Streptococcal Protein Research Articles

Genetic Algorithms Applied to Thermodynamic Rational Design of Mimetic Antibodies Based on the GB1 Domain of Streptococcal Protein G: An Atomistic Simulation Study.

The development of mimetic antibodies (MA) capable of combining the high affinity and selectivity of antibodies with the small size of the peptides has enormous potential for applications in current biotechnology. In this work, we demonstrate that in silico MA design is possible through genetic algorithms (GA) developed from shell scripts capable of combining software commonly used for atomistic simulation. Our results demonstrate that, using the GB1 domain of the streptococcal G protein as a model, it is possible to optimize the molecular recognition capacity of a large MA population in a few generations. In the first case, GA was able to generate 10 MA with binding free energy (BFE) less than the vascular endothelial cell growth factor conjugated with the fms-type tyrosine kinase receptor. In the second case, it generated 13 MA with BFE less than that of the hepatitis C-E2 viral envelope conjugate with the antibody. Through the GA developed in this work, we demonstrate the use of a new protocol, capable of guiding experimental methods for the design of bioactive peptides that can assist in the development of new therapeutic molecules.

Antibody responses to collagen peptides and streptococcal collagen-like 1 proteins in acute rheumatic fever patients.

Acute rheumatic fever (ARF) is a serious post-infectious immune sequelae of Group A streptococcus (GAS). Pathogenesis remains poorly understood, including the events associated with collagen autoantibody generation. GAS express streptococcal collagen-like proteins (Scl) that contain a collagenous domain resembling human collagen. Here, the relationship between antibody reactivity to GAS Scl proteins and human collagen in ARF was investigated. Serum IgG specific for a representative Scl protein (Scl1.1) together with collagen-I and collagen-IV mimetic peptides were quantified in ARF patients (n=36) and healthy matched controls (n=36). Reactivity to Scl1.1 was significantly elevated in ARF compared to controls (P < 0.0001) and this was mapped to the collagen-like region of the protein, rather than the N-terminal non-collagenous region. Reactivity to collagen-1 and collagen-IV peptides was also significantly elevated in ARF cases (P < 0.001). However, there was no correlation between Scl1.1 and collagen peptide antibody binding, and hierarchical clustering of ARF cases by IgG reactivity showed two distinct clusters, with Scl1.1 antigens in one and collagen peptides in the other, demonstrating that collagen autoantibodies are not immunologically related to those targeting Scl1.1. Thus, anti-collagen antibodies in ARF appear to be generated as part of the autoreactivity process, independent of any mimicry with GAS collagen-like proteins.

Estimating the Directional Flexibility of Proteins from Equilibrium Thermal Fluctuations.

The directional flexibility of proteins is an equilibrium molecular property which is accessible to both experiment and computation. Single molecule force spectroscopy (SMFS) experiments report effective directional spring constants to describe the collective anisotropic response of a protein structure to mechanical pulling forces applied along selected axes. On the other hand, computational methods have thus far employed either indirect force based nonequilibrium simulations or coarse-grained elastic network models (ENM) to predict protein directional spring constants. Here, we examine the ability of equilibrium atomistic Molecular Dynamics (MD) simulations to estimate the directional flexibility and mechanical anisotropy of proteins. MD-derived effective directional spring constants are found to correlate well with SMFS spring constants (ρ2 = 0.97-0.99; Adj R2 = 0.92-0.99) and unfolding forces (ρ2 = 0.85-0.97; Adj R2 = 0.63-0.91) for five different globular proteins. Specifically, the computed spring constants reproduce the mechanical anisotropy reported by SMFS along five different directions of green fluorescence protein (GFP) and six directions of the immunoglobulin-binding B1 domain of streptococcal protein G (GB1). Further, protein dynamics as captured in MD can be translated into spring constants which can distinguish the N-C directional flexibility of ubiquitin (Ub) from two structurally homologous small ubiquitin-like modifier (SUMO1 and SUMO2) isoforms. We apply our computational framework to study the mechanical anisotropy of Ub along the seven lysine-C-term directions which are functionally relevant. We show that Ub possesses two distinct flexibility scales along these directions which roughly differ by an order of magnitude. Further, our studies reveal that the mechanical anisotropy of Ub is modified in contrasting ways by the binding of two partner proteins (UBCH5A and UEV) which attach and recognize these biomolecular tag proteins. On the basis of equilibrium MD benchmarks for flexibility along 2485 bond vectors in Ub, we propose and validate a new covariance-propagation scheme to extract spring constants from ENM normal modes. We also critically examine the ability of ENM to predict directional flexibility of proteins and suggest modifications to improve these intuitive and scalable descriptions.

