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Traditional monoclonal antibodies such as Trastuzumab encounter limitations when treating Human Epidermal Growth Factor Receptor 2 (HER2)-positive breast cancer, particularly in cases that develop resistance. This study introduces plant-derived anti-HER2 variable fragments of camelid heavy chain domain (VHH) fragment crystallizable region (Fc) KEDL(K) antibody as a potent alternative for overcoming these limitations. A variety of biophysical techniques, in vitroassays, and in vivoexperiments uncover the antibody’s nanoscale binding dynamics with transmembrane HER2 on living cells. Single-molecule force spectroscopy reveals the rapid formation of two robust bonds, exhibiting approximately 50 pN force resistance and bond lifetimes in the second range. The antibody demonstrates a specific affinity for HER2-positive breast cancer cells, including those that are Trastuzumab-resistant. Moreover, in immune-deficient mice, the plant-derived anti-HER2 VHH-FcK antibody exhibits superior antitumor activity, especially against tumors that are resistant to Trastuzumab. These findings underscore the plant-derived antibody’s potential as an impactful immunotherapeutic strategy for treating Trastuzumab-resistant HER2-positive breast cancer.

There is concern about the potential for transgene contamination in wild type (WT) plants via pollen-mediated gene flow from transgenic plants. In this study, we investigate pollen-mediated gene flow using tobacco transgenic lines carrying recombinant protein GA733-FcK, which is a putative candidate for producing colorectal cancer vaccine. Transgenic and WT plants were grown in greenhouse, with WT plants placed 0.3, 1, 5, 10, and 20 m away from transgenic plants. Seeds were harvested from randomly selected WT plants and sown on germination media supplemented with or without kanamycin. At 30 days after sowing, none of the WT seedlings produced true leaves and roots when grown on selective media. Polymerase chain reaction analysis revealed that GA733-FcK and nptIIgenes were expressed in shoots of transgenic plants but not of WT plants. Expression of GA733-FcK and nptIIproteins was also abolished in WT leaves when compared to that of transgenic plants. Our findings suggested that there is low risk of pollen-mediated gene flow from transgenic plants expressing GA733-FcK when grown in greenhouse conditions.

ABSTRACTGenomic imprinting is the process of epigenetic modification whereby genes are expressed in a parent-of-origin dependent manner; it plays an important role in normal growth and development. Parthenogenetic embryos contain only the maternal genome. Parthenogenetic embryonic stem cells could be useful for studying imprinted genes. In humans, mature cystic ovarian teratomas originate from parthenogenetic activation of oocytes; they are composed of highly differentiated mature tissues containing all three germ layers. To establish human parthenogenetic induced pluripotent stem cell lines (PgHiPSCs), we generated parthenogenetic fibroblasts from ovarian teratoma tissues. We compared global DNA methylation status of PgHiPSCs with that of biparental human induced pluripotent stem cells by using Illumina Infinium HumanMethylation450 BeadChip array. This analysis identified novel single imprinted CpG sites. We further tested DNA methylation patterns of two of these sites using bisulfite sequencing and described novel candidate imprinted CpG sites. These results confirm that PgHiPSCs are a powerful tool for identifying imprinted genes and investigating their roles in human development and diseases.

Plants have emerged as one of the most attractive systems for producing human therapeutic proteins against viral diseases. These include diagnostic reagents, vaccines, and antibodies. This process is known as molecular biofarming. The objective of this study was to develop and evaluate tobacco and Arabidopsis as plant platforms for producing human anti-rabies monoclonal antibody (mAb). Both tobacco and Arabidopsis transgenic plants were generated by Agrobacterium-mediated transformation. Purification of mAb SO57K from each plant was performed with ammonium sulfate-mediated precipitation and protein A affinity columns. SDS–PAGE analysis showed that the purity of mAb SO57K obtained from each transgenic plant was similar, whereas Arabidopsis showed approximately twofold greater protein expression than tobacco. The N-glycosylation was not significantly different between proteins from the two plant species, with both showing oligo-mannose glycan structures. The mAbs SO57 derived from both the model plants had similar neutralizing efficacy against target virus strain CVS-11. Taken together, tobacco and Arabidopsis are both promising platforms for producing a human anti-rabies mAb.

This study investigated the effects of nitrogen (NH4+) in the soil on the expression of prostatic acid phosphatase (PAP)-IgA Fc and PAP-IgA FcK recombinant proteins in transgenic plants, and on plant growth in the greenhouse. Plant height and leaf length were greater in soil A (NH4+: 51 mg·L-1) than in soil B (NH4+: 1 mg·L-1). In soil B, the plant leaves turned yellow, with chlorophyll concentrations that were significantly lower than the chlorophyll concentration of leaves from plants grown in soil A. The expression level of the PAP-IgA Fc and PAP-IgA FcK proteins and the protein dimerization rates were lower in soil B when compared with soil A. In addition, PAP-IgA Fc and PAP-IgA FcK expression and dimerization levels were significantly lower in transgenic plants grown in soil B, when compared with transgenic plants grown in soil A. Soil characteristics such as hydrogen ion concentration (pH), electric conductivity (EC), and bulk density did not differ between soil A and B. Thus, appropriate soil NH4+concentrations are essential for optimized plant growth and enhanced expression of dimerized PAP-IgA Fc and PAP-IgA FcK recombinant proteins in transgenic plants.

