Genetic Fusion Research Articles

PlexinA1 (PLXNA1) as a novel scaffold protein for the engineering of extracellular vesicles.

Extracellular vesicles (EVs) had been described as a next-generation drug delivery system, due to the compelling evidence that they can facilitate the transfer of a variety of biomolecules between cells. The most frequently used strategy for loading protein cargoes is the endogenous engineering of EVs through genetic fusion of the protein of interest (POI) and scaffold proteins with high EV-sorting ability. However, the lack of scaffold proteins had become a major issue hindering the promotion of this technology. Herein, we proposed novel screening criteria that relax the inclusion requirement of candidate scaffold proteins and eventually identified a new scaffold protein, PLXNA1. The truncated PLXNA1 not only inherits the high EV-sorting ability of its full-length counterpart but also allows the fusion expression of POI in both outer surface and luminal areas, individually or simultaneously. In conclusion, our screening criteria expanded the range of potential scaffold proteins. The identified scaffold protein PLXNA1 showed great potential in developing therapeutic EVs.

Establishing the green algae Chlamydomonas incerta as a platform for recombinant protein production.

Chlamydomonas incerta, a genetically close relative of the model green alga Chlamydomonas reinhardtii, shows significant potential as a host for recombinant protein expression. Because of the close genetic relationship between C. incerta and C. reinhardtii, this species offers an additional reference point for advancing our understanding of photosynthetic organisms, and also provides a potential new candidate for biotechnological applications. This study investigates C. incerta's capacity to express three recombinant proteins: the fluorescent protein mCherry, the hemicellulose-degrading enzyme xylanase, and the plastic-degrading enzyme PHL7. We have also examined the capacity to target protein expression to various cellular compartments in this alga, including the cytosol, secretory pathway, cytoplasmic membrane, and cell wall. When compared directly with C. reinhardtii, C. incerta exhibited a distinct but notable capacity for recombinant protein production. Cellular transformation with a vector encoding mCherry revealed that C. incerta produced approximately 3.5 times higher fluorescence levels and a 3.7-fold increase in immunoblot intensity compared to C. reinhardtii. For xylanase expression and secretion, both C. incerta and C. reinhardtii showed similar secretion capacities and enzymatic activities, with comparable xylan degradation rates, highlighting the industrial applicability of xylanase expression in microalgae. Finally, C. incerta showed comparable PHL7 activity levels to C. reinhardtii, as demonstrated by the in vitro degradation of a polyester polyurethane suspension, Impranil® DLN. Finally, we also explored the potential of cellular fusion for the generation of genetic hybrids between C. incerta and C. reinhardtii as a means to enhance phenotypic diversity and augment genetic variation. We were able to generate genetic fusion that could exchange both the recombinant protein genes, as well as associated selectable marker genes into recombinant offspring. These findings emphasize C. incerta's potential as a robust platform for recombinant protein production, and as a powerful tool for gaining a better understanding of microalgal biology.

Research on Fracture Parameter Optimization Method Based on Generative Adversarial Network in Small Sample Size

Abstract This study addresses the optimization of fracturing parameters in the fractured gas reservoirs of the Tarim Basin, especially under the challenge of small sample sizes and high data acquisition costs. It proposes an innovative method based on Generative Adversarial Networks (GAN). Traditional fracturing technology parameter optimization often leads to model overfitting and poor generalization due to limited data. This research adopts GAN for data augmentation to generate additional training samples, thereby improving the training effect of machine learning models. The combination of genetic algorithms and model fusion techniques further optimizes production capacity prediction and fracturing parameter adjustment. Application of this method to actual data from the Tarim Basin shows that it effectively enhances the accuracy of parameter optimization and the generalization ability of the model, providing significant guidance for gas reservoir development under similar complex geological conditions.

Tyrosinase-Mediated Conjugation for Antigen Display on Ferritin Nanoparticles.

