Lactobacillus plantarum is a food-grade microorganism with industrial and medical relevance belonging to the group of lactic acid bacteria (LAB). Traditional strategies for obtaining gene deletion variants in this organism are mainly vector-based double-crossover methods, which are inefficient and laborious. A feasible possibility to solve this problem is the recombineering, which greatly expands the possibilities for engineering DNA molecules in vivo in various organisms.

Cell-derived small extracellular vesicles have been exploited as potent drug vehicles. However, significant challenges hamper their clinical translation, including inefficient cytosolic delivery, poor target-specificity, low yield, and inconsistency in production. Here, we report a bioinspired material, engineered fusogen and targeting moiety co-functionalized cell-derived nanovesicle (CNV) called eFT-CNV, as a drug vehicle. We show that universal eFT-CNVs can be produced by extrusion of genetically modified donor cells with high yield and consistency. We demonstrate that bioinspired eFT-CNVs can efficiently and selectively bind to targets and trigger membrane fusion, fulfilling endo-lysosomal escape and cytosolic drug delivery. We find that, compared to counterparts, eFT-CNVs significantly improve the treatment efficacy of drugs acting on cytosolic targets. We believe that our bioinspired eFT-CNVs will be promising and powerful tools for nanomedicine and precision medicine.

Aluminum phosphate adjuvants play a critical role in human inactivated and subunit prophylactic vaccines. However, a major challenge is that the underlying mechanism of immune stimulation remains poorly understood, which impedes the further optimal design and application of more effective adjuvants in vaccine formulations. To address this, a library of amorphous aluminum hydroxyphosphate nanoparticles (AAHPs) is engineered with defined surface properties to explore the specific mechanism of adjuvanticity at the nano-bio interface. The results demonstrate that AAHPs could induce cell membrane perturbation and downstream inflammatory responses, with positively-charged particles showing the most significantly enhanced immunostimulation potentials compared to the neutral or negatively-charged particles. In a vaccine using Staphylococcus aureus (S. aureus) recombinant protein as antigens, the positively-charged particles elicit long-lasting and enhanced humoral immunity, and provide protection in S. aureus sepsis mice models. In addition, when formulated with human papillomavirus type 18 virus-like particles, it is demonstrated that particles with positive charges outperform in promoting serum antigen-specific antibody productions. This study shows that engineering AAHPs with well-controlled physicochemical properties enable the establishment of a structure-activity relationship that is critical to instruct the design of suitable engineered nanomaterial-based adjuvants within vaccine formulations for the benefits of human health.

Genetically engineered mesenchymal stem cells (MSCs), as non-viral gene delivery platforms, are rapidly evolving in tumor therapy due to their low immunogenicity and natural tumor-homing capacity. Methods: In this paper, we selected reconstituted high-density lipoprotein (rHDL), a lipoprotein-bioinspired nanovector with specific binding ability to scavenger receptor B type I (SR-BI) expressed on MSCs, as a transfection agent to genetically modify MSCs. pDNA encoding tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) was used as a functional gene to be transfected into the nucleus of MSCs for TRAIL expression. Lauric acid-coupled polyethyleneimine (PEI-LA) as an amphiphilic cationic polymer was synthesized to electrostatically bind to pDNA, and then incorporated into rHDL to form rHDL/PEI-LA/pDNA nanoparticles. Results: The nanoparticles exhibited homogenous particle size and excellent serum stability in vitro. Meanwhile, this SR-BI-targeted rHDL performed efficient intracellular gene delivery, specific lysosome-independent mechanism of cellular uptake and high transfection of pDNA towards MSCs. Moreover, high TRAIL expression in MSCs was detected after rHDL-mediated transfection. In vitro and in vivo results indicated that genetically engineered MSCs could accurately target to B16F10 cells, thereby producing significant apoptosis-inducing effect on aggressive melanoma. Conclusion: TRAIL-expressing MSCs engineered by rHDL nanovector was an efficient and hypotoxic method for stem cells-based pulmonary melanoma metastasis-targeting therapy.

