A Multipathway Phosphopeptide Standard for Rapid Phosphoproteomics Assay Development.

Recent advances in methodology have made phosphopeptide analysis a tractable problem for many proteomics researchers. There are now a wide variety of robust and accessible enrichment strategies to generate phosphoproteomes while free or inexpensive software tools for quantitation and site localization have simplified phosphoproteome analysis workflow tremendously. As a research group under the Association for Biomolecular Resource Facilities umbrella, the Proteomics Standards Research Group has worked to develop a multipathway phosphopeptide standard based on a mixture of heavy-labeled phosphopeptides designed to enable researchers to rapidly develop assays. This mixture contains 131 mass spectrometry vetted phosphopeptides specifically chosen to cover as many known biologically interesting phosphosites as possible from seven different signaling networks: AMPK signaling, death and apoptosis signaling, ErbB signaling, insulin/insulin-like growth factor-1 signaling, mTOR signaling, PI3K/AKT signaling, and stress (p38/SAPK/JNK) signaling. Here, we describe a characterization of this mixture spiked into a HeLa tryptic digest stimulated with both epidermal growth factor and insulin-like growth factor-1 to activate the MAPK and PI3K/AKT/mTOR pathways. We further demonstrate a comparison of phosphoproteomic profiling of HeLa performed independently in five labs using this phosphopeptide mixture with data-independent acquisition. Despite different experimental and instrumentation processes, we found that labs could produce reproducible, harmonized datasets by reporting measurements as ratios to the standard, while intensity measurements showed lower consistency between labs even after normalization. Our results suggest that widely available, biologically relevant phosphopeptide standards can act as a quantitative "yardstick" across laboratories and sample preparations enabling experimental designs larger than a single laboratory can perform. Raw data files are publicly available in the MassIVE dataset MSV000090564.

NCCN Guidelines® Insights: Survivorship, Version 1.2023.

The NCCN Guidelines for Survivorship are intended to help healthcare professionals address the complex and varied needs of cancer survivors. The NCCN Guidelines provide screening, evaluation, and treatment recommendations for psychosocial and physical problems resulting from adult-onset cancer and its treatment; recommendations to help promote healthy behaviors and immunizations in survivors; and a framework for care coordination. These NCCN Guidelines Insights summarize recent guideline updates and panel discussions pertaining to sleep disorders, fatigue, and cognitive function in cancer survivors.

Automating UbiFast for High-throughput and Multiplexed Ubiquitin Enrichment.

Robust methods for deep-scale enrichment and site-specific identification of ubiquitylation sites are necessary for characterizing the myriad roles of protein ubiquitylation. To this end we previously developed UbiFast, a sensitive method for highly multiplexed ubiquitylation profiling where K-ϵ-GG peptides are enriched with anti-K-ε-GG antibody and labeled on-antibody with isobaric labeling reagents for sample multiplexing. Here, we present robotic automation of the UbiFast method using a magnetic bead-conjugated K-ε-GG antibody (mK-ε-GG) and a magnetic particle processor. We report the identification of ∼20,000 ubiquitylation sites from a TMT10-plex with 500 µg input per sample processed in ∼2 h. Automation of the UbiFast method greatly increased the number of identified and quantified ubiquitylation sites, improved reproducibility, and significantly reduced processing time. The automated method also significantly reduced variability across process replicates compared with the manual method. The workflow enables processing of up to 96 samples in a single day making it suitable to study ubiquitylation in large sample sets. Here we demonstrate the applicability of the method to profile small amounts of tissue using breast cancer patient-derived xenograft (PDX) tissue samples.

Novel Antibodies for the Simple and Efficient Enrichment of Native O-GlcNAc Modified Peptides.