Ferritin (Ft) nanoparticles have become versatile platforms for displaying antigens, being a promising technology for vaccine development. While genetic fusion has traditionally been the preferred method for antigen display, concerns about improper folding and steric hindrance that may compromise vaccine efficacy or stability have prompted alternative approaches. Bioconjugation offers the advantage of preserving native protein structure and function, with recent advancements improving efficiency and specificity. In this study, we used tyrosinase (TYR) to bioconjugate the receptor binding domain of the SARS-CoV-2 spike protein, tagged with a tyrosine (RBD-Y), to native cysteines on Ft, resulting in RBD-Y-Ft nanoparticles. We quantified available cysteines on ferritin using Ellman's assay and monitored their reduction during the reactions. Denaturing analytics (via SDS-PAGE, Western blot, and LC-TOF-MS) confirmed the formation of RBD-Y-Ft monomers with an expected molecular weight of 46 kDa. Mass photometry and HPLC estimated a molecular weight of RBD-Y-Ft nanoparticles of 680 kDa, which was higher than that of nonfunctionalized ferritin (480 kDa), indicating successful binding of up to eight RBD-Y antigens per 24-mer Ft nanoparticle. This work enhances our understanding of how Ft nanoparticles can be engineered to present antigens, leveraging them as a robust scaffold for producing tailored-made candidate vaccines in a timely manner.

Targeted Therapies in Pancreatic Cancer: A New Era of Precision Medicine.

Pancreatic ductal adenocarcinoma (PDAC), a leading cause of cancer mortality in the United States, presents significant treatment challenges due to its late diagnosis and poor prognosis. Despite advances, the five-year survival rates remain dismally low, with only a fraction of patients eligible for potentially curative surgical interventions. This review aims to comprehensively examine the current landscape of targeted therapies in PDAC, focusing on recent developments in precision medicine approaches. We explore various molecular targets, including KRAS mutations, DNA damage repair deficiencies, mismatch repair pathway alterations, and rare genetic fusions. The review discusses emerging therapies, such as PARP inhibitors, immune checkpoint inhibitors, and novel targeted agents, like RET and NTRK inhibitors. We analyze the results of key clinical trials and highlight the potential of these targeted approaches in specific patient subgroups. Recent developments in PDAC research have emphasized precision oncology, facilitated by next-generation sequencing and the identification of genetic and epigenetic alterations. This approach tailors treatments to individual genetic profiles, improving outcomes and reducing side effects. Significant strides have been made in classifying PDAC into various subtypes, enhancing therapeutic precision. The identification of specific mutations in genes like KRAS, along with advancements in targeted therapies, including small molecule inhibitors, offers new hope. Furthermore, emerging therapies targeting DNA repair pathways and immunotherapeutic strategies also show promising results. As research evolves, integrating these targeted therapies with conventional treatments might improve survival rates and quality of life for PDAC patients, underscoring the shift towards a more personalized treatment paradigm.

Engineering a Bifunctional Fusion Purine/Pyrimidine Nucleoside Phosphorylase for the Production of Nucleoside Analogs.

Nucleoside phosphorylases (NPs) are pivotal enzymes in the salvage pathway, catalyzing the reversible phosphorolysis of nucleosides to produce nucleobases and α-D-ribose 1-phosphate. Due to their efficiency in catalyzing nucleoside synthesis from purine or pyrimidine bases, these enzymes hold significant industrial importance in the production of nucleoside-based drugs. Given that the thermodynamic equilibrium for purine NPs (PNPs) is favorable for nucleoside synthesis-unlike pyrimidine NPs (PyNPs, UP, and TP)-multi-enzymatic systems combining PNPs with PyNPs, UPs, or TPs are commonly employed in the synthesis of nucleoside analogs. In this study, we report the first development of two engineered bifunctional fusion enzymes, created through the genetic fusion of purine nucleoside phosphorylase I (PNP I) and thymidine phosphorylase (TP) from Thermus thermophilus. These fusion constructs, PNP I/TP-His and TP/PNP I-His, provide an innovative one-pot, single-step alternative to traditional multi-enzymatic synthesis approaches. Interestingly, both fusion enzymes retain phosphorolytic activity for both purine and pyrimidine nucleosides, demonstrating significant activity at elevated temperatures (60-90 °C) and within a pH range of 6-8. Additionally, both enzymes exhibit high thermal stability, maintaining approximately 80-100% of their activity when incubated at 60-80 °C over extended periods. Furthermore, the transglycosylation capabilities of the fusion enzymes were explored, demonstrating successful catalysis between purine (2'-deoxy)ribonucleosides and pyrimidine bases, and vice versa. To optimize reaction conditions, the effects of pH and temperature on transglycosylation activity were systematically examined. Finally, as a proof of concept, these fusion enzymes were successfully employed in the synthesis of various purine and pyrimidine ribonucleoside and 2'-deoxyribonucleoside analogs, underscoring their potential as versatile biocatalysts in nucleoside-based drug synthesis.

