A Cancer Nanovaccine for Co-Delivery of Peptide Neoantigens and Optimized Combinations of STING and TLR4 Agonists.

Immune checkpoint blockade (ICB) has revolutionized cancer treatment and led to complete and durable responses, but only for a minority of patients. Resistance to ICB can largely be attributed to insufficient number and/or function of antitumor CD8+ T cells in the tumor microenvironment. Neoantigen targeted cancer vaccines can activate and expand the antitumor T cell repertoire, but historically, clinical responses have been poor because immunity against peptide antigens is typically weak, resulting in insufficient activation of CD8+ cytotoxic T cells. Herein, we describe a nanoparticle vaccine platform that can overcome these barriers in several ways. First, the vaccine can be reproducibly formulated using a scalable confined impingement jet mixing method to coload a variety of physicochemically diverse peptide antigens and multiple vaccine adjuvants into pH-responsive, vesicular nanoparticles that are monodisperse and less than 100 nm in diameter. Using this approach, we encapsulated synergistically acting adjuvants, cGAMP and monophosphoryl lipid A (MPLA), into the nanocarrier to induce a robust and tailored innate immune response that increased peptide antigen immunogenicity. We found that incorporating both adjuvants into the nanovaccine synergistically enhanced expression of dendritic cell costimulatory markers, pro-inflammatory cytokine secretion, and peptide antigen cross-presentation. Additionally, the nanoparticle delivery increased lymph node accumulation and uptake of peptide antigen by dendritic cells in the draining lymph node. Consequently, nanoparticle codelivery of peptide antigen, cGAMP, and MPLA enhanced the antigen-specific CD8+ T cell response and delayed tumor growth in several mouse models. Finally, the nanoparticle platform improved the efficacy of ICB immunotherapy in a murine colon carcinoma model. This work establishes a versatile nanoparticle vaccine platform for codelivery of peptide neoantigens and synergistic adjuvants to enhance responses to cancer vaccines.

Clinical Implementation of "Plan of the Day" Strategy in Definitive Radiation Therapy of Cervical Cancer: Online Adaptation to Address the Challenge of Organ Filling Reproducibility.

PURPOSE: Definitive pelvic intensity modulated radiation therapy (IMRT) in cervical cancer is susceptible to geographic miss due to daily positional and volumetric variations in target and organs at risk. Hence, despite evidence of reduced acute and late treatment-related toxicities, implementation of image-guided IMRT (IG-IMRT) with a reasonable safety margin to encompass organ motion is challenging. METHODS AND MATERIALS: In this prospective, nonrandomized phase 2 study, patients with cervical cancer International Federation of Gynecology and Obstetrics (2009) stage IB2-IIIB between the ages of 18 and 65 years were treated with definitive pelvic chemoradiotherapy with a prespecified organ (bladder and rectum) filling protocol. Reproducibility of organ filling was assessed along with the implementation of daily comprehensive adaptive image-guided radiotherapy (IGRT), with a library of 3 IMRT (volumetric modulated arc therapy) plans with incremental expansions of clinical target volume (CTV) to planning target volume (PTV) (primary) margins (small, 0.7 cm; adequate, 1 cm; and large, 1.5 cm) and a backup motion robust 3-dimensional conformal radiotherapy plan; the appropriate plan is chosen based on pretreatment cone beam computed tomography (CBCT) ("plan of the day" approach). RESULTS: Fifty patients with a median age of 49 years (IQR, 45-56 years) received definitive radiation therapy (45-46 Gy in 23-25 fractions to pelvis, with simultaneous integrated boost to gross nodes in 15 patients) with the aforementioned IGRT protocol. In the analysis of 1171 CBCT images (in 1184 treatment sessions), the mean planning computed tomography (CT) and CBCT bladder volumes were 417 and 373 cc, respectively. Significant interfractional variation in bladder volume was noted with a mean absolute dispersion of 29.5% with respect to planning CT; significant influential random factors were postchemotherapy sessions (P ≤ .001), pre-CBCT protocol duration (P = .001), and grades of chemotherapy induced nausea vomiting (P = .001). Significantly higher variation in bladder filling was noted in patients with older age (P = .014) and larger planning CT bladder volume (P ≤ .001). Time trend analysis of fraction-wise bladder volume revealed an absolute systemic reduction of 16.3% in bladder volume means from the first to the fifth week. Variation in rectal diameter was much less pronounced, with 19.2% mean dispersion and without any significant factors affecting it. Although in 19% and 2% of sessions large IMRT PTV and 3-dimensional conformal radiotherapy were necessary to cover the primary target, respectively, reduction in treated volume was possible in 43% of sessions with small PTV selection instead of standard adequate PTV (36% sessions). Plan of the day selection had a moderate to strong correlation with nonabsolute dispersion of bladder filling (Spearman ρ =0.4; P = .001) and a weak (but significant) correlation with grades of acute toxicities. The planned protocol was well tolerated with no radiation-induced local grade 3 toxicity. CONCLUSIONS: Interfractional variation in organ filling (especially bladder) is inevitable despite fixed pretreatment protocol in definitive settings (intact cervix). Despite the logistical challenges, adaptive IGRT in the form of plan of the day based on incremental CTV-to-PTV margins is a relatively simple and feasible strategy to minimize geometric uncertainties in radical IG-IMRT of cervical cancer.

