Protein Binding Capacity Research Articles

Dominant negative OTULIN-related autoinflammatory syndrome.

OTU deubiquitinase with linear linkage specificity (OTULIN) regulates inflammation and cell death by deubiquitinating linear ubiquitin chains generated by the linear ubiquitin chain assembly complex (LUBAC). Biallelic loss-of-function mutations causes OTULIN-related autoinflammatory syndrome (ORAS), while OTULIN haploinsuffiency has not been associated with spontaneous inflammation. However, herein, we identify two patients with the heterozygous mutation p.Cys129Ser in OTULIN. Consistent with ORAS, we observed accumulation of linear ubiquitin chains, increased sensitivity to TNF-induced death, and dysregulation of inflammatory signaling in patient cells. While the C129S mutation did not affect OTULIN protein stability or binding capacity to LUBAC and linear ubiquitin chains, it did ablate OTULIN deubiquitinase activity. Loss of activity facilitated the accumulation of autoubiquitin chains on LUBAC. Altered ubiquitination of LUBAC inhibits its recruitment to the TNF receptor signaling complex, promoting TNF-induced cell death and disease pathology. By reporting the first dominant negative mutation driving ORAS, this study expands our clinical understanding of OTULIN-associated pathology.

A PadR family transcriptional repressor regulates the transcription of chromate efflux transporter in Enterobacter sp. Z1.

Chromium is a prevalent toxic heavy metal, and chromate [Cr(VI)] exhibits high mutagenicity and carcinogenicity. The presence of the Cr(VI) efflux protein ChrA has been identified in strains exhibiting resistance to Cr(VI). Nevertheless, certain strains of bacteria that are resistant to Cr(VI) lack the presence of ChrB, a known regulatory factor. Here, a PadR family transcriptional repressor, ChrN, has been identified as a regulator in the response of Enterobacter sp. Z1(CCTCC NO: M 2019147) to Cr(VI). The chrN gene is cotranscribed with the chrA gene, and the transcriptional expression of this operon is induced by Cr(VI). The binding capacity of the ChrN protein to Cr(VI) was demonstrated by both the tryptophan fluorescence assay and Ni-NTA purification assay. The interaction between ChrN and the chrAN operon promoter was validated by reporter gene assay and electrophoretic mobility shift assay. Mutation of the conserved histidine residues His14 and His50 resulted in loss of ChrN binding with the promoter of the chrAN operon. This observation implies that these residues are crucial for establishing a DNA-binding site. These findings demonstrate that ChrN functions as a transcriptional repressor, modulating the cellular response of strain Z1 to Cr(VI) exposure.

Preparation of a macroporous anion exchange chromatographic medium by polyamine grafting and evaluation of its protein-adsorption behavior

The macroporous anion exchange chromatographic medium (FastSep-PAA) was prepared through grafting polyallylamine (PAA) onto polyacrylate macroporous microspheres (FastSep-epoxy). The effects of the synthesis conditions, including the PAA concentration, reaction time, and reaction solution pH, on the ion exchange (IC) of the medium were investigated in detail. When the PAA concentration, reaction time, and reaction solution pH were increased, the IC of the medium increased, and optimal synthesis conditions were then selected in combination with changes of protein binding capacity. A scanning electron microscope was used to examine the surface morphology of the medium. The medium possessed high pore connectivity. Furthermore, the pore structure of the medium was preserved after the grafting of PAA onto the macroporous microspheres. This finding demonstrates that the density of the PAA ligands does not appear to have any discernible impact on the structure of the medium; that is, no difference in the structure of the medium is observed before and after the grafting of PAA onto the microspheres. The pore size and pore-size distribution of the medium before and after grafting were determined by mercury intrusion porosimetry and the nitrogen adsorption method to investigate the relationship between pore size (measured in the range of 300-1000 nm) and protein adsorption. When the pore size of the medium was increased, its protein binding capacity did not exhibit any substantial decrease. An increase in pore size may hasten the mass transfer of proteins within the medium. Among the media prepared, that with a pore size of 400 nm exhibited the highest dynamic-binding capacity (DBC: 70.3 g/L at 126 cm/h). The large specific surface area of the medium and its increased number of protein adsorption sites appeared to positively influence its DBC. When the flow rate was increased, the protein DBC decreased in media with original pore sizes of less than 700 nm. In the case of the medium with an original pore size of 1000 nm, the protein DBC was independent of the flow rate. The protein DBC decreased by 3.5% when the flow rate was increased from 126 to 628 cm/h. In addition, the protein DBC was maintained at 57.7 g/L even when the flow velocity was 628 cm/h. This finding reveals that the diffusion rate of protein molecules at this pore size is less restricted and that the prepared medium has excellent mass-transfer performance. These results confirm that the macroporous polymer anion exchange chromatographic medium developed in this study has great potential for the high-throughput separation of proteins.

