Ultrasound-Guided Percutaneous Cryoneurolysis of the Antebrachial Cutaneous Nerves for the Management of Prolonged Acute Post-Surgical Forearm Pain: A Report of Two Cases.

Prolonged acute postsurgical pain (PAPSP) contributes to the development of chronic postsurgical pain, impaired rehabilitation, longer hospital stays, and decreased quality of life. For upper extremity analgesia, the duration of postoperative pain management with continuous brachial plexus peripheral nerve blocks is limited due to the risk of infection. Ultrasound-guided percutaneous cryoneurolysis provides extended analgesia and avoids the risks and inconveniences of indwelling catheters. We present 2 cases of PAPSP of the forearm effectively managed by the use of ultrasound-guided percutaneous cryoneurolysis to treat the medial, lateral, and posterior antebrachial cutaneous nerves.

Biomechanical strength of triceps tendon repairs: systematic review and meta-regression analysis of human cadaveric studies.

PURPOSE: It is unclear which triceps tendon repair constructs and techniques produce the strongest biomechanical performance while minimizing the risk of gap formation and repair failure. We aimed to determine associations of construct and technique variables with the biomechanical strength of triceps tendon repairs. PubMed, Embase, Cochrane Library, Web of Science, Scopus, and ClinicalTrials.gov were systematically searched for peer-reviewed studies on biomechanical strength of triceps tendon repairs in human cadavers. 6 articles met the search criteria. Meta-regression was performed on the pooled dataset (123 specimens). Outcomes of interest included gap formation, failure mode, and ultimate failure load. Covariates were fixation type; number of implants; and number of sutures. Stratification by covariates was performed. We found no association between fixation type and ultimate failure load; however, suture anchor fixation was associated with less gap formation compared with transosseous direct repair (ß = - 1.1; 95% confidence interval [CI]:- 2.2, - 0.04). A greater number of implants was associated with smaller gap formation (ß = - 0.77; 95% CI: - 1.3, - 0.28) while a greater number of sutures was associated with higher ultimate failure load ( ß= 3; 95% CI: 21, 125). In human cadaveric models, the number of sutures used in triceps tendon repairs may be more important than the fixation type or number of implants for overall strength. If using a transosseous direct repair approach to repair triceps tendon tears, surgeons may choose to use more sutures in their repair in order to balance the risk of larger gap formation when compared to indirect repair techniques. LEVEL OF EVIDENCE: Level III.

Expanding the adenovirus toolbox: reporter viruses for studying the dynamics of human adenovirus replication.

To streamline standard virological assays, we developed a suite of nine fluorescent or bioluminescent replication competent human species C5 adenovirus reporter viruses that mimic their parental wild-type counterpart. These reporter viruses provide a rapid and quantitative readout of various aspects of viral infection and replication based on EGFP, mCherry, or NanoLuc measurement. Moreover, they permit real-time non-invasive measures of viral load, replication dynamics, and infection kinetics over the entire course of infection, allowing measurements that were not previously possible. This suite of replication competent reporter viruses increases the ease, speed, and adaptability of standard assays and has the potential to accelerate multiple areas of human adenovirus research.IMPORTANCEIn this work, we developed a versatile toolbox of nine HAdV-C5 reporter viruses and validated their functions in cell culture. These reporter viruses provide a rapid and quantitative readout of various aspects of viral infection and replication based on EGFP, mCherry, or NanoLuc measurement. The utility of these reporter viruses could also be extended for use in 3D cell culture, organoids, live cell imaging, or animal models, and provides a conceptual framework for the development of new reporter viruses representing other clinically relevant HAdV species.

Up-regulated PLA2G10 in cancer impairs T cell infiltration to dampen immunity.

