Engineering Proteins for Cell Entry.

Proteins are essential for life, as they participate in all vital processes in the body. In the past decade, delivery of active proteins to specific cells and organs has attracted increasing interest. However, most proteins cannot enter the cytoplasm due to the cell membrane acting as a natural barrier. To overcome this challenge, various proteins have been engineered to acquire cell-penetrating capacity by mimicking or modifying natural shuttling proteins. In this review, we provide an overview of the different types of engineered cell-penetrating proteins such as cell-penetrating peptides, supercharged proteins, receptor-binding proteins, and bacterial toxins. We also discuss some strategies for improving endosomal escape such as pore formation, the proton sponge effect, and hijacking intracellular trafficking pathways. Finally, we introduce some novel methods and technologies for designing and detecting engineered cell-penetrating proteins.

Protein-based delivery systems for RNA delivery.

RNA-based therapeutics have emerged as promising approaches to modulate gene expression and generate therapeutic proteins or antigens capable of inducing immune responses to treat a variety of diseases, such as infectious diseases, cancers, immunologic disorders, and genetic disorders. However, the efficient delivery of RNA molecules into cells poses significant challenges due to their large molecular weight, negative charge, and susceptibility to degradation by RNase enzymes. To overcome these obstacles, viral and non-viral vectors have been developed, including lipid nanoparticles, viral vectors, proteins, dendritic macromolecules, among others. Among these carriers, protein-based delivery systems have garnered considerable attention due to their potential to address specific issues associated with nanoparticle-based systems, such as liver accumulation and immunogenicity. This review provides an overview of currently marketed RNA drugs, underscores the significance of RNA delivery vector development, delineates the essential characteristics of an ideal RNA delivery vector, and introduces existing protein carriers for RNA delivery. By offering valuable insights, this review aims to serve as a reference for the future development of protein-based delivery vectors for RNA therapeutics.

Unstructured polypeptides as a versatile drug delivery technology.

Although polyethylene glycol (PEG), or "PEGylation" has become a widely applied approach for improving the efficiency of drug delivery, the immunogenicity and non-biodegradability of this synthetic polymer have prompted an evident need for alternatives. To overcome these caveats and to mimic PEG -or other natural or synthetic polymers- for the purpose of drug half-life extension, unstructured polypeptides are designed. Due to their tunable length, biodegradability, low immunogenicity and easy production, unstructured polypeptides have the potential to replace PEG as the preferred technology for therapeutic protein/peptide delivery. This review provides an overview of the evolution of unstructured polypeptides, starting from natural polypeptides to engineered polypeptides and discusses their characteristics. Then, it is described that unstructured polypeptides have been successfully applied to numerous drugs, including peptides, proteins, antibody fragments, and nanocarriers, for half-life extension. Innovative applications of unstructured peptides as releasable masks, multimolecular adaptors and intracellular delivery carriers are also discussed. Finally, challenges and future perspectives of this promising field are briefly presented. STATEMENT OF SIGNIFICANCE: Polypeptide fusion technology simulating PEGylation has become an important topic for the development of long-circulating peptide or protein drugs without reduced activity, complex processes, and kidney injury caused by PEG modification. Here we provide a detailed and in-depth review of the recent advances in unstructured polypeptides. In addition to the application of enhanced pharmacokinetic performance, emphasis is placed on polypeptides as scaffolders for the delivery of multiple drugs, and on the preparation of reasonably designed polypeptides to manipulate the performance of proteins and peptides. This review will provide insight into future application of polypeptides in peptide or protein drug development and the design of novel functional polypeptides.

Microenvironment-responsive anti-PD-L1 × CD3 bispecific T-cell engager for solid tumor immunotherapy.

Bispecific T-cell Engager (BiTE) antibodies can redirect T-cells to tumor cells, and turn on the targeted lysis of tumor cells. However, BiTE has been challenging in solid tumors due to short plasma half-life, "off-target" effect, and immunosuppression via PD-1/PD-L1 axis. This study designed a safe, long-acting, and highly effective Protease-Activated PSTAGylated BiTE, named PAPB, which includes a shielding polypeptide domain (PSTAG), a protease-activated linker, and a BiTE core. The BiTE core consists of two scFvs targeting PD-L1 and CD3. BiTE core bound PD-L1 and CD3 in a dose-dependent manner, and PAPB can release BiTE core in response to MMP2 in the tumor microenvironment to exert antitumor activity. The plasma half-life of PAPB in mice was significantly prolonged from 2.46 h to 6.34 h of the BiTE core. In mice bearing melanoma (A375) xenografts, PAPB significantly increased infiltration of T lymphocytes in tumor tissue, and inhibited tumor proliferation without activating T-cells in the peripheral blood. Overall, the engineering protein PAPB could be a promising drug candidate for solid tumor immunotherapy.

