PNAS Research Articles

Cross-Linking Mass Spectrometry Analysis of Metastable Compact Structures in Intrinsically Disordered Proteins.

Protein assembly into beta-sheet-rich amyloids is a common phenomenon in neurodegenerative diseases including Alzheimer's (AD) and Parkinson's (PD). The proteins implicated in amyloid deposition are often intrinsically disordered proteins (IDPs) and are characterized by not folding into a defined globular conformation. The amyloidogenic properties of IDPs are determined by the presence of short sequence elements, referred to as amyloid motifs, that drive ordered aggregation (Thompson MJ, Sievers SA, Karanicolas J et al. Proc Natl Acad Sci USA 103(11):4074-8, 2006; Goldschmidt L, Teng PK, Riek R et al. Proc Natl Acad Sci USA 107(8):3487-92, 2010]. The microtubule-associated protein tau adopts amyloid assemblies in over 20 different diseases commonly referred to as tauopathies. However, native tau is aggregation-resistant despite encoding at least three amyloid motifs (Chen D, Drombosky KW, Hou Z et al. Nat Commun 10(1):2493, 2019). Recent cryogenic electron microscopy (cryo-EM) structures of tau amyloid fibrils isolated from patient brains showed the involvement of amyloid motifs in the fibril core (Fitzpatrick AWP, Falcon B, He S et al. Nature 547(7662):185-90, 2017; Falcon B, Zhang W, Murzin AG et al. Nature 561(7721):137-40, 2018; Zhang W, Tarutani A, Newell KL et al. Nature 580(7802):283-7, 2020). How does tau change from an aggregation-resistant state to an aggregation-prone state? Consistent with the fibril structures, we hypothesize that tau must change conformation to expose the amyloid motifs that allow self-association into beta-sheet-rich aggregates. This would suggest that the amyloid motifs are likely buried in natively folded tau to prevent self-assembly. We developed an approach that couples cross-linking mass spectrometry (XL-MS) with temperature denaturation to probe the loss of contacts as a proxy to measure protein unfolding with sequence resolution. Using thismethod, we demonstrated that disease-associated mutations in tau located near an amyloid motif disrupt the protective local structure, promote amyloid motif exposure, and thus lead to aggregation (Chen D, Drombosky KW, Hou Z et al. Nat Commun 10(1):2493, 2019). In this chapter, we describe the detailed protocol for this approach. We anticipate that our protocol can be generalized to other IDPs and will help discover critical structural elements to better understand important biological questions including protein aggregation.

Structural Analysis of SOD1 Fibrils with Mass Spectrometry, Limited Proteolysis, and Atomic Force Microscopy (AFM).

This protocol describes a method to purify SOD1 in Saccharomyces cerevisiae to characterize using ICP-MS and AFM, to agitate and fibrillate for aggregation of SOD1. The human SOD1 (hSOD1) is a 32-kDa homodimer, with one copper- and one zinc-binding site per 153-amino acid subunit. Misfolded protein aggregates are often correlated with diseases known as amyloidosis, including ALS, Alzheimer's, Parkinson's, and prion disease (Valentine and Hart, Proc Natl Acad Sci USA 100: 3617-3622, 2003; Tanzi and Bertram, Cell 120: 545-555, 2005; Soto and Pritzkow, Nat Neurosci 21:1332-1340, 2018; Sarafian et al., J Neurosci Res 95:1871-1887, 2017). Proteinaceous aggregates containing hSOD1 have frequently been found in the spinal cords of ALS patients.

An Optimized Stress Granule Detection Method: Investigation of UBQLN2 Effect on Stress Granule Formation.

Stress granules (SGs) are cytosolic ribonucleoprotein granules that form via a liquid-liquid phase separation in response to environmental stresses such as heat, oxidative, and osmotic changes. Due to the condensation of low complexity, hydrophobic regions in core SG components in these highly dynamic granules, defects in SG maintenance and formation have been linked to toxic aggregate formation in neurodegenerative diseases such as amyotrophic lateral sclerosis and frontotemporal dementia. However, efforts to dissect mechanisms regulating SG formation and maintenance have been limited by methods of tracking protein-SG localization. Here we describe a method for detecting and quantifying recruitment of cytosolically enriched proteins to SGs by indirect immunofluorescence microscopy. Using this method, we tracked the transient recruitment of the cytosolically enriched ubiquitin-like protein, ubiquilin 2 (UBQLN2), and a number of other factors into SGs, demonstrating its utility (Alexander et al., Proc Natl Acad Sci U S A 115:E11485-E11494, 2018).

Ca2+ signaling in plant manganese uptake: CPK21/23 kinases phosphorylate and activate manganese transporter NRAMP1

This brief article highlights the results of Fu et al. (Proc Natl Acad Sci USA 119:e2204574119, 2022), who recently found that manganese (Mn) deficiency triggers long-lasting multicellular Ca2+ oscillations in the elongation zone (EZ) of Arabidopsis roots and revealed a Ca2+-CPK21/23-NRAMP1 axis as an important mechanism for plant tolerance and adaptation to low Mn.

Spatial dynamics of a vegetation model with uptake-diffusion feedback in an arid environment.