T cells are often absent from human cancer tissues during both spontaneously induced immunity and therapeutic immunotherapy, even in the presence of a functional T cell-recruiting chemokine system, suggesting the existence of T cell exclusion mechanisms that impair infiltration. Using a genome-wide in vitro screening platform, we identified a role for phospholipase A2 group 10 (PLA2G10) protein in T cell exclusion. PLA2G10 up-regulation is widespread in human cancers and is associated with poor T cell infiltration in tumor tissues. PLA2G10 overexpression in immunogenic mouse tumors excluded T cells from infiltration, resulting in resistance to anti-PD-1 immunotherapy. PLA2G10 can hydrolyze phospholipids into small lipid metabolites, thus inhibiting chemokine-mediated T cell mobility. Ablation of PLA2G10's enzymatic activity enhanced T cell infiltration and sensitized PLA2G10-overexpressing tumors to immunotherapies. Our study implicates a role for PLA2G10 in T cell exclusion from tumors and suggests a potential target for cancer immunotherapy.

The UK Divide: Does Having a Pembrolizumab-Chemotherapy Option in Head and Neck Cancer Matter? Real-world Experience of First-line Palliative Pembrolizumab Monotherapy and Pembrolizumab-Chemotherapy Combination in Scotland.

AIMS: The Scottish Medical Consortium recently approved first-line pembrolizumab monotherapy or in combination with chemotherapy for head and neck squamous cell carcinoma in the palliative setting, contrasting with the decision made by the National Institute for Health and Care Excellence, who approved monotherapy alone in England and Wales. The aim of this study was to provide real-world performance data for first-line pembrolizumab-containing treatments for head and neck squamous cell carcinoma in the palliative setting in Scotland. MATERIALS AND METHODS: We analysed the electronic records of patients who started pembrolizumab-containing treatment between 1 March 2020 and 30 September 2021. Outcomes included overall survival, progression-free survival (PFS), the duration of response and the disease control rate. Data were compared with the KEYNOTE-048 study and clinical factors were evaluated for association with survival. RESULTS: Our cohort included 91 patients (median follow-up 10.8 months). Patient characteristics were similar to those in the KEYNOTE-048 study, although our cohort had a higher proportion of patients with newly diagnosed, non-metastatic disease. For patients receiving monotherapy (n = 76), 12- and 24-month overall survival were 45% and 27%, respectively. For patients receiving pembrolizumab-chemotherapy (n = 15), 12-month overall survival was 60% (24-month overall survival had not yet been reached). Experiencing one or more immune-related adverse event (irAE; versus no irAEs), of any grade, was associated with favourable overall survival and PFS for patients receiving monotherapy in both univariable Log-rank analysis (median overall survival 17.4 months versus 8.6 months, respectively, P = 0.0033; median PFS 10.9 months versus 3.0 months, respectively, P < 0.0001) and multivariable analysis (Cox proportional hazards regression: overall survival hazard ratio 0.31, P = 0.0009; PFS hazard ratio 0.17, P < 0.0001). CONCLUSION: Our real-world data support the KEYNOTE-048 study findings and the value of combination treatment options. Additionally, our data show that irAEs of any grade, as reported in routine clinical records, are associated with better outcomes in this patient group, adding to the growing body of evidence showing that irAEs are generally a positive marker of programmed death-ligand 1 (PD-L1) inhibitor response.

From cup to dish: how to make and use endometrial organoid and stromal cultures derived from menstrual fluid.