Application of postmortem MRI for identification of medulla oblongata contusion as a cause of death: a case report.

Whiplash injury is common in traffic accidents, and severe whiplash is characterized by cervical spinal cord injuries with cervical dislocation or fracture, that can be diagnosed by postmortem computed tomography (PMCT), postmortem magnetic resonance (PMMR), or conventional autopsy. However, for cervical spinal cord injury without fracture and dislocation, PMMR can be more informative because it provides higher resolution of soft tissues. We report the case of a 29-year-old male who died immediately following a traffic accident, in which the vehicle hit an obstacle at a high speed, causing deformation of the bumper and severe damage of the vehicle body. PMCT indicated no significant injuries or diseases related to death, but PMMR showed patchy abnormal signals in the medulla oblongata, and the lower edge of the cerebellar tonsil was herniated out of the foramen magnum. The subsequent pathological and histological results confirmed that death was caused by medulla oblongata contusion combined with cerebellar tonsillar herniation. Our description of this case of a rare but fatal whiplash injury in which there was no fracture or dislocation provides a better understanding of the potentially fatal consequences of cervical spinal cord whiplash injury without fracture or dislocation and of the underlying lethal mechanisms. Compared with PMCT, PMMR provides important diagnostic information in forensic practice for the identification of soft tissue injuries, and is therefore an important imaging modality for diagnosis of whiplash injury when there is no fracture or dislocation.

Early detection of emergency events from social media: a new text clustering approach.

Emergency events require early detection, quick response, and accurate recovery. In the era of big data, social media users can be seen as social sensors to monitor real-time emergency events. This paper proposed an integrated approach to detect all four kinds of emergency events early, including natural disasters, man-made accidents, public health events, and social security events. First, the BERT-Att-BiLSTM model is used to detect emergency-related posts from massive and irrelevant data. Then, the 3 W attribute information (what, where, and when) of the emergency event is extracted. With the 3 W attribute information, we create an unsupervised dynamical event clustering algorithm based on text similarity and combine it with the supervised logistical regression model to cluster posts into different events. Experiments on Sina Weibo data demonstrate the superiority of the proposed framework. Case studies on some real emergency events show that the proposed framework has good performance and high timeliness. Practical applications of the framework are also discussed, followed by future directions for improvement.

Rrp14 controls rRNA transcription via facilitating the translocation of Pol5 into the nucleolus.

Rrp14 is a conserved protein that plays an important role in rRNA processing and ribosomal biogenesis. In Schizosaccharomyces pombe, the rrp14 gene is split into SPAC8C9.10 c (rrp14) and SPBC947.07 (rrp1402). Although the SPAC8C9.10 c gene is not essential for S. pombe survival, deletion of the gene causes the yeast cells to grow sick and to exhibit decreased rRNA transcription. We identified a novel Pol5 protein that physically interacts with the Rrp14 protein. Taking advantage of the Pil1 co-tethering assay, we found that Rrp14 facilitates the nucleolus translocation of Pol5, and the 7-RINAWN-12 motif of the Rrp14 protein is responsible for the interaction between Pol5 and Rrp14. Since deletion of the 7-RINAWN-12 motif affects rRNA transcription, we thus propose that Rrp14 affects rRNA transcription by facilitating the nucleolus translocation of Pol5.

Research on insulator defect detection algorithm of transmission line based on CenterNet.

The reliability of the insulator has directly affected the stable operation of electric power system. The detection of defective insulators has always been an important issue in smart grid systems. However, the traditional transmission line detection method has low accuracy and poor real-time performance. We present an insulator defect detection method based on CenterNet. In order to improve detection efficiency, we simplified the backbone network. In addition, an attention mechanism is utilized to suppress useless information and improve the accuracy of network detection. In image preprocessing, the blurring of some detected images results in the samples being discarded, so we use super-resolution reconstruction algorithm to reconstruct the blurred images to enhance the dataset. The results show that the AP of the proposed method reaches 96.16% and the reasoning speed reaches 30FPS under the test condition of NVIDIA GTX 1080 test conditions. Compared with Faster R-CNN, YOLOV3, RetinaNet and FSAF, the detection accuracy of proposed method is greatly improved, which fully proves the effectiveness of the proposed method.

Molecular modeling and rational design of hydrocarbon-stapled/halogenated helical peptides targeting CETP self-binding site: Therapeutic implication for atherosclerosis.