Abstract 1642: NuMA exclusive post-translational modification in epithelial cancer cells is a novel target for cell eradication

Abstract The post-translational modification of proteins implicated in mitosis in healthy and in cancer epithelial cells was measured by 2-D gels analysis. The post-translational modifications of NuMA in a variety of human epithelial cancer cells was prevented by treatment with a small phenanthridine, PJ34, while not impaired in similarly treated healthy epithelial cells. NuMA in the cancer cells was phosphorylated by pim1 kinase, and was polyADP-ribosylated by Tankyrase1. These post-translational modifications are inhibited by PJ34. Tankyrase1 is not expressed in healthy epithelial cells, and pim1 kinase was hardly expressed in these cells. Preventing the post-translational modification of NuMA in the epithelial cancer cells also suppressed the protein binding capacity of NuMA. NuMA clustering in the mitotic spindle poles, which stabilizes the mitotic spindle, and is required for the alignment of chromosomes in the spindle mid-zone was impaired. In epithelial cancer cells treated with PJ34, the mitotic spindle poles were distorted, extra-centrosomes were not clustered in multi-centrosomal cancer cells, and chromosomes were dispersed. Chromosomes dispersion violated mitosis termination. Mitosis arrested in the anaphase led to mitotic catastrophe cell death. PJ34 treated epithelial cancer cells were eradicated during 72-96 hours, regardless of their mutations, while treated healthy epithelial cells were not affected, and continued to proliferate. Citation Format: Malka Cohen-Armon. NuMA exclusive post-translational modification in epithelial cancer cells is a novel target for cell eradication [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1642.

Screening Peptide Drug Candidates To Neutralize Whole Viral Agents: A Case Study with Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2).

The COVID-19 pandemic revealed the need for therapeutic and pharmaceutical molecule development in a short time with different approaches. Although boosting immunological memory by vaccination was the quickest and robust strategy, still medication is required for the immediate treatment of a patient. A popular approach is the mining of new therapeutic molecules. Peptide-based drug candidates are also becoming a popular avenue. To target whole pathogenic viral agents, peptide libraries can be employed. With this motivation, we have used the 12mer M13 phage display library for selecting SARS-CoV-2 targeting peptides as potential neutralizing molecules to prevent viral infections. Panning was applied with four iterative cycles to select SARS-CoV-2 targeting phage particles displaying 12-amino acid-long peptides. Randomly selected peptide sequences were synthesized by a solid-state peptide synthesis method. Later, selected peptides were analyzed by the quartz crystal microbalance method to characterize their molecular interaction with SARS-CoV-2's S protein. Finally, the neutralization activity of the selected peptides was probed with an in-house enzyme-linked immunosorbent assay. The results showed that scpep3, scpep8, and scpep10 peptides have both binding and neutralizing capacity for S1 protein as a candidate for therapeutic molecule. The results of this study have a translational potential with future in vivo and human studies.

Barley AGO4 proteins show overlapping functionality with distinct small RNA-binding properties in heterologous complementation

Key messageBarley AGO4 proteins complement expressional changes of epigenetically regulated genes in Arabidopsis ago4-3 mutant and show a distinct affinity for the 5′ terminal nucleotide of small RNAs, demonstrating functional conservation and divergence.The function of Argonaute 4 (AGO4) in Arabidopsis thaliana has been extensively characterized; however, its role in monocots, which have large genomes abundantly supplemented with transposable elements (TEs), remains elusive. The study of barley AGO4 proteins can provide insights into the conserved aspects of RNA-directed DNA methylation (RdDM) and could also have further applications in the field of epigenetics or crop improvement. Bioinformatic analysis of RNA sequencing data identified two active AGO4 genes in barley, HvAGO4a and HvAGO4b. These genes function similar to AtAGO4 in an Arabidopsis heterologous complementation system, primarily binding to 24-nucleotide long small RNAs (sRNAs) and triggering methylation at specific target loci. Like AtAGO4, HvAGO4B exhibits a preference for binding sRNAs with 5′ adenine residue, while also accepting 5′ guanine, uracil, and cytosine residues. In contrast, HvAGO4A selectively binds only sRNAs with a 5′ adenine residue. The diverse binding capacity of barley AGO4 proteins is reflected in TE-derived sRNAs and in their varying abundance. Both barley AGO4 proteins effectively restore the levels of extrachromosomal DNA and transcript abundancy of the heat-activated ONSEN retrotransposon to those observed in wild-type Arabidopsis plants. Our study provides insight into the distinct binding specificities and involvement in TE regulation of barley AGO4 proteins in Arabidopsis by heterologous complementation.