A pH-Responsive Protein Assembly through Clustering of a Charge-Tunable Single Amino Acid Repeat.

Specific targeting of tumor cells is a key to achieving high therapeutic efficacy while minimizing off-target side effects. As a general approach to targeting diverse tumor cells, considerable attention has been paid to the tumor microenvironment, particularly its slightly acidic pH (6.5-6.8). However, existing pH-sensitive nanomaterials, based on organic polymers and proteins, often lack sufficient pH sensitivity and specificity. Here, we demonstrate a strategy to construct a pH-responsive protein assembly through clustering of a single amino acid repeat as a charge-tunable moiety. As a proof of concept, a histidine peptide with varying lengths was displayed on the surface of a ferritin assembly composed of 24 subunits by genetic fusion to a subunit. The resulting self-assembled ferritin particles, termed "pHerricle (pH-responsive ferritin particle)", were shown to exhibit a specific binding to tumor cells in response to pH changes through cooperative effects of histidine peptides. Increasing the histidine peptide length from 0 to 12 residues increased the pHerricle's cell-binding capacity by 21-fold and allowed modulation of the targetable pH range. General applicability as a tumor cell-targeting platform was shown by specific delivery of a cytotoxic cargo by the pHerricle into tumor cells of various origins in a pH-dependent manner.

An Investigation into the In Vitro Targeted Killing of CD44-Expressing Triple-Negative Breast Cancer Cells Using Recombinant Photoimmunotherapeutics Compared to Auristatin-F-Based Antibody-Drug Conjugates.

Triple-negative breast cancer (TNBC) is the deadliest form of breast cancer with limited treatment options. The persistence of highly tumorigenic CD44-expressing subpopulation referred to as cancer stem cells (CSCs), endowed with the self-renewal capacity, has been associated with therapeutic resistance, hence clinical relapses. To mitigate these undesired events, targeted immunotherapies using antibody-photoconjugate (APC) or antibody-drug conjugate (ADC), were developed to specifically release cytotoxic payloads within targeted cells overexpressing cognate antigen receptors. Therefore, an αCD44(scFv)-SNAP-tag antibody fusion protein was engineered through genetic fusion of a single-chain antibody fragment (scFv) to a SNAPf-tag fusion protein, capable of self-conjugating with benzylguanine-modified light-sensitive near-infrared (NIR) phthalocyanine dye IRDye700DX (BG-IR700) or the small molecule toxin auristatin-F (BG-AURIF). Binding of the αCD44(scFv)-SNAPf-IR700 photoimmunoconjugate to antigen-positive cells was demonstrated by confocal microscopy and flow cytometry. By switching to NIR irradiation, CD44-expressing TNBC was selectively killed through induced phototoxic activities. Likewise, the αCD44(scFv)-SNAPf-AURIF immunoconjugate was able to selectively accumulate within targeted cells and significantly reduced cell viability through antimitotic activities at nano- to micromolar drug concentrations. This study provides an in vitro proof-of-concept for a future strategy to selectively destroy light-accessible superficial CD44-expressing TNBC tumors and their metastatic lesions which are inaccessible to therapeutic light.

Development of a Clonal and High-Yield Mammalian Cell Line for the Manufacturing of a Hyperactive Human DNase I with Extended Plasma Half-Life Using PASylation® Technology.