Structural similarities between SAM and ATP recognition motifs and detection of ATP binding in a SAM binding DNA methyltransferase.

S-adenosylmethionine (SAM) is a ubiquitous co-factor that serves as a donor for methylation reactions and additionally serves as a donor of other functional groups such as amino and ribosyl moieties in a variety of other biochemical reactions. Such versatility in function is enabled by the ability of SAM to be recognized by a wide variety of protein molecules that vary in their sequences and structural folds. To understand what gives rise to specific SAM binding in diverse proteins, we set out to study if there are any structural patterns at their binding sites. A comprehensive analysis of structures of the binding sites of SAM by all-pair comparison and clustering, indicated the presence of 4 different site-types, only one among them being well studied. For each site-type we decipher the common minimum principle involved in SAM recognition by diverse proteins and derive structural motifs that are characteristic of SAM binding. The presence of the structural motifs with precise three-dimensional arrangement of amino acids in SAM sites that appear to have evolved independently, indicates that these are winning arrangements of residues to bring about SAM recognition. Further, we find high similarity between one of the SAM site types and a well known ATP binding site type. We demonstrate using in vitro experiments that a known SAM binding protein, HpyAII.M1, a type 2 methyltransferase can bind and hydrolyse ATP. We find common structural motifs that explain this, further supported through site-directed mutagenesis. Observation of similar motifs for binding two of the most ubiquitous ligands in multiple protein families with diverse sequences and structural folds presents compelling evidence at the molecular level in favour of convergent evolution.

Rationally Designed Novel Phenyloxazoline Synthase Inhibitors: Chemical Synthesis and Biological Evaluation to Accelerate the Discovery of New Antimycobacterial Antibiotics.

The uncontrolled spread of drug-resistant tuberculosis (DR-TB) clinical cases necessitates the urgent discovery of newer chemotypes with novel mechanisms of action. Here, we report the chemical synthesis of rationally designed novel transition-state analogues (TSAs) by targeting the cyclization (Cy) domain of phenyloxazoline synthase (MbtB), a key enzyme of the conditionally essential siderophore biosynthesis pathway. Following bio-assay-guided evaluation of TSA analogues preferentially in iron-deprived and iron-rich media to understand target preferentiality against a panel of pathogenic and non-pathogenic mycobacteria strains, we identified a hit, i.e., TSA-5. Molecular docking, dynamics, and MMPBSA calculations enabled us to comprehend TSA-5's stable binding at the active site pocket of MbtB_Cy and the results imply that the MbtB_Cy binding pocket has a strong affinity for electron-withdrawing functional groups and contributes to stable polar interactions between enzyme and ligand. Furthermore, enhanced intracellular killing efficacy (8 µg/mL) of TSA-5 against Mycobacterium aurum in infected macrophages is noted in comparison to moderate in vitro antimycobacterial efficacy (64 µg/mL) against M. aurum. TSA-5 also demonstrates whole-cell efflux pump inhibitory activity against Mycobacterium smegmatis. Identification of TSA-5 by focusing on the modular MbtB_Cy domain paves the way for accelerating novel anti-TB antibiotic discoveries.

