Transcriptome-Based Treatment for Melanoma With Brain Metastasis: A Case Report.

Malignant melanoma with brain metastasis has a high mortality rate. New approaches for diagnosis and treatment are urgently required to improve prognosis. Here we present a 60-year-old male with metastatic melanoma to the brain. Using a transcriptomics pipeline, we analyzed whole blood and resected tumor tissue, identifying enriched gene expression biomarkers and pathways - including seven upregulated ( BRAF, CDK4, EIF1AX, IK, NRAS, PIK3R2, and TP53) and 11 downregulated (CASP8, CDK10, CDKN2A, CTLA4, GNA11, HERC2, IRF4, MC1R, PLA2G6, RREB1, and TPCN2) genes in the blood (across 15 pathways), showing 14% enrichment, and 16 upregulated (CCND1, CDK4, CTLA4, EIF1AX, IK, IRF4, MITF, NRAS, PIK3CB, PIK3R2, PMEL, RREB1, SLC45A2, SOX10, TYR, and TYRP1) and three downregulated ( GNA11, KITLG, and PLA2G6) genes in tissue (across 17 pathways), showing 33% enrichment, with five shared markers and 12 shared pathways. The model connected CDK4 pathway overactivity observed in both samples to inhibitors like ribociclib, abemaciclib, and palbociclib as putative treatments. By enabling objective personalized therapy selection, this approach shows great promise for advancing patient outcomes.

Dual Checkpoint Aptamer Immunotherapy: Unveiling Tailored Cancer Treatment Targeting CTLA-4 and NKG2A.

Recent strides in immunotherapy have illuminated the crucial role of CTLA-4 and PD-1/PD-L1 pathways in contemporary oncology, presenting both promises and challenges in response rates and adverse effects. This study employs a computational biology tool (in silico approach) to craft aptamers capable of binding to dual receptors, namely, inhibitory CTLA4 and NKG2A, thereby unleashing both T and NK cells and enhancing CD8+ T and NK cell functions for tumor cell lysis. Computational analysis highlighted AYA22T-R2-13 with HADDOCK scores of -78.2 ± 10.2 (with CTLA4), -60.0 ± 4.2 (with NKG2A), and -77.5 ± 5.6 (with CD94/NKG2A). Confirmation of aptamer binding to targeted proteins was attained via ELISA and flow cytometry methods. In vitro biological functionality was assessed using lactate dehydrogenase (LDH) cytotoxicity assay. Direct and competitive assays using ELISA and flow cytometry demonstrated the selective binding of AYA22T-R2-13 to CTLA4 and NKG2A proteins, as well as to the cell surface receptors of IL-2-stimulated T cells and NK cells. This binding was inhibited in the presence of competition from CTLA4 or NKG2A proteins. Remarkably, the blockade of CTLA4 or NKG2A by AYA22T-R2-13 augmented human CD8 T cell- and NK cell-mediated tumor cell lysis in vitro. Our findings highlight the precise binding specificity of AYA22T-R2-13 for CTLA4-B7-1/B7-2 (CD80/CD86) or CD94/NKG2A-HLA-E interactions, positioning it as a valuable tool for immune checkpoint blockade aptamer research in murine tumor models. These in vitro studies establish a promising foundation for further enhancing binding capacity and establishing efficacy and safety in animal models. Consequently, our results underscore the potential of AYA22T-R2-13 in cancer immunotherapy, offering high specificity, low toxicity, and the potential for cost-effective production.

T helper (Th) cell profiles and cytokines/chemokines in characterization, treatment, and monitoring of autoimmune diseases.