The detail understanding of physiological/biochemical characteristics of individual laccase isoenzymes in fungi is necessary for fundamental and application purposes, but our knowledge is still limited for most of fungi due to difficult to express laccases heterologously. In this study, two novel laccase genes, named lac3 and lac4, encoding proteins of 547 and 532-amino acids preceded by 28 and 16-residue signal peptides, respectively, were cloned from the edible basidiomycete Coprinus comatus. They showed 70% identity but much lower homology with other fungal laccases at protein level (less than 58%). Two novel laccase isoenzymes were successfully expressed in Pichia pastoris by fusing an additional 10 amino acids (Thr-Pro-Phe-Pro-Pro-Phe-Asn-Thr-Asn-Ser) tag at N-terminus, and the volumetric activities could be dramatically enhanced from undetectable level to 689 and 1465 IU/l for Lac3 and Lac4, respectively. Both laccases possessed the lowest Km and highest kcat/Km value towards syringaldazine, followed by ABTS, guaiacol and 2,6-dimethylphenol similar as the low redox potential laccases from other microorganisms. Lac3 and Lac4 showed resistant to SDS, and retained 31.86% and 43.08% activity in the presence of 100 mM SDS, respectively. Lac3 exhibited higher decolorization efficiency than Lac4 for eleven out of thirteen different dyes, which may attribute to the relatively higher catalytic efficiency of Lac3 than Lac4 (in terms of kcat/Km) towards syringaldazine and ABTS. The mild synergistic decolorization by two laccases was observed for triphenylmethane dyes but not for anthraquinone and azo dyes.

Eimeria acervulina (E. acervulina) causes coccidiosis in poultry which persists as economic pain worldwide. Most damage to the intestinal mucosa results from apoptosis of the infected intestinal epithelial cells. The Microneme protein 3 (MIC3) protein is a key virulence factor in some parasites involved in host cell apoptosis inhibition. Here, we studied whether and how MIC3 affects the apoptosis in E. acervulina infected chicken duodenal epithelial cells. Through flow cytometry (FCM), we found that the presence of merozoites and the overexpression of MIC3 significantly decreased apoptosis and the activity of caspase-3 in chicken duodenal epithelial cells at 4, 6, and 8 h post merozoite infection (P < 0.01). Silencing the Casitas B-lineage lymphoma (CBL) protein, a host receptor for MIC3 with shRNA was shown to promote apoptosis in the chicken duodenal epithelial cells. The early apoptotic rate of host cells in the lentiviral-MIC3 group was significantly lower than that in the lentiviral-MIC3 + shRNA CBL group at 4 h after MIC3 expression (P < 0.01), and it was moderately decreased in the lentiviral-MIC3 + shRNA CBL group compared with that in the shRNA CBL group. Our data indicated that MIC3 inhibited early apoptosis of E. acervulina infected chicken duodenal epithelial cells by targeting host receptor-CBL protein. These findings unveiled one of the mechanisms of how intracellular parasites affect the apoptosis of infected host cells, which provided a deeper understanding of their pathogenesis.

The increasing availability of large-scale protein-protein interaction data has made it possible to understand the basic components and organization of cell machinery from the network level. The arising challenge is how to analyze such complex interacting data to reveal the principles of cellular organization, processes and functions. Many studies have shown that clustering protein interaction network is an effective approach for identifying protein complexes or functional modules, which has become a major research topic in systems biology. In this review, recent advances in clustering methods for protein interaction networks will be presented in detail. The predictions of protein functions and interactions based on modules will be covered. Finally, the performance of different clustering methods will be compared and the directions for future research will be discussed.

The lack of tools to identify causative variants from sequencing data greatly limits the promise of precision medicine. Previous studies suggest that one-third of disease-associated alleles alter splicing. We discovered that the alleles causing splicing defects cluster in disease-associated genes (for example, haploinsufficient genes). We analyzed 4,964 published disease-causing exonic mutations using a massively parallel splicing assay (MaPSy), which showed an 81% concordance rate with splicing in patient tissue. Approximately 10% of exonic mutations altered splicing, mostly by disrupting multiple stages of spliceosome assembly. We present a large-scale characterization of exonic splicing mutations using a new technology that facilitates variant classification and keeps pace with variant discovery.