Antibodies against posttranslational modifications (PTMs) such as lysine acetylation, ubiquitin remnants, or phosphotyrosine have resulted in significant advances in our understanding of the fundamental roles of these PTMs in biology. However, the roles of a number of PTMs remain largely unexplored due to the lack of robust enrichment reagents. The addition of N-acetylglucosamine to serine and threonine residues (O-GlcNAc) by the O-GlcNAc transferase (OGT) is a PTM implicated in numerous biological processes and disease states but with limited techniques for its study. Here, we evaluate a new mixture of anti-O-GlcNAc monoclonal antibodies for the immunoprecipitation of native O-GlcNAcylated peptides from cells and tissues. The anti-O-GlcNAc antibodies display good sensitivity and high specificity toward O-GlcNAc-modified peptides and do not recognize O-GalNAc or GlcNAc in extended glycans. Applying this antibody-based enrichment strategy to synaptosomes from mouse brain tissue samples, we identified over 1300 unique O-GlcNAc-modified peptides and over 1000 sites using just a fraction of sample preparation and instrument time required in other landmark investigations of O-GlcNAcylation. Our rapid and robust method greatly simplifies the analysis of O-GlcNAc signaling and will help to elucidate the role of this challenging PTM in health and disease.

Exploring the Microbiome-Wide Lysine Acetylation, Succinylation, and Propionylation in Human Gut Microbiota.

Lysine acylations are important post-translational modifications that are present in both eukaryotes and prokaryotes and regulate diverse cellular functions. Our knowledge of the microbiome lysine acylation remains limited due to the lack of efficient analytical and bioinformatics methods for complex microbial communities. Here, we show that the serial enrichment using motif antibodies successfully captures peptides containing lysine acetylation, propionylation, and succinylation from human gut microbiome samples. A new bioinformatic workflow consisting of an unrestricted database search confidently identified >60,000 acetylated, and ∼20,000 propionylated and succinylated gut microbial peptides. The characterization of these identified modification-specific metaproteomes, i.e., meta-PTMomes, demonstrates that lysine acylations are differentially distributed in microbial species with different metabolic capabilities. This study provides an analytical framework for the study of lysine acylations in the microbiome, which enables functional microbiome studies at the post-translational level.

Targeted Quantification of Peptides Using Miniature Mass Spectrometry.

Proteomics by mass spectrometry (MS) allows for the identification of amino acid/peptide sequences in complex mixtures. Peptide analysis and quantitation enables screening of protein biomarkers and targeted protein biomarker analysis for clinical applications. Whereas miniature mass spectrometers have primarily demonstrated point-of-care analyses with simple procedures aiming at drugs and lipids, it would be interesting to explore their potential in analyzing proteins and peptides. In this work, we adapted a miniature MS instrument for peptide analysis. A mass range as wide as 100-2000 m/z was achieved for obtaining peptide spectra using this instrument with dual linear ion traps. MS2 and MS3 can be performed to analyze a wide range of peptides. The parameters of pressure, electric potentials, and solution conditions were optimized to analyze peptides with molecular weights between 900 and 1800 Da. The amino acid sequences were identified using both beam-type and in-trap collision-induced dissociation, and the results were comparable to those obtained by a commercial quadrupole time-of-flight mass spectrometer. With product ion monitoring scan mode, peptide quantitation was performed with a limit of detection of 20 nM achieved for the Met peptide. The method developed has also been applied to the analysis of the trypsin-digested cell lysate of SKBR3 cells with a low expression level of the Met gene.

Measures of Effectiveness, Efficiency, and Quality of Telemedicine in the Management of Alcohol Abuse, Addiction, and Rehabilitation: Systematic Review.