Vegetation patterns with a variety of structures is amazing phenomena in arid or semi-arid areas, which can identify the evolution law of vegetation and are typical signals of ecosystem functions. Many achievements have been made in this respect, yet the mechanisms of uptake-diffusion feedback on the pattern structures of vegetation is not fully understood. To well reveal the influences of parameters perturbation on the pattern formation of vegetation, we give a comprehensive analysis on a vegetation-water model in the forms of reaction-diffusion equation which is posed by Zelnik et al.(Proc Natl Acad Sci 112:12,327-12,331, 2015). We obtain the exact parameters range for stationary patterns and show the dynamical behaviors near the bifurcation point based on nonlinear analysis. It is found that the model has the properties of spot, labyrinth and gap patterns. Moreover, water diffusion rate prohibits the growth of vegetation while shading parameter promotes the increase of vegetation biomass. Our results show that gradual transitions from uniform state to gap pattern can occur for suitable value of parameters which may induce the emergence of desertification.

Effect of caffeine on mitochondrial biogenesis in the skeletal muscle - A narrative review.

Caffeine is one of the most widely used substances as recreational drug for performance-enhancement in sport, underpinned by a strong evidence base. Although the effects of caffeine are widely investigated within the scope of performance physiology, the molecular effects of caffeine within skeletal muscle remain unclear. Evidence from invitro and invivo models suggest that caffeine regulates the glucose metabolism in the skeletal muscle. Moreover, caffeine seems to stimulate CaMKII, PPARδ/β, AMPK and PGC1α, classical markers of exercise-adaptations, including mitochondrial biogenesis and mitochondrial content. This review summarizes evidence to suggest caffeine-effects within skeletal muscle fibers, focusing on the putative role of caffeine on mitochondrial biogenesis to explore whether caffeine supplementation might be a strategy to enhance mitochondrial biogenesis.

Default mode and fronto-parietal network associations with IQ development across childhood in autism

BackgroundIntellectual disability affects approximately one third of individuals with autism spectrum disorder (autism). Yet, a major unresolved neurobiological question is what differentiates autistic individuals with and without intellectual disability. Intelligence quotients (IQs) are highly variable during childhood. We previously identified three subgroups of autistic children with different trajectories of intellectual development from early (2–3½ years) to middle childhood (9–12 years): (a) persistently high: individuals whose IQs remained in the normal range; (b) persistently low: individuals whose IQs remained in the range of intellectual disability (IQ < 70); and (c) changers: individuals whose IQs began in the range of intellectual disability but increased to the normal IQ range. The frontoparietal (FPN) and default mode (DMN) networks have established links to intellectual functioning. Here, we tested whether brain regions within the FPN and DMN differed volumetrically between these IQ trajectory groups in early childhood.MethodsWe conducted multivariate distance matrix regression to examine the brain regions within the FPN (11 regions x 2 hemispheres) and the DMN (12 regions x 2 hemispheres) in 48 persistently high (18 female), 108 persistently low (32 female), and 109 changers (39 female) using structural MRI acquired at baseline. FPN and DMN regions were defined using networks identified in Smith et al. (Proc Natl Acad Sci U S A 106:13040–5, 2009). IQ trajectory groups were defined by IQ measurements from up to three time points spanning early to middle childhood (mean age time 1: 3.2 years; time 2: 5.4 years; time 3: 11.3 years).ResultsThe changers group exhibited volumetric differences in the DMN compared to both the persistently low and persistently high groups at time 1. However, the persistently high group did not differ from the persistently low group, suggesting that DMN structure may be an early predictor for change in IQ trajectory. In contrast, the persistently high group exhibited differences in the FPN compared to both the persistently low and changers groups, suggesting differences related more to concurrent IQ and the absence of intellectual disability.ConclusionsWithin autism, volumetric differences of brain regions within the DMN in early childhood may differentiate individuals with persistently low IQ from those with low IQ that improves through childhood. Structural differences in brain networks between these three IQ-based subgroups highlight distinct neural underpinnings of these autism sub-phenotypes.

Inhibiting TGF-β to increase response rates to chemoradiotherapy in rectal cancer.