Autoimmune diseases (AD) consist of a spectrum of disease entities whose etiologies are very complex and still not well understood. Every individual has the potential for developing AD under appropriate conditions because the body contains lymphocytes that are potentially reactive with self-antigens. The aims of this study are to (1) explore the flow cytometry method to identify the frequency of various circulating CD4+ T helper (Th) cell-subsets, including Th1, Th2, Th9, Th17, Th17.1, and Th22; (2) In parallel, to examine multiplex ELISA method for pathogenic inflammatory cytokines/chemokines, and (3) To assess the correlation of expression of T cell-subsets with serum cytokines/chemokines and understand its clinical importance with available AD treatments. We analyzed Th17, Th17.1, Th22, Th2, Th1, and Th9 Th cell populations and compared the concentrations of 67 cytokines/chemokines in healthy as well as AD-diagnosed patients. We observed that patients with autoimmune markers had significantly elevated percentages of naïve (Th17, Th22, and Th9) as well as memory (Th17 and Th22) Th cell-subsets, along with increased concentrations of cytokines/chemokines (Eotaxin, TNFß, and FABP4). The percentage of Th cell-subsets correlated positively or negatively with the production of cytokines/chemokines of patients diagnosed with AD. Our study demonstrates that the naïve and memory Th cell-subsets with positive correlations to cytokines/chemokines show new diagnostic markers to predict the patients' outcome, while the negative correlation of cytokines/chemokines shows the response to autoimmune therapies. Our findings of Th cell-subsets by flow cytometry and cytokines/chemokines by multiplex ELISA suggest that CCR6+ Th cell-subsets (Th17, Th17.1, Th22, and Th9) contribute to our understanding of the pathogenesis of AD and identify the new onset of AD from the autoimmune spectrum. Our findings highlight the importance of CCR6+ as a possible marker in the characterization, treatment, and monitoring of AD.

New High-Affinity Thrombin Aptamers for Advancing Coagulation Therapy: Balancing Thrombin Inhibition for Clot Prevention and Effective Bleeding Management with Antidote.

Thrombin is a key enzyme involved in blood clotting, and its dysregulation can lead to thrombotic diseases such as stroke, myocardial infarction, and deep vein thrombosis. Thrombin aptamers have the potential to be used as therapeutic agents to prevent or treat thrombotic diseases. Thrombin DNA aptamers developed in our laboratory exhibit high affinity and specificity to thrombin. In vitro assays have demonstrated their efficacy by significantly decreasing Factor II activity and increasing PT and APTT times in both plasma and whole blood. Aptamers AYA1809002 and AYA1809004, the two most potent aptamers, exhibit high affinity for their target, with affinity constants (Kd) of 10 nM and 13 nM, respectively. Furthermore, the in vitro activity of these aptamers displays dose-dependent behavior, highlighting their efficacy in a concentration-dependent manner. In vitro stability assessments reveal that the aptamers remain stable in plasma and whole blood for up to 24 h. This finding is crucial for their potential application in clinical settings. Importantly, the thrombin inhibitory activity of the aptamers can be reversed by employing reverse complement sequences, providing a mechanism to counteract their anticoagulant effects when necessary to avoid excessive bleeding. These thrombin aptamers have been determined to be safe, with no observed mutagenic or immunogenic effects. Overall, these findings highlight the promising characteristics of these newly developed thrombin DNA aptamers, emphasizing their potential for therapeutic applications in the field of anticoagulation therapy. Moreover, the inclusion of an antidote in the coagulation therapy regimen can improve patient safety, ensure greater therapeutic efficacy, and minimize risk during emergency situations.

Highly efficacious and safe neutralizing DNA aptamer of SARS-CoV-2 as an emerging therapy for COVID-19 disease.

BACKGROUND: The paucity of SARS-CoV-2-specific virulence factors has greatly hampered the therapeutic management of patients with COVID-19 disease. Although available vaccines and approved therapies have shown tremendous benefits, the continuous emergence of new variants of SARS-CoV-2 and side effects of existing treatments continue to challenge therapy, necessitating the development of a novel effective therapy. We have previously shown that our developed novel single-stranded DNA aptamers not only target the trimer S protein of SARS-CoV-2, but also block the interaction between ACE2 receptors and trimer S protein of Wuhan origin, Delta, Delta plus, Alpha, Lambda, Mu, and Omicron variants of SARS-CoV-2. We herein performed in vivo experiments that administer the aptamer to the lungs by intubation as well as in vitro studies utilizing PBMCs to prove the efficacy and safety of our most effective aptamer, AYA2012004_L. METHODS: In vivo studies were conducted in transgenic mice expressing human ACE2 (K18hACE2), C57BL/6J, and Balb/cJ. Flow cytometry was used to check S-protein expressing pseudo-virus-like particles (VLP) uptake by the lung cells and test the immuogenicity of AYA2012004_L. Ames test was used to assess mutagenicity of AYA2012004_L. RT-PCR and histopathology were used to determine the biodistribution and toxicity of AYA2012004_L in vital organs of mice. RESULTS: We measured the in vivo uptake of VLPs by lung cells by detecting GFP signal using flow cytometry. AYA2012004_L specifically neutralized VLP uptake and also showed no inflammatory response in mice lungs. In addition, AYA2012004_L did not induce inflammatory response in the lungs of Th1 and Th2 mouse models as well as human PBMCs. AYA2012004_L was detectable in mice lungs and noticeable in insignificant amounts in other vital organs. Accumulation of AYA2012004_L in organs decreased over time. AYA2012004_L did not induce degenerative signs in tissues as seen by histopathology and did not cause changes in the body weight of mice. Ames test also certified that AYA2012004_L is non-mutagenic and proved it to be safe for in vivo studies. CONCLUSIONS: Our aptamer is safe, effective, and can neutralize the uptake of VLPs by lung cells when administered locally suggesting that it can be used as a potential therapeutic agent for COVID-19 management.