ABSTRACTSpermatogonial stem cells (SSCs), which are unipotent stem cells in the testes that give rise to sperm, can be converted into germline-derived pluripotent stem (gPS) by self-induction. The androgenetic imprinting pattern of SSCs is maintained even after their reprogramming into gPS cells. In this study, we used an in vitroneural differentiation model to investigate whether the imprinting patterns are maintained or altered during differentiation. The androgenetic patterns of H19, Snrpn, and Mestwere maintained even after differentiation of gPS cells into NSCs (gPS-NSCs), whereas the fully unmethylated status of Ndnin SSCs was altered to somatic patterns in gPS cells and gPS-NSCs. Thus, our study demonstrates epigenetic alteration of genomic imprinting during the induction of pluripotency in SSCs and neural differentiation, suggesting that gPS-NSCs can be a useful model to study the roles of imprinted genes in brain development and human neurodevelopmental disorders.

The aim of this study was to investigate the mechanisms involved in the apoptosis of HeLa cells due to 2,3-dehydrosilybin (DHS) treatment. DHS treatment over 24 h significantly inhibited cell viability and induced apoptosis in a dose-dependent manner. It also triggered the cleavage of caspase-8, caspase-9, caspase-3, and PARP, and significantly increased caspase-3 activity in a dose-dependent manner. Moreover, it triggered the depolarization of the mitochondrial membrane potential (Δψm), the release of cytochrome cinto the cytosol, the cleavage of Bid, and the downregulation of Bcl-2 in a dose-dependent manner. Furthermore, z-VAD-fmk (a pan-caspase inhibitor) and z-IETD-fmk (a specific caspase-8 inhibitor) abolished the DHS-induced activation of the caspase-8, -9, and -3, cleavage of PARP, the depolarization of Δψm, the release of cytochrome c, the cleavage of Bid, and the downregulation of Bcl-2. Taken together, these results suggest that DHS-induced apoptosis is mediated by a caspase-dependent pathway in human HeLa cells.

Transgenic plants offer a source for the sustainable, safe, and large-scale production of therapeutic recombinant proteins. In this study, both murine anti-colorectal cancer mAb (mAbMC) and human anti-rabies mAb 57 (mAbHR), expressed in a single plant were investigated for their cancer cell binding activity and rabies virus neutralization activity, respectively. Transgenic plants, expressing murine anti-colorectal cancer mAb CO17-1A (mAbMC) and human anti-rabies mAb 57 (mAbHR), respectively, were crossed to reproduce F1transgenic plant, expressing both mAbs. PCR and immunoblot analyses demonstrated that heavy (HC) and light chain (LC) genes of mAbMC and mAbHR were present, and that both mAbs were expressed in F1transgenic lines, respectively. Quantitative immunoblot for purified mAb also showed the presence of both mAbs in F1transgenic lines. However, Cell ELISA analysis showed that in mAbPC and mAbPR purified from the F1transgenic lines (mAbPC×R), the binding activity to SW948 human colorectal carcinoma cells was lower than mAbMC, and in vitro mAbMC, mixed with mAbHR (mAbMHC+R). The in vitro rabies virus neutralization assay demonstrated that the mAbPC×R, from the F1 transgenic plants, had lower bioactivity against rabies virus than mAbH57, and mAbMHC+R. N-glycan structure analysis revealed that mAbMC and mAbHR had Golgi type (94 and 14%) and ER type (6 and 86%), respectively, and the purified mAbs from the F1 transgenic plants had Golgi type (75%) and ER type (25%). These results indicate that the F1 transgenic plant produced both mAbPC and mAbPR; however, the HC and LC proteins of each anti-rabies virus and anti-colorectal cancer mAbs were assembled randomly, resulting in chimerism in HC and LC assembly for mAb.