Diseases impacting the female reproductive tract pose a critical health concern. The establishment of in vitro models to study primary endometrial cells is crucial to understanding the mechanisms that contribute to normal endometrial function and the origins of diseases. Established protocols for endometrial stromal cell culture have been in use for decades but recent advances in endometrial organoid culture have paved the way to allowing study of the roles of both epithelial and stromal endometrial cells in vitro. Due to inter-individual variability, primary cell cultures must be established from numerous persons. Generally, endometrial epithelial and stromal cells can be isolated from an endometrial biopsy, however, this is collected in a clinical setting by an invasive transcervical procedure. Our goal was to develop a non-invasive method for the isolation of paired endometrial epithelial organoids and stromal cells from menstrual fluid collected from individual women, based on recent reports describing the isolation of endometrial epithelial organoids or endometrial stromal cells from menstrual fluid. Participants recruited by the NIEHS Clinical Research Unit were provided with a menstrual cup and instructed to collect on the heaviest day of their menstrual period. Endometrial tissue fragments in the menstrual fluid samples were washed to remove blood, minced, and digested with proteinases. Following digestion, the solution was strained to separate epithelial fragments from stromal cells. Epithelial fragments were washed, resuspended in Matrigel, and plated for organoid formation. Stromal cells were separated from residual red blood cells using a Ficoll gradient and then plated in a flask. Once established, estrogen responsiveness of endometrial epithelial organoids was assessed and the decidual response of stromal cells was evaluated. Following treatments, qPCR was performed on organoids for genes induced by estradiol and on stromal cells for genes induced by decidualization. In this manner, the relative responsiveness of paired organoid and stroma cell cultures isolated from each woman could be assessed. In conclusion, we can isolate both epithelial and stromal cells from a single menstrual fluid sample, allowing us to establish organoids and cells in a paired manner. This protocol can greatly enhance our knowledge of the role of epithelial and stromal cells alone and in coordination.

Development of a targeted hydrophilic interaction liquid chromatography-tandem mass spectrometry based lipidomics platform applied to a coronavirus disease severity study.

The importance of lipids seen in studies of metabolism, cancer, the recent COVID-19 pandemic and other diseases has brought the field of lipidomics to the forefront of clinical research. Quantitative and comprehensive analysis is required to understand biological interactions among lipid species. However, lipidomic analysis is often challenging due to the various compositional structures, diverse physicochemical properties, and wide dynamic range of concentrations of lipids in biological systems. To study the comprehensive lipidome, a hydrophilic interaction liquid chromatography-tandem mass spectrometry (HILIC-MS/MS)-based screening method with 1200 lipid features across 19 (sub)classes, including both nonpolar and polar lipids, has been developed. HILIC-MS/MS was selected due to its class separation property and fatty acyl chain level information. 3D models of class chromatographic retention behavior were established and evaluations of cross-class and within-class interferences were performed to avoid over-reporting these features. This targeted HILIC-MS/MS method was fully validated, with acceptable analytical parameters in terms of linearity, precision, reproducibility, and recovery. The accurate quantitation of 608 lipid species in the SRM 1950 NIST plasma was achieved using multi-internal standards per class and post-hoc correction, extending current databases by providing lipid concentrations resolved at fatty acyl chain level. The overall correlation coefficients (R2) of measured concentrations with values from literature range from 0.64 to 0.84. The applicability of the developed targeted lipidomics method was demonstrated by discovering 520 differential lipid features related to COVID-19 severity. This high coverage and targeted approach will aid in future investigations of the lipidome in various disease contexts.

Identification of 2-Sulfonyl/Sulfonamide Pyrimidines as Covalent Inhibitors of WRN Using a Multiplexed High-Throughput Screening Assay.

Werner syndrome protein (WRN) is a multifunctional enzyme with helicase, ATPase, and exonuclease activities that are necessary for numerous DNA-related transactions in the human cell. Recent studies identified WRN as a synthetic lethal target in cancers characterized by genomic microsatellite instability resulting from defects in DNA mismatch repair pathways. WRN's helicase activity is essential for the viability of these high microsatellite instability (MSI-H) cancers and thus presents a therapeutic opportunity. To this end, we developed a multiplexed high-throughput screening assay that monitors exonuclease, ATPase, and helicase activities of full-length WRN. This screening campaign led to the discovery of 2-sulfonyl/sulfonamide pyrimidine derivatives as novel covalent inhibitors of WRN helicase activity. The compounds are specific for WRN versus other human RecQ family members and show competitive behavior with ATP. Examination of these novel chemical probes established the sulfonamide NH group as a key driver of compound potency. One of the leading compounds, H3B-960, showed consistent activities in a range of assays (IC50 = 22 nM, KD = 40 nM, KI = 32 nM), and the most potent compound identified, H3B-968, has inhibitory activity IC50 ∼ 10 nM. These kinetic properties trend toward other known covalent druglike molecules. Our work provides a new avenue for screening WRN for inhibitors that may be adaptable to different therapeutic modalities such as targeted protein degradation, as well as a proof of concept for the inhibition of WRN helicase activity by covalent molecules.