Noninvasive nasopharyngeal proteomics of COVID-19 patient identify abnormalities related to complement and coagulation cascade and mucosal immune system.

Serum or plasma have been the primary focus of proteomics studies for COVID-19 to identity biomarkers and potential drug targets. The nasal mucosal environment which consists of lipids, mucosal immune cells, and nasal proteome, has been largely neglected but later revealed to have critical role combating SARS-CoV-2 infection. We present a bottom-up proteomics investigation of the host response to SARS-CoV-2 infection in the nasopharyngeal environment, featuring a noninvasive approach using proteins in nasopharyngeal swabs collected from groups of 76 SARS-CoV-2 positive and 76 negative patients. Results showed that 31 significantly down-regulated and 6 up-regulated proteins were identified (p < 0.05, log2 FC > 1.3) in SARS-CoV-2 positive patient samples as compared to the negatives; these proteins carry potential value as markers for the early detection of COVID-19, disease monitoring, as well as be drug targets. The down-regulation of coagulation factor 5 indicates a thrombotic abnormality in COVID-19 patients and the decreased IgG4 suggests an abnormal immune response at the point of entry in human nasopharyngeal environment, which is in consistent with KEGG and GO pathway analysis. Our study also demonstrated that mass spectrometry proteomics analysis of nasopharyngeal swabs can be used as a powerful early approach to evaluate host response to SARS-CoV-2 viral infection.

The Cry4B toxin of Bacillus thuringiensis subsp. israelensis kills Permethrin-resistant Anopheles gambiae, the principal vector of malaria.

Resurgence of malaria has been attributed, in part, to the development of resistance by Anopheles gambiae, a principal vector of the disease, to various insecticidal compounds such as Permethrin. Permethrin, a neurotoxicant, is widely used to impregnate mosquito nets. An alternative strategy to control mosquitoes is the use of Bacillus thuringiensis subsp. israelensis (Bti) because there is no observable resistance in the field to the bacterium. Bti kills mosquitoes by targeting cadherin molecules residing in the midgut epithelium of larvae of the insect. Cry proteins (Cry4A, Cry4B, Cry10A and Cry11A) produced by the bacterium during the sporulation phase of its life cycle bind to the cadherin molecules, which serve as receptors for the proteins. These Cry proteins have variable specificity to a variety of mosquitoes, including Culex and Aedes as well as Anopheles. Importantly, selective mosquitocidal action is occasioned by binding of the respective Cry toxins to cadherins distinctive to individual mosquito species. Differential fractionation of the four Cry proteins from a novel Bti isolate (M1) and cloning and expression of their genes in Escherichia coli revealed that Cry4B is the only Cry protein that exerts insecticidal action against An. gambiae. Indeed, it does so against a Permethrin-resistant strain of the mosquito. The other three Cry proteins are ineffective. Multiple sequence alignments of the four Cry proteins revealed a divergent sequence motif in the Cry4B toxin, which most likely determines binding of the toxin to its cognate receptor, BT-R3, in An. gambiae and to its specific toxicity. A model showing Cry4B toxin binding to BT-R3 is presented.

Cytotoxicity of the Bacillus thuringiensis Cry4B toxin is mediated by the cadherin receptor BT-R3 of Anopheles gambiae.