With the rapid increase in the incidence of rectal cancer in young adults, 1 Bailey CE Hu CY You YN et al. Increasing disparities in the age-related incidences of colon and rectal cancers in the United States, 1975–2010. JAMA Surg. 2015; 150: 17-22 Crossref PubMed Scopus (528) Google Scholar interest in organ preservation as an oncologically safe 2 Garcia-Aguilar J Patil S Gollub MJ et al. Organ preservation in patients with rectal adenocarcinoma treated with total neoadjuvant therapy. J Clin Oncol. 2022; (published online April 28.)https://doi.org/10.1200/JCO.22.00032 Crossref PubMed Scopus (28) Google Scholar , 3 Smith JJ Strombom P Chow OS et al. Assessment of a watch-and-wait strategy for rectal cancer in patients with a complete response after neoadjuvant therapy. JAMA Oncol. 2019; 5: e185896 Crossref PubMed Scopus (208) Google Scholar and cost-effective strategy to improve patients' quality of life has also been growing. 4 Miller JA Wang H Chang DT Pollom EL Cost-effectiveness and quality-adjusted survival of watch and wait after complete response to chemoradiotherapy for rectal cancer. J Natl Cancer Inst. 2020; 112: 792-801 Crossref PubMed Scopus (12) Google Scholar Studies evaluating novel radiosensitisers in rectal cancer are receiving attention as a consequence of the need to develop personalised treatment strategies to increase response rates while also facilitating organ preservation. The TGF-β pathway has intrigued scientists and clinicians for decades, 5 Barnard JA Beauchamp RD Coffey RJ Moses HL Regulation of intestinal epithelial cell growth by transforming growth factor type beta. Proc Natl Acad Sci USA. 1989; 86: 1578-1582 Crossref PubMed Scopus (316) Google Scholar and targeting this complex signalling network has now been implicated as a potential way to improve response to radiotherapy. 6 Batlle E Massagué J Transforming growth factor-β signaling in immunity and cancer. Immunity. 2019; 50: 924-940 Summary Full Text Full Text PDF PubMed Scopus (781) Google Scholar However, decades of research into TGF-β inhibitors—both as a monotherapy and in combination with radiotherapy—have shown mixed clinical efficacy, probably due to the complex biology of TGF-β and the context-dependent effects of its inhibition. 6 Batlle E Massagué J Transforming growth factor-β signaling in immunity and cancer. Immunity. 2019; 50: 924-940 Summary Full Text Full Text PDF PubMed Scopus (781) Google Scholar , 7 Andarawewa KL Paupert J Pal A Barcellos-Hoff MH New rationales for using TGFbeta inhibitors in radiotherapy. Int J Radiat Biol. 2007; 83: 803-811 Crossref PubMed Scopus (58) Google Scholar Chest CT scan plus x-ray versus chest x-ray for the follow-up of completely resected non-small-cell lung cancer (IFCT-0302): a multicentre, open-label, randomised, phase 3 trialThe addition of thoracic CT scans during follow-up, which included clinic visits and chest x-rays after surgery, did not result in longer survival among patients with NSCLC. However, it did enable the detection of more cases of early recurrence and second primary lung cancer, which are more amenable to curative-intent treatment, supporting the use of CT-based follow-up, especially in countries where lung cancer screening is already implemented, alongside with other supportive measures. Full-Text PDF

Skin proteomic analysis of immune activation associated with regression of melanoma metastases induced by diphencyprone

To the Editor: Diphencyprone (DPCP), a hapten that causes delayed-type hypersensitivity reactions, has been used to treat warts, alopecia areata, and cutaneous metastases in melanoma patients.1Damian D.L. Saw R.P. Thompson J.F. Topical immunotherapy with diphencyprone for in transit and cutaneously metastatic melanoma.J Surg Oncol. 2014; 109: 308-313https://doi.org/10.1002/jso.23506Crossref PubMed Scopus (51) Google Scholar Previously, our group performed transcriptomic analysis of skin biopsies from melanoma metastases treated with topical DPCP, revealing significant increases in Th1-related genes but not in genes of other major T-cell subsets.2Gulati N. Garcet S. Fuentes-Duculan J. et al.Molecular profiling of immune activation associated with regression of melanoma metastases induced by diphencyprone.J Invest Dermatol. 2016; 136: 2101-2103https://doi.org/10.1016/j.jid.20 16.06.611Crossref PubMed Scopus (0) Google Scholar While gene expression profiling has long been used as a proxy for protein expression, correlation between messenger RNA (mRNA) and protein levels is inconsistent due to factors such as post-transcriptional regulation. Therefore, to more precisely ascertain the mechanisms involved in immune-mediated tumor regression induced by DPCP, we performed proteomic analysis of skin biopsies from 5 patients with cutaneous melanoma metastases treated with DPCP twice weekly for 7 to 14 weeks (Supplemental Material and Methods and Supplementary Table I, available via Mendeley at https://doi.org/10.17632/f5ttw4mygy.1). In this study, we assessed 96 proteins using the Olink immuno-oncology panel for the 5 patients who underwent molecular profiling. All patients had expected skin inflammation and at least partial regression of skin metastases, without systemic side effects. There were 10 proteins (interleukin 6 [IL-6], IL-12, IL-18, TIE2, HGF, PDGFB, TNFSF14, PD-L2, MMP7, MMP12) significantly upregulated (P < .05) in post-treatment versus pretreatment metastases (Fig 1), including biomarkers associated with Th1 response (IL-12), innate immunity (IL-6), and immune checkpoints (PD-L2) (P < .05). All of these proteins had progressively increased expression in cutaneous metastases with repeated DPCP applications, and similar increases were observed in nonlesional skin of these patients following a single DPCP application. Prior work has shown that administration of IL-18 in mice increases activated T cells and natural killer cells that contribute to effective antimelanoma immunity,3Zhou T. Damsky W. Weizman O.E. et al.IL-18BP is a secreted immune checkpoint and barrier to IL-18 immunotherapy.Nature. 2020; 583: 609-614https://doi.org/10.1038/s41586-020-2422-6Crossref PubMed Scopus (93) Google Scholar so the significant upregulation of this protein in our study may contribute to the successful skin metastasis regression observed in our patients. TNFSF14 also likely plays an active role in immune-mediated regression, as in vivo studies have demonstrated an association between the expression of this protein in melanoma metastasis lesions and proliferation of T cells.4Mortarini R. Scarito A. Nonaka D. et al.Constitutive expression and costimulatory function of LIGHT/TNFSF14 on human melanoma cells and melanoma-derived microvesicles.Cancer Res. 15 2005; 65: 3428-3436https://doi.org/10.1158/0008-5472.CAN-04-3239Crossref PubMed Scopus (35) Google Scholar Additionally, there were 40 proteins significantly upregulated in pretreatment metastases versus untreated nonlesional skin (P < .05) (Fig 1), suggesting baseline immune activation in melanoma metastases. Upon DPCP treatment, changes in nonlesional skin positively correlated with those in skin metastases (Supplementary Fig 1, available via Mendeley at https://doi.org/10.17632/f5ttw4mygy.1; Spearman correlation = 0.61, P < .0001). We performed a correlation analysis between the proteomic data and the previously published mRNA data,2Gulati N. Garcet S. Fuentes-Duculan J. et al.Molecular profiling of immune activation associated with regression of melanoma metastases induced by diphencyprone.J Invest Dermatol. 2016; 136: 2101-2103https://doi.org/10.1016/j.jid.20 16.06.611Crossref PubMed Scopus (0) Google Scholar which both were completed on the same tissue samples, to assess the relationship between these 2 “omic” approaches (Fig 2). Correlation was poor (r = −0.1, P = .4012), including for proteins that were significantly upregulated by proteomics (IL-6 and TNFSF14) (both P < .05). Gene expression from mRNA is variably correlated with protein expression, the latter being influenced by many regulatory processes prior to translation. Another explanation for the discrepancy observed between mRNA and protein expression includes the shorter half-life of mRNA molecules compared to proteins.5Mauger D.M. Cabral B.J. Presnyak V. et al.mRNA structure regulates protein expression through changes in functional half-life.Proc Natl Acad Sci U S A. 2019; 116: 24075-24083https://doi.org/10.1073/pnas.1908052116Crossref PubMed Scopus (152) Google Scholar Despite our limited sample size, we were able to observe statistically significant protein expression changes, and this study is, to our knowledge, the first to assess the proteomic signature of successful immune-mediated regression of cutaneous melanoma metastases induced by a topical immunomodulator. Given the lack of correlation between mRNA and protein expression, along with the relatively greater feasibility of measuring protein compared to gene expression levels, our study demonstrates that proteomic analysis can give unique and potentially complementary insights into immune reactions induced by topical immunotherapy. None disclosed.