BACKGROUND: More than 18 million Americans are currently suffering from alcohol use disorder (AUD): a compulsive behavior of alcohol use as a result of a chronic, relapsing brain disease. With alcohol-related injuries being one of the leading causes of preventable deaths, there is a dire need to find ways to assist those suffering from alcohol dependence. There still exists a gap in knowledge as to the potential of telemedicine in improving health outcomes for those patients suffering from AUD. OBJECTIVE: The purpose of this systematic review was to evaluate the measures of effectiveness, efficiency, and quality that result from the utilization of telemedicine in the management of alcohol abuse, addiction, and rehabilitation. METHODS: This review was conducted utilizing the Preferred Reporting Items for Systematic Reviews and Meta-Analyses guidelines. The articles used in this analysis were gathered using keywords inclusive of both telemedicine and alcohol abuse, which were then searched in the Cumulative Index to Nursing and Allied Health Literature, Cochrane, and MEDLINE (PubMed) databases. A total of 22 articles were chosen for analysis. RESULTS: The results indicated that telemedicine reduced alcohol consumption. Other common outcomes included reduced depression (4/35, 11%), increased patient satisfaction (3/35, 9%), increase in accessibility (3/35, 9%), increased quality of life (2/35, 6%), and decreased cost (1/35, 3%). Interventions included mobile health (11/22, 50%), electronic health (6/22, 27%), telephone (3/33, 14%), and 2-way video (2/22, 9%). Studies were conducted in 3 regions: the United States (13/22, 59%), the European Union (8/22, 36%), and Australia (1/22, 5%). CONCLUSIONS: Telemedicine was found to be an effective tool in reducing alcohol consumption and increasing patients' accessibility to health care services or health providers. The group of articles for analysis suggested that telemedicine may be effective in reducing health care costs and improving the patient's quality of life. Although telemedicine shows promise as an effective way to manage alcohol-related disorders, it should be further investigated before implementation.

Identification of Rpl29 as a major substrate of the lysine methyltransferase Set7/9.

Set7/9 (also known as Set7, Set9, Setd7, and Kmt7) is a lysine methyltransferase that catalyzes the methylation of multiple substrates, including histone H3 and non-histone proteins. Although not essential for normal development and physiology, Set7/9-mediated methylation events play important roles in regulating cellular pathways involved in various human diseases, making Set7/9 a promising therapeutic target. Multiple Set7/9 inhibitors have been developed, which exhibit varying degrees of potency and selectivity in vitro However, validation of these compounds in vivo has been hampered by the lack of a reliable cellular biomarker for Set7/9 activity. Here, we report the identification of Rpl29, a ribosomal protein abundantly expressed in all cell types, as a major substrate of Set7/9. We show that Rpl29 lysine 5 (Rpl29K5) is methylated exclusively by Set7/9 and can be demethylated by Lsd1 (also known as Kdm1a). Rpl29 is not a core component of the ribosome translational machinery and plays a regulatory role in translation efficiency. Our results indicate that Rpl29 methylation has no effect on global protein synthesis but affects Rpl29 subcellular localization. Using an Rpl29 methylation-specific antibody, we demonstrate that Rpl29K5 methylation is present ubiquitously and validate that (R)-PFI-2, a Set7/9 inhibitor, efficiently reduces Rpl29K5 methylation in cell lines. Thus, Rpl29 methylation can serve as a specific cellular biomarker for measuring Set7/9 activity.

Quantitative Profiling of Post-translational Modifications by Immunoaffinity Enrichment and LC-MS/MS in Cancer Serum without Immunodepletion.

A robust method was developed and optimized for enrichment and quantitative analysis of posttranslational modifications (PTMs) in serum/plasma samples by combining immunoaffinity purification and LC-MS/MS without depletion of abundant proteins. The method was used to survey serum samples of patients with acute myeloid leukemia (AML), breast cancer (BC), and nonsmall cell lung cancer (NSCLC). Peptides were identified from serum samples containing phosphorylation, acetylation, lysine methylation, and arginine methylation. Of the PTMs identified, lysine acetylation (AcK) and arginine mono-methylation (Rme) were more prevalent than other PTMs. Label-free quantitative analysis of AcK and Rme peptides was performed for sera from AML, BC, and NSCLC patients. Several AcK and Rme sites showed distinct abundance distribution patterns across the three cancer types. The identification and quantification of posttranslationally modified peptides in serum samples reported here can be used for patient profiling and biomarker discovery research.

A Community-Based "Street Team" Tobacco Cessation Intervention by and for Youth and Young Adults.