A ClO− triggered phenol derivative that induced mitochondrial damage for tumor therapy

Mitochondrial proteome is comprised of more than 1000 different proteins, whose synergistic ensured the integrity and homeostasis of the proteome, thus provided a guarantee for the rapid and accurate regulation of mitochondrial activities. Based on this, mitochondrial proteins have become a compelling focus for the treatment and intervention of tumors. Phenol derivatives exhibited superior protein binding capacities and showed efficient antitumor activity through their interaction with specific target proteins. Selective delivery of phenol derivatives to the mitochondria can be achieved through lipophilic cations modification. However, lack stability of phenol hydroxyl group under physiological conditions, taken into account, an ClO−-activated mitochondria-targeting prodrug RCl was constructed in the present study. RCl was delivered into the mitochondria and rapidly released ROH when struck by the presence of high ClO− levels, the released ROH act on mitochondrial proteins, resulting in mitochondrial dysfunction, ATP inhibition and apoptosis of cancer cells. The vivo antitumor experiment results showed that the RCl displayed significantly antitumor efficacy in vivo which provided the rationale to the further development of anticancer drugs targeting mitochondria.

Discovery of a small-molecule NDR1 agonist for prostate cancer therapy.

Prostatic cancer (PCa) is a common malignant neoplasm in men worldwide. Most patients develop castration-resistant prostate cancer (CRPC) after treatment with androgen deprivation therapy (ADT), usually resulting in death. Therefore, investigating new therapeutic targets and drugs for PCa patients is urgently needed. Nuclear Dbf2-related kinase 1 (NDR1), also known as STK38, is a serine/threonine kinase in the NDR/LATS kinase family that plays a critical role in cellular processes, including immunity, inflammation, metastasis, and tumorigenesis. It was reported that NDR1 inhibited the metastasis of prostate cancer cells by suppressing epithelial-mesenchymal transition (EMT), and decreased NDR1 expression might lead to a poorer prognosis, suggesting the enormous potential of NDR1 in antitumorigenesis. In this study, we characterized a small-molecule agonist named aNDR1, which specifically bound to NDR1 and potently promoted NDR1 expression, enzymatic activity and phosphorylation. aNDR1 exhibited drug-like properties, such as favorable stability, plasma protein binding capacity, cell membrane permeability, and PCa cell-specific inhibition, while having no obvious effect on normal prostate cells. Meanwhile, aNDR1 exhibited good antitumor activity both in vitro and in vivo. aNDR1 inhibited proliferation and migration of PCa cells and promoted apoptosis of PCa cells in vitro. We further found that aNDR1 inhibited subcutaneous tumors and lung metastatic nodules in vivo, with no obvious toxicity to the body. In summary, our study presents a potential small-molecule lead compound that targets NDR1 for clinical therapy of PCa patients.

N123I mutation in the ALV-J receptor-binding domain region enhances viral replication ability by increasing the binding affinity with chNHE1.