Unmasking Schizophrenia: Synaptic Pruning in Adolescence Reveals a Latent Physiological Vulnerability in Prefrontal Recurrent Networks

Theories of the pathophysiology in schizophrenia are converging on the synapse. This special issue of Biological Psychiatry collects papers from leading researchers that suggest a hypothesis that multiple factors spanning many levels lead to synaptic dysfunction in recurrent circuits in the prefrontal cortex (PFC). The hypothesis (Figure 1) suggests that the defect within the PFC is established during early development but remains latent through childhood. Pruning of excitatory synapses during adolescence, resulting in lower synaptic density in persons with schizophrenia, triggers the emergence of symptoms. The lower synaptic density results in deficits in cognitive processes, including working memory, because overpruned neural circuits cannot support the relevant computations ( 1 Averbeck B.B. Pruning recurrent neural networks replicates adolescent changes in working memory and reinforcement learning. Proc Natl Acad Sci U S A. 2022; 119e2121331119 Crossref Scopus (0) Google Scholar ). Additionally, excessive prefrontal pruning leads to striatal hyperdopaminergia through increased activation of midbrain dopamine neurons by prefrontal input. The decreased synaptic density following adolescent pruning could be due to genetic or epigenetic effects that lead to an inability to build stable synapses or excessive pruning of established synapses. The excessive pruning could follow from overactive pruning mechanisms or from decreased excitatory synaptic activity due to synaptic changes local to the PFC or prolonged decreases in thalamic input across development. All of these factors may converge to alter spike timing dynamics in prefrontal recurrent circuits in adolescence, reducing synchronous neural activity and accelerating synaptic disconnection via activity-dependent mechanisms ( 2 Zick J.L. Crowe D.A. Blackman R.K. Schultz K. Bergstrand D.W. DeNicola A.L. et al. Disparate insults relevant to schizophrenia converge on impaired spike synchrony and weaker synaptic interactions in prefrontal local circuits. Curr Biol. 2022; 32: 14-25.e4 Abstract Full Text Full Text PDF PubMed Scopus (0) Google Scholar ). Further, synaptic pruning is mediated by components of the immune system, and in utero immune challenge may affect adolescent pruning. Decreased synaptic activity can also lead to increased pruning as pruning removes weak synapses. Thus, multiple effects converging on the synapse could lead to a PFC with fewer excitatory synaptic connections, and this decreased recurrent connectivity could lead to cognitive deficits and increased striatal dopamine and psychosis.