Comparative transcriptomic and weighted gene co-expression network analysis to identify the core genes in the cultivars of Musa acuminata under both infected and chemical perturbated conditions.

Banana is a high nutrient crop, which ranks fourth in terms of gross value production. Fusarium wilt of banana, caused by Fusarium oxysporum f. sp. cubense tropical race 4 (FocTR4), is considered the most destructive disease leading to the complete loss of production of the Cavendish cultivars Berangan, Brazilian and Williams, which are vulnerable to the infection of FocTR4. However, the treatment with benzothiadiazole, a synthetic salicylic analog, is aimed to induce resistance in plants. Thus, the treatments pertaining to the banana plants subjected to the Foc infection within the chosen cultivars were compared with chemically treated samples obtained at different time intervals for a short duration (0-4 days). The integrated omics analyses considering the parameters of WGCNA, functional annotation, and protein-protein interactions revealed that many pathways have been negatively influenced in Cavendish bananas under FocTR4 infections and the number of genes influenced also increased over time in Williams cultivar. Furthermore, elevation in immune response and resistance genes were also observed in the roots of the Cavendish banana.

Synergistic Role of NK Cells and Monocytes in Promoting Atherogenesis in Severe COVID-19 Patients.

Clinical data demonstrate an increased predisposition to cardiovascular disease (CVD) following severe COVID-19 infection. This may be driven by a dysregulated immune response associated with severe disease. Monocytes and vascular tissue resident macrophages play a critical role in atherosclerosis, the main pathology leading to ischemic CVD. Natural killer (NK) cells are a heterogenous group of cells that are critical during viral pathogenesis and are known to be dysregulated during severe COVID-19 infection. Their role in atherosclerotic cardiovascular disease has recently been described. However, the contribution of their altered phenotypes to atherogenesis following severe COVID-19 infection is unknown. We demonstrate for the first time that during and after severe COVID-19, circulating proinflammatory monocytes and activated NK cells act synergistically to increase uptake of oxidized low-density lipoprotein (Ox-LDL) into vascular tissue with subsequent foam cell generation leading to atherogenesis despite recovery from acute infection. Our data provide new insights, revealing the roles of monocytes/macrophages, and NK cells in COVID-19-related atherogenesis.

Mitochondrial Magnesium is the cationic rheostat for MCU-mediated mitochondrial Ca2+ uptake.

Calcium (Ca2+) uptake by mitochondria is essential in regulating bioenergetics, cell death, and cytosolic Ca2+ transients. Mitochondrial Calcium Uniporter (MCU) mediates the mitochondrial Ca2+ uptake. MCU is a heterooligomeric complex with a pore-forming component and accessory proteins required for channel activity. Though MCU regulation by MICUs is unequivocally established, there needs to be more knowledge of whether divalent cations regulate MCU. Here we set out to understand the mitochondrial matrix Mg2+-dependent regulation of MCU activity. We showed Mrs2 as the authentic mammalian mitochondrial Mg2+ channel using the planar lipid bilayer recordings. Using a liver-specific Mrs2 KO mouse model, we showed that decreased matrix [Mg2+] is associated with increased MCU activity and matrix Ca2+ overload. The disruption of Mg2+dependent MCU regulation significantly prompted mitochondrial permeability transition pore opening-mediated cell death during tissue IR injury. Our findings support a critical role for mMg2+ in regulating MCU activity and attenuating mCa2+ overload.

