Biological Research最新文献

Animal pharming involves producing recombinant protein drugs using transgenic animals. The United States Food and Drug Administration (FDA) has approved certain drugs produced in the milk of transgenic Rabbits. Traditionally, these pharming Rabbits have been developed using conventional transgenic technology, which often results in an unpredictable success rate, uncontrollable transgene insertion sites, varying copy numbers, and generally low recombinant protein yields, typically 1-2 g/L or lower. We hypothesized that utilizing the promoter of a native major milk protein gene to drive transgene expression could significantly enhance yield. To test this, we developed a rabbit line that expresses tdTomato under the control of the CSN2 gene promoter, responsible for encoding β-casein, the most abundant protein in Rabbit milk. We successfully generated knock-in founder Rabbits using CRISPR/Cas9-mediated knock-in technology, augmented by the homology-directed repair (HDR)-promoting small molecule RS-1. These founder Rabbits were able to transmit the knock-in allele to their offspring, producing both heterozygous and homozygous tdTomato knock-in Rabbits. Remarkably, the recombinant protein yield reached 15-20 g/L in the milk of homozygous animals. Our work demonstrates a promising strategy to enhance recombinant protein production in Rabbit pharming.

Epilepsy is a chronic neurological disorder characterized by a propensity for seizures due to an imbalance between excitatory and inhibitory brain activity. This condition also induces neuroinflammation, which contributes to disease progression. Given that hemichannels (HCs) permeabilize the cell membrane of glia playing a critical role in neuroinflammation, we investigated the antiepileptic potential of Boldo (Peumus boldus M.), an endemic Chilean tree containing several bioactive molecules including boldine, a HC inhibitor. Mice were treated with pulverized Boldo leaves, the antiseizure medication valproate, or a combination of both for 5 days. Seizure severity was assessed in a pentylenetetrazole-induced kindling mouse model. Using the dye uptake technique, we evaluated the membrane permeability in hippocampal astrocytes, microglia, and neurons. Additionally, we analyzed astroglial and microglial reactivity and measured levels of pro-inflammatory cytokines (IL-1β, IL6, and TNF-α). Both Boldo and valproate significantly reduced seizure severity. However, distinct mechanisms were observed. Valproate administration increased dye uptake in control animals and enhanced glial reactivity, corroborating its established ability to stimulate hemichannel activity. Conversely, Boldo treatment, either alone or in conjunction with valproate, reduced these parameters, consistent with its HC-blocking properties. Importantly, Boldo was more effective than valproate in reducing plasmatic levels of inflammatory and oxidative stress markers. These findings indicate that Boldo, by inhibiting these HCs, could provide a valuable therapeutic strategy to mitigate neuroinflammation in epilepsy, highlighting the clinical potential of this readily available medicinal herb.

Background: Osteosarcoma (OS) is the most common non-hematogenous primary malignancy in the bone. Due to several origins of OS, 30-40% OS patients would experience recurrence and metastasis, with a 5-year survival rate of 20-30%. Mesenchymal stem cells (MSCs) transform into OS cells during the differentiation into osteoblasts, and circular RNA (circRNA) hsa_circ_0003611 might contribute to the differentiation of MSCs into osteoblasts. However, the role of hsa_circ_0003611 in the transformation of MSCs into OS cells is largely unknown. This study aims to investigate whether hsa_circ_0003611 tunes the transformation of MSCs into OS cells.

Methods: Here, human bone marrow mesenchymal stem cells (hBMSCs) with hsa_circ_0003611 stably silenced was constructed. Moreover, protein-RNA interaction was detected by RNA immunoprecipitation (RIP), and N⁶-methyladenosine (m⁶A) modification of hsa_circ_0003611 was determined using methylated RNA immunoprecipitation (MeRIP).

Results: The present study reveals that hsa_circ_0003611 level is almost absent in OS cells compared to that in osteoblasts and MSCs. Moreover, hsa_circ_0003611 silence enhances the transformation of MSCs into OS cells in vitro and triggered tumorigenicity of MSCs for OS in vivo. Mechanistically, silence of hsa_circ_0003611 promotes the transformation of MSCs into OS cells by activating MYC proto-oncogene, bHLH transcription factor (MYC) via insulin like growth factor 2 mRNA binding protein 3 (IGF2BP3). Moreover, hsa_circ_0003611 silence improves MYC mRNA stability by facilitating the association between IGF2BP3 and MYC mRNA in MSCs. Furthermore, m⁶A modification disrupts the association between hsa_circ_0003611 and IGF2BP3 to enhance the association between IGF2BP3 and MYC mRNA in MSCs.

Conclusion: In summary, these findings highlight the role of hsa_circ_0003611 in the transformation of MSCs into OS cells and provide novel targets and strategies for OS treatment.