Dietary tryptophan metabolite released by intratumoral Lactobacillus reuteri facilitates immune checkpoint inhibitor treatment.

The use of probiotics by cancer patients is increasing, including among those undergoing immune checkpoint inhibitor (ICI) treatment. Here, we elucidate a critical microbial-host crosstalk between probiotic-released aryl hydrocarbon receptor (AhR) agonist indole-3-aldehyde (I3A) and CD8 T cells within the tumor microenvironment that potently enhances antitumor immunity and facilitates ICI in preclinical melanoma. Our study reveals that probiotic Lactobacillus reuteri (Lr) translocates to, colonizes, and persists within melanoma, where via its released dietary tryptophan catabolite I3A, it locally promotes interferon-γ-producing CD8 T cells, thereby bolstering ICI. Moreover, Lr-secreted I3A was both necessary and sufficient to drive antitumor immunity, and loss of AhR signaling within CD8 T cells abrogated Lr's antitumor effects. Further, a tryptophan-enriched diet potentiated both Lr- and ICI-induced antitumor immunity, dependent on CD8 T cell AhR signaling. Finally, we provide evidence for a potential role of I3A in promoting ICI efficacy and survival in advanced melanoma patients.

Everolimus downregulates STAT3/HIF-1α/VEGF pathway to inhibit angiogenesis and lymphangiogenesis in TP53 mutant head and neck squamous cell carcinoma (HNSCC).

TP53 mutant head and neck squamous cell carcinoma (HNSCC) patients exhibit poor clinical outcomes with 50-60% recurrence rates in advanced stage patients. In a recent phase II clinical trial, adjuvant therapy with everolimus (mTOR inhibitor) significantly increased 2-year progression-free survival in p53 mutated patients. TP53-driven mTOR activation in solid malignancies causes upregulation of HIF-1α and its target, downstream effector VEGF, by activating STAT3 cell signaling pathway. Here, we investigated the effects of everolimus on the STAT3/HIF-1α/VEGF pathway in TP53 mutant cell lines and xenograft models. Treatment with everolimus significantly inhibited cell growth in vitro and effectively reduced the growth of TP53 mutant xenografts in a minimal residual disease (MRD) model in nude mice. Everolimus treatment was associated with significant downregulation of STAT3/HIF-1α/VEGF pathway in both models. Further, treatment with everolimus was associated with attenuation in tumor angiogenesis and lymphangiogenesis as indicated by decreased microvessel density of vascular and lymphatic vessels in HN31 and FaDu xenografts. Everolimus downregulated the STAT3/HIF-1α/VEGF pathway to inhibit growth and in vitro tube formation of HMEC-1 (endothelial) and HMEC-1A (lymphatic endothelial) cell lines. Our studies demonstrated that everolimus inhibits the growth of TP53 mutant tumors by inhibiting angiogenesis and lymphangiogenesis through the downregulation of STAT3/HIF-1α/VEGF signaling.

"Is it worth living?" psychosocial challenges of childhood sexual abuse survivors aging with HIV.