The human colorectal carcinoma-associated GA733 antigen epithelial cell adhesion molecule (EpCAM) was initially described as a cell surface protein selectively expressed in some myeloid cancers. Gangliosides are sialic acid-containing glycosphingolipids involved in inflammation and oncogenesis. We have demonstrated that treatment with anti-EpCAM mAb and RAW264.7 cells significant inhibited the cell growth in SW620 cancer cells, but neither anti-EpCAM mAb nor RAW264.7 cells alone induced cytotoxicity. The relationship between ganglioside expression and the anti-cancer effects of anti-EpCAM mAb and RAW264.7 was investigated by high-performance thin-layer chromatography. The results demonstrated that expression of GM1 and GD1a significantly increased in the ability of anti-EpCAM to inhibit cell growth in SW620 cells. Anti-EpCAM mAb treatment increased the expression of anti-apoptotic proteins such as Bcl-2, but the expression of pro-apoptotic proteins Bax, TNF-a, caspase-3, cleaved caspase-3, and cleaved caspase-8 were unaltered. We observed that anti-EpCAM mAb significantly inhibited the growth of colon tumors, as determined by a decrease in tumor volume and weight. The expression of anti-apoptotic protein was inhibited by treatment with anti-EpCAM mAb, whereas the expression of pro-apoptotic proteins was increased. These results suggest that GD1a and GM1 were closely related to anticancer effects of anti-EpCAM mAb. In light of these results, further clinical investigation should be conducted on anti-EpCAM mAb to determine its possible chemopreventive and/or therapeutic efficacy against human colon cancer.

Glycosphingolipids including gangliosides play important regulatory roles in cell proliferation and differentiation. UDP-glucose:ceramide glucosyltransferase (Ugcg) catalyze the initial step in glycosphingolipids biosynthesis pathway. In this study, Ugcg expression was reduced to approximately 80% by short hairpin RNAs (shRNAs) to evaluate the roles of glycosphingolipids in proliferation and neural differentiation of mouse embryonic stem cells (mESCs). HPTLC/immunofluorescence analyses of shRNA-transfected mESCs revealed that treatment with Ugcg-shRNA decreased expression of major gangliosides, GM3 and GD3. Furthermore, MTT and Western blot/immunofluorescence analyses demonstrated that inhibition of the Ugcg expression in mESCs resulted in decrease of cell proliferation (P < 0.05) and decrease of activation of the ERK1/2 (P < 0.05), respectively. To further investigate the role of glycosphingolipids in neural differentiation, the embryoid bodies formed from Ugcg-shRNA transfected mESCs were differentiated into neural cells by treatment with retinoic acid. We found that inhibition of Ugcg expression did not affect embryoid body (EB) differentiation, as judged by morphological comparison and expression of early neural precursor cell marker, nestin, in differentiated EBs. However, RT-PCR/immunofluorescence analyses showed that expression of microtubule- associated protein 2 (MAP-2) for neurons and glial fibrillary acidic protein (GFAP) for glial cells was decreased in neural cells differentiated from the shRNA-transfected mESCs. These results suggest that glycosphingolipids are involved in the proliferation of mESCs through ERK1/2 activation, and that glycosphingolipids play roles in differentiation of neural precursor cells derived from mESCs.

A gene encoding attacin E, an inducible antibacterial protein from Hyalophora cecropia pupae, was cloned into the pRSETB Escherichia coli expression vector under the control of the T7 promoter. The resulting vector, pRSETBAtt, produced a fusion protein in E. coli JM109 of attacin with an N-terminal peptide containing six histidine residues in tandem. Fusion attacin was purified from cell lysates (6–9 mg l−1) by Ni2+-Sepharose affinity chromatography. Purified attacin protein was used as antigen to produce polyclonal antibody to detect attacin expressed in transgenic apple. Antibody capture immunoassay and immunoblot assays indicated that polyclonal antisera derived from fusion attacin had specific immunoreaction against attacins in the hemolymph of immunized pupae and attacin expressed in transgenic apple lines similar to native attacin antisera. Attacin expressed in transgenic apple could be quantified using immunoblot assays with the fusion attacin polyclonal antibody. [ABSTRACT FROM AUTHOR]

In this study, recombinant Fc-fused Prostate acid phosphatase (PAP) proteins were produced in transgenic plants. PAP was fused to immunoglobulin (Ig) A and M Fc domain (PAP-IgA Fc and PAP-IgM Fc), which were tagged to the ER retention sequence KDEL to generate PAP-IgA FcK and PAP-IgM FcK. Agrobacteriummediated transformation was performed to produce transgenic tobacco plants expressing four recombinant proteins. Genomic PCR and RT-PCR analyses confirmed the transgene insertion and mRNA transcription of PAP-IgA Fc, PAP-IgM Fc, PAP-IgA FcK, and PAP-IgM FcK in tobacco plant leaves. Western blot confirmed the expression of PAP-IgA Fc, PAP-IgM Fc, PAP-IgA FcK, and PAP-IgM FcK proteins. SEC-HPLC and Bio-TEM analyses were performed to confirm the size and shape of the plant-derived recombinant PAP-Fc fusion proteins. In mice experiments, the plant-derived IgA and IgM Fc fused proteins induced production of total IgGs including IgG1 against PAP. This result suggests that IgA and IgM Fc fusion can be applied to produce recombinant PAP proteins as a prostate cancer vaccine in plant expression system. [BMB Reports 2023; 56(7): 392-397].

The BMB Reports would like to correct in the ACKNOWLEDGEMENTS of BMB Rep. 45(12), 713-718 titled "Ganglioside GM1 influences the proliferation rate of mouse induced pluripotent stem cells".