An effective pandemic treaty requires accountability

An effective pandemic treaty requires accountability

Applying C:N ratio to assess the rationality of estimates of carbon sequestration in terrestrial ecosystems and nitrogen budgets

Carbon (C) sequestration in terrestrial ecosystems needs to ensure the reactive nitrogen (Nr) supply. However, the organic C:N ratio is rarely considered in both estimates of C sequestration in terrestrial ecosystems and N budget at regional and global scale. Here we propose an approach for extracting Nr sequestrated in terrestrial ecosystems with C (termed as Nrc) from N budget and then assessing the rationality of estimates of C sequestration in terrestrial ecosystems and N budgets by using the sequestrated organic C:N ratio as a criterion. We extracted Nrc from the N budget of IPCC AR5 at global scale and Chinese N budget (Proc Natl Acad Sci (USA) 112:8792, 2015) at regional scale based on the assumptions that there is no net Nr accumulation in agricultural products and no net Nr (except N2O) accumulation in the atmosphere at annual temporal scale, and N2O is not involved in biological processes. By taking the C sequestration in terrestrial ecosystems from C budget of IPCC AR5 (2.1 Pg C/yr during 2000–2009) to assess the N budget from the same report, the organic C:N ratios calculated by the Nrc extracted from both the upper ranges and averages of Nr input and output in global N budgets of IPCC AR5 fell outside the rational range of organic C:N ratio, but the organic C:N ratio (=31) calculated by the Nrc (68.1 Tg N/yr) extracted from the lower ranges of the N budget fell very well into the range of organic C:N ratio in global terrestrial ecosystems. Nrc extracted from Chinese N budget was 11.6 Tg N/yr in 2010, which could be combined with C into Chinese terrestrial ecosystems in a range between 0.14–0.66 Pg C/yr with a robust estimate of 0.36 Pg C/yr by applying the global terrestrial ecosystem sequestrated organic C:N ratio to calculation. The results indicate that the proposed approach for extracting Nrc from N budget works well. Checking the estimated organic C:N ratio in terrestrial ecosystems will help to assess whether the C and/or N budgets are rational or not.Graphical abstract

Abstract 5632: Combinatorial epigenetic targeting of PRC2 complexes upregulates MHC antigen presentation components in melanoma and gastrointestinal cancer cells

Abstract Low tumor immunogenicity is associated with immune escape, immunotherapy resistance, and poor patient survival [1-3]. Major histocompatibility complex (MHC) antigen presentation pathways (APPs) play a crucial role in promoting tumor T-cell recognition and initiating antitumoral immune responses. However, epigenetic silencing of APPs is a common mechanism in various cancers, including melanoma and gastrointestinal malignancies [3,4]. In the current investigation, mouse melanoma (B16/OVA) and human colon cancer (LOVO and HCT116), small intestine cancer (HUTU80), and normal colonic epithelial (CCD841) cell lines were treated with epigenetic drug candidates. As reported [5], a decrease in histone H3K27 methylation marks via polycomb repressive complex 2 (PRC2) inhibitors, as well as altered histone acetylation status from histone deacetylase 3 (HDAC3)-specific inhibition, increased the expression of APP components, based on immunoblotting and RT-qPCR experiments. Normal colonic epithelial cells showed resistance to the treatments, indicating cancer cell-specific effects of the test agents. In B16/OVA cells, flow cytometry and enzyme linked immunosorbent assays corroborated that HDAC3 and/or PRC2 inhibitor combinations increased cell surface occupancy of MHC-I complexes and increased CD8+ T-cell activation. These findings have implications for the clinical application of next-generation combinatorial epigenetic agents [6,7], targeted towards increased tumor immunogenicity and enhanced efficacy of new immunoepigenetic strategies. Supported in part by NCI grant CA122959, by the John S. Dunn Foundation, and by a Chancellor’s Research Initiative.

Abstract 1764: Development of an alphalex™-auristatin low pH targeting conjugate for the treatment of solid tumors

Abstract Auristatins such as monomethyl auristatin E (MMAE) are a class of high potency microtubule targeting compounds that have an extremely narrow therapeutic window. Targeting potent auristatins to the tumor is the only feasible method of unlocking the clinical potential of such toxic molecules. While there are currently four marketed antibody-drug conjugates (ADCs) featuring auristatins, these ADCs face the same fundamental issues - tumor restriction by target antigen and the potential for off target release of payload. Alphalex࣪ is a tumor targeting technology consisting of a unique variant of a family of pH-Low Insertion Peptides (pHLIP®) that target acidic cell surfaces (references 1-2), a cleavable small molecule linker, and an anti-cancer agent warhead. Alphalex࣪ thereby allows for antigen independent targeting of the tumor and enables intracellular delivery of the warhead by leveraging the low pH microenvironment of the tumor, a universal feature common to all tumors due to the Warburg effect. Here we report the preclinical efficacy, safety, and antigen-independent tumor-targeting properties of alphalex࣪ conjugated to MMAE. We demonstrate the ability of alphalex࣪-MMAE to display potent in vitro and in vivo efficacy in colorectal, non-small cell lung, and prostate carcinoma cell lines. We further show that alphalex࣪-MMAE efficiently and safely delivers efficacious levels of MMAE selectively to tumor and demonstrates extreme plasma stability, with 0.02% warhead release over 24h in the rat. Based on the excellent preclinical safety and efficacy profile of alphalex࣪-MMAE, Cybrexa will move forward with the goal of initiating IND-enabling studies in 2022.