Ru(II)-catalyzed synthesis of poly-substituted furans via intermolecular oxidative annulation reaction of ethyl 3-oxo-3-phenylpropanoates with aryl alkynes/heteroaryl alkynes.

The oxidative annulation reaction of ethyl 3-oxo-3-phenylpropanoates with internal alkynes proceeds efficiently in the presence of a Ru(II)-catalyst, a copper oxidant and an additive such as AgSbF6 to give poly-substituted furans, which offers a novel method for the selective construction of poly-substituted furans. The reaction has wider substrate scope with simple starting materials, and the desired tetrasubstituted furans were prepared in good to excellent yields.

Hybrid Deep Learning Approach for Stress Detection Using Decomposed EEG Signals.

Stress has an impact, not only on a person's physical health, but also on the ability to perform at the workplace in daily life. The well-established relation between psychological stress and its pathogeneses highlights the need for detecting psychological stress early, in order to prevent disease advancement and to save human lives. Electroencephalography (EEG) signal recording tools are widely used to collect these psychological signals/brain rhythms in the form of electric waves. The aim of the current research was to apply automatic feature extraction to decomposed multichannel EEG recordings, in order to efficiently detect psychological stress. The traditional deep learning techniques, namely the convolution neural network (CNN), long short-term memory (LSTM), bidirectional long short-term memory (BiLSTM), gated recurrent unit (GRU) and recurrent neural network (RNN) models, have been frequently used for stress detection. A hybrid combination of these techniques may provide improved performance, and can handle long-term dependencies in non-linear brain signals. Therefore, this study proposed an integration of deep learning models, called DWT-based CNN, BiLSTM, and two layers of a GRU network, to extract features and classify stress levels. Discrete wavelet transform (DWT) analysis was used to remove the non-linearity and non-stationarity from multi-channel (14 channel) EEG recordings, and to decompose them into different frequency bands. The decomposed signals were utilized for automatic feature extraction using the CNN, and the stress levels were classified using BiLSTM and two layers of GRU. This study compared five combinations of the CNN, LSTM, BiLSTM, GRU and RNN models with the proposed model. The proposed hybrid model performed better in classification accuracy compared to the other models. Therefore, hybrid combinations are appropriate for the clinical intervention and prevention of mental and physical problems.

Dendritic cell therapy augments antitumor immunity triggered by CDK4/6 inhibition and immune checkpoint blockade by unleashing systemic CD4 T-cell responses.

BACKGROUND: Cyclin-dependent kinase 4/6 inhibitors (CDK4/6i) combined with endocrine therapy are a mainstay treatment for hormone receptor-positive breast cancer. While their principal mechanism is inhibition of cancer cell proliferation, preclinical and clinical evidence suggests that CDK4/6i can also promote antitumor T-cell responses. However, this pro-immunogenic property is yet to be successfully harnessed in the clinic, as combining CDK4/6i with immune checkpoint blockade (ICB) has not shown a definitive benefit in patients. METHOD: We performed an in-depth analysis of the changes in the tumor immune microenvironment and systemic immune modulation associated with CDK4/6i treatment in muring breast cancer models and in patients with breast cancer using high dimensional flow cytometry and RNA sequencing. Gain and loss of function in vivo experiments employing cell transfer and depletion antibody were performed to uncover immune cell populations critical for CDK4/6i-mediated stimulation of antitumor immunity. RESULTS: We found that loss of dendritic cells (DCs) within the tumor microenvironment resulting from CDK4/6 inhibition in bone marrow progenitors is a major factor limiting antitumor immunity after CDK4/6i and ICB. Consequently, restoration of DC compartment by adoptively transferring ex vivo differentiated DCs to mice treated with CDK4/6i and ICB therapy enabled robust tumor inhibition. Mechanistically, the addition of DCs promoted the induction of tumor-localized and systemic CD4 T-cell responses in mice receiving CDK4/6i-ICB-DC combination therapy, as characterized by enrichment of programmed cell death protein-1-negative T helper (Th)1 and Th2 cells with an activated phenotype. CD4 T-cell depletion abrogated the antitumor benefit of CDK4/6i-ICB-DC combination, with outgrowing tumors displaying an increased proportion of terminally exhausted CD8 T cells. CONCLUSIONS: Our findings suggest that CDK4/6i-mediated DC suppression limits CD4 T-cell responses essential for the sustained activity of CD8 T cells and tumor inhibition. Furthermore, they imply that restoring DC-CD4 T-cell crosstalk via DC transfer enables effective breast cancer immunity in response to CDK4/6i and ICB treatment.