Most tobacco users initiate use as youth or young adults. To promote tobacco cessation for this group and encourage non-users' engagement in tobacco control efforts, a community-based organization developed a "Street Team" brief outreach intervention that enlisted youth and young adults to encourage their peers to stop tobacco use through a brief intervention. Street Team members provided education, a Quit Kit, and referrals to cessation resources at a total of 27 community events over a four-year period. Tobacco users (n = 279) completed assessments of tobacco use, quit intention, and quit self-efficacy at baseline. Self-reports of cessation outcomes including past week abstinence were assessed 1-, 3-, and 6-months post-intervention. Perceptions of the intervention were gathered from Street Team members (n = 28) and intervention participants post-intervention. T-tests and χ2-tests were used to compare those who completed at least one follow-up assessment to those lost to follow-up. Time effects were analyzed using fixed effect models. Missing = using analyses indicate 16.1, 18.6, and 12.5% 7-day quit rate at 1-, 3-, and 6-months follow-up. Feedback from intervention participants indicate the intervention was acceptable and that discussions with Street Team members and provision of quit kits motivated tobacco users to consider quitting. All Street Team members responded positively to their participation in the intervention. This Street Team approach for youth and young adults is promising as an effective approach to the promotion of tobacco cessation among users and engagement and empowerment in tobacco control efforts among non-users.

Immunoaffinity enrichment and mass spectrometry analysis of protein methylation.

Protein methylation is a common posttranslational modification that mostly occurs on arginine and lysine residues. Arginine methylation has been reported to regulate RNA processing, gene transcription, DNA damage repair, protein translocation, and signal transduction. Lysine methylation is best known to regulate histone function and is involved in epigenetic regulation of gene transcription. To better study protein methylation, we have developed highly specific antibodies against monomethyl arginine; asymmetric dimethyl arginine; and monomethyl, dimethyl, and trimethyl lysine motifs. These antibodies were used to perform immunoaffinity purification of methyl peptides followed by LC-MS/MS analysis to identify and quantify arginine and lysine methylation sites in several model studies. Overall, we identified over 1000 arginine methylation sites in human cell line and mouse tissues, and ∼160 lysine methylation sites in human cell line HCT116. The number of methylation sites identified in this study exceeds those found in the literature to date. Detailed analysis of arginine-methylated proteins observed in mouse brain compared with those found in mouse embryo shows a tissue-specific distribution of arginine methylation, and extends the types of proteins that are known to be arginine methylated to include many new protein types. Many arginine-methylated proteins that we identified from the brain, including receptors, ion channels, transporters, and vesicle proteins, are involved in synaptic transmission, whereas the most abundant methylated proteins identified from mouse embryo are transcriptional regulators and RNA processing proteins.

Death or neurodevelopmental impairment at 18 to 22 months corrected age in a randomized trial of early dexamethasone to prevent death or chronic lung disease in extremely low birth weight infants.

OBJECTIVE: To evaluate the incidence of death or neurodevelopmental impairment (NDI) at 18-22 months corrected age in subjects enrolled in a trial of early dexamethasone treatment to prevent death or chronic lung disease in extremely low birth weight infants. STUDY DESIGN: Evaluation of infants at 18-22 months corrected age included anthropomorphic measurements, a standard neurological examination, and the Bayley Scales of Infant Development-II, including the Mental Developmental Index and the Psychomotor Developmental Index. NDI was defined as moderate or severe cerebral palsy, Mental Developmental Index or Psychomotor Developmental Index <70, blindness, or hearing impairment. RESULTS: Death or NDI at 18-22 months corrected age was similar in the dexamethasone and placebo groups (65% vs 66%, P = .99 among those with known outcome). The proportion of survivors with NDI was also similar, as were mean values for weight, length, and head circumference and the proportion of infants with poor growth (50% vs 41%, P = .42 for weight less than 10th percentile); 49% of infants in the placebo group received treatment with corticosteroid compared with 32% in the dexamethasone group (P = .02). CONCLUSION: The risk of death or NDI and rate of poor growth were high but similar in the dexamethasone and placebo groups. The lack of a discernible effect of early dexamethasone on neurodevelopmental outcome may be due to frequent clinical corticosteroid use in the placebo group.

Activating alleles of JAK3 in acute megakaryoblastic leukemia.