Cumulative evidence from several pre-clinical studies suggests that restoration of plasma DNase activity in a thrombo-inflammatory state may improve clinical outcomes. Following injury, hyperactivated immune cells release large amounts of granular proteins together with DNA, which often accumulate in the surrounding environment in so-called neutrophil extracellular traps (NETs). Degradation of excess NETs by systemic DNase administration offers a promising therapeutic approach to ameliorate inflammation and dissolve intravascular clots. In order to expand the therapeutic utility of human DNase I, a variant of the enzyme was developed that has both a prolonged systemic half-life and a higher catalytic activity compared to Dornase alfa (Pulmozyme®), the recombinant form of DNase I approved for inhaled therapy of cystic fibrosis. The hyperactive enzyme was "PASylated" by genetic fusion with a strongly hydrophilic and biodegradable PAS-polypeptide to increase its hydrodynamic volume and retard kidney filtration. A stable TurboCell™ CHO-K1-based cell line was generated which is suitable for the future production of PASylated DNase I according to good manufacturing practice (GMP). Furthermore, a robust bioprocess strategy was devised and an effective downstream process was developed. The final protein product is characterized by excellent purity, favorable physicochemical properties, a 14-fold higher DNA-degrading activity than Dornase alfa and a sustained pharmacokinetic profile, with a 22-fold slower clearance in rats.

Engineered reversible inhibition of SpyCatcher reactivity enables rapid generation of bispecific antibodies

The precise regulation of protein function is essential in biological systems and a key goal in chemical biology and protein engineering. Here, we describe a straightforward method to engineer functional control into the isopeptide bond-forming SpyTag/SpyCatcher protein ligation system. First, we perform a cysteine scan of the structured region of SpyCatcher. Except for two known reactive and catalytic residues, none of these mutations abolish reactivity. In a second screening step, we modify the cysteines with disulfide bond-forming small molecules. Here we identify 8 positions at which modifications strongly inhibit reactivity. This inhibition can be reversed by reducing agents. We call such a reversibly inhibitable SpyCatcher “SpyLock”. Using “BiLockCatcher”, a genetic fusion of wild-type SpyCatcher and SpyLock, and SpyTagged antibody fragments, we generate bispecific antibodies in a single, scalable format, facilitating the screening of a large number of antibody combinations. We demonstrate this approach by screening anti-PD-1/anti-PD-L1 bispecific antibodies using a cellular reporter assay.

Optimal Allocation of Water Resources in Canal Systems Based on the Improved Grey Wolf Algorithm

Xinjiang is located in the arid region of northwestern China, and agriculture accounts for an absolute share of total water use. Resource-based, engineering, structural, and managed water shortages coexist. Therefore, it is of great significance to vigorously develop water conservation technology and improve the efficiency of water transmission and distribution in canal systems. This research aims at addressing the problems of difficult manual regulation and the overall optimization of the final canal system, low-water-resource utilization efficiency, and management efficiency. Taking the branch-double two-stage canal system of Dongfeng branch canal in Mangxiang, Jinghe irrigation district, as a case study, and the rotation irrigation group and irrigation duration as decision variables, canal distribution is modeled with the goal of minimizing seepage losses. The improved grey wolf algorithm combined with particle swarm optimization is used for the first time and compared with the traditional grey wolf algorithm, genetic particle swarm optimization fusion algorithm, and northern goshawk algorithm. The results show that (1) on the basis of meeting the water discharge capacity and water demand requirements of the canal system, the diversion time of the water distribution scheme obtained by using the improved grey wolf algorithm is shortened from 11 d to 8.91 d compared with the traditional empirical water distribution scheme. (2) The improved grey wolf algorithm converges to the optimal value within 10 generations compared to the remaining methods, and the total water leakage is reduced from 16.15 × 104 m3 to 11.75 × 104 m3. (3) The number of gate adjustments is reduced, and the canal gates are opened and closed at the same time within each rotational irrigation group. The grey wolf algorithm improved by its combination with particle swarm has stronger optimization ability and convergence, which can better meet the requirements of efficient water resource allocation in irrigation canal systems, as well as a high application value.