The ion channel CALHM6 controls bacterial infection-induced cellular cross-talk at the immunological synapse.

Membrane ion channels of the calcium homeostasis modulator (CALHM) family promote cell-cell crosstalk at neuronal synapses via ATP release, where ATP acts as a neurotransmitter. CALHM6, the only CALHM highly expressed in immune cells, has been linked to the induction of natural killer (NK) cell anti-tumour activity. However, its mechanism of action and broader functions in the immune system remain unclear. Here, we generated Calhm6-/- mice and report that CALHM6 is important for the regulation of the early innate control of Listeria monocytogenes infection in vivo. We find that CALHM6 is upregulated in macrophages by pathogen-derived signals and that it relocates from the intracellular compartment to the macrophage-NK cell synapse, facilitating ATP release and controlling the kinetics of NK cell activation. Anti-inflammatory cytokines terminate CALHM6 expression. CALHM6 forms an ion channel when expressed in the plasma membrane of Xenopus oocytes, where channel opening is controlled by a conserved acidic residue, E119. In mammalian cells, CALHM6 is localised to intracellular compartments. Our results contribute to the understanding of neurotransmitter-like signal exchange between immune cells that fine-tunes the timing of innate immune responses.

Synthesis of highly substituted isoquinolines/isoquinolones by ruthenium (II)-catalyzed reaction of benzyl/α-methyl benzyl/benzoyl isocyanates with diaryl alkynes.

An efficient and novel method has been developed for the synthesis of highly substituted isoquinolines/isoquinolones by Ru(II)-catalyzed intermolecular oxidative annulation of benzyl/benzoyl isocyanates with diaryl alkynes in the presence of Cs2CO3 as base and Cu(OTf)2 as an oxidant at 120 °C for 1 h.

Computational identification and characterization of vascular wilt pathogen (Fusarium oxysporum f. sp. lycopersici) CAZymes in tomato xylem sap.

Fusarium oxysporum f. sp. lycopersici is a devastating plant pathogenic fungi known for wilt disease in the tomato plant and secrete cell wall degrading enzymes. These enzymes are collectively known as carbohydrate-active enzymes (CAZymes), crucial for growth, colonization and pathogenesis. Therefore, the present study was aimed to identify and annotate pathogen CAZymes in the xylem sap of a susceptible tomato variety using downstream proteomics and meta servers. Further, structural elucidation and conformational stability analysis of the selected CAZyme families were done through homology modeling and molecular dynamics simulation. Among all the fungal proteins identified, the carbohydrate metabolic process was found to be enriched. Most of the annotated CAZymes belonged to the hydrolase and oxidoreductase families, and 90% were soluble and extracellular. Moreover, using a publically available interactome database, interactions were observed between the families acting on chitin, hemicellulose and pectin. Subsequently, important catalytic residues were identified in the candidate CAZymes belonging to carbohydrate esterase (CE8) and glycosyl hydrolase (GH18 and GH28). Further, essential dynamics after molecular simulation of 100 ns revealed the overall behavior of these CAZymes with distinct global minima and transition states in CE8. Thus, our study identified some of the CAZyme families that assist in pathogenesis and growth through host cell wall deconstruction with further structural insight into the selected CAZyme families.Communicated by Ramaswamy H. Sarma.