Streptococcal protein SIC activates monocytes and induces inflammation

SummaryStreptococcus pyogenes is a major bacterial pathogen in the human population and isolates of the clinically important M1 serotype secrete protein Streptococcal inhibitor of complement (SIC) known to interfere with human innate immunity. Here we find that SIC from M1 bacteria interacts with TLR2 and CD14 on monocytes leading to the activation of the NF-κB and p38 MAPK pathways and the release of several pro-inflammatory cytokines (e.g. TNFα and INFγ). In human plasma, SIC binds clusterin and histidine-rich glycoprotein, and whole plasma, and these two purified plasma proteins enhanced the activation of monocytes by SIC. Isolates of the M55 serotype secrete an SIC homolog, but this protein did not activate monocytes. M1 isolates are common in cases of invasive S. pyogenes infections characterized by massive inflammation, and the results of this study indicate that the pro-inflammatory property of SIC contributes to the pathology of these severe clinical conditions.

Method for efficient soluble expression and purification of recombinant hyperactive Tn5 transposase

Efficient preparation of libraries is the key step of next-generation sequencing (NGS) methods. Tn5 transposase enables simple, robust and highly efficient tagmentation-based library construction. Here, we report a simple and reliable expression and purification strategy based on fusing Tn5 to the small B1 immunoglobulin binding domain of Streptococcal protein G (GB1) and high affinity 10× His tag. The purified recombinant Tn5 showed high DNA tagmentation activity and ultra-low nucleic acid contamination. This method greatly cuts the costs of Tn5-based NGS library construction and is beneficial to the development of new NGS methods.

Improving the diagnosis of bovine tuberculosis using gold nanoparticles conjugated with purified protein derivative: special regard to staphylococcal protein A and streptococcal protein G.

Different ancillary immunodiagnostic tests were traditionally-established for diagnosis of bovine tuberculosis (BTB) either cellular or humoral as tuberculin skin test (TST), gamma interferon (INF-γ), and indirect enzyme-linked immunosorbent assay (iELISA). These tests had been consumed more time and expensive, and needed sophisticated equipment. To dissolve these problems, serological diagnosis depending on humoral immunity is the aim of this work. Herein, slide-based agglutination test was chosen as a rapid and simple field test based on purified protein derivative (PPD) antigen in addition to some supplementation materials such as Staphylococcal protein A (SPA) and Streptococcal protein G (SPG) to improve detection of BTB antibody in serum samples. Gold nanoparticles (Au NPs) were synthesized by gamma ray, and after complete characterization, the synthesized Au NPs were spherical, small-sized, and stable without any impurities. Addition of such supplementation reagents for serodiagnosis of tuberculosis is of paramount important for the detection of serum antibodies against tuberculosis (TB) and it was considered an easily simple and possible way for improving TB diagnosis. In this work, 70 animals tested positive for TST as well as 20 animals tested negative for TST were used for the diagnosis of BTB depending on humoral immune response based on PPD slide agglutination test using reporter regents (SPA and/or SPG) either native or recombinant. The agglutination density was recorded and read in 4 degrees of positivity with scores ranging from negative (-) to very strong reaction (++++) occurred in different times of agglutination. Groups showed 100% positive reactivates employed in Exp. 1, 2, and 3 with differentiation of slide agglutination test density and was rated from moderate positivity (2+) to very strong (4+), with predominant positivity in density of (3+). Pink-colored intensity is associated with the strengthened reactions between PPD-conjugated Au NPs and serum antibody of each tested samples, which allows for visual rapid, simple, and effective attractive diagnosis of BTB. The specificity and sensitivity of the serological tests were characterized. TST offers the highest sensitivity (83.6%) among the other immunoassays, while the lowest specificity was recorded in TST (57.4%). SPA/SPG offers the best performance in term of combined sensitivity and specificity (performance index) of 175.4. Therefore, the development and uses of detection reagent (such as SPA and/or SPG) slide co-agglutination test (COAT), either native or recombinant (rSPA/SPG) for the detection of TB antibodies based on PPD antigen, as well as the uses of Au NPs rSPA/SPG as detection conjugate based on the same antigen, were also performed as a simple, rapid, sensitive, specific, eco-friendly, and low cost, which shows a great potential in field and lab diagnosis of BTB. So, high reduction in reagents that yields reactions similarly as traditional techniques was needed.

