Reductive prodrug and AIE copolymer nanoparticle for monitoring and chemotherapy.

Polymeric micelle systems for drug delivery, monitor and chemotherapy have gained significant attention, and reductive polymeric micelle systems have become particularly attractive due to their controlled release behavior without additional assistance. However, there are challenges in accurately controlling drug and probe release from the nanoparticles and determining the loading content of drug and probe. To address these issues, we have developed a reduction-responsive Pt(IV) prodrug-based polymeric delivery system that can be dynamically monitored using aggregation-induced emission luminogens (AIE) based bioprobes. These polymeric micelle can self-assemble into nanoparticles and release both bio-active Pt(II) drug and bio-probe upon reduction activation. TPE molecules released in the inner endo/lysosomal microenvironment aggregate and fluoresce upon irradiation, thus allowing real-time tracking of drug biodistribution without additional contrast agents. Advantages of this system include position-specific chemical bond cleavage, control of platinum content, and monitoring of drug reduction and biodistribution.

[Preparation of HSV-IgM human-mouse chimeric antibody and development of stable recombinant cell line].

In order to achieve large-scale production of HSV-IgM (HSV1, HSV2) human-mouse chimeric antibody in vitro, the gene sequence of the corresponding hybridoma cell was harvested by RNA ligase-mediated rapid amplification of cDNA ends (RLM-RACE) technique to clone the chimeric antibody into eukaryotic expression vectors, and express the target proteins in CHO-S cells. At the same time, the screening process of stable cell lines was optimized, and the pressure conditions of pool construction stage and monoclonal screening stage were explored. Finally, the target protein was purified by protein L affinity purification method and the biological activity was detected. The recombinant IgM antibodies, HSV1 and HSV2, weighted at 899 kDa and 909 kDa respectively, were prepared. The optimal screening pressure was 20P200M (the first phase of pressure) and 50P1000M (the second phase of pressure). The final titer for the monoclonal expression of HSV1-IgM and HSV2-IgM was 1 620 mg/L and 623 mg/L, respectively. This study may facilitate the development of quality control products of HSV1 and HSV2 IgM series recombinant antibodies as well as efficient expression of IgM subtype antibodies in vitro.

Non-neutralizing monoclonal antibody targeting VP2 EF loop of Coxsackievirus A16 can protect mice from lethal attack via Fc-dependent effector mechanism.

Coxsackievirus A16 (CA16), a main causative agent of hand, foot, and mouth disease (HFMD), has become a serious public health concern in the Asia-Pacific region. Here, we generated an anti-CA16 monoclonal antibody, DMA2017, derived from an epidemic strain CA16. Surprisingly, although DMA2017 could not neutralize the original and circulating CA16 strains in vitro, the passive transfer of DMA2017 (10 µg/g) could protect suckling mice from a lethal challenge with CA16 in vivo. Then, we confirmed the protective effect of DMA2017 relies on the Fc-dependent effector functions, such as antibody-dependent cellular cytotoxicity (ADCC). The linear epitope of DMA2017 was mapped by phage display technique to a conserved patch spanning residues 143-148 (NSHPPY) of the VP2 EF-loop of CA16. DMA2017 could inhibit the binding of the antibodies present in the sera of naturally infected children to CA16, indicating that the epitope of DMA2017 is immunodominant for CA16. Our results confirm, for the first time, that a potential preventive and therapeutic effect could be mediated by a non-neutralizing antibody elicited against CA16. These findings bring a hitherto understudied protective role of non-neutralizing antibodies during viral infections into the spotlight and provide a new perspective on the design and evaluation of CA16 vaccines.

Hepatitis B virus pathogenesis relevant immunosignals uncovering amino acids utilization related risk factors guide artificial intelligence-based precision medicine.