BCL-xL inhibition potentiates cancer therapies by redirecting the outcome of p53 activation from senescence to apoptosis.

Cancer therapies trigger diverse cellular responses, ranging from apoptotic death to acquisition of persistent therapy-refractory states such as senescence. Tipping the balance toward apoptosis could improve treatment outcomes regardless of therapeutic agent or malignancy. We find that inhibition of the mitochondrial protein BCL-xL increases the propensity of cancer cells to die after treatment with a broad array of oncology drugs, including mitotic inhibitors and chemotherapy. Functional precision oncology and omics analyses suggest that BCL-xL inhibition redirects the outcome of p53 transcriptional response from senescence to apoptosis, which likely occurs via caspase-dependent down-modulation of p21 and downstream cytostatic proteins. Consequently, addition of a BCL-2/xL inhibitor strongly improves melanoma response to the senescence-inducing drug targeting mitotic kinase Aurora kinase A (AURKA) in mice and patient-derived organoids. This study shows a crosstalk between the mitochondrial apoptotic pathway and cell cycle regulation that can be targeted to augment therapeutic efficacy in cancers with wild-type p53.

Comparative transcriptome profiling and weighted gene co-expression network analysis to identify core genes in maize (Zea mays L.) silks infected by multiple fungi.

Maize (Zea mays L.) is the third most popular Poaceae crop after wheat and rice and used in feed and pharmaceutical sectors. The maize silk contains bioactive components explored by traditional Chinese herbal medicine for various pharmacological activities. However, Fusarium graminearum, Fusarium verticillioides, Trichoderma atroviride, and Ustilago maydis can infect the maize, produce mycotoxins, hamper the quantity and quality of silk production, and further harm the primary consumer's health. However, the defense mechanism is not fully understood in multiple fungal infections in the silk of Z. mays. In this study, we applied bioinformatics approaches to use the publicly available transcriptome data of Z. mays silk affected by multiple fungal flora to identify core genes involved in combatting disease response. Differentially expressed genes (DEGs) were identified among intra- and inter-transcriptome data sets of control versus infected Z. mays silks. Upon further comparison between up- and downregulated genes within the control of datasets, 4,519 upregulated and 5,125 downregulated genes were found. The DEGs have been compared with genes in the modules of weighted gene co-expression network analysis to relevant specific traits towards identifying core genes. The expression pattern of transcription factors, carbohydrate-active enzymes (CAZyme), and resistance genes was analyzed. The present investigation is supportive of our findings that the gene ontology, immunity stimulus, and resistance genes are upregulated, but physical and metabolic processes such as cell wall organizations and pectin synthesis were downregulated respectively. Our results are indicative that terpene synthase TPS6 and TPS11 are involved in the defense mechanism against fungal infections in maize silk.

A molecular signature of lung-resident CD8+ T cells elicited by subunit vaccination.

Natural infection as well as vaccination with live or attenuated viruses elicit tissue resident, CD8+ memory T cell (Trm) response. Trm cells so elicited act quickly upon reencounter with the priming agent to protect the host. These Trm cells express a unique molecular signature driven by the master regulators-Runx3 and Hobit. We previously reported that intranasal instillation of a subunit vaccine in a prime boost vaccination regimen installed quick-acting, CD8+ Trm cells in the lungs that protected against lethal vaccinia virus challenge. It remains unexplored whether CD8+ Trm responses so elicited are driven by a similar molecular signature as those elicited by microbes in a real infection or by live, attenuated pathogens in conventional vaccination. We found that distinct molecular signatures distinguished subunit vaccine-elicited lung interstitial CD8+ Trm cells from subunit vaccine-elicited CD8+ effector memory and splenic memory T cells. Nonetheless, the transcriptome signature of subunit vaccine elicited CD8+ Trm resembled those elicited by virus infection or vaccination. Clues to the basis of tissue residence and function of vaccine specific CD8+ Trm cells were found in transcripts that code for chemokines and chemokine receptors, purinergic receptors, and adhesins when compared to CD8+ effector and splenic memory T cells. Our findings inform the utility of protein-based subunit vaccination for installing CD8+ Trm cells in the lungs to protect against respiratory infectious diseases that plague humankind.