Tyrosine kinases are aberrantly activated in numerous malignancies, including acute myeloid leukemia (AML). To identify tyrosine kinases activated in AML, we developed a screening strategy that rapidly identifies tyrosine-phosphorylated proteins using mass spectrometry. This allowed the identification of an activating mutation (A572V) in the JAK3 pseudokinase domain in the acute megakaryoblastic leukemia (AMKL) cell line CMK. Subsequent analysis identified two additional JAK3 alleles, V722I and P132T, in AMKL patients. JAK3(A572V), JAK3(V722I), and JAK3(P132T) each transform Ba/F3 cells to factor-independent growth, and JAK3(A572V) confers features of megakaryoblastic leukemia in a murine model. These findings illustrate the biological importance of gain-of-function JAK3 mutations in leukemogenesis and demonstrate the utility of proteomic approaches to identifying clinically relevant mutations.

PTMScan direct: identification and quantification of peptides from critical signaling proteins by immunoaffinity enrichment coupled with LC-MS/MS.

Proteomic studies of post-translational modifications by metal affinity or antibody-based methods often employ data-dependent analysis, providing rich data sets that consist of randomly sampled identified peptides because of the dynamic response of the mass spectrometer. This can complicate the primary goal of programs for drug development, mutational analysis, and kinase profiling studies, which is to monitor how multiple nodes of known, critical signaling pathways are affected by a variety of treatment conditions. Cell Signaling Technology has developed an immunoaffinity-based LC-MS/MS method called PTMScan Direct for multiplexed analysis of these important signaling proteins. PTMScan Direct enables the identification and quantification of hundreds of peptides derived from specific proteins in signaling pathways or specific protein types. Cell lines, tissues, or xenografts can be used as starting material. PTMScan Direct is compatible with both SILAC and label-free quantification. Current PTMScan Direct reagents target key nodes of many signaling pathways (PTMScan Direct: Multipathway), serine/threonine kinases, tyrosine kinases, and the Akt/PI3K pathway. Validation of each reagent includes score filtering of MS/MS assignments, filtering by identification of peptides derived from expected targets, identification of peptides homologous to expected targets, minimum signal intensity of peptide ions, and dependence upon the presence of the reagent itself compared with a negative control. The Multipathway reagent was used to study sensitivity of human cancer cell lines to receptor tyrosine kinase inhibitors and showed consistent results with previously published studies. The Ser/Thr kinase reagent was used to compare relative levels of kinase-derived phosphopeptides in mouse liver, brain, and embryo, showing tissue-specific activity of many kinases including Akt and PKC family members. PTMScan Direct will be a powerful quantitative method for elucidation of changes in signaling in a wide array of experimental systems, combining the specificity of traditional biochemical methods with the high number of data points and dynamic range of proteomic methods.

Multi-prong quality improvement approach for increasing mother's own milk use for very low birth weight infants.

OBJECTIVE: Evaluate a single center quality improvement (QI) collaborative designed to increase the provision of mother's own milk (MOM) at discharge to premature infants through evidence-based practices while targeting perinatal health disparities. DESIGN: This QI initiative was designed for preterm infants admitted to a single-center NICU within 24 h of life. Interventions were implemented between March 2022 and June 2022. MOM provision rates were compared between baseline (August 2021-February 2022), and after interventions (March 2022-December 2022). RESULTS: The percentage of mothers who discontinued pumping during the infant hospitalization decreased from 49% to 35% (p < 0.01). Infant discharge diet with MOM improved from 36% to 58% (p < 0.001). Pump frequency at two weeks increased from 4.0 ± 2.6 to 5.1 ± 2.4 (p = 0.026). CONCLUSION: Our collaborative increased the percentage of preterm infants receiving MOM at discharge and reduced the number of mothers who discontinue pumping during the NICU hospitalization.

Evaluation of the Effect of Lorlatinib on CYP2B6, CYP2C9, UGT, and P-Glycoprotein Substrates in Patients with Advanced Non-Small Cell Lung Cancer.