Safflower Picking Trajectory Planning Strategy Based on an Ant Colony Genetic Fusion Algorithm

Safflower Picking Trajectory Planning Strategy Based on an Ant Colony Genetic Fusion Algorithm

Efficacy of genotype-matched vaccine against re-emerging genotype V Japanese encephalitis virus

ABSTRACT Japanese encephalitis (JE), caused by the Japanese encephalitis virus (JEV), is a highly threatening disease with no specific treatment. Fortunately, the development of vaccines has enabled effective defense against JE. However, re-emerging genotype V (GV) JEV poses a challenge as current vaccines are genotype III (GIII)-based and provide suboptimal protection. Given the isolation of GV JEVs from Malaysia, China, and the Republic of Korea, there is a concern about the potential for a broader outbreak. Under the hypothesis that a GV-based vaccine is necessary for effective defense against GV JEV, we developed a pentameric recombinant antigen using cholera toxin B as a scaffold and mucosal adjuvant, which was conjugated with the E protein domain III of GV by genetic fusion. This GV-based vaccine antigen induced a more effective immune response in mice against GV JEV isolates compared to GIII-based antigen and efficiently protected animals from lethal challenges. Furthermore, a bivalent vaccine approach, inoculating simultaneously with GIII- and GV-based antigens, showed protective efficacy against both GIII and GV JEVs. This strategy presents a promising avenue for comprehensive protection in regions facing the threat of diverse JEV genotypes, including both prevalent GIII and GI as well as emerging GV strains.

Genetically Fusing Order-Promoting and Thermoresponsive Building Blocks to Design Hybrid Biomaterials.

The unique biophysical and biochemical properties of intrinsically disordered proteins (IDPs) and their recombinant derivatives, intrinsically disordered protein polymers (IDPPs) offer opportunities for producing multistimuli-responsive materials; their sequence-encoded disorder and tendency for phase separation facilitate the development of multifunctional materials. This review highlights the strategies for enhancing the structural diversity of elastin-like polypeptides (ELPs) and resilin-like polypeptides (RLPs), and their self-assembled structures via genetic fusion to ordered motifs such as helical or beta sheet domains. In particular, this review describes approaches that harness the synergistic interplay between order-promoting and thermoresponsive building blocks to design hybrid biomaterials, resulting in well-structured, stimuli-responsive supramolecular materials ordered on the nanoscale.

Enhanced Immunogenicity and Protective Effects against SARS-CoV-2 Following Immunization with a Recombinant RBD-IgG Chimeric Protein.

The unprecedented global impact caused by SARS-CoV-2 imposed huge health and economic challenges, highlighting the urgent need for safe and effective vaccines. The receptor-binding domain (RBD) of SARS-CoV-2 is the major target for neutralizing antibodies and for vaccine formulations. Nonetheless, the low immunogenicity of the RBD requires the use of alternative strategies to enhance its immunological properties. Here, we evaluated the use of a subunit vaccine antigen generated after the genetic fusing of the RBD with a mouse IgG antibody. Subcutaneous administration of RBD-IgG led to the extended presence of the protein in the blood of immunized animals and enhanced RBD-specific IgG titers. Furthermore, RBD-IgG immunized mice elicited increased virus neutralizing antibody titers, measured both with pseudoviruses and with live original (Wuhan) SARS-CoV-2. Immunized K18-hACE2 mice were fully resistant to the lethal challenge of the Wuhan SARS-CoV-2, demonstrated by the control of body-weight loss and virus loads in their lungs and brains. Thus, we conclude that the genetic fusion of the RBD with an IgG molecule enhanced the immunogenicity of the antigen and the generation of virus-neutralizing antibodies, supporting the use of IgG chimeric antigens as an approach to improve the performance of SARS-CoV-2 subunit vaccines.