Insights on Aggregation of Hen Egg-White Lysozyme from Raman Spectroscopy and MD Simulations.

Protein misfolding and aggregation play a significant role in several neurodegenerative diseases. In the present work, the spontaneous aggregation of hen egg-white lysozyme (HEWL) in an alkaline pH 12.2 at an ambient temperature was studied to obtain molecular insights. The time-dependent changes in spectral peaks indicated the formation of ß sheets and their effects on the backbone and amino acids during the aggregation process. Introducing iodoacetamide revealed the crucial role of intermolecular disulphide bonds amidst monomers in the aggregation process. These findings were corroborated by Molecular Dynamics (MD) simulations and protein-docking studies. MD simulations helped establish and visualize the unfolding of the proteins when exposed to an alkaline pH. Protein docking revealed a preferential dimer formation between the HEWL monomers at pH 12.2 compared with the neutral pH. The combination of Raman spectroscopy and MD simulations is a powerful tool to study protein aggregation mechanisms.

Role of Charged Amino Acids in Sullying the Fluorescence of Tryptophan or Conjugated Dansyl Probe in Monomeric Proteins.

When Trp/dansyl probe conjugated to a monomeric protein is photoexcited, it is assumed that all emitted fluorescence originates solely from them. In this work, we show that hidden unconventional intrinsic chromophores (called ProCharTS) that originate from confined charge clusters in the protein can contaminate Trp/dansyl emission. Previous work has shown that charge recombination among charge-separated excited states of monomeric proteins, rich in charged residues, can emit weak luminescence (300-700 nm) overlapping with ProCharTS absorption (250-800 nm) and Trp (300-400 nm) and dansyl (400-600 nm) emission. We examine how this overlap taints the fluorescence arising from Trp/dansyl. We compared the effect of dense aqueous solutions of amino acids, Lys/Glu/Asp/Arg/His, on the fluorescence intensity decay/spectrum of N-acetyl-l-tryptophan amide (NATA). Significant broadening on the red side of Trp emission spectrum was observed solely in the presence of lysine, which appeared to be the most potent in altering the mono-exponential fluorescence decay of NATA. Interestingly, NATA in the presence of proteins α3C and dehydrin (DHN1), which are rich in Lys residues, showed substantial deviation from mono-exponential fluorescence decay in contrast to PEST wt and Symfoil-4P pv2, which lack Lys residues. Remarkably, Trp emission spectra among charge-rich proteins like α3W, PEST M1, and DHN1 CW1 were altered on the red side of Trp emission. Emission spectrum of dansyl-labeled human serum albumin (HuSA) was broadened and its fluorescence quenched with gradual addition of excess unlabeled HuSA, which displays bountiful ProCharTS luminescence. Our results unveil the additive influence of ProCharTS luminescence on Trp/dansyl emission with no measurable evidence of energy transfer.

Treatment with soluble CD24 attenuates COVID-19-associated systemic immunopathology.