Background: Although immune microenvironment-related chemokines, extracellular matrix (ECM), and intrahepatic immune cells are reported to be highly involved in hepatitis B virus (HBV)-related diseases, their roles in diagnosis, prognosis, and drug sensitivity evaluation remain unclear. Here, we aimed to study their clinical use to provide a basis for precision medicine in hepatocellular carcinoma (HCC) via the amalgamation of artificial intelligence. Methods: High-throughput liver transcriptomes from Gene Expression Omnibus (GEO), NODE (https://www.bio.sino.org/node), the Cancer Genome Atlas (TCGA), and our in-house hepatocellular carcinoma patients were collected in this study. Core immunosignals that participated in the entire diseases course of hepatitis B were explored using the "Gene set variation analysis" R package. Using ROC curve analysis, the impact of core immunosignals and amino acid utilization related gene on hepatocellular carcinoma patient's clinical outcome were calculated. The utility of core immunosignals as a classifier for hepatocellular carcinoma tumor tissue was evaluated using explainable machine-learning methods. A novel deep residual neural network model based on immunosignals was constructed for the long-term overall survival (LS) analysis. In vivo drug sensitivity was calculated by the "oncoPredict" R package. Results: We identified nine genes comprising chemokines and ECM related to hepatitis B virus-induced inflammation and fibrosis as CLST signals. Moreover, CLST was co-enriched with activated CD4+ T cells bearing harmful factors (aCD4) during all stages of hepatitis B virus pathogenesis, which was also verified by our hepatocellular carcinoma data. Unexpectedly, we found that hepatitis B virus-hepatocellular carcinoma patients in the CLSThighaCD4high subgroup had the shortest overall survival (OS) and were characterized by a risk gene signature associated with amino acids utilization. Importantly, characteristic genes specific to CLST/aCD4 showed promising clinical relevance in identifying patients with early-stage hepatocellular carcinoma via explainable machine learning. In addition, the 5-year long-term overall survival of hepatocellular carcinoma patients can be effectively classified by CLST/aCD4 based GeneSet-ResNet model. Subgroups defined by CLST and aCD4 were significantly involved in the sensitivity of hepatitis B virus-hepatocellular carcinoma patients to chemotherapy treatments. Conclusion: CLST and aCD4 are hepatitis B virus pathogenesis-relevant immunosignals that are highly involved in hepatitis B virus-induced inflammation, fibrosis, and hepatocellular carcinoma. Gene set variation analysis derived immunogenomic signatures enabled efficient diagnostic and prognostic model construction. The clinical application of CLST and aCD4 as indicators would be beneficial for the precision management of hepatocellular carcinoma.

[Effects of different signal peptides on the secretion of human-mouse chimeric CMV-IgM].

In order to prepare human-mouse chimeric cytomegalovirus-immunoglobulin M (CMV-IgM) in vitro and study the effects of different signal peptides on the secretion of CMV-IgM, genes were amplified from hybridoma cell line using RLM-RACE to construct the expression vector of chimeric CMV-IgM. Then, the signal peptide of SigF itself was replaced by five different secreted signal peptides (SigA-SigE) by PCR method, and the CHO cell was chosen as host cell for in vitro expression. SDS-PAGE, SEC-HPLC and ELISA experiments were carried out to evaluate the protein expression level and immunoreactivity of the purified CMV-IgM. A 910 kDa recombinant protein was successfully prepared and signal peptides (SigA-SigE) had an increased expressed CMV-IgM, which were 6.72, 5.19, 1.44, 1.85 and 1.98 times higher than that of the CMV 6# cell signal peptide SigF. In summary, this work provides a theoretical basis for the development of human-mouse chimeric CMV-IgM, and a novel route to increase the expression level of CMV-IgM.

Genetic variations of TAP1 gene exon 3 affects gene expression and Escherichia coli F18 resistance in piglets.

Firstly, our research group identified Sutai pigs' phenotypes that exhibited extreme resistance and susceptibility to the Escherichia coli F18 respectively, and then eight ETEC (Enterotoxigenic Escherichia coli) F18-resistant piglets and eight ETEC F18-sensitive piglets were selected. Then, the TAP1 (Transporter associated with antigen processing) mRNA relative expression levels were analyzed in 11 tissues of the resistant and susceptible phenotypes. Simultaneously, we detected the genetic variations in exon 3 of the TAP1 gene and evaluated the TAP1 mRNA expression levels among the different genotype pigs to study the effects of the genetic variation on gene expression, and the E. coli F18 resistance. The results revealed higher expression levels in the resistant genotypes than that in the susceptible genotypes in 11 tissues, with significant differences in the spleen, lymph node, lung, thymus, duodenum and jejunum. Furthermore, a G729A mutation was identified in the TAP1 gene exon 3, and this mutation deviates from Hardy-Weinberg equilibrium (p < 0.01). The TAP1 mRNA levels in GG genotype were significantly higher than that in the other two genotypes, with significant differences in the liver, lung, kidney, thymus, lymph node, duodenum and jejunum tissues. We speculated that high expression of the TAP1 gene might confer resistance against the E. coli F18, the G729A mutation had a significant effect on the mRNA expression, and individuals with the GG genotype possessed a stronger ability to resist the E. coli F18 infection.

Genetic variation in exon 10 of the BPI gene is associated with Escherichia coli F18 susceptibility in Sutai piglets.