Older adults living with HIV (OALH) undergo challenges such as comorbidities, social isolation, and "double stigma" associated with their HIV and aging statuses. Simultaneously, research has shown that experiences of childhood sexual abuse (CSA) continue to impact the quality of life across the lifespan and may pose unique hardships for older adults. Despite the high prevalence of trauma among people living with HIV, research examining the psychosocial challenges of OALH with a CSA history is scant. To address this gap in the literature, this study aimed to explore psychosocial challenges among OALH who are CSA survivors using a qualitative approach. Twenty-four in-depth, semi-structured interviews were completed with OALH (age 50 years and older) who reported histories of CSA. Multiple coders and an inductive coding process were employed for data analysis. Four main themes regarding psychosocial challenges emerged from the analysis: (1) depression and suicidal ideation, (2) fear and anxiety, (3) social support issues, and (4) memory issues. The authors discuss the implications of these findings and the importance of trauma-informed treatment for these individuals.

Adenoviruses in medicine: innocuous pathogen, predator, or partner.

The consequences of human adenovirus (HAdV) infections are generally mild. However, despite the perception that HAdVs are harmless, infections can cause severe disease in certain individuals, including newborns, the immunocompromised, and those with pre-existing conditions, including respiratory or cardiac disease. In addition, HAdV outbreaks remain relatively common events and the recent emergence of more pathogenic genomic variants of various genotypes has been well documented. Coupled with evidence of zoonotic transmission, interspecies recombination, and the lack of approved AdV antivirals or widely available vaccines, HAdVs remain a threat to public health. At the same time, the detailed understanding of AdV biology garnered over nearly 7 decades of study has made this group of viruses a molecular workhorse for vaccine and gene therapy applications.

Role of Serine Coordination in the Structural and Functional Protection of the Nitrogenase P-Cluster.

Nitrogenase catalyzes the multielectron reduction of dinitrogen to ammonia. Electron transfer in the catalytic protein (MoFeP) proceeds through a unique [8Fe-7S] cluster (P-cluster) to the active site (FeMoco). In the reduced, all-ferrous (PN) state, the P-cluster is coordinated by six cysteine residues. Upon two-electron oxidation to the P2+ state, the P-cluster undergoes conformational changes in which a highly conserved oxygen-based residue (a Ser or a Tyr) and a backbone amide additionally ligate the cluster. Previous studies of Azotobacter vinelandii (Av) MoFeP revealed that when the oxygen-based residue, ßSer188, was mutated to a noncoordinating residue, Ala, the P-cluster became redox-labile and reversibly lost two of its eight Fe centers. Surprisingly, the Av strain with a MoFeP variant that lacked the serine ligand (Av ßSer188Ala MoFeP) displayed the same diazotrophic growth and in vitro enzyme turnover rates as wild-type Av MoFeP, calling into question the necessity of this conserved ligand for nitrogenase function. Based on these observations, we hypothesized that ßSer188 plays a role in protecting the P-cluster under nonideal conditions. Here, we investigated the protective role of ßSer188 both in vivo and in vitro by characterizing the ability of Av ßSer188Ala cells to grow under suboptimal conditions (high oxidative stress or Fe limitation) and by determining the tendency of ßSer188Ala MoFeP to be mismetallated in vitro. Our results demonstrate that ßSer188 (1) increases Av cell survival upon exposure to oxidative stress in the form of hydrogen peroxide, (2) is necessary for efficient Av diazotrophic growth under Fe-limiting conditions, and (3) may protect the P-cluster from metal exchange in vitro. Taken together, our findings suggest a structural adaptation of nitrogenase to protect the P-cluster via Ser ligation, which is a previously unidentified functional role of the Ser residue in redox proteins and adds to the expanding functional roles of non-Cys ligands to FeS clusters.

The MASTL-ENSA-PP2A/B55 axis modulates cisplatin resistance in oral squamous cell carcinoma.

