novel TREM2 ligands and map their binding sites. Cholecalciferol, the unmodified parent form of vitamin D3, emerged as a top candidate. Structural modeling indicated that cholecalciferol binds TREM2 at …

Aging Reduces Motivation Through Decreased Bdnf Expression In The Ventral Tegmental Area, Hanyue Cecilia Lei, Kyle E Parker, Chao-Cheng Kuo, Carla M Yuede, Jordan G Mccall, Shin-Ichiro Imai

2020-Current year OA Pubs

Age-associated reduced motivation is a hallmark of neuropsychiatric disorders in the elderly. In our rapidly aging societies, it is critical to keep motivation levels high enough to promote healthspan and lifespan. However, how motivation is reduced during aging remains unknown. Here, we used multiple mouse models to evaluate motivation and related affective states in young and old mice. We also compared the effect of social isolation, a common stressor in aged populations, to those of aging. We found that both social isolation and aging decreased motivation in mice, but that Bdnf expression in the ventral tegmental area (VTA) was selectively …

Finding Significant Hits In Networks: A Network-Based Tool For Analyzing Gene-Level P-Values To Identify Significant Genes Missed By Standard Methods, Sandeep Acharya, Vaha Akbary Moghaddam, Wooseok J Jung, Yu S Kang, Shu Liao, Michael A Province, Michael R Brent

2020-Current year OA Pubs

Finding Significant Hits in Networks (FISHNET) uses prior biological knowledge, represented as gene interaction networks and gene function annotations, to identify genes that do not meet the genome-wide significance threshold but replicate, nonetheless. Its input is gene-level P-values from any source, including omicsWAS, aggregation of genome-wide association studies P-values, CRISPR screens, or differential expression analysis. It is based on the idea that genes whose P-values are low purely by chance are distributed randomly across networks and functions, so genes with suggestive P-values that cluster in densely connected subnetworks and share common functions are less likely to reflect chance and more …

Emerging Options And Future Strategies In Immunotherapy For Advanced Lung Squamous-Cell Carcinoma, Y Wang, R Rosell, F R Hirsch, S Lu, R Govindan, K Park, S Peters, J J Zhang

2020-Current year OA Pubs

Advanced-stage lung squamous-cell carcinoma (LUSC) remains a therapeutic challenge. Although immune checkpoint inhibitors (ICIs) have revolutionized LUSC treatment, only a small subset of LUSC patients can achieve durable clinical benefits, underscoring the need for novel strategies. Emerging immunotherapy strategies have demonstrated early evidence of promise for LUSC patients, particularly multi-specific antibodies and fusion proteins, antibody-drug conjugates, adoptive cell therapies, and cancer vaccines. Additionally, advances in multi-omics and artificial intelligence offer potential for precise immunotherapy by deciphering the complex tumor-immune microenvironment and predicting therapeutic responses. The future of LUSC treatment lies in rational multi-target and biological therapies to boost immune cytotoxicity, …

Signal Peptidase Complex Mediates Rotavirus Vp7 Processing And Virion Assembly, Xuejiao Zhu, Enkai Li, Liliana Sanchez-Tacuba, Wandy Beatty, Bin Li, Siyuan Ding

2020-Current year OA Pubs

For viruses that replicate in the proximity of or bud at the endoplasmic reticulum (ER) associated membranes, proper processing of their glycoproteins is critical for successful infection. Rotavirus outer capsid protein VP7 is an ER-resident protein. However, its N-terminal signal peptide is removed by an unknown proteolytic mechanism. In this study, we leveraged tandem affinity purification followed by high-resolution mass spectrometry to profile host proteins that interact with VP7. We identified members of the signal peptidase complex (SPC) family as important host factors that facilitate rotavirus infection. CRISPR knockout or siRNA knockdown of distinct SPC subunits resulted in significant decrease …

Active Bone Marrow Dose Reconstruction In A Large-Scale Cohort Study Of Cancer Patients Treated With Photon Radiotherapy, Keith T Griffin, Kishan J Pithadia, Yeon Soo Yeom, Lior Braunstein, Kelly L Bolton, Lindsay M Morton, Choonsik Lee

2020-Current year OA Pubs

BACKGROUND: Previous studies have investigated the dose-response relationship between external beam radiotherapy (EBRT) and leukemia by reconstructing the mean active bone marrow (ABM) dose as part of the exposure assessment. However, no prior study has leveraged electronic medical records (EMR) to reconstruct ABM dose and dose-volume in an EBRT patient population.

PURPOSE: To support future studies on the relationship between radiation exposure and adverse health effects among EBRT patients, we demonstrate methods to retrospectively calculate ABM dose and dose-volume metrics in a large patient cohort using EMR.

METHODS: We retrieved complete EMR for 639 individuals (five pediatric and 634 adult) …

Intraocular Amyloidosis Presenting With Secondary Glaucoma: A Clinical-Pathologic Report And Literature Review, Joseph B Lin, James C Liu, Rajendra S Apte, George J Harocopos

2020-Current year OA Pubs

PURPOSE: We describe a case of intraocular amyloidosis presenting as secondary glaucoma, highlighting the clinical presentation and pathologic correlates. We also review the medical literature on all prior reported cases and discuss the diagnostic challenges pertinent to this particular case.

OBSERVATIONS: We report the case of a 72-year-old man presenting with medically intractable elevations of intraocular pressure. On clinical examination, the anterior segment was unremarkable, but there were significant vitreous opacities. The patient underwent tube shunt surgery, and a simultaneous aqueous humor biopsy was acellular and nondiagnostic. A subsequent pars plana vitrectomy demonstrated amyloid aggregates on histology. Mass spectrometric analysis …