We demonstrate for the first time the selective cytotoxicity of Bacillus thuringiensis subsp. israelensis Cry4B toxin mediated by BT-R3 using a cell-based system, which employs High Five insect cells stably expressing BT-R3. Discovery and validation of BT-R3 as the Cry4B receptor was accomplished using a web-based computational pipeline platform that facilitates high-throughput insecticidal target identification utilizing the Anopheles gambiae genome. Once the Cry4B toxin binds to the BT-R3 receptor, a cell death pathway is manifested by sequential cytological changes that include membrane blebbing, cell swelling and lysis. Cry4B toxin associates with cell membrane in both oligomeric and monomeric forms. Monomeric toxin binds specifically to BT-R3 whereas oligomer interacts with cell membrane non-specifically. Cytotoxicity and cell death are the direct result of binding of toxin monomer to BT-R3. The oligomeric form of Cry4B toxin is not involved in cell death. Both the location of the toxin-binding region within BT-R3 and its structural motif are critical to the binding affinity and specificity of the toxin. The toxin-binding region of BT-R3 appears to be located in EC11, the most membrane proximal EC module within the extracellular domain. It is characterized by the presence of two highly conserved amino acid sequences within their N- and C-termini that flank EC11. These sequences represent signature motifs that mark the toxin-binding function in BT-R3. The two sequences form two adjacent ß-strands within the ß-barrel of EC11, the positioning of which is a hallmark of all Cry toxin receptors thus far reported.

Bacillus thuringiensis: a genomics and proteomics perspective.

Bacillus thuringiensis (Bt) is a unique bacterium in that it shares a common place with a number of chemical compounds which are used commercially to control insects important to agriculture and public health. Although other bacteria, including B. popilliae and B. sphaericus, are used as microbial insecticides, their spectrum of insecticidal activity is quite limited compared to Bt. Importantly, Bt is safe for humans and is the most widely used environmentally compatible biopesticide worldwide. Furthermore, insecticidal Bt genes have been incorporated into several major crops, rendering them insect resistant, and thus providing a model for genetic engineering in agriculture.This review highlights what the authors consider the most relevant issues and topics pertaining to the genomics and proteomics of Bt. At least one of the authors (L.A.B.) has spent most of his professional life studying different aspects of this bacterium with the goal in mind of determining the mechanism(s) by which it kills insects. The other authors have a much shorter experience with Bt but their intellect and personal insight have greatly enriched our understanding of what makes Bt distinctive in the microbial world. Obviously, there is personal interest and bias reflected in this article notwithstanding oversight of a number of published studies. This review contains some material not published elsewhere although several ideas and concepts were developed from a broad base of scientific literature up to 2010.

Susceptibility of Manduca sexta to Cry1Ab toxin of Bacillus thuringiensis correlates directly to developmental expression of the cadherin receptor BT-R(1).

The cadherin receptor BT-R(1), localized in the midgut epithelium of the tobacco hornworm, Manduca sexta, is coupled to programmed oncotic-like cell death, which is triggered by the univalent binding of the Cry1Ab toxin of Bacillus thuringiensis (Bt) to the receptor. Kinetic analysis of BT-R(1) expression during larval development reveals that the density of BT-R(1) on the midgut surface increases dramatically along with an equivalent rise in the concentration of Cry1Ab toxin molecules needed to kill each of the five larval stages of the insect. The increase in the number of BT-R(1) molecules per midgut surface area requires additional toxin molecules to kill older versus younger larvae, as evidenced by the corresponding LC(50) values. Based on these observations, we developed a mathematical model to quantify both the expression of BT-R(1) and the susceptibility of M. sexta larvae to the Cry1Ab toxin. Interestingly, the toxin-receptor ratio remains constant during larval development regardless of larval size and mass. This ratio apparently is critical for insecticidal activity and the decrease in toxin effectiveness during larval development is due primarily to the number of effective toxins and available receptors in the larval midgut. Evidently, susceptibility of M. sexta to the Cry1Ab toxin of Bt correlates directly to the developmental expression of BT-R(1) in this insect.

Enhanced exocytosis of the receptor BT-R(1) induced by the Cry1Ab toxin of Bacillus thuringiensis directly correlates to the execution of cell death.