The cytosolic hyperoxidation-sensitive and -robust Leishmania peroxiredoxins cPRX1 and cPRX2 are both dispensable for parasite infectivity

Typical two-cysteine peroxiredoxins (2-Cys-PRXs) are H2O2-metabolizing enzymes whose activity relies on two cysteine residues. Protists of the family Trypanosomatidae invariably express one cytosolic 2-Cys-PRX (cPRX1). However, the Leishmaniinae sub-family features an additional isoform (cPRX2), almost identical to cPRX1, except for the lack of an elongated C-terminus with a Tyr-Phe (YF) motif. Previously, cytosolic PRXs were considered vital components of the trypanosomatid antioxidant machinery. Here, we shed new light on the properties, functions and relevance of cPRXs from the human pathogen Leishmania infantum. We show first that LicPRX1 is sensitive to inactivation by hyperoxidation, mirroring other YF-containing PRXs participating in redox signaling. Using genetic fusion constructs with roGFP2, we establish that LicPRX1 and LicPRX2 can act as sensors for H2O2 and oxidize protein thiols with implications for signal transduction. Third, we show that while disrupting the LicPRX-encoding genes increases susceptibility of L. infantum promastigotes to external H2O2in vitro, both enzymes are dispensable for the parasites to endure the macrophage respiratory burst, differentiate into amastigotes and initiate in vivo infections. This study introduces a novel perspective on the functions of trypanosomatid cPRXs, exposing their dual roles as both peroxidases and redox sensors. Furthermore, the discovery that Leishmania can adapt to the absence of both enzymes has significant implications for our understanding of Leishmania infections and their treatment. Importantly, it questions the conventional notion that the oxidative response of macrophages during phagocytosis is a major barrier to infection and the suitability of cPRXs as drug targets for leishmaniasis.

Genetic fusion of CCL11 to antigens enhances antigenicity in nucleic acid vaccines and eradicates tumor mass through optimizing T-cell response

Nucleic acid vaccines have shown promising potency and efficacy for cancer treatment with robust and specific T-cell responses. Improving the immunogenicity of delivered antigens helps to extend therapeutic efficacy and reduce dose-dependent toxicity. Here, we systematically evaluated chemokine-fused HPV16 E6/E7 antigen to improve the cellular and humoral immune responses induced by nucleotide vaccines in vivo. We found that fusion with different chemokines shifted the nature of the immune response against the antigens. Although a number of chemokines were able to amplify specific CD8 + T-cell or humoral response alone or simultaneously. CCL11 was identified as the most potent chemokine in improving immunogenicity, promoting specific CD8 + T-cell stemness and generating tumor rejection. Fusing CCL11 with E6/E7 antigen as a therapeutic DNA vaccine significantly improved treatment effectiveness and caused eradication of established large tumors in 92% tumor-bearing mice (n = 25). Fusion antigens with CCL11 expanded the TCR diversity of specific T cells and induced the infiltration of activated specific T cells, neutrophils, macrophages and dendritic cells (DCs) into the tumor, which created a comprehensive immune microenvironment lethal to tumor. Combination of the DNA vaccine with anti-CTLA4 treatment further enhanced the therapeutic effect. In addition, CCL11 could also be used for mRNA vaccine design. To summarize, CCL11 might be a potent T cell enhancer against cancer.

High-throughput screening of catalytically active inclusion bodies using laboratory automation and Bayesian optimization