The subgroup J avian leukosis virus (ALV-J), a retrovirus, uses its gp85 protein to bind to the receptor, the chicken sodium hydrogen exchanger isoform 1 (chNHE1), facilitating viral invasion. ALV-J is the main epidemic subgroup and shows noteworthy mutations within the receptor-binding domain (RBD) region of gp85, especially in ALV-J layer strains in China. However, the implications of these mutations on viral replication and transmission remain elusive. In this study, the ALV-J layer strain JL08CH3-1 exhibited a more robust replication ability than the prototype strain HPRS103, which is related to variations in the gp85 protein. Notably, the gp85 of JL08CH3-1 demonstrated a heightened binding capacity to chNHE1 compared to HPRS103-gp85 binding. Furthermore, we showed that the specific N123I mutation within gp85 contributed to the enhanced binding capacity of the gp85 protein to chNHE1. Structural analysis indicated that the N123I mutation primarily enhanced the stability of gp85, expanded the interaction interface, and increased the number of hydrogen bonds at the interaction interface to increase the binding capacity between gp85 and chNHE1. We found that the N123I mutation not only improved the viral replication ability of ALV-J but also promoted viral shedding in vivo. These comprehensive data underscore the notion that the N123I mutation increases receptor binding and intensifies viral replication.

Anionic extracellular polymeric substances extracted from seawater-adapted aerobic granular sludge.

Anionic polymers, such as heparin, have been widely applied in the chemical and medical fields, particularly for binding proteins (e.g., fibroblast growth factor 2 (FGF-2) and histones). However, the current animal-based production of heparin brings great risks, including resource shortages and product contamination. Recently, anionic compounds, nonulosonic acids (NulOs), and sulfated glycoconjugates were discovered in the extracellular polymeric substances (EPS) of aerobic granular sludge (AGS). Given the prevalence of anionic polymers, in marine biofilms, it was hypothesized that the EPS from AGS grown under seawater condition could serve as a raw material for producing the alternatives to heparin. This study aimed to isolate and enrich the anionic fractions of EPS and evaluate their potential application in the chemical and medical fields. The AGS was grown in a lab-scale reactor fed with acetate, under the seawater condition (35 g/L sea salt). The EPS was extracted with an alkaline solution at 80 °C and fractionated by size exclusion chromatography. Its protein binding capacity was evaluated by native gel electrophoresis. It was found that the two highest molecular weight fractions (438- > 14,320 kDa) were enriched with NulO and sulfate-containing glycoconjugates. The enriched fractions can strongly bind the two histones involved in sepsis and a model protein used for purification by heparin-column. These findings demonstrated possibilities for the application of the extracted EPS and open up a novel strategy for resource recovery. KEY POINTS: • High MW EPS from seawater-adapted AGS are dominant with sulfated groups and NulOs • Fifty-eight percent of the EPS is high MW of 68-14,320 kDa • EPS and its fractions can bind histones and fibroblast growth factor 2.

Study of the reusability and stability of nylon nanofibres as an antibody immobilisation surface.

In the case of a biological threat, early, rapid, and specific detection is critical. In addition, ease of handling, use in the field, and low-cost production are important considerations. Immunological devices are able to respond to these needs. In the design of these immunological devices, surface antibody immobilisation is crucial. Nylon nanofibres have been described as a very good option because they allow for an increase in the surface-to-volume ratio, leading to an increase in immunocapture efficiency. In this paper, we want to deepen the study of other key points, such as the reuse and stability of these nanofibres, in order to assess their profitability. On the one hand, the reusability of nanofibres has been studied using different stripping treatments at different pH values on the nylon nanofibres with well-oriented antibodies anchored by protein A/G. Our study shows that stripping with glycine buffer pH 2.5 allows the nanofibres to be reused as long as protein A/G has been previously anchored, leaving both nanofibre and protein A/G unchanged. On the other hand, we investigated the stability of the nylon nanofibres. To achieve this, we analysed any loss of immunocapture ability of well-oriented antibodies anchored both to the nylon nanofibres and to a specialised surface with high protein binding capacity. The nanofibre immunocapture system maintained an unchanged immunocapture ability for a longer time than the specialised planar surface. In conclusion, nylon nanofibres seem to be a very good choice as an antibody immobilisation surface, offering not only higher immunocapture efficiency, but also more cost efficiency as they are reusable and stable.

Short-time ozone treatment promotes protease-mediated destruction of B cell allergen epitopes by altering the structural characteristics of whey protein.