Abstract 5598: Engineered hypoimmune allogeneic CAR T cells as potential off-the-shelf CAR T cell immunotherapies

Abstract Off-the-shelf CAR T cells may offer advantages over autologous strategies, including ease of manufacturing, improved quality control with avoidance of malignant contamination and T cell dysfunction, and the ability to generate a final product from healthy T cells. However, host-versus-graft immune response against histoincompatible T cells prevents the expansion and persistence of allogeneic CAR T cells and mitigates the efficacy of this approach. A major challenge is that, while HLA deletion can result in adaptive immune evasion, innate reactivity is enhanced. While T cells express CD47, we demonstrate here that CD47 expression above endogenous levels is important for immune evasion. We describe here the engineering of human immune evasive CAR T cells building on our previously described hypoimmune technology (Nat Biotechnol 2019;37(3):252-258 and Proc Natl Acad Sci U S A 2021;118(28):e2022091118). The goal is to achieve improved rates of durable complete remissions by improving allogeneic CD19CAR persistence, since it has been shown that autologous CAR T cells have greater durability over years than allogeneic CAR T cells. Human T cells from healthy donors were obtained by leukapheresis. To generate hypoimmune CD19CAR T cells, gene editing was used to eliminate HLA-I/II and TCR expression and lentiviral transduction was used to express CD47 and CD19CAR containing a 4-1BB costimulatory domain to generate hypoimmune CD19CAR T cells. Control CD19CAR T cells were unmanipulated, i.e., unedited, except for lentiviral transduction used to express CD19CAR. Hypoimmune CD19CAR T cells persist in allogeneic humanized mice and lack T cell activation measured using bioluminescence imaging and ELISPOT analysis, respectively. In contrast, transplantation of control CD19CAR T cells generated from the same human donor resulted in rejection (ELISPOT mean 59 and 558 spot frequencies for hypoimmune CD19CAR T cells and control CD19CAR T cells, respectively; p&amp;lt;0.0001 unpaired t-test). Innate immune cell assays show that CD47 overexpression protects hypoimmune CD19CAR T cells from NK cell and macrophage killing. A blocking antibody against CD47 made the hypoimmune CD19CAR T cells susceptible to macrophage and NK cell killing, confirming the importance of CD47 overexpression to evade innate immune clearance. Importantly, CD47 seemed to provide protection from all NK cell populations while other tested NK cell inhibitory molecules (such as HLA-E/G, PD-L1) seemed to prevent NK cell killing of only certain subpopulations rather than primary NK cells in total. Hypoimmune CD19CAR T cells retain their antitumor activity in the Nalm-6 B cell leukemia model in vitro and in vivo comparable to control CD19CAR T cells derived from various donors. Thus, hypoimmune edits seem to not impact CD19CAR T cell activity and have the potential to provide universal CAR T cells that are able to persist without immunosuppression. Citation Format: Xiaomeng Hu, Kathy White, Corie Gattis, Ryan Clarke, Sam Landry, Ron Basco, Eleonore Tham, Emily Luo, Andrew Tucker, Christopher Bandoro, Elaine Chu, Chi Young, Karl Manner, Priscilla Nho, Ben Lam, Pascal Beauchesne, Aaron Foster, William E. Dowdle, Edward J. Rebar, Terry J. Fry, Sonja Schrepfer. Engineered hypoimmune allogeneic CAR T cells as potential off-the-shelf CAR T cell immunotherapies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5598.

An application of the splitting-up method for the computation of a neural network representation for the solution for the filtering equations

The filtering equations govern the evolution of the conditional distribution of a signal process given partial, and possibly noisy, observations arriving sequentially in time. Their numerical approximation plays a central role in many real-life applications, including numerical weather prediction [Llopis et al. (SIAM J Sci Comput 40(3):A1544–A1565, 2018), Galanis et al. (Geophysicae 24(10): 2451–2460, 2006)], finance [Brigo and Hanzon (Insurance Math Econom 22(1):53–64, 1998), Date and Ponomareva (IMA J Manag Math 22(3): 195–211, 2011), Crisan and Rozovskii (The Oxford handbook of nonlinear filtering, 2011)] and engineering [Myötyri et al. (Reliability Eng Syst Saf 91(2):200–208, 2005)]. One of the classical approaches to approximate the solution of the filtering equations is to use a PDE inspired method, called the splitting-up method, initiated by Gyongy, Krylov, LeGland, among other contributors, see e.g., Gyöngy and Krylov (Stochastic inequalities and applications, Progr. Probab. 56:301–321, 2003), Le Gland(Stochastic partial differential equations and their applications (Charlotte, NC, 1991), Lect. Notes Control Inf. Sci. 176:177–187, 1992). This method, and other PDE based approaches, have particular applicability for solving low-dimensional problems. In this work we combine this method with a neural network representation inspired by [Han et al. (Proc Natl acad Sci 115(34):8505–8510, 2018)]. The new methodology is used to produce an approximation of the unnormalised conditional distribution of the signal process. We further develop a recursive normalisation procedure to recover the normalised conditional distribution of the signal process. The new scheme can be iterated over multiple time steps whilst keeping its asymptotic unbiasedness property intact. We test the neural network approximations with numerical approximation results for the Kalman and Benes filter.