Platinum-based chemotherapy is the standard first-line treatment for oral squamous cell carcinoma (OSCC) that is inoperable, recurrent, or metastatic. Platinum sensitivity is a major determinant of patient survival in advanced OSCC. Here, we investigated the involvement of MASTL, a cell cycle kinase that mediates ENSA/ARPP19 phosphorylation and PP2A/B55 inhibition, in OSCC therapy. Interestingly, upregulation of MASTL and ENSA/ARPP19, and downregulation of PP2A/B55, were common in OSCC. MASTL expression was in association with poor patient survival. In established OSCC cell lines, upregulation of MASTL and ENSA, and downregulation of B55 genes, correlated with cisplatin resistance. We further confirmed that stable expression of MASTL in OSCC cells promoted cell survival and proliferation under cisplatin treatment, in an ENSA-dependent manner. Conversely, deletion of MASTL or ENSA, or overexpression of B55α, sensitized cisplatin response, consistent with increased DNA damage accumulation, signaling, and caspase activation. Moreover, GKI-1, the first-in-class small molecule inhibitor of MASTL kinase, phenocopied MASTL depletion in enhancing the outcome of cisplatin treatment in OSCC cells, at a dose substantially lower than that needed to disrupt mitotic entry. Finally, GKI-1 exhibited promising efficacy in a mouse tumor xenograft model, in conjunction with cisplatin therapy.

The role of progesterone receptor isoforms in the myometrium.

Myometrial contraction is stringently controlled throughout pregnancy and parturition. Progesterone signaling, effecting through the progesterone receptor (PR), is pivotal in modulating uterine activity. Evidence has shown that two major PR isoforms, PR-A and PR-B, have distinct activities on gene regulation, and the ratio between these isoforms determines the contractility of the myometrium at different gestational stages. Herein, we focus on the regulation of PR activity in the myometrium, especially the differential actions of the two PR isoforms, which maintain uterine quiescence during pregnancy and regulate the switch to a contractile state at the onset of labor. To demonstrate the PR regulatory network and its mechanisms of actions on myometrial activity, we summarized the findings into three parts: Regulation of PR Expression and Isoform Levels, Progesterone Receptor Interacting Factors, and Biological Processes Regulated by Myometrial Progesterone Receptor Isoforms. Recent genomic and epigenomic data, from human specimens and mouse models, are recruited to support the existing knowledge and offer new insights and future directions in myometrial biology.

The future of oncology policy.

To ensure the previous progress seen in cancer survival rates continues as we move through the 21st Century it is important to determine future effective policy related to oncology healthcare delivery and funding. Recent successes with, for example, the COVID vaccine response, the decision-making agility exhibited by governments and healthcare systems and the effective use of telehealth and real-world evidence highlight the progress that can be made with pooled efforts and innovative thinking. This shared approach is the basis for the European Beating Cancer Plan which outlines action points for governments and health systems for the period 2021-2025. It focuses on a whole government approach, centred on patients, maximising the potential of new technologies and insights across policy areas including employment, education, transport and taxation, enabling the tackling of cancer drivers in schools, workplaces, research labs, towns and cities and rural communities. Despite the plan there are still concerns that oncology policy has not adequately responded to the pace of innovation and the unique challenges generated by innovative oncological technologies. There needs to be focus on: gaining consensus on the most appropriate methods to assess and price combination therapies and cell and gene therapies, developing effective outcome-based payment models for personalised medicine and developing consensus on the ideal approach for multiple indication pricing. Finally, future policy needs to ensure pharmaceutical companies and other research organisations are adequately rewarded for innovation to ensure continued R&D and the development of innovative oncological products.

Tet2 deficiency drives liver microbiome dysbiosis triggering Tc1 cell autoimmune hepatitis.