Binding characteristics of staphylococcal protein A and streptococcal protein G for fragment crystallizable portion of human immunoglobulin G

In the wide array of physiological processes, protein–protein interactions and their binding are the most basal activities for achieving adequate biological metabolism. Among the studies on binding proteins, the examination of interactions between immunoglobulin G (IgG) and natural immunoglobulin-binding ligands, such as staphylococcal protein A (spA) and streptococcal protein G (spG), is essential in the development of pharmaceutical science, biotechnology, and affinity chromatography. The widespread utilization of IgG-spA/spG binding characteristics has allowed researchers to investigate these molecular interactions. However, the detailed binding strength of each ligand and the corresponding binding mechanisms have yet to be fully investigated. In this study, the authors analyzed the binding strengths of IgG–spA and IgG–spG complexes and identified the mechanisms enabling these bindings using molecular dynamics simulation, steered molecular dynamics, and advanced Poisson–Boltzmann Solver simulations. Based on the presented data, the binding strength of the spA ligand was found to significantly exceed that of the spG ligand. To find out which non-covalent interactions or amino acid sites have a dominant role in the tight binding of these ligands, further detailed analyses of electrostatic interactions, hydrophobic bonding, and binding free energies have been performed. In investigating their binding affinity, a relatively independent and different unbinding mechanism was found in each ligand. These distinctly different mechanisms were observed to be highly correlated to the protein secondary and tertiary structures of spA and spG ligands, as explicated from the perspective of hydrogen bonding.

Small Bispecific Affinity Proteins for Simultaneous Target Binding and Albumin-Associated Half-Life Extension

Albumin-binding fusion partners are frequently used as a means for the in vivo half-life extension of small therapeutic molecules that would normally be cleared very rapidly from circulation. However, in applications where small size is key, fusion to an additional molecule can be disadvantageous. Albumin-derived affinity proteins (ADAPTs) are a new type of scaffold proteins based on one of the albumin-binding domains of streptococcal protein G, with engineered binding specificities against numerous targets. Here, we engineered this scaffold further and showed that this domain, as small as 6 kDa, can harbor two distinct binding surfaces and utilize them to interact with two targets simultaneously. These novel ADAPTs were developed to possess affinity toward both serum albumin as well as another clinically relevant target, thus circumventing the need for an albumin-binding fusion partner. To accomplish this, we designed a phage display library and used it to successfully select for single-domain bispecific binders toward a panel of targets: TNFα, prostate-specific antigen (PSA), C-reactive protein (CRP), renin, angiogenin, myeloid-derived growth factor (MYDGF), and insulin. Apart from successfully identifying bispecific binders for all targets, we also demonstrated the formation of the ternary complex consisting of the ADAPT together with albumin and each of the five targets, TNFα, PSA, angiogenin, MYDGF, and insulin. This simultaneous binding of albumin and other targets presents an opportunity to combine the advantages of small molecules with those of larger ones allowing for lower cost of goods and noninvasive administration routes while still maintaining a sufficient in vivo half-life.

Sensitivity enhancement by sequential data acquisition for 13C-direct detection NMR

13C-direct detection NMR has several advantages compared to proton detection, including a tendency to relax slower and wider chemical shift range. However, the sensitivity of 13C-direct detection is much lower than that of proton detection because of its lower gyromagnetic ratio. In addition, a virtual decoupling procedure is often performed to remove peak splitting in the 13C-direct detection axis, which further reduces the sensitivity to 1/√2. In this study, to enhance the sensitivity of 13C-direct detection experiments, we developed a HCACO-type new pulse sequence in which anti-phase (AP) and in-phase (IP) signals are acquired sequentially in a single scan. The developed experiment was tested on an amino acid (valine) and two proteins (streptococcal protein G B1 domain (GB1) and α-synuclein). The AP and IP spectra were successfully obtained in all cases. Using these spectra, IPAP virtual decoupling was performed, and peak splitting was successfully removed. The sensitivity of the experiment was increased by 1.43, 1.26 and 1.26 times for valine, GB1 and α-synuclein, respectively, compared to the conventional HCACO experiment. In addition, we developed another HCACO-type pulse sequence, where AP and IP signals are simultaneously acquired in a single FID. The sensitivity of the experiment was increased by 1.40 and 1.35 times for valine and GB1, respectively. These methods are potentially applicable to other 13C-direct detection experiments that measure one-bond correlations and will further extend the utility of the 13C-direct detection method, especially for structural analyses of intrinsically disordered proteins.