Gene alterations are recognized as important events in acute myeloid leukemia (AML) progression. Studies on hematopoiesis of altered genes contribute to a better understanding on their roles in AML progression. Our previous work reported a DEAH box helicase 15 (DHX15) R222G mutation in AML patients, and we showed DHX15 overexpression is associated with poor prognosis in AML patients. In this work, we further study the role of dhx15 in zebrafish developmental hematopoiesis by generating dhx15-/- zebrafish using transcription activator-like effector nuclease technology. Whole-mount in situ hybridization (WISH) analysis showed hematopoietic stem/progenitor cells were dramatically perturbed when dhx15 was deleted. Immunofluorescence staining indicated inhibited hematopoietic stem/progenitor cell (HSPC) proliferation instead of accelerated apoptosis were detected in dhx15-/- zebrafish. Furthermore, our data showed that HSPC defect is mediated through the unfolded protein response (UPR) pathway. DHX15 R222G mutation, a recurrent mutation identified in AML patients, displayed a compromised function in restoring HSPC failure in dhx15-/- ; Tg (hsp: DHX15 R222G) zebrafish. Collectively, this work revealed a vital role of dhx15 in the maintenance of definitive hematopoiesis in zebrafish through the unfolded protein respone pathway. The study of DHX15 and DHX15 R222G mutation could hold clinical significance for evaluating prognosis of AML patients with aberrant DHX15 expression.

Human oligodendrocyte progenitor cells (hOPCs) persist into adulthood as an abundant precursor population capable of division and differentiation. The transcriptional mechanisms that regulate hOPC homeostasis remain poorly defined. Herein, we identify paired related homeobox protein 1 (PRRX1) in primary PDGFαR+ hOPCs. We show that enforced PRRX1 expression results in reversible G1/0 arrest. While both PRRX1 splice variants reduce hOPC proliferation, only PRRX1a abrogates migration. hOPC engraftment into hypomyelinated shiverer/rag2 mouse brain is severely impaired by PRRX1a, characterized by reduced cell proliferation and migration. PRRX1 induces a gene expression signature characteristic of stem cell quiescence. Both IFN-γ and BMP signaling upregulate PRRX1 and induce quiescence. PRRX1 knockdown modulates IFN-γ-induced quiescence. In mouse brain, PRRX1 mRNA was detected in non-dividing OPCs and is upregulated in OPCs following demyelination. Together, these data identify PRRX1 as a regulator of quiescence in hOPCs and as a potential regulator of pathological quiescence.

Protein essentiality is usually accepted to be a conditional trait and strongly affected by cellular environments. However, existing computational methods often do not take such characteristics into account, preferring to incorporate all available data and train a general model for all cell lines. In addition, the lack of model interpretability limits further exploration and analysis of essential protein predictions.

Proteins play an important role in life activities and are the basic units for performing functions. Accurately annotating functions to proteins is crucial for understanding the intricate mechanisms of life and developing effective treatments for complex diseases. Traditional biological experiments struggle to keep pace with the growing number of known proteins. With the development of high-throughput sequencing technology, a wide variety of biological data provides the possibility to accurately predict protein functions by computational methods. Consequently, many computational methods have been proposed. Due to the diversity of application scenarios, it is necessary to conduct a comprehensive evaluation of these computational methods to determine the suitability of each algorithm for specific cases. In this study, we present a comprehensive benchmark, BeProf, to process data and evaluate representative computational methods. We first collect the latest datasets and analyze the data characteristics. Then, we investigate and summarize 17 state-of-the-art computational methods. Finally, we propose a novel comprehensive evaluation metric, design eight application scenarios and evaluate the performance of existing methods on these scenarios. Based on the evaluation, we provide practical recommendations for different scenarios, enabling users to select the most suitable method for their specific needs. All of these servers can be obtained from https://csuligroup.com/BEPROF and https://github.com/CSUBioGroup/BEPROF.