BACKGROUND AND OBJECTIVE: Lorlatinib is a tyrosine kinase inhibitor approved for the treatment of advanced anaplastic lymphoma kinase-positive non-small cell lung cancer. This study assessed the effect of steady-state lorlatinib on the metabolic enzymes cytochrome P450 (CYP) 2B6, CYP2C9, and uridine 5'-diphospho-glucuronosyltransferase (UGT) and the P-glycoprotein (P-gp) transporter. METHODS: Thirty-two patients received a single oral dose of a probe drug on Day - 2 to determine the pharmacokinetics of the probe drug alone. Starting on Day 1, patients received 100 mg oral lorlatinib daily. On Day 15, a single oral dose of the probe drug was administered concurrently with lorlatinib. Pharmacokinetic parameters for these probe substrates were assessed. RESULTS: Plasma exposures of all probe substrates were reduced by lorlatinib compared with the probe alone. The greatest reduction in area under the plasma concentration-time curve from time zero to infinity (AUC∞) and maximum (peak) plasma drug concentration (Cmax) (67% and 63% decrease, respectively) was observed with the P-gp probe substrate fexofenadine. Lorlatinib coadministration also decreased the AUC∞ and Cmax of bupropion (CYP2B6 probe substrate) by 25% and 27%, tolbutamide (CYP2C9 probe substrate) by 43% and 15%, and acetaminophen (UGT probe substrate) by 45% and 28%, respectively. CONCLUSIONS: Lorlatinib is a net moderate inducer of P-gp and a weak inducer of CYP2B6, CYP2C9, and UGT after steady state is achieved with daily dosing. Medications that are P-gp substrates with a narrow therapeutic window should be avoided in patients taking lorlatinib; no dose modifications are needed with substrates of CYP2B6, CYP2C9, or UGT. CLINICALTRIALS: gov: NCT01970865.

Radiation-induced loss of cell surface CD47 enhances immune-mediated clearance of human papillomavirus-positive cancer.

The increasing incidence of human papillomavirus (HPV) related oropharyngeal squamous cell carcinoma (OSSC) demands development of novel therapies. Despite presenting at a more advanced stage, HPV(+) oropharyngeal squamous cell carcinoma (OSCC) have a better prognosis than their HPV(-) counterparts. We have previously demonstrated that clearance of HPV(+) OSCC during treatment with radiation and chemotherapy requires an immune response which is likely responsible for the improved clinical outcomes. To further elucidate the mechanism of immune-mediated clearance, we asked whether radiation therapy induces tumor cell changes that allow the body to recognize and aid in tumor clearance. Here, we describe a radiation-induced change in tumor surface protein expression that is critical for immune-mediated clearance. Radiation therapy decreases surface expression of CD47, a self-marker. CD47 is frequently overexpressed in head and neck squamous cell carcinoma and radiation induces a decrease of CD47 in a dose-dependent manner. We show that both in vitro and in vivo tumor cell CD47 protein levels are restored over time after sublethal radiation exposure and that protein levels on adjacent, normal tissues remain unaffected. Furthermore, reduction of tumor cell CD47 increases phagocytosis of these cells by dendritic cells and leads to increased interferon gamma and granzyme production from mixed lymphocytes. Finally, decreasing tumor cell CD47 in combination with standard radiation and chemotherapy results in improved immune-mediated tumor clearance in vivo. These findings help define an important mechanism of radiation-related immune clearance and suggest that decreasing CD47 specifically on tumor cells may be a good therapeutic target for HPV related disease.

Subtherapeutic linezolid concentrations in a patient with morbid obesity and methicillin-resistant Staphylococcus aureus pneumonia: case report and review of the literature.