Cry1Ab toxin produced by Bacillus thuringiensis exerts insecticidal action upon binding to BT-R(1), a cadherin receptor localized in the midgut epithelium of the tobacco hornworm Manduca sexta. The univalent binding of toxin to receptor transmits a death signal into the cell and turns on a multi-step signal transduction pathway involving adenylyl cyclase (AC) and protein kinase A (PKA), which drives the biochemical events that culminate in oncotic cell death. Here, we report that cell killing by the Cry1Ab toxin is a dynamic episode in which the toxin promotes exocytotic transport of BT-R(1) from intracellular membrane vesicles to the plasma membrane. The resultant dramatic increase in BT-R(1) displayed on the surface of toxin-treated cells effects the recruitment and concomitant binding of additional toxin monomers which, in turn, amplifies the original signal in a cascade-like manner. Blocking the activation of AC/PKA signal transduction by either EDTA or PKAi inhibits exocytotic trafficking of BT-R(1) and prevents cell death. Moreover, the exocytosis inhibitor Exo1 blocks translocation of receptor and progression of cell death alike. Obviously, movement of BT-R(1) is mediated by toxin-induced signal transduction and amplification of this signaling apparently is critical to the execution of cell death.

Univalent binding of the Cry1Ab toxin of Bacillus thuringiensis to a conserved structural motif in the cadherin receptor BT-R1.

The Cry1Ab toxin produced by Bacillus thuringiensis (Bt) exerts insecticidal action upon binding to BT-R1, a cadherin receptor localized in the midgut epithelium of the tobacco hornworm Manduca sexta [Dorsch, J. A., Candas, M., Griko, N. B., Maaty, W. S., Midboe, E. G., Vadlamudi, R. K., and Bulla, L. A., Jr. (2002) Cry1A toxins of Bacillus thuringiensis bind specifically to a region adjacent to the membrane-proximal extracellular domain of BT-R1 in Manduca sexta: involvement of a cadherin in the entomopathogenicity of Bacillus thuringiensis, Insect Biochem. Mol. Biol. 32, 1025-1036]. BT-R1 represents a family of invertebrate cadherins whose ectodomains (ECs) are composed of multiple cadherin repeats (EC1 through EC12). In the present work, we determined the Cry1Ab toxin binding site in BT-R1 in the context of cadherin structural determinants. Our studies revealed a conserved structural motif for toxin binding that includes two distinct regions within the N- and C-termini of EC12. These regions are characterized by unique sequence signatures that mark the toxin-binding function in BT-R1 as well as in homologous lepidopteran cadherins. Structure modeling of EC12 discloses the conserved motif as a single broad interface that holds the N- and C-termini in close proximity. Binding of toxin to BT-R1, which is univalent, and the subsequent downstream molecular events responsible for cell death depend on the conserved motif in EC12.

A mechanism of cell death involving an adenylyl cyclase/PKA signaling pathway is induced by the Cry1Ab toxin of Bacillus thuringiensis.

Many pathogenic organisms and their toxins target host cell receptors, the consequence of which is altered signaling events that lead to aberrant activity or cell death. A significant body of literature describes various molecular and cellular aspects of toxins associated with bacterial invasion, colonization, and host cell disruption. However, there is little information on the molecular and cellular mechanisms associated with the insecticidal action of Bacillus thuringiensis (Bt) Cry toxins. Recently, we reported that the Cry1Ab toxin produced by Bt kills insect cells by activating a Mg(2+)-dependent cytotoxic event upon binding of the toxin to its receptor BT-R(1). Here we show that binding of Cry toxin to BT-R(1) provokes cell death by activating a previously undescribed signaling pathway involving stimulation of G protein (G(alphas)) and adenylyl cyclase, increased cAMP levels, and activation of protein kinase A. Induction of the adenylyl cyclase/protein kinase A pathway is manifested by sequential cytological changes that include membrane blebbing, appearance of ghost nuclei, cell swelling, and lysis. The discovery of a toxin-induced cell death pathway specifically linked to BT-R(1) in insect cells should provide insights into how insects evolve resistance to Bt and into the development of new, safer insecticides.

The proteomics of sickle cell disease: profiling of erythrocyte membrane proteins by 2D-DIGE and tandem mass spectrometry.