Optogenetic stimulation of cholinergic neuronal endings in the celiac‐superior mesenteric ganglion complex suppresses inflammation in murine endotoxemia

The vagus nerve plays a key role in the regulation of inflammation within the inflammatory reflex (Annu Rev Immunol, 2018, 36:783). In this major physiological neuroimmunoregulatory mechanism, efferent vagus nerve cholinergic axons interact with catecholaminergic neurons of the splenic nerve in the celiac‐superior mesenteric ganglion (CSMG) complex(Proc Natl Acad Sci USA, 2020, 117(47):29803).Previous studies utilizing electrical and optogenetic neuromodulation have demonstrated that vagus nerve cholinergic signalling suppresses pro‐inflammatory cytokine levels during endotoxemia and other inflammatory conditions. However, the specific role of cholinergic axonal terminals in the CSMG complex in this regulation remained enigmatic. To provide insight, we investigated the effect of selective optogenetic stimulation of cholinergic neuronal endings in the CSMG complex on cytokine and chemokine levels during murine endotoxemia. We used transgenic female 10–14‐week‐old mice expressing channelrhodopsin 2 coupled with yellow fluorescent protein on cholinergic, i.e., choline acetyltransferase positive neurons (ChAT‐ChR2‐eYFP). Anesthetized mice were subjected to abdominal surgery and the CSMG complex was localized medial to the upper pole left kidney and along the branch of the abdominal aorta, above the left renal vein. Blue laser light stimulation at 473nm was delivered in square pulses for 10mins at 20Hz, 25% duty cycle through an optic fiber cannula placed in contact with the CSMG complex. 30mins following optogenetic stimulation or sham stimulation, lipopolysaccharide (0.25mg/kg) was injected intraperitoneally and 90mins later mice were euthanized and blood, and spleen were collected and processed for cytokine and chemokines. Optogenetic stimulation of the CSMG complex in ChAT‐ChR2 mice (n=14) suppressed serum pro‐inflammatory cytokines and chemokines compared with sham stimulation (n=13), including TNF(401.7 ± 102.9 pg/ml vs 766.1 ± 207.2 pg/ml; P&lt;0.0001) and MIP‐1α (6,523 ± 3,041 pg/ml vs 10,672 ± 3,915 pg/ml, P=0.0066), while the serum levels of the anti‐inflammatory cytokine IL‐10 were significantly increased (30,779 ± 10,035 pg/ml vs 22,979 ± 9,669 pg/ml, P=0.0427). There was also a significant reduction in splenic TNF levels in the stimulated vs the sham group(P=0.0023). Of note, optogenetic stimulation of the CSMG complex in littermate mice that do not express ChR2 on cholinergic neurons did not alter serum and splenic cytokine levels. These results provide the first experimental evidence that selective stimulation of cholinergic neuronal endings in the CSMG complex attenuates cytokine and chemokine levels. Future studies examining the specific contribution of vagal and sympathetic preganglionic cholinergic neurons in the CSMG complex in the regulation of immune responses will be instrumental for developing new targeted therapeutic anti‐inflammatory approaches.

Field performance metric(s) optimization

Introduction: In contrast to methods for monitoring physical vital signs, such as pulse, respiration rate, and body temperature, the ability to monitor molecules indicative of health or performance status has greatly lagged. Specifically, until recently, technologies able to monitor specific molecules in the body in real-time were limited to the detection of only a few metabolites (glucose, lactate, and oxygen) and no hormones or protein biomarkers. This is because all prior molecular monitoring technologies relied on the specific chemical or enzymatic reactivity of their targets to generate a signal, rendering it impossible to generalize them to new targets. Methods: Human technological prowess notwithstanding, the ability to monitor arbitrary molecules in the body continuous and in real-time is a solved problem: the human body, for example, utilizes hundreds of receptors that respond quantitatively to specific molecular cues without exploiting their chemical or enzymatic reactivity. To achieve this, nature has invented two critical “tricks.” The first is the combinatorial chemistry of biopolymers (proteins, nucleic acids), with which it can generate high-affinity, high-specificity receptors against almost any water-soluble target. The second is the use of conformation-linked signalling, in which the binding of a target molecule alters the shape of the receptor, producing in turn an easily detected output1,2. Motivated by these arguments, recent advances in biosensors have exploited the same two tricks. Results: Using the combinatorial chemistry of biomolecules to generate high-performance receptors and our growing understanding of biomolecular physics to re-engineering them such that they undergo binding-induced conformational changes, recent advances in the field of biosensors has enabled, for the first time, the high-frequency, real-time measurement of specific molecules in living bodies via an approach that is agnostic to the reactivity of the target and thus can be generalized to new targets3,4. Discussion/conclusions: The newly achieved ability to monitor arbitrary molecules continuously and in real-time in the human body provides unprecedented opportunities to both identify the biomarkers correlative of health and performance status and to monitor them in real-time. Acknowledgements: Biosensor research in Prof. Plaxco’s group at UCSB has been supported by the U.S. Office of Naval Research and the NIH. References 1Lubin AA, Plaxco KW. Folding-based electrochemical biosensors: the case for responsive nucleic acid architectures. Acc Chem Res 2010; 43:496–505. https://doi.org/10.1021/ar900165x 2Plaxco KW, Soh HT. Switch based biosensors: a new approach towards real-time, in vivo molecular detection. Trends in Biotech 2011; 29:1-5. https://doi.org/10.1016/j.tibtech.2010.10.005 3Arroyo-Curras N, Somerson J, Vieira P, et al. Real-time measurement of small molecules in awake, ambulatory animals. Proc Natl Acad Sci USA 2017; 114:645–650. https://doi.org/10.1073/pnas.1613458114 4Idili A, Gerson J, Kippin T, et al. Seconds-resolved, in situ measurements of plasma phenylalanine disposition in living rats. Anal Chem 2021; 93:4023-4032. https://doi.org/10.1021/acs.analchem.0c05024