BackgroundIn recent years, the production of inclusion bodies that retain substantial catalytic activity was demonstrated. These catalytically active inclusion bodies (CatIBs) are formed by genetic fusion of an aggregation-inducing tag to a gene of interest via short linker polypeptides. The resulting CatIBs are known for their easy and cost-efficient production, recyclability as well as their improved stability. Recent studies have outlined the cooperative effects of linker and aggregation-inducing tag on CatIB activities. However, no a priori prediction is possible so far to indicate the best combination thereof. Consequently, extensive screening is required to find the best performing CatIB variant.ResultsIn this work, a semi-automated cloning workflow was implemented and used for fast generation of 63 CatIB variants with glucose dehydrogenase of Bacillus subtilis (BsGDH). Furthermore, the variant BsGDH-PT-CBDCell was used to develop, optimize and validate an automated CatIB screening workflow, enhancing the analysis of many CatIB candidates in parallel. Compared to previous studies with CatIBs, important optimization steps include the exclusion of plate position effects in the BioLector by changing the cultivation temperature. For the overall workflow including strain construction, the manual workload could be reduced from 59 to 7 h for 48 variants (88%). After demonstration of high reproducibility with 1.9% relative standard deviation across 42 biological replicates, the workflow was performed in combination with a Bayesian process model and Thompson sampling. While the process model is crucial to derive key performance indicators of CatIBs, Thompson sampling serves as a strategy to balance exploitation and exploration in screening procedures. Our methodology allowed analysis of 63 BsGDH-CatIB variants within only three batch experiments. Because of the high likelihood of TDoT-PT-BsGDH being the best CatIB performer, it was selected in 50 biological replicates during the three screening rounds, much more than other, low-performing variants.ConclusionsAt the current state of knowledge, every new enzyme requires screening for different linker/aggregation-inducing tag combinations. For this purpose, the presented CatIB toolbox facilitates fast and simplified construction and screening procedures. The methodology thus assists in finding the best CatIB producer from large libraries in short time, rendering possible automated Design-Build-Test-Learn cycles to generate structure/function learnings.

Plant-Produced Viral Nanoparticles as a Functionalized Catalytic Support for Metabolic Engineering.

Substrate channeling could be very useful for plant metabolic engineering; hence, we propose that functionalized supramolecular self-assembly scaffolds can act as enzymatic hubs able to perform reactions in close contiguity. Virus nanoparticles (VNPs) offer an opportunity in this context, and we present a functionalization strategy to display different enzymes on the outer surface of three different VNPs produced in plants. Tomato bushy stunt virus (TBSV) and Potato virus X (PVX) plant viruses were functionalized by the genetic fusion of the E-coil peptide coding sequence to their respective coat proteins genes, while the enzyme lichenase was tagged with the K-coil peptide. Immobilized E-coil VNPs were able to interact in vitro with the plant-produced functionalized lichenase, and catalysis was demonstrated by employing a lichenase assay. To prove this concept in planta, the Hepatitis B core (HBc) virus-like particles (VLPs) were similarly functionalized by genetic fusion with the E-coil sequence, while acyl-activating enzyme 1, olivetolic acid synthase, and olivetolic acid cyclase enzymes were tagged with the K-coil. The transient co-expression of the K-coil-enzymes together with E-coil-VLPs allowed the establishment of the heterologous cannabinoid precursor biosynthetic pathway. Noteworthy, a significantly higher yield of olivetolic acid glucoside was achieved when the scaffold E-coil-VLPs were employed.

Advances towards genome-based acute myeloid leukemia classification: A comparative analysis of WHO-HAEM4R, WHO-HAEM5, and International Consensus Classification.

Two recent guidelines, the 5th edition of the World Health Organization Classification of Haematolymphoid Tumours (WHO-HAEM5) and the International Consensus Classification (ICC), were published to refine the diagnostic criteria of acute myeloid leukemia (AML). They both consider genomic features more extensively and expand molecularly defined AML subtypes. In this study, we compared the classifications of 1135 AML cases under both criteria. According to WHO-HAEM5 and ICC, the integration of whole transcriptome sequencing, targeted gene mutation screening, and conventional cytogenetic analysis identified defining genetic abnormalities in 89% and 90% of AML patients, respectively. The classifications displayed discrepancies in 16% of AML cases after being classified using the two guidelines, respectively. Both new criteria significantly reduce the number of cases defined by morphology and differentiation. However, their clinical implementation heavily relies on comprehensive and sophisticated genomic analysis, including genome and transcriptome levels, alongside the assessment of pathogenetic somatic and germline variations. Discrepancies between WHO-HAEM5 and ICC, such as the assignment of RUNX1 mutations, the rationality of designating AML with mutated TP53 as a unique entity, and the scope of rare genetic fusions, along with the priority of concurrent AML-defining genetic abnormalities, are still pending questions requiring further research for more elucidated insights.