Quantitative changes in the red blood cell membrane proteome in sickle cell disease were analyzed using the two-dimensional fluorescence difference gel electrophoresis 2D-DIGE technique. From over 500 analyzed two-dimensional gel spots, we found 49 protein gel spots whose content in sickle cell membranes were changed by at least 2.5-fold as compared to control cells. In 38 cases we observed an increase and in 11 cases a decrease in content in the sickle cell membranes. The proteins of interest were identified by in-gel tryptic digestion followed by liquid chromatography in line with tandem mass spectrometry. From 38 analyzed gel spots, we identified 44 protein forms representing different modifications of 22 original protein sequences. The majority of the identified proteins fall into small groups of related proteins of the following five categories: actin accessory proteins--four proteins, components of lipid rafts--two proteins, scavengers of oxygen radicals--two proteins, protein repair participants--six proteins, and protein turnover components--three proteins. The number of proteins whose content in sickle RBC membrane is decreased is noticeably smaller, and most are either components of lipid rafts or actin accessory proteins. Elevated content of protein repair participants as well as oxygen radical scavengers may reflect the increased oxidative stress observed in sickle cells.

Selective antagonism to the cadherin BT-R1 interferes with calcium-induced adhesion of epithelial membrane vesicles.

BT-R(1) is a member of the cadherin superfamily of proteins and is expressed in the midgut epithelium of Manduca sexta during larval development. Previously, we showed that calcium ions influence the structure and stability of BT-R(1) on brush border membrane vesicles (BBMVs) prepared from M. sexta midgut epithelium. In the present study, the effects of calcium and Cry1Ab toxin, produced by Bacillus thuringiensis, on the adhesive properties of BBMVs were investigated. Addition of calcium to a suspension of BBMVs promoted adhesion and aggregation of the vesicles. Treatment of BBMVs with trypsin or lowering the pH (pH 4.0) of the BBMV suspension abolished calcium-induced vesicle aggregation, whereas treatment with deglycosylating enzymes did not affect the aggregation of vesicles, indicating that adhesion and clustering of BBMVs involves protein-protein interactions. Preincubation of BBMVs with Cry1Ab toxin, which specifically binds to BT-R(1) with high affinity and disrupts the midgut epithelium of M. sexta, caused a 50% decrease in calcium-induced vesicle aggregation. The inhibitory effects of the Cry1Ab toxin on BBMV aggregation was blocked completely when the toxin was preincubated with a peptide containing the toxin-binding site of BT-R(1). Cry3A toxin, which is similar in molecular structure to Cry1Ab but does not bind to BT-R(1) and is not toxic to M. sexta larvae, did not affect BBMV aggregation. The results of this study demonstrate that the adhesive function of BT-R(1) is compromised by the Cry1Ab toxin, which acts as a selective antagonist, and supports the notion that BT-R(1) is critical in preserving the integrity of larval midgut epithelium in M. sexta.

Proteolytic cleavage of the developmentally important cadherin BT-R1 in the midgut epithelium of Manduca sexta.

BT-R1 (M(r) = 210 kDa) represents a new type of insect cadherin that is expressed specifically in the midgut epithelium during growth and development of Manduca sexta larvae. It also is a target receptor for the Cry1A toxins of the entomopathogenic bacterium Bacillus thuringiensis. Expression of BT-R1, which varies during larval development, correlates with the abundance of the protein and with the differential cleavage of the molecule at each developmental stage. The cleavage of BT-R1 is calcium dependent, and consequently, Ca2+ directly influences the structural integrity of BT-R1. Indeed, removal of calcium ions by chelating agents promotes cleavage of the BT-R1 ectodomain, resulting in formation of fragments that are similar to those observed during larval development. Partial purification of proteins from brush border membrane vesicles (BBMVs) by gel filtration chromatography hinders the cleavage of BT-R1 in the presence of EDTA and EGTA, indicating that there is specific proteolytic activity associated with the BBMV. This specific proteolytic cleavage of BT-R1 not only alters the integrity of BT-R1 but it most likely is implicated in cell adhesion events during differentiation and development of M. sexta midgut epithelium. We propose a model for calcium-dependent protection of BT-R1 as well as a cleavage pattern that may modulate the molecular interactions and adhesive properties of its ectodomain. Molecular characterization of such a protection mechanism should lead to a better understanding of how the function of specific cadherins is modulated during tissue differentiation and insect development.