Inferring Protein Sequence-Function Relationships with Large-Scale Positive-Unlabeled Learning.

Machine learning can infer how protein sequence maps to function without requiring a detailed understanding of the underlying physical or biological mechanisms. It is challenging to apply existing supervised learning frameworks to large-scale experimental data generated by deep mutational scanning (DMS) and related methods. DMS data often contain high-dimensional and correlated sequence variables, experimental sampling error and bias, and the presence of missing data. Notably, most DMS data do not contain examples of negative sequences, making it challenging to directly estimate how sequence affects function. Here, we develop a positive-unlabeled (PU) learning framework to infer sequence-function relationships from large-scale DMS data. Our PU learning method displays excellent predictive performance across ten large-scale sequence-function datasets, representing proteins of different folds, functions, and library types. The estimated parameters pinpoint key residues that dictate protein structure and function. Finally, we apply our statistical sequence-function model to design highly stabilized enzymes.

Validation of DBFOLD: An efficient algorithm for computing folding pathways of complex proteins.

Atomistic simulations can provide valuable, experimentally-verifiable insights into protein folding mechanisms, but existing ab initio simulation methods are restricted to only the smallest proteins due to severe computational speed limits. The folding of larger proteins has been studied using native-centric potential functions, but such models omit the potentially crucial role of non-native interactions. Here, we present an algorithm, entitled DBFOLD, which can predict folding pathways for a wide range of proteins while accounting for the effects of non-native contacts. In addition, DBFOLD can predict the relative rates of different transitions within a protein's folding pathway. To accomplish this, rather than directly simulating folding, our method combines equilibrium Monte-Carlo simulations, which deploy enhanced sampling, with unfolding simulations at high temperatures. We show that under certain conditions, trajectories from these two types of simulations can be jointly analyzed to compute unknown folding rates from detailed balance. This requires inferring free energies from the equilibrium simulations, and extrapolating transition rates from the unfolding simulations to lower, physiologically-reasonable temperatures at which the native state is marginally stable. As a proof of principle, we show that our method can accurately predict folding pathways and Monte-Carlo rates for the well-characterized Streptococcal protein G. We then show that our method significantly reduces the amount of computation time required to compute the folding pathways of large, misfolding-prone proteins that lie beyond the reach of existing direct simulation. Our algorithm, which is available online, can generate detailed atomistic models of protein folding mechanisms while shedding light on the role of non-native intermediates which may crucially affect organismal fitness and are frequently implicated in disease.

An Orthogonal Fusion Tag for Efficient Protein Purification.

In this chapter, we present an efficient method for stringent protein purification facilitated by a dual affinity tag referred to as ABDz1, which is based on a 5kDa albumin-binding domain from Streptococcal Protein G. The small fusion tag enables an orthogonal affinity purification approach based on two successive and highly specific affinity purification steps. This approach is enabled by native binding of ABDz1 to human serum albumin and engineered binding to Staphylococcal Protein A, respectively. The ABDz1-tag can be fused to either terminus of a protein of interest and the purification steps can be carried out using standard laboratory equipment.

Cell immunocapture microfluidic chip based on high-affinity recombinant protein binders

Cell immunocapture microfluidic devices represent a rapidly developing field with many potential applications in medical diagnostics. The core of such approach lies in the cell binding to antibody coated surfaces through their surface receptors. Here we show, that the small recombinant protein binders (PBs) can be used for this purpose as well, with the advantage of their constructional flexibility, possibility of fusion with range of tags and cheap mass production. For this purpose, two different PBs derived from Albumin Binding Domain (ABDwt) of streptococcal protein G, so called REX and ARS ligands with proved high affinity and selectivity to the human interleukin-23 (IL-23R) and IL-17 receptor A were used. Four PBs variants recognizing two different epitopes on two different receptors and two PBs variants binding to the same epitope on one receptor but having different peptide spacer with Avitag sequence necessary for their immobilization on sensor surface were tested for cell-capture efficiency. The glass microfluidic Y-system with planar immunocapture channel working in so-called stop-flow dynamic regime was designed. Up to 60–74% immunocapture efficiency of model THP-1 cells on REX/ARS surfaces and practically no cell binding on control ABDwt surfaces was achieved. Moreover, the specific immunocapture of THP-1 cells from mixture with IL-17RA negative DU-145 cells was demonstrated. We discuss the role of the epitope, affinity and immobilization spacer of PBs as well as the influence of stop-flow dynamic regime on the effectivity of THP-1 cell immunocapture. Results can be further exploited in design of microfluidic devices for rare cells immunocapture.