The human plasma cholesteryl ester transfer protein (CETP) collects triglycerides from very-/low-density lipoproteins (V/LDL) and exchanges them for cholesteryl esters from high-density lipoproteins (HDL), which has recognized as an important therapeutic target for atherosclerosis. The protein has a C-terminal amphipathic α-helix that serves as self-binding peptide to fulfill biological function by dynamically binding to/unbinding from its cognate site (termed self-binding site) in the same protein. Previously, we successfully derived and halogenated the helical peptide to competitively disrupt the self-binding behavior of CETP C-terminal tail. However, the halogenated peptides have only a limited affinity increase as compared to native helical peptide (∼3-fold), thus exhibiting only a moderate competitive potency. Here, instead of optimizing the direct intermolecular interaction of peptide with CETP self-binding site we attempt to further improve the peptide competitive potency by reducing its conformational flexibility with hydrocarbon-stapling technique. Computational analysis reveals that the helical peptide has large intrinsic disorder in unbound free state, which would incur a considerable entropy penalty upon rebinding to the self-binding site. All-hydrocarbon bridge is designed and optimized on native and halogenated peptides in terms of the helical pattern and binding mode of self-binding peptide. Dynamics simulation and circular dichroism indicate that the stapling can considerably reduce peptide disorder in free state. Energetics calculation and fluorescence assay conform that the binding affinity of stapled/halogenated peptides is improved substantially (by > 5-fold), thus exhibiting an effective competition potency with native peptide for the self-binding site. Structural examination suggests that the binding modes and nonbonded interactions of native and halogenated peptides are not influenced essentially due to the stapling.

Pharmaceutical Inhibition of Neddylation as Promising Treatments for Various Cancers.

BACKGROUND: Neddylation is an important post-translational modification of proteins, in which a NEDD8 (neural-precursor-cell-expressed developmentally down-regulated 8) is covalently introduced onto the substrate proteins to regulate their functions and homeostasis. As neddylation is frequently up-regulated in various cancers, its interference was proposed as a promising therapy of related diseases. OBJECTIVE: The recent advances in developing neddylation interfering agents were summarized to provide an overview of current achievements and perspectives for future development. METHODS: Reports on neddylation interfering agents were acquired from Pubmed as well as the EPO and clinicaltrials.gov websites, which were subsequently analyzed and summarized according to targets, chemical structures and biological activities. RESULTS: Neddylation as a sophisticated procedure comprises proteolytic processing of NEDD8 precursor, deploying conjugating enzymes E1 (NAE), E2 (UBE2M and UBE2F) and various E3, as well as translocating NEDD8 along these conjugating enzymes sequentially and finally to substrate proteins. Among these nodes, NAE, UBE2M and the interaction between UBE2M-DCN1 have been targeted by small molecules, metal complexes, peptides and RNAi. A NAE inhibitor pevonedistat (MLN4924) is currently under evaluation in clinical trials for the treatment of various cancers. CONCLUSION: With multiple inhibitory approaches of neddylation being introduced, the development of neddylation interference as a novel cancer therapy is significantly boosted recently, although its efficacy and the best way to achieve that are still to be demonstrated in clinical trials.

Immunoproteomic analysis of the excretory-secretory products of Trichinella pseudospiralis adult worms and newborn larvae.

BACKGROUND: The nematode Trichinella pseudospiralis is an intracellular parasite of mammalian skeletal muscle cells and exists in a non-encapsulated form. Previous studies demonstrated that T. pseudospiralis could induce a lower host inflammatory response. Excretory-secretory (ES) proteins as the most important products of host-parasite interaction may play the main functional role in alleviating host inflammation. However, the ES products of T. pseudospiralis early stage are still unknown. The identification of the ES products of the early stage facilitates the understanding of the molecular mechanisms of the immunomodulation and may help finding early diagnostic markers. RESULTS: In this study, we used two-dimensional gel electrophoresis (2-DE)-based western blotting coupled with matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF/TOF-MS/MS) to separate and identify the T. pseudospiralis adult worms ES products immunoreaction-positive proteins. In total, 400 protein spots were separated by 2-DE. Twenty-eight protein spots were successfully identified using the sera from infected pigs and were characterized to correlate with 12 different proteins of T. pseudospiralis, including adult-specific DNase II-10, poly-cysteine and histidine-tailed protein isoform 2, serine protease, serine/threonine-protein kinase ULK3, enolase, putative venom allergen 5, chymotrypsin-like elastase family member 1, uncharacterized protein, peptidase inhibitor 16, death-associated protein 1, deoxyribonuclease II superfamily and golgin-45. Bioinformatic analyses showed that the identified proteins have a wide diversity of molecular functions, especially deoxyribonuclease II (DNase II) activity and serine-type endopeptidase activity. CONCLUSIONS: Early candidate antigens from the ES proteins of T. pseudospiralis have been screened and identified. Our results suggest these proteins may play key roles during the T. pseudospiralis infection and suppress the host immune response. Further, they are the most likely antigen for early diagnosis and the development of a vaccine against the parasite.