OBJECTIVE: To report a case of subtherapeutic linezolid concentrations in a patient with morbid obesity. CASE SUMMARY: A 34-year-old male with morbid obesity (265 kg, body mass index 82 kg/m(2)) was admitted for severe sepsis due to respiratory failure requiring emergent intubation and treatment of community-acquired pneumonia. Admission tracheal aspirate culture revealed methicillin-resistant Staphylococcus aureus (MRSA) for which vancomycin was prescribed. Therapy subsequently was changed to linezolid, because the patient's clinical status worsened, with significant hypoxia (partial pressure of arterial oxygen/fraction of inspired oxygen [PaO2/FiO2] ratio 145), increasing leukocytosis (white blood cell count from 10,800/µL on admission to 15,400/µL on hospital day 6), and persistent fever (38.3 °C). After 48 hours of linezolid monotherapy, the patient remained febrile with continued leukocytosis, worsening hypoxemia, and a persistently positive MRSA culture from a repeat endotracheal aspirate. Linezolid serum concentrations were obtained and vancomycin was reinstituted, after which the patient began to improve (afebrile, improving PaO2/FiO2 ratio, decreasing leukocytosis). On hospital day 12, the patient removed his endotracheal tube, and a sputum sample was obtained for culture. The patient's clinical status subsequently declined, prompting addition of cefepime to his antibiotic regimen. This sputum culture revealed not only MRSA, but also quinolone-resistant Escherichia coli. After completing treatment for both organisms the patient was discharged home. DISCUSSION: Limited data on linezolid dosing in the morbidly obese population show lower serum drug concentrations than those in nonobese patients, but no clinical failure has been reported when treating MRSA skin and soft tissue infections or MRSA tracheitis. In our patient, low steady-state linezolid serum concentrations (peak 4.13 µg/mL [reference 15-27] and trough 1.27 µg/mL [reference 2-9]) were thought to contribute to his poor clinical response. CONCLUSIONS: To our knowledge, this is the first report of subtherapeutic linezolid concentrations correlated with decreased clinical effectiveness when during treatment of MRSA pneumonia in a patient with morbid obesity.

Using Mitra sampling to support first-in-human pharmacokinetic evaluations for PF-07059013.

Aim: A sensitive and selective method for the determination of PF-07059013 in dried blood collected by Mitra™ tips was developed and qualified from 50 to 50,000 ng/ml. Materials & methods: PF-07059013 is isolated from 10 µl of human dried blood by extraction with methanol and analyzed by HPLC-MS/MS. Results & conclusions: In addition to routine validation elements, impact of hematocrit and Mitra tip's lot-to-lot variation on assay accuracy were evaluated. The qualified method was used in one clinical study with excellent performance. Correlation coefficient between blood concentrations obtained from liquid-incurred blood samples and dried-incurred blood samples is 0.95. Clinical Trial Registration: NCT04323124 (ClinicalTrials.gov).

The Association Between Facility Affiliations and Revenue Generation in Skilled Nursing Facilities - An Exploratory Study.

Background: Although hospitals have been the traditional setting for interventional and rehabilitative care, skilled nursing facilities (SNFs) can offer a high-quality and less costly alternative than hospitals. Unfortunately, the financial health of SNFs is often a matter of concern. To partially address these issues, SNF leaders have increased engagement in a number of affiliations to assist in improving quality and reducing operational costs, including Accountable Care Organizations (ACOs), Health Information Exchanges (HIEs), and participation in Bundled Payment for Care Improvement (BPCI) programs. What is not well understood is what impact these affiliations have on the financial viability of the host organizations. Given these factors, this study aims to identify what association, if any, exists between SNF affiliations and revenue generation. Methods: Data from calendar year 2022 for n=13,447 SNFs in the US were assessed using multivariate regression analysis. We evaluated two separate dependent measures of revenue generation capacity: net patient revenue per bed and net patient revenue per discharge and considered three unique facility affiliations including (1) ACOs, (2) HIEs, and (3) BPCI participants. Results: Six multivariable linear regressions revealed that ACO affiliation is negatively associated with revenue generation on both dependent measures, while HIE affiliation and BPCI participation reflected mixed results. Conclusion: A better understanding of the financial impact of SNFs' affiliations may prove insightful. By carefully considering the value of each affiliation, and how each is applicable to any given market, policymakers, funding agencies, and facility leaders may be able to better position SNFs for more sustainable financial performance in a challenging economic environment.