Fulminant Necrotizing Fasciitis And Myositis With Streptococcal Toxic Shock Syndrome In A Patient With Rheumatoid Arthritis On Tocilizumab: A Case Report

Introduction: We present the case of a 44-year man with a fulminant necrotizing myositis and fasciitis and streptococcal toxic shock syndrome, the patient being under treatment with tocilizumab and methotrexate for rheumatoid arthritis. Case presentation: He presented to the emergency department with diarrhea, flu-like symptoms and intense progressive pain in the right thigh after having shoveled while doing building work at home. His condition deteriorated within hours, he developed severe rhabdomyolysis, multiple organ dysfunction syndrome, disseminated intravascular coagulopathy and died, despite early treatment. Discussion: A great index of suspicion for severe necrotizing soft tissue infections is necessary in patients on tocilizumab or other biologic immunosuppressive agents. The possible mechanisms in this setting include lowering CRP involved in innate immune responses, decrement of neutrophil count and function, increasing iron availability for infection, as well as high expression of vimentin, the streptococcal binding protein, by regenerating muscle fibers after trauma. Diarrhea due to endotoxins and flu-like symptoms may be early and sometimes they may be the only presenting signs of severe group A streptococcal infection in patients with blunt trauma on biologics. Low initial CRP and white blood cell count may be misleading. A rapid streptococcal strip test may help anticipate and possibly prevent a deleterious evolution in such patients. Conclusion: Biologics may modify the clinical presentation of NF, the LRINEC score, and the pathology decision algorithm. A great index of suspicion for severe soft tissue infections is required in patients on biologics with blunt trauma, especially when presenting with flu-like symptoms, diarrhea, or vomiting.

Streptococcal Serine-Rich Repeat Proteins in Colonization and Disease.

Glycosylation of proteins, previously thought to be absent in prokaryotes, is increasingly recognized as important for both bacterial colonization and pathogenesis. For mucosal pathobionts, glycoproteins that function as cell wall-associated adhesins facilitate interactions with mucosal surfaces, permitting persistent adherence, invasion of deeper tissues and transition to disease. This is exemplified by Streptococcus pneumoniae and Streptococcus agalactiae, which can switch from being relatively harmless members of the mucosal tract microbiota to bona fide pathogens that cause life-threatening diseases. As part of their armamentarium of virulence factors, streptococci encode a family of large, glycosylated serine-rich repeat proteins (SRRPs) that facilitate binding to various tissue types and extracellular matrix proteins. This minireview focuses on the roles of S. pneumoniae and S. agalactiae SRRPs in persistent colonization and the transition to disease. The potential of utilizing SRRPs as vaccine targets will also be discussed.

Exploring the sequence fitness landscape of a bridge between protein folds.

Most foldable protein sequences adopt only a single native fold. Recent protein design studies have, however, created protein sequences which fold into different structures apon changes of environment, or single point mutation, the best characterized example being the switch between the folds of the GA and GB binding domains of streptococcal protein G. To obtain further insight into the design of sequences which can switch folds, we have used a computational model for the fitness landscape of a single fold, built from the observed sequence variation of protein homologues. We have recently shown that such coevolutionary models can be used to design novel foldable sequences. By appropriately combining two of these models to describe the joint fitness landscape of GA and GB, we are able to describe the propensity of a given sequence for each of the two folds. We have successfully tested the combined model against the known series of designed GA/GB hybrids. Using Monte Carlo simulations on this landscape, we are able to identify pathways of mutations connecting the two folds. In the absence of a requirement for domain stability, the most frequent paths go via sequences in which neither domain is stably folded, reminiscent of the propensity for certain intrinsically disordered proteins to fold into different structures according to context. Even if the folded state is required to be stable, we find that there is nonetheless still a wide range of sequences which are close to the transition region and therefore likely fold switches, consistent with recent estimates that fold switching may be more widespread than had been thought.