Electrical Stimulation of the Dorsal Motor Nucleus of the Vagus Attenuates Serum Pro‐Inflammatory Cytokine Levels in Murine Models of Endotoxemia and Sepsis

The inflammatory reflex is a vagus nerve‐mediated and brainstem‐integrated physiological mechanism that regulates immunity and inflammation (Annu Rev Immunol, 2018, 36:783). The efferent vagus nerve originates in brainstem nuclei, including the dorsal motor nucleus of the vagus (DMN). Recently, it was shown that optogenetic stimulation of cholinergic DMN neurons inhibits serum TNF levels during endotoxemia (Proc Natl Acad Sci USA, 2020, 117(47):29803). However, the effects of electrical DMN stimulation on cytokine levels in inflammatory conditions have not been previously evaluated. To provide insight, we examined the effect of electrical DMN stimulation in mice with endotoxemia and cecal ligation and puncture (CLP)‐induced polymicrobial sepsis. In the endotoxemia model, 10–12‐week‐old male C57BL/6 mice (n=14‐20 per group) were anesthetized using isoflurane and the head was positioned on a stereotaxic frame. A surgical intervention and a stereotaxic‐guided approach were used to insert a concentric bipolar electrode into the left DMN. Electrical stimulation (50 μA, 30 Hz, 260 μs) or sham stimulation was performed for five mins, followed by lipopolysaccharide (LPS, 0.5 mg/kg) i.p. administration. The mice were euthanized 90 mins after LPS and blood (via cardiac puncture) was collected and processed for cytokine analysis. In the CLP model, DMN stimulation or sham stimulation was performed as described above immediately after subjecting mice (n=14‐20 per group) to CLP, i.e., following a midline laparotomy in anesthetised mice, the cecum was isolated and the distal end was ligated and punctured. 24h later, the mice were euthanised and blood was collected and processed for analysis. In mice with endotoxemia, electrical DMN stimulation significantly decreased serum TNF (p = 0.0069) and IL‐6 (p = 0.0427) levels compared with sham stimulation. In addition, electrical DMN stimulation increased the serum levels of the anti‐inflammatory cytokine IL‐10 (p = 0.0284). In mice with CLP, electrical DMN stimulation resulted in significantly lower serum TNF levels compared with sham stimulation (p = 0.0291). These results indicate for the first time that electrical DMN stimulation alters circulating pro‐inflammatory and anti‐inflammatory levels in murine endotoxemia and sepsis. These findings are of interest for further development of bioelectronic approaches targeting DMN for therapeutic benefit in conditions, characterised by immune dysregulation and inflammation.

Enhancing Residency Recruitment Processes to Facilitate a Diverse Work Force in Anesthesiology

Committee News| May 2022 Enhancing Residency Recruitment Processes to Facilitate a Diverse Work Force in Anesthesiology Sujatha Ramachandran, MBBS, MACM; Sujatha Ramachandran, MBBS, MACM Search for other works by this author on: This Site PubMed Google Scholar Camille Fontaine, MD; Camille Fontaine, MD Search for other works by this author on: This Site PubMed Google Scholar Tracey Straker, MD, MS, MPH, CBA, FASA Tracey Straker, MD, MS, MPH, CBA, FASA Search for other works by this author on: This Site PubMed Google Scholar ASA Monitor May 2022, Vol. 86, 36–37. https://doi.org/10.1097/01.ASM.0000830860.56287.f1 Views Icon Views Article contents Figures & tables Video Audio Supplementary Data Peer Review Share Icon Share Facebook Twitter LinkedIn MailTo Cite Icon Cite Get Permissions Search Site Citation Sujatha Ramachandran, Camille Fontaine, Tracey Straker; Enhancing Residency Recruitment Processes to Facilitate a Diverse Work Force in Anesthesiology. ASA Monitor 2022; 86:36–37 doi: https://doi.org/10.1097/01.ASM.0000830860.56287.f1 Download citation file: Ris (Zotero) Reference Manager EasyBib Bookends Mendeley Papers EndNote RefWorks BibTex toolbar search Search Dropdown Menu toolbar search search input Search input auto suggest filter your search All ContentAll PublicationsASA Monitor Search Advanced Search Topics: internship and residency, mentors, workforce Building a diverse workforce of physician anesthesiologists should be paramount for hospitals and health care systems. More inclusive preoperative staff would better reflect current demographics nationwide, help address health care disparities, encourage minority patient participation in decision-making, improve patient outcomes and satisfaction, and foster greater innovation in medicine (JAMA Intern Med 2014;174:289-91; Proc Natl Acad Sci U S A 2020;117:9284-91). Despite these indisputable advantages of physician workforce diversity, the percentage of people identifying as belonging to racial/ethnic minority groups in medicine has continued to remain below that of the general U.S. population (JAMA Netw Open 2019;2:e1910490; N Engl J Med 2019;380:803-5). In anesthesiology, the percentage of under-represented minority (URM) physicians is around 11%. Medical schools are documenting upward trends in Black and brown female graduates (asamonitor.pub/3tLSg8M; JAMA Intern Med 2015;175:1706-8). As per the Association of American Medical Colleges (AAMC), between 1986... You do not currently have access to this content.