Our aim was to investigate the effect of the porcine bactericidal/permeability-increasing protein (BPI) on the susceptibility to enterotoxigenic Escherichia coli F18 (ETEC F18). Specifically, we wanted to determine whether the HpaII restriction polymorphism in exon 10 of BPI mediates susceptibility to ETEC F18. Thirty verified ETEC F18-resistant and thirty susceptible Sutai (Duroc×Taihu) piglets were identified using the receptor binding assay. Exon 10 of the BPI gene produced the AA, BB, and AB genotypes after HpaII digestion. The genotype distribution among ETEC F18-resistant piglets was significantly different from that among susceptible piglets. Among piglets with the AA genotype, 90% were ETEC F18-resistant; this percentage of resistant piglets was significantly higher than the percentage of resistant piglets with the AB (57.1%) and BB genotypes (17.4%). There was high expression only in the tissues of the duodenum and jejunum, wherein the expression levels in the ETEC F18-resistant group were significantly higher than those in the susceptible group (P<0.05). The average expression levels in individuals with the AA genotype were significantly higher than those in individuals with the AB or BB genotype (P<0.05), while the results of Western blot show the same evidences as real time PCR. These results indicate that the upregulation of porcine BPI gene expression in the small intestines plays a direct role in resistance to ETEC F18 infection. The AA genotype for the HpaII site in exon 10 of the porcine BPI gene was demonstrated to be an anti-ETEC F18 marker and could be used for selective breeding to enhance ETEC F18 resistance.

Characterization and expression of bone morphogenetic protein 4 gene in postnatal pigs.

Bone morphogenetic protein 4 (BMP4) is involved in animal embryonic development and reproductive physiology. The human and murine BMP4 genes have been isolated and characterized. The objectives of this study were to: (1) characterize the full mRNA and genomic sequence for porcine BMP4, and (2) examine BMP4 gene expression in 10 tissues of postnatal female pigs. Using RT-PCR, RACE and general PCR techniques, a 1,626 bp DNA including the full coding region of BMP4 was isolated and identified as a homologue of human BMP4 transcript variant (TV)-c. The porcine TV-c contained 3 exons and astride 3.6 kb in the isolated 7.8 kb porcine BMP4 genome. The In silicon cloning identified other three forms of mRNAs, including the homologues of human TV-1, TV-a and a novel variant related to human TV-3 (TV-3p). The porcine TV-c, TV-1 and TV-3p bear internal ribosome entry sites (IRES) in 5' untranslated region (UTR), while there are two ARE elements in the 3'UTR. The full genomic sequence of porcine BMP4 gene showed 81.38, 76.23 and 64.00% identity with that of bovine, human and murine, respectively. The expression of BMP4 mRNA was determined by RT-PCR in 7, 14, and 28 day old female piglets and non-gestational sows. The results showed that porcine BMP4 occurred in all 10 examined tissues (heart, lung, liver, kidney, ovary, spleen, spinal medulla, brain, duodenum and thymus). The mRNA expression levels were relatively higher in lung and kidney in 7 day old piglets, thymus in 14 day old piglets, and spleen in 28 day old piglets, respectively, while the higher expressions were detected in liver of non-gestational pigs (P < 0.05). Moreover, the mRNA amounts both in 7 day old piglets and sows were generally higher than those in 14 and 28 day old piglets in nearly all examined tissues, except in thymus. It is concluded that the structure of porcine BMP4 gene is highly conservative with other mammalian BMP4 genes, but some differences may present in the regulation of gene expression. BMP4 mRNAs are expressed in postnatal pigs, and is spatiotemporally regulated.

[Cloning and bioinformatic analysis of bone morphological protein 7 (BMP7) in rabbit].

On the basis of known partial coding DNA sequence (GenBank accession No. AF413111), we tried to clone the full mRNA of rabbit BMP7 gene by sub-cloning and RACE methods. Then after, bioinformatic analysis on the acquired sequences were conducted. The sequencing results showed that the all cloned sequences could be assembled into a 1 654 bp long DNA fragment, which contained the near full coding cDNA for the propetide, full coding cDNA for the mature protein, and full length of 3'UTR of rabbit BMP7. The newly cloned sequences extended the 5'and 3'ends of known partial DNA sequence by 395 bp and 628 bp, respectively. Sequence comparisons revealed that the full length of coding cDNA of rabbit BMP7 was 91.89% and 89.32% identical to that of human and mouse, while the deduced amino acids was 96.51% and 96.01% identical, respectively. The 3'UTR of rabbit BMP7 was 446 bp in length, 57.38% and 45.57% identical to that of human or mouse, respectively. In addition, there were two closely arranged AATAAA sites in the 3'UTR of rabbit BMP7 gene, as well as in human BMP7. The deduced mature protein of rabbit BMP7 had the seven conservative cysteines and a TGF-b family signature, which were the characters of all BMPs. Our results suggested that the main part of rabbit BMP7 had been successively cloned. The alternation of polyadenization sites in the 3'UTR of rabbit BMP7 maybe related to the posttranscriptional regulation of the gene.