Establishment of a fluorescence staining method for Schistosoma japonicum miracidia

Currently the diagnosis of schistosomiasis is mainly determined by observing the presence of eggs in host stool samples. Because of the overwhelming number of impurities in the stool, eggs are rarely observed. Therefore, the stool hatching method is used to observe the miracidia in the water. However, the miracidia of Schistosoma japonicum are small and difficult to detect, and missed detection is likely to occur when the infection level is low. In this study, recombinant streptococcal protein G-enhanced green fluorescent protein (rSPG-EGFP) was expressed, purified, and used as a fluorescence staining reagent for miracidia. A preliminary miracidium fluorescence staining method based on antigen and antibody bindingwas established by combining recombinant protein staining with the stool hatching method. The stool hatching method was used to collect the miracidia of S. japonicum, and Schistosoma-positive serum and the recombinant protein were mixed to assess the feasibility of fluorescence staining of miracidia. The miracidia of S. japonicum and Schistosoma turkestanicum were incubated with S. japonicum-positive serum and S. turkestanicum-positive serum, respectively, to identify miracidia species. When the fluorescence staining method was used to observe living miracidia, the miracidiawere labelled by the recombinant protein, and their motility status was not affected.

Abstract CT241: Trial in progress: IFx-Hu2.0 (plasmid DNA coding for Emm55 streptococcal antigen in a cationic polymer) phase I first in human study for unresectable stage III or stage IV cutaneous melanoma

Abstract Background: Anti-PD-1 and anti-CTLA4 immune-based therapeutics have vastly improved melanoma survival rates, but many patients still do not benefit from these therapies. IFx-Hu2.0 is a plasmid DNA encoding the streptococcal membrane protein, Emm55, contained within a cationic polymer. Preliminary correlative laboratory data from the first in human IFx-Hu2.0 phase I feasibility study for use as an intralesional melanoma therapy is reported. No clinical data will be presented. The melanoma tumor microenvironment, pre- and post-treatment, was analyzed on FFPE tissues collected from the first three patients with a panel consisting of CD3, CD8, FOXP3, PD1, PDL1, SOX10 and DAPI. The numbers of SOX10+ melanoma cells tended to decrease with treatment (Patient 1: 16.7% SOX10+ cells pre-treatment, 0.3% SOX10+ cells post treatment; Patient 2: 47.1% pre/6.0% post; Patient 3: 49.0% pre/52.7% post). Multi-parameter analysis revealed that when the numbers of SOX10+ cells decreased, there were an increased number of T cells in the tumor environment. This observation also held true for the whole section for patient 2. In this patient, there were 0.3% CD3+CD8+ T cells prior to treatment but this number increased to 15.2% after treatment. Interestingly, the numbers of FOXP3 regulatory T cells also increased in this patient suggesting the formation of an immune response. Taken together, IFx-Hu2.0 is proposed to create an immune response in melanoma patients and is being tested in a first in human trial. Methods: Up to six patients (&amp;gt;18 years) with unresectable stage III or IV cutaneous melanoma will have cutaneous melanoma lesions injected with IFx-Hu2.0 to test the safety, tolerability, and feasibility of intralesional IFx-Hu2.0 injection. The subjects must have at least one lesion available for injection and another biopsiable lesion. This trial is unique as a cutaneous lesion as small as 3 mm may be injected. To be eligible, subjects should have been considered for anti-PD-1 therapy, TVEC and/or BRAF inhibition as indicated in the FDA labels for those agents. Small brain metastases (1 cm) are permitted assuming the subjects receive concurrent radiation at a site distant from intralesional injection with a greater than 3-month life expectancy. These patients may not be receiving any other systemic therapies, may not have uncontrolled hepatitis/HIV infection, and may not have a history of immunosuppression as is seen in patients who have undergone organ transplantation. For correlative studies, 4 tubes of peripheral blood will be collected at the beginning of the study and at the dose limiting toxicity (DLT) assessment visit 28 days later. Trial subjects will have the option of continued dosing every 3 weeks if they have a response to therapy. Success of the study is defined as being able to treat 5/6 subjects without DLT at 28 days. Three of the six planned trial subjects have been enrolled as of January 2020 (Clinical trial registry number: NCT03655756). Citation Format: Joseph Markowitz, Andrew Brohl, Amod A. Sarnaik, Zeynep Eroglu, Deanryan B. De Aquino, Nikhil Khushalani, Ahmad Tarhini, Vernon K. Sondak, Shari Pilon-Thomas. Trial in progress: IFx-Hu2.0 (plasmid DNA coding for Emm55 streptococcal antigen in a cationic polymer) phase I first in human study for unresectable stage III or stage IV cutaneous melanoma [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr CT241.