Atherosclerosis (AS) is recognized as the original cause of most cardiovascular and cerebrovascular diseases. The dual-protein (DP) nutrition that consists of soy protein and whey protein is reported to be associated with a reduction in AS; however, the relationship between DP and AS remains ambiguous. Therefore, this study aimed to verify the effect of DP on AS and explore the optimal DP intake to improve AS. ApoE-/- mice were administrated with low- (LDP), middle- (MDP), and high-dose (HDP) DP. The MDP group exhibited significant improvements in AS. In terms of lipid metabolism, the levels of plasma total triglyceride and LDL-C and the mRNA expression levels of Cyp7a1 and PCSK9 were markedly tuned in the MDP group. In addition, the MDP treatment group had a substantially lower inflammatory response and better intestinal barrier function than LDP and HDP groups. The species richness demonstrated by the Chao1 index was distinctly increased in the MDP group, and the relative abundance of intestinal-permeability-protective microbes Blautia and Akkermansia was significantly elevated. In summary, an adequate intake of DP was able to counteract atherosclerosis development in ApoE-/- mice, and this study provides a scientific theoretical basis for the application of DP in the food and pharmaceutical fields.

Contact-dependent growth inhibition is a mechanism of interbacterial competition mediated by delivery of the C-terminal toxin domain of CdiA protein (CdiA-CT) into neighboring bacteria. The CdiA-CT of enterohemorrhagic Escherichia coli EC869 (CdiA-CTEC869) cleaves the 3'-acceptor regions of specific tRNAs in a reaction that requires the translation factors Tu/Ts and GTP. Here, we show that CdiA-CTEC869 has an intrinsic ability to recognize a specific sequence in substrate tRNAs, and Tu:Ts complex promotes tRNA cleavage by CdiA-CTEC869. Uncharged and aminoacylated tRNAs (aa-tRNAs) were cleaved by CdiA-CTEC869 to the same extent in the presence of Tu/Ts, and the CdiA-CTEC869:Tu:Ts:tRNA(aa-tRNA) complex formed in the presence of GTP. CdiA-CTEC869 interacts with domain II of Tu, thereby preventing the 3'-moiety of tRNA to bind to Tu as in canonical Tu:GTP:aa-tRNA complexes. Superimposition of the Tu:GTP:aa-tRNA structure onto the CdiA-CTEC869:Tu structure suggests that the 3'-portion of tRNA relocates into the CdiA-CTEC869 active site, located on the opposite side to the CdiA-CTEC869 :Tu interface, for tRNA cleavage. Thus, CdiA-CTEC869 is recruited to Tu:GTP:Ts, and CdiA-CT:Tu:GTP:Ts recognizes substrate tRNAs and cleaves them. Tu:GTP:Ts serves as a reaction scaffold that increases the affinity of CdiA-CTEC869 for substrate tRNAs and induces a structural change of tRNAs for efficient cleavage by CdiA-CTEC869.

Yellow mealworm meal (MWM) as a protein feedstuff in the broiler diet was investigated based on the growth performance, hematological characteristics, carcass, and meat quality of broiler chicks. A total of 700 one-day-old Ross 308 male broiler chicks were assigned to five dietary MWM treatments containing 0%, 2%, 4%, and 8% dried MWM or 10.48% fresh mealworm (corresponding to 4% dried MWM). For each treatment, there were seven pens with 20 chicks each. The nutritional profile of dried MWM is comparable to all conventional protein feedstuffs. MWM significantly increased BW and ADG (linear and quadratic, p < 0.05), and FCR was best at 4% MWM inclusion level (quadratic, p < 0.10) for broiler chicks during the starter phase. The predicted MWM levels for optimal starter BW and ADG were 4.13% and 3.84%. Hematological characteristics of broiler chicks fed on the MWM diet did not differ or showed small change within the physiological range. A fresh 10.48% mealworm diet significantly reduced the blood LZM for the grower. Broiler Chicks fed on fresh 10.48% mealworm had a significantly reduced abdominal fat percentage compared to the 4% dried MWM counterparts. MWM did not significantly affect meat quality. Taken together, MWM inclusion in broiler diet is acceptable as a protein feedstuff, and a 4% level could stimulate early growth in the starter phase.