A novel processing method combining short-time ozone pretreatment with hydrolysis has been developed to reduce whey protein allergenicity. The results showed that ozone treatment altered the whey protein spatial structure, initially increasing the surface hydrophobicity index, and then decreasing due to polymer formation as the time increased. Under the optimized conditions of alkaline protease-mediated hydrolysis, a 10-second pre-exposure to ozone significantly promoted the reduction in the IgE binding capacity of whey protein without compromising the hydrolysis efficiency. Compared with whey protein, the degranulation of KU812 cells stimulated by this hydrolysate decreased by 20.54%, 17.99%, and 22.80% for IL-6, β-hexosaminidase, and histamine, respectively. In vitro simulated gastrointestinal digestion confirmed increased digestibility and reduced allergenicity. Peptidomics identification revealed that short-time ozonation exposed allergen epitopes, allowing alkaline protease to target these epitopes more effectively, particularly those associated with α-lactalbumin. These findings suggest the promising application of this processing method in mitigating the allergenicity of whey protein.

Dynamic and static control of the off-target interactions of antisense oligonucleotides using toehold chemistry

Off-target interactions between antisense oligonucleotides (ASOs) with state-of-the-art modifications and biological components still pose clinical safety liabilities. To mitigate a broad spectrum of off-target interactions and enhance the safety profile of ASO drugs, we here devise a nanoarchitecture named BRace On a THERapeutic aSo (BROTHERS or BRO), which is composed of a standard gapmer ASO paired with a partially complementary peptide nucleic acid (PNA) strand. We show that these non-canonical ASO/PNA hybrids have reduced non-specific protein-binding capacity. The optimization of the structural and thermodynamic characteristics of this duplex system enables the operation of an in vivo toehold-mediated strand displacement (TMSD) reaction, effectively reducing hybridization with RNA off-targets. The optimized BROs dramatically mitigate hepatotoxicity while maintaining the on-target knockdown activity of their parent ASOs in vivo. This technique not only introduces a BRO class of drugs that could have a transformative impact on the extrahepatic delivery of ASOs, but can also help uncover the toxicity mechanism of ASOs.

Structural and functional prediction of WsMYB34 transcription factor in Withania somnifera (L.) Dunal by deciphering its role in NaCl-induced secondary metabolism

In the present study, a MYB transcription factor from Withania somnifera, was identified i.e., WsMYB34, known for development and stress tolerance in plants. MYB transcription factors have been widely implicated in conferring stress tolerance in medicinal plants. To validate this notion, we analyzed the impact of different concentrations of NaCl on W. somnifera and hypothesized the potentiality of MYB protein in regulating the expression of secondary metabolite-related genes by docking approach. Since structural and functional characteristics of R2R3 type MYB TF in W. somnifera remain largely unknown, in-silico analysis was exploited to understand conserved binding motifs, phylogeny, 3-D structure, and role in combating stress. Based on these outcomes, WsMYB34 was found to be an R2R3 type MYB protein, which is phylogenetically close to that of Capsicum baccatum (CbMYB34). In addition, in-silico interaction analysis depicted the binding capacity of WsMYB34 protein on the promoter region of a triterpenoid synthesising gene viz CYP85A69 (having CAACTG cis-regulatory sequence). The comparative microarray analysis also offered the role of MYB34 in abiotic stress tolerance in model plants. Exposure to NaCl imposed increased withanolide and flavonoid contents and significant up-regulation of key genes for withanolide and WsMYB34 gene by 0.93–3.68 and 2.61–3.47-folds respectively. The enhanced expressivity of transcript encoding WsMYB34 indicated its possible role in inducing terpenes, flavonoids, anthocyanins, and trichome organogenesis to provide defence against salt. In addition, NaCl caused a reduction in morphology and photosynthesis of W. somnifera. However, enhanced contents of phenolics, proline, MDA, and free radical scavenging activities were observed.

Effects of spike proteins on angiotensin converting enzyme 2 (ACE2)

The Coronavirus Disease 2019 (COVID-19) pandemic was caused by Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), which enters host cells through interactions of its spike protein to Angiotensin-Converting Enzyme 2 (ACE2). ACE2 is a peptidase that cleaves Angiotensin II, a critical pathological mediator. This study investigated if the spike protein binding to ACE2 compromises its peptidase activity. Spike/ACE2 Binding Assays suggested that spike proteins of SARS-CoV-2, SARS-CoV and MERS-CoV, but not HKU1, bind to ACE2. S1 and receptor-binding domain (RBD), but not S2, extracellular domain (ECD) or CendR domain, bind to ACE2. While glycosylated spike proteins prepared in HEK293 cells bind to ACE2, non-glycosylated proteins produced in E. coli do not. Cysteine residues of the spike protein expressed in HEK293 cells are fully oxidized, while those of the protein expressed in E. coli are reduced. The deglycosylation of HEK cell-produced protein attenuates the ACE2 binding, while the oxidation of the E. coli protein does not promote the binding. The S1 protein of SARS-CoV-2 enhances the ACE2 peptidase activity, while SARS-CoV, MERS-CoV or HKU1 does not. The ACE2 activity is enhanced by RBD, but not ECD or CendR. In contrast to distinct ACE2 binding capacities of proteins expressed in HEK293 cells and in E. coli, spike proteins expressed in both systems enhance the ACE2 activity. Thus, the spike protein of SARS-CoV-2, but not other coronaviruses, enhances the ACE2 peptidase activity through its RBD in a glycosylation-independent manner.