The triggers that drive interferon-γ (IFNγ)-producing CD8 T cell (Tc1 cell)-mediated autoimmune hepatitis (AIH) remain obscure. Here, we show that lack of hematopoietic Tet methylcytosine dioxygenase 2 (Tet2), an epigenetic regulator associated with autoimmunity, results in the development of microbiota-dependent AIH-like pathology, accompanied by hepatic enrichment of aryl hydrocarbon receptor (AhR) ligand-producing pathobionts and rampant Tc1 cell immunity. We report that AIH-like disease development is dependent on both IFNγ and AhR signaling, as blocking either reverts ongoing AIH-like pathology. Illustrating the critical role of AhR-ligand-producing pathobionts in this condition, hepatic translocation of the AhR ligand indole-3-aldehyde (I3A)-releasing Lactobacillus reuteri is sufficient to trigger AIH-like pathology. Finally, we demonstrate that I3A is required for L. reuteri-induced Tc1 cell differentiation in vitro and AIH-like pathology in vivo, both of which are restrained by Tet2 within CD8 T cells. This AIH-disease model may contribute to the development of therapeutics to alleviate AIH.

The endometrial transcriptomic response to pregnancy is altered in cows after uterine infection.

Pregnancy induces changes in the transcriptome of the bovine endometrium from 15 days after insemination. However, pregnancy is less likely to occur if cows had a postpartum bacterial infection of the uterus, even after the resolution of disease. We hypothesized that uterine bacterial infection alters the endometrial transcriptomic signature of pregnancy after the resolution of disease. To examine the endometrial transcriptomic signature of pregnancy, cows were inseminated 130 days after intrauterine infusion of pathogenic Escherichia coli and Trueperella pyogenes, subsequently endometrium was collected 16 days after insemination for RNA sequencing. We found 171 pregnancy regulated genes in cows 146 days after bacterial infection. When comparing our findings with previous studies that described the endometrial transcriptomic signature of pregnancy in healthy cows, 24 genes were consistently differentially expressed in pregnancy, including MX1, MX2 and STAT1. However, 12 pregnancy regulated genes were found only in the endometrium of healthy cows, including ISG15 and TRANK1. Furthermore, 28 pregnancy regulated genes were found only in the endometrium of cows following bacterial infection and these were associated with altered iNOS, TLR, and IL-7 signaling pathways. Although 94 predicted upstream regulators were conserved amongst the studies, 14 were found only in the endometrium of pregnant healthy cows, and 5 were found only in cows following bacterial infection, including AIRE, NFKBIA, and DUSP1. In conclusion, there were both consistent and discordant features of the endometrial transcriptomic signature of pregnancy 146 days after intrauterine bacterial infusion. These findings imply that there is an essential transcriptomic signature of pregnancy, but that infection induces long-term changes in the endometrium that affect the transcriptomic response to pregnancy.

A DMS Shotgun Lipidomics Workflow Application to Facilitate High-Throughput, Comprehensive Lipidomics.

Differential mobility spectrometry (DMS) is highly useful for shotgun lipidomic analysis because it overcomes difficulties in measuring isobaric species within a complex lipid sample and allows for acyl tail characterization of phospholipid species. Despite these advantages, the resulting workflow presents technical challenges, including the need to tune the DMS before every batch to update compensative voltages settings within the method. The Sciex Lipidyzer platform uses a Sciex 5500 QTRAP with a DMS (SelexION), an LC system configured for direction infusion experiments, an extensive set of standards designed for quantitative lipidomics, and a software package (Lipidyzer Workflow Manager) that facilitates the workflow and rapidly analyzes the data. Although the Lipidyzer platform remains very useful for DMS-based shotgun lipidomics, the software is no longer updated for current versions of Analyst and Windows. Furthermore, the software is fixed to a single workflow and cannot take advantage of new lipidomics standards or analyze additional lipid species. To address this multitude of issues, we developed Shotgun Lipidomics Assistant (SLA), a Python-based application that facilitates DMS-based lipidomics workflows. SLA provides the user with flexibility in adding and subtracting lipid and standard MRMs. It can report quantitative lipidomics results from raw data in minutes, comparable to the Lipidyzer software. We show that SLA facilitates an expanded lipidomics analysis that measures over 1450 lipid species across 17 (sub)classes. Lastly, we demonstrate that the SLA performs isotope correction, a feature that was absent from the original software.