The T cell-dependent (TD) antibody response involves the generation of high affinity, immunoglobulin heavy chain class-switched antibodies that are generated through germinal center (GC) response. This process is controlled by coordinated transcriptional and post-transcriptional gene regulatory mechanisms. RNA-binding proteins (RBPs) have emerged as critical players in post-transcriptional gene regulation. Here we demonstrate that B cell-specific deletion of RBP hnRNP F leads to diminished production of class-switched antibodies with high affinities in response to a TD antigen challenge. B cells deficient in hnRNP F are characterized by defective proliferation and c-Myc upregulation upon antigenic stimulation. Mechanistically, hnRNP F directly binds to the G-tracts of Cd40 pre-mRNA to promote the inclusion of Cd40 exon 6 that encodes its transmembrane domain, thus enabling appropriate CD40 cell surface expression. Furthermore, we find that hnRNP A1 and A2B1 can bind to the same region of Cd40 pre-mRNA but suppress exon 6 inclusion, suggesting that these hnRNPs and hnRNP F might antagonize each-other's effects on Cd40 splicing. In summary, our study uncovers an important posttranscriptional mechanism regulating the GC response.

Androgen receptor-targeted therapy improves survival in castration-resistant prostate cancer (CRPC). However, almost all patients with CRPC eventually develop secondary resistance to these drugs. Therefore, alternative therapeutic approaches for incurable metastatic CRPC are urgently needed. Unfolded protein response (UPR) is regarded as a cytoprotective mechanism that removes misfolded proteins in rapidly proliferating tumor cells. However, acute activation of the UPR directly leads to tumor cell death. This study has shown that WJ-644A, a novel small molecule activator of UPR, potently inhibited the proliferation of prostate cancer cells and caused tumor regression with a good safety profile in multiple animal models. Mechanistically, we have identified that WJ-644A induced cell methuosis and autophagy upon UPR activation. Our study not only identifies the UPR as an actionable target for CRPC treatment, but also establishes WJ-644A as a novel UPR activator that has potential therapeutic value for CRPC.

Several studies were conducted to explore the prognostic significance of podocalyxin-like protein (PODXL) expression in various cancers, with contradictory. This study aims to summarize the prognostic significance of PODXL expression in cancers. PubMed, the Cochrane Library and Embase were completely retrieved. The prospective or retrospective studies focusing on the prognostic role of PODXL expression in cancers were eligible. The endpoints were overall survival (OS), disease-specific survival (DSS) and disease-free survival (DFS).12 studies involving a total of 5,309 patients were identified. The results indicated that high PODXL expression was significantly associated with worse OS when compared to the low PODXL expression (HR=1.76, 95%CI=1.53-2.04, p<0.00001; I2=41%, p=0.08). And similar results were detected in the subgroup analysis of analysis model, ethnicity, sample size, tumor type and antibody type. And the results also showed that high PODXL expression was obviously related to shorter DSS (HR=2.47, 95%CI=1.53-3.99, p=0.0002; I2=66%, p=0.03) and DFS (HR=2.12, 95%CI=1.58-2.85, p<0.00001; I2=19%, p=0.29). In conclusion, it was revealed that high PODXL expression is an unfavorable predictor of OS, DSS and DFS in patients with cancers, and high PODXL expression is a promising prognostic biomarker for cancers, especially for patients in European.

Human epididymis protein 4 (HE4) is a novel cancer biomarker. This study evaluates the prognostic role of HE4 in determining the survival of endometrial cancer patients.

The epithelial to mesenchymal transition (EMT) contributes to fibrosis during silicosis. Zinc finger CCCH-type containing 4 protein (ZC3H4) is a novel CCCH-type zinc finger protein that activates inflammation in pulmonary macrophages during silicosis. However, whether ZC3H4 is involved in EMT during silicosis remains unclear. In this study, we investigated the circular ZC3H4 (circZC3H4) RNA/microRNA-212 (miR-212) axis as the upstream molecular mechanism regulating ZC3H4 expression and the downstream mechanism by which ZC3H4 regulates EMT as well as its accompanying migratory characteristics.