Background: Polyploidization is associated with progression of cancer, making cancer cells more dangerous. The common polyploid cancer cells constitute a considerable part of tumors (up to 56% in metastases). The giant polyploid cancer cells (PGCC), which appear under severe stress caused by treatment when the majority of cells die, present an enigmatic phenomenon both in fundamental and practical sense because they develop treatment resistance.

Results: Using transcriptome meta-analysis, we studied different types of polyploid cancer cells and found that in common polyploid cancer cells, the genes of unicellular (UC) origin and stemness are upregulated (compared to diploid cancer cells). At that, the upregulated UC genes show a higher local and global protein interactome centrality than the upregulated stemness genes, suggesting that the UC interactome attractor is a driving force behind this backward movement along the evodevo axis. Surprisingly, PGCC show the opposite picture. There occurs the suppression of UC and stemness genes with the upregulation of multicellular genes (especially those involved in intercellular communication), suggesting a reversal towards multicellular (MC) state. This effect is enhanced in PGCC's early progeny but diminished in the late progeny, indicating its transient nature. PGCC of different origin (breast, ovarian, prostate cancers), induced by different stresses (radiation or drugs with various mechanisms of action), show a similar behavior. The first principal component of transcriptome profiles, which is common for all cell types (initial cancer cells, PGCC, early and late progeny) and contains the major part of expression variance, is also directed along the gene evolutionary age axis.

Conclusions: While the common polyploid cancer cells comply with the 'serial atavism' model of oncogenesis, PGCC present a unique phenomenon of the short-term return to multicellularity probably associated with collective acquisition of resistance to treatment. Our analysis revealed also the evolutionary origin of the main differences in gene expression, emphasizing the importance of gene age axis in transcriptome analyses. The deep evolutionary basis of variation in gene expression across and within cell types might become a general framework for interrelated problems of cell and cancer biology and regenerative medicine.

Background: Protein kinase CK2 is known to exist as a tetramer of two catalytic α- or α'- subunits and two non-catalytic β-subunits, or as multimers of this tetramer. Moreover, CK2α (CSNK2A1) and CK2α' (CSNK2A2) are also active in the absence of CK2β (CSNK2B). Very little is known about specific functions of the individual subunits of protein kinase CK2.

Results: In order to study the effects of CK2α and CK2α' on gene expression, we used the Mus musculus pancreatic α-cell line αTC1 and two derivatives with either CK2α (KO1 cells) or CK2α' (KO2 cells) expression knocked-out by CRISPR/Cas technology. We found numerous genes deregulated in both KO1 and KO2 cells compared to the parental cells. Applying stringent thresholds, 266 genes were found down-regulated and 153 genes up-regulated in KO1 cells, 233 genes were found down-regulated and 84 genes up-regulated in KO2 cells. Dozens of genes were found deregulated in a similar fashion in both KO1 and KO2 cells. We found altered expression of genes involved in the differentiation of pancreatic cells, including Hox genes, and in the regulation of glucagon synthesis or secretion. Moreover, many of the deregulated genes play an important role in developmental processes and in neuronal cell biology.

Conclusion: Our findings reveal individual and shared functions of the CK2α and CK2α' catalytic subunits, in particular regarding their involvement in regulating gene expression.

Methionine serves as an essential amino acid regulating de novo protein synthesis and redox homeostasis. Previous studies have established adverse impacts of methionine restriction and deprivation on semen quality, but effects on early spermatogenesis remain poorly characterized. In this study, a methionine dietary model (0.86%, 0.17%, 0%) was used to investigate the role of methionine in early spermatogenesis. The results indicated that methionine deprivation caused spermatogenesis defects by inhibiting spermatogonial proliferation and increasing apoptosis. Further studies showed that methionine deprivation downregulated mitochondrial function-related genes (Gpx4, Fis1 and Gstm1), but upregulated ISR- (Atf4, Chac1 and Ddit3) and DNA damage response-related genes (Cdkn1a, Chek2 and Atm). Meanwhile, methionine deprivation caused mitochondrial dysfunction characterized by mitochondrial membrane potential depolarization, ROS accumulation, and MitoSOX accumulation. Methionine deprivation also caused an obvious increase in DNA damage response proteins (γH2AX, p-CHK2 and p-p53) and pro-apoptotic proteins (PUMA, BAX and c-PARP1), but suppressed anti-apoptotic protein BCL2. Furthermore, NAC effectively reversed the proliferation deficiency of GC-1 cells caused by methionine deprivation. Collectively, these findings suggest that methionine deprivation triggers ISR activation, which subsequently induces spermatogonial apoptosis via oxidative stress and the CHK2-p53/p21 signaling cascade. This study highlights the critical role of methionine in early spermatogenesis, provides mechanistic insights for optimizing dietary interventions and addresses related reproductive disorders.