Enhancing protein trapping efficiency of graphene oxide-polybutylene succinate nanofiber membrane via molecular imprinting

Filtration of biological liquids has been widely employed in biological, medical, and environmental investigations due to its convenience; many could be performed without energy and on-site, particularly protein separation. However, most available membranes are universal protein absorption or sub-fractionation due to molecule sizes or properties. SPMA, or syringe-push membrane absorption, is a quick and easy way to prepare biofluids for protein evaluation. The idea of initiating SPMA was to filter proteins from human urine for subsequent proteomic analysis. In our previous study, we developed nanofiber membranes made from polybutylene succinate (PBS) composed of graphene oxide (GO) for SPMA. In this study, we combined molecular imprinting with our developed PBS fiber membranes mixed with graphene oxide to improve protein capture selectivity in a lock-and-key fashion and thereby increase the efficacy of protein capture. As a model, we selected albumin from human serum (ABH), a clinically significant urine biomarker, for proteomic application. The nanofibrous membrane was generated utilizing the electrospinning technique with PBS/GO composite. The PBS/GO solution mixed with ABH was injected from a syringe and transformed into nanofibers by an electric voltage, which led the fibers to a rotating collector spinning for fiber collection. The imprinting process was carried out by removing the albumin protein template from the membrane through immersion of the membrane in a 60% acetonitrile solution for 4 h to generate a molecular imprint on the membrane. Protein trapping ability, high surface area, the potential for producing affinity with proteins, and molecular-level memory were all evaluated using the fabricated membrane morphology, protein binding capacity, and quantitative protein measurement. This study revealed that GO is a controlling factor, increasing electrical conductivity and reducing fiber sizes and membrane pore areas in PBS-GO-composites. On the other hand, the molecular imprinting did not influence membrane shape, nanofiber size, or density. Human albumin imprinted membrane could increase the PBS-GO membrane’s ABH binding capacity from 50 to 83%. It can be indicated that applying the imprinting technique in combination with the graphene oxide composite technique resulted in enhanced ABH binding capabilities than using either technique individually in membrane fabrication. The suitable protein elution solution is at 60% acetonitrile with an immersion time of 4 h. Our approach has resulted in the possibility of improving filter membranes for protein enrichment and storage in a variety of biological fluids.

Interaction of Blenoxane and Congeners Bleomycins A2 and B2 with Human Plasma Proteins Using Circular Dichroism Spectroscopy.

Bleomycin is a glycopeptide congeners' family of antitumor antibiotics employed for the treatment of several types of tumors such as squamous cell carcinomas and malignant lymphomas. The general chemical structure is constituted by three main portions: (i) a metal binding domain that is recognized to be responsible for the DNA cleavage activity; (ii) a DNA binding domain via the 1-4' bithiazole moiety; and (iii) a carbohydrate domain thought to be responsible for the accumulation of bleomycin in some cancer cells. To date, a limited number of protein interactions with bleomycin have been studied, but the plasma binding has not yet been determined. Here, we explore this aspect of the protein binding capacity of bleomycin to the two most abundant plasma proteins, human serum albumin (HSA) and α1-acid glycoprotein (AGP), which are known to bind and to be carriers of many drug molecules using spectroscopic techniques, such as circular dichroism, UV-vis absorbance, and fluorescence. The results showed that bleomycin binds to plasma proteins with an order-of-magnitude higher affinity for AGP than HSA. This is particularly important as AGP is an acute phase protein and is overexpressed in cancer patients. This should be taken into consideration as it could affect the therapeutic effect of the bleomycin dosage.