Streptococcal protein G based fluorescent universal probes and biosynthetic mimetics for Fumonisin B1 immunochromatographic assay

Immunochromatographic assay (ICA) plays an important role in the detection of chemical pollutants in food. During the construction of conventional ICA for small molecules, labeling the antibody directly may effect the binding properties of antibody to target antigen. In addition, chemical synthesis of hapten-conjugtes, especially for toxic haptens, may face problems such as complex operations, high inter-batch error, and environmental pollution. In this work, using the detection for mycotoxin fumonisin B1 (FB1) as a model system, the immunochromatographic assay based on fluorescent universal probes and biosynthetic mimetics was established. Biosynthetic mimotope peptide-MBP fusion protein (F15-MBP) was used as a substitute of chemical synthetic FB1-conjugates. Quantum dots nanobeads (QBs) labeled streptococcal protein G (SPG, which can bind to the fragement crytallizable of antibody) was used as a universal signal probe. The antibody and biosynthetic mimetics of coating antigen were sprayed on the conjugate pad and test line, respectively. Under the conditions of optimization, the visual limit detection (vLOD) for FB1 can reach up to 125 pg/mL within 10 min, compared with conventional chemical synthesized FB1-conjugates, when there was a drop of nearly 25 times. The spiked sample (wheat, rice, and corn) with FB1 can be found when the concentration is greater than 1.25 μg/kg. The specificity, repeatability, and stability are confirmed using nagative samples and in accordance with spiked experiments. Furthermore, the novel concept might provide potential applications to a general method for multiplex immunochromatographic assay of various toxic small molecules without steps for labeling antibodies and avoiding synthesis of toxic mycotoxin conjugates.

Etiopathogenetic, Morphological, Diagnostic and Therapeutic Aspects of Acute Glomerulonephritis: Current Status

The review provides up-to-date information on the etiological factors and pathogenic mechanisms of development, morphological changes, clinical and laboratory manifestations of acute glomerulonephritis, as well as therapeutic possibilities for its treatment. The review provides up-to-date information on the etiological factors and pathogenetic mechanisms of development, morphological changes, clinical and laboratory manifestations of acute glomerulonephritis, as well as therapeutic possibilities for its treatment. The epidemiological issues of acute post-streptococcal glomerulonephritis are presented, indicating the characteristics of the effects of nephritogenic strains of Streptococcal infection. The immunopathological reactions of the body in acute glomerulonephritis to the causative agent of the disease and its antigens with the development of an imbalance of subpopulations of T-lymphocytes, the nephritogenic potential of streptococcal proteins, a marker of active proliferation of mesangiocytes, C3 and C4 fractions of complement, and also the renin-angiotensin-aldosterone system are shown. The article emphasizes that the informational content of serological tests in acute glomerulonephritis increases while assessing the C3 and C4 fractions of serum complement. The pathological effects of angiotensin II and aldosterone on the renal tissue with the chronization of acute glomerulonephritis, the development of nephrotic proteinuria and a rapid decrease in renal function are presented. The data of a direct correlation between the severity of histological changes and the clinical manifestations of acute glomerulonephritis, possibly the prognosis, are shown. Presented are modern data on the assessment of the main clinical manifestations of acute glomerulonephritis (urinary syndrome, edema syndrome and arterial hypertension). When discussing the treatment of acute glomerulonephritis, the controversial issues of the use of antibiotic therapy, preventive tonsillectomy are noted. The literature data on the treatment options for edematous syndrome and arterial hypertension using thiazide and loop diuretics, calcium antagonists, beta-blockers, angiotensin converting enzyme inhibitors, angiotensin II receptor blockers are presented. The issues of immunosuppressive therapy using glucorticoids, as well as prognostic criteria for acute glomerulonephritis are discussed.