BACKGROUND: Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) causes coronavirus disease 2019 (COVID-19) through direct lysis of infected lung epithelial cells, which releases damage-associated molecular patterns and induces a pro-inflammatory cytokine milieu causing systemic inflammation. Anti-viral and anti-inflammatory agents have shown limited therapeutic efficacy. Soluble CD24 (CD24Fc) blunts the broad inflammatory response induced by damage-associated molecular patterns via binding to extracellular high mobility group box 1 and heat shock proteins, as well as regulating the downstream Siglec10-Src homology 2 domain-containing phosphatase 1 pathway. A recent randomized phase III trial evaluating CD24Fc for patients with severe COVID-19 (SAC-COVID; NCT04317040) demonstrated encouraging clinical efficacy. METHODS: Using a systems analytical approach, we studied peripheral blood samples obtained from patients enrolled at a single institution in the SAC-COVID trial to discern the impact of CD24Fc treatment on immune homeostasis. We performed high dimensional spectral flow cytometry and measured the levels of a broad array of cytokines and chemokines to discern the impact of CD24Fc treatment on immune homeostasis in patients with COVID-19. RESULTS: Twenty-two patients were enrolled, and the clinical characteristics from the CD24Fc vs. placebo groups were matched. Using high-content spectral flow cytometry and network-level analysis, we found that patients with severe COVID-19 had systemic hyper-activation of multiple cellular compartments, including CD8+ T cells, CD4+ T cells, and CD56+ natural killer cells. Treatment with CD24Fc blunted this systemic inflammation, inducing a return to homeostasis in NK and T cells without compromising the anti-Spike protein antibody response. CD24Fc significantly attenuated the systemic cytokine response and diminished the cytokine coexpression and network connectivity linked with COVID-19 severity and pathogenesis. CONCLUSIONS: Our data demonstrate that CD24Fc rapidly down-modulates systemic inflammation and restores immune homeostasis in SARS-CoV-2-infected individuals, supporting further development of CD24Fc as a novel therapeutic against severe COVID-19.

TREATMENT WITH SOLUBLE CD24 ATTENUATES COVID-19-ASSOCIATED SYSTEMIC IMMUNOPATHOLOGY.

BACKGROUND: SARS-CoV-2 causes COVID-19 through direct lysis of infected lung epithelial cells, which releases damage-associated molecular patterns (DAMPs) and induces a pro-inflammatory cytokine milieu causing systemic inflammation. Anti-viral and anti-inflammatory agents have shown limited therapeutic efficacy. Soluble CD24 (CD24Fc) is able to blunt the broad inflammatory response induced by DAMPs in multiple models. A recent randomized phase III trial evaluating the impact of CD24Fc in patients with severe COVID-19 demonstrated encouraging clinical efficacy. METHODS: We studied peripheral blood samples obtained from patients enrolled at a single institution in the SAC-COVID trial (NCT04317040) collected before and after treatment with CD24Fc or placebo. We performed high dimensional spectral flow cytometry analysis of peripheral blood mononuclear cells and measured the levels of a broad array of cytokines and chemokines. A systems analytical approach was used to discern the impact of CD24Fc treatment on immune homeostasis in patients with COVID-19. FINDINGS: Twenty-two patients were enrolled, and the clinical characteristics from the CD24Fc vs. placebo groups were matched. Using high-content spectral flow cytometry and network-level analysis, we found systemic hyper-activation of multiple cellular compartments in the placebo group, including CD8+ T cells, CD4+ T cells, and CD56+ NK cells. By contrast, CD24Fc-treated patients demonstrated blunted systemic inflammation, with a return to homeostasis in both NK and T cells within days without compromising the ability of patients to mount an effective anti-Spike protein antibody response. A single dose of CD24Fc significantly attenuated induction of the systemic cytokine response, including expression of IL-10 and IL-15, and diminished the coexpression and network connectivity among extensive circulating inflammatory cytokines, the parameters associated with COVID-19 disease severity. INTERPRETATION: Our data demonstrates that CD24Fc treatment rapidly down-modulates systemic inflammation and restores immune homeostasis in SARS-CoV-2-infected individuals, supporting further development of CD24Fc as a novel therapeutic against severe COVID-19. FUNDING: NIH.