Fe3O4@SiO2@NiAl-LDH microspheres implication in separation, kinetic and structural properties of phenylalanine dehydrogenase

Fe3O4@SiO2@NiAl-LDH three-components microsphere contains a Fe3O4@SiO2 magnetic core and a layered double hydroxide with nickel cation provide the binding ability to (His)-tagged-protein and exhibits high performance in protein separation and purification. The morphology and chemistry of the synthesized Fe3O4@SiO2@NiAl-LDH microspheres were characterized by energy-dispersive X-ray spectroscopy (EDX), scanning electron microscopy (SEM), X-ray diffraction (XRD), Fourier transform infrared (FTIR), vibrating sample magnetometer (VSM), Dynamic light scattering (DLS). Purified enzyme was assesed with SDS-PAGE (sodium dodecyl sulfate–polyacrylamide gel electrophoresis and intrinsic fluorescence spectroscopy. In this study, the separation of phenylalanine dehydrogenase (PheDH) by Fe3O4@SiO2@NiAl -LDH was performed and the effect of microsphere was investigated on the kinetic and structural properties of PheDH. After purification, kinetic parameters such as Km, Vmax, Kcat, kcat/Km, optimum temperature, thermal stability, and and activation energy were evaluated and compared according to the mentioned methods. The interaction between the enzyme and the microsphere displayed a high performance in protein binding capacity. The results also revealed that the kinetic parameters of the enzyme changed in a dose-dependent manner in the presence of a microsphere. Moreover, the results of intrinsic fluorescence and Circular Dichroism (CD) confirmed the structural changes of the protein in the interaction with the microsphere.

Comprehensive analysis of circular RNAs in nasopharyngeal cancer.

Nasopharyngeal cancer (NPC) is a type of epithelial malignancy that is positive for Epstein-Barr virus (EBV) and affects several populations worldwide. Due to the high rates of relapse and metastasis following primary treatment, there is an urgent need to identify new candidates for NPC therapy. Recently, circular RNA (circRNA) has emerged as a promising target for cancer diagnosis and prevention. This study aimed to study the circRNAs enriched in NPC patients, and further analyze potential signaling pathways involved. A new bioinformatic tool named psirc was used to analyze RNA-sequencing datasets from NPC patients and normal specimens to study the NPC-enriched circRNAs. We identified and quantified the full-length circRNA in these samples and found the top 10 enriched circRNAs in NPC patients compared to control samples. Furthermore, we selected the most enriched circRNA, circEEF1A1_E8B1, and studied its protein coding ability, microRNA and RNA-binding protein (RBP) binding capacity. We also constructed a protein-protein interaction (PPI) network for its binding proteins and extracted hub genes. Finally, we conducted survival analysis for these hub genes in head and neck cancer patients. In summary, our study has revealed the presence of previously unidentified circRNAs that are enriched in NPC patients. Through an analysis of their molecular functions, we have advanced our understanding of the potential role of circRNAs in NPC development.

BMP-2 releasing mineral-coated microparticle-integrated hydrogel system for enhanced bone regeneration.

Introduction: Large bone defects (LBD) caused by trauma, infection, and tumor resection remain a significant clinical challenge. Although therapeutic agents such as bone morphogenetic protein-2 (BMP-2), have shown substantial potency in various clinical scenarios, their uncontrollable release kinetics has raised considerable concern from the clinical viewpoint. Mineral-coated microparticle (MCM) has shown its excellent biologics loading and delivery potential due to its superior protein-binding capacity and controllable degradation behaviors; thus, it is conceivable that MCM can be combined with hydrogel systems to enable optimized BMP-2 delivery for LBD healing. Methods: Herein, BMP-2 was immobilized on MCMs via electrostatic interaction between its side chains with the coating surface. Subsequently, MCM@BMP-2 is anchored into a hydrogel by the crosslinking of chitosan (CS) and polyethylene glycol (PEG). Results and Discussion: This microparticle-hydrogel system exhibits good biocompatibility, excellent vascularization, and the sustained release of BMP-2 in the bone defect. Furthermore, it is observed that this microsphere-hydrogel system accelerates bone formation by promoting the expression of osteogenesis-related proteins such as RUNX2, osteopontin, and osteocalcin in bone marrow mesenchymal stem cells (BMSCs). Thus, this newly developed multifunctional microparticle-hydrogel system with vascularization, osteogenesis, and sustained release of growth factor demonstrates an effective therapeutic strategy toward LBD.