Diploid Cells Research Articles

Analysis of cellular effects by continuous exposure AT low concentration of tritium.

This study investigated the induction of DNA double-strand breaks (DSBs) in the hTERT-immortalized normal human diploid epithelial cells (RPE1-hTERT) continuously exposed to 6000Bq/ml of tritiated water (HTO) and organically bound tritium (OBT). The relationship of the DSBs induction with the intracellular amount as well as the localization of tritium was also examined. Tritium-labeled thymidine (3H-Thy) and palmitic acid (3H-PA) were used as OBT. The average number of DSBs, which were indicated as co-localized foci of 53BP1 with phosphorylated H2AX, per cell was higher in the order of treatments with 3H-Thy, 3H-PA, and HTO. This order was consistent with that of tritium localization in the insoluble nuclear fraction but not with the intracellular amount of tritium. In addition to the intracellular amount of tritium, we showed that the subcellular localization of tritium is an important factor in cellular effects stimulated by DSBs.

Inter-chromosomal contacts demarcate genome topology along a spatial gradient.

Non-homologous chromosomal contacts (NHCCs) between different chromosomes participate considerably in gene and genome regulation. Due to analytical challenges, NHCCs are currently considered as singular, stochastic events, and their extent and fundamental principles across cell types remain controversial. We develop a supervised and unsupervised learning algorithm, termed Signature, to call NHCCs in Hi-C datasets to advance our understanding of genome topology. Signature reveals 40,282 NHCCs and their properties across 62 Hi-C datasets of 53 diploid human cell types. Genomic regions of NHCCs are gene-dense, highly expressed, and harbor genes for cell-specific and sex-specific functions. Extensive inter-telomeric and inter-centromeric clustering occurs across cell types [Rabl's configuration] and 61 NHCCs are consistently found at the nuclear speckles. These constitutive 'anchor loci' facilitate an axis of genome activity whilst cell-type-specific NHCCs act in discrete hubs. Our results suggest that non-random chromosome positioning is supported by constitutive NHCCs that shape genome topology along an off-centered spatial gradient of genome activity.

Polyploidy mitigates the impact of DNA damage while simultaneously bearing its burden

Polyploidy is frequently enhanced under pathological conditions, such as tissue injury and cancer in humans. Polyploidization is critically involved in cancer evolution, including cancer initiation and the acquisition of drug resistance. However, the effect of polyploidy on cell fate remains unclear. In this study, we explored the effects of polyploidization on cellular responses to DNA damage and cell cycle progression. Through various comparisons based on ploidy stratifications of cultured cells, we found that polyploidization and the accumulation of genomic DNA damage mutually induce each other, resulting in polyploid cells consistently containing more genomic DNA damage than diploid cells under both physiological and stress conditions. Notably, despite substantial DNA damage, polyploid cells demonstrated a higher tolerance to its impact, exhibiting delayed cell cycle arrest and reduced secretion of inflammatory cytokines associated with DNA damage-induced senescence. Consistently, in mice with ploidy tracing, hepatocytes with high ploidy appeared to potentially persist in the damaged liver, while being susceptible to DNA damage. Polyploidy acts as a reservoir of genomic damage by mitigating the impact of DNA damage, while simultaneously enhancing its accumulation.

Mitochondrial dysfunction matures Ras-induced early senescence to full senescence with a proinflammatory senescence-associated secretory phenotype in the fish cell line, EPC

Fish cell lines differ from most mammalian diploid cell lines by the fact that cellular senescence is not readily induced. Previously, we demonstrated that the absence of the p16 gene in the fish genome prevents cells from reaching full senescence even when Ras is activated. Drosophila also lacks p16; however, early senescence triggered by Ras activation progresses to full senescence and is accompanied by a proinflammatory senescence-associated secretory phenotype (SASP), due to mitochondrial deficiency. It is unclear whether mitochondrial deficiency can also induce the maturation of Ras-induced early senescence (RIS) to full senescence along with a proinflammatory SASP in fish cell lines. Here, we investigated whether mitochondrial dysfunction induced by carbonyl cyanide 3-chlorophenylhydrazone (CCCP) in concert with activated Ras results in full senescence and whether this is accompanied by a proinflammatory SASPs in the EPC fish cell line. We found that although EPC cells with mitochondrial dysfunction exhibited a proinflammatory SASP, this did not result in permanent cell proliferation arrest or the upregulation of endogenous Ras expression. These findings suggest that other factors must act in concert with mitochondrial dysfunction to induce full senescence. The proliferation of EPC cells overexpressing a constitutively active mutant of H-Ras (H-RasV12) was markedly reduced, irrespective of CCCP treatment. These findings suggest that there are similarities between the cellular senescence observed in fish and Drosophila cells lacking the p16 gene. However, it should be noted that fish cells differ from Drosophila cells in that mitochondrial dysfunction alone can induce proinflammatory SASP factors.

Kinesins regulate the heterogeneity in centrosome clustering after whole-genome duplication.

After whole-genome duplication (WGD), tetraploid cells can undergo multipolar mitosis or pseudo-bipolar mitosis with clustered centrosomes. Kinesins play a crucial role in regulating spindle formation. However, the contribution of kinesin expression levels to the heterogeneity in centrosome clustering observed across different cell lines after WGD remains unclear. We identified two subsets of cell lines: "BP" cells efficiently cluster extra centrosomes for pseudo-bipolar mitosis, and "MP" cells primarily undergo multipolar mitosis after WGD. Diploid MP cells contained higher levels of KIF11 and KIF15 compared with BP cells and showed reduced sensitivity to centrosome clustering induced by KIF11 inhibitors. Moreover, partial inhibition of KIF11 or depletion of KIF15 converted MP cells from multipolar to bipolar mitosis after WGD. Multipolar spindle formation involved microtubules but was independent of kinetochore-microtubule attachment. Silencing KIFC1, but not KIFC3, promoted multipolar mitosis in BP cells, indicating the involvement of specific kinesin-14 family members in counteracting the forces from KIF11/KIF15 after WGD. These findings highlight the collective role of KIF11, KIF15, and KIFC1 in determining the polarity of the mitotic spindle after WGD.

Steroidal constituents in the whole plants of Helleborus niger and their cytotoxic activity in vitro

Phytochemical investigation of the whole plants of Helleborus niger L. (Ranunculaceae) resulted in the isolation of five undescribed compounds, including one bufadienolide (1), two bufadienolide rhamnosides (2 and 3), and two ecdysteroids (12 and 13), along with eight known compounds (4–11). The chemical structures of 1–3, 12, and 13 were determined by spectroscopic studies, including 2D NMR, and chromatographic and spectroscopic analyses of the hydrolyzed products. Compounds 1–13 were evaluated for their cytotoxic activity against HL-60 human leukemia cells, A549 human lung adenocarcinoma cells, SBC-3 human small-cell lung cancer cells, and TIG-3 human normal diploid lung cells. Compounds 1–12 showed cytotoxic activity against HL-60, A549, and SBC-3 cells, with IC50 values ranging from 0.0016 to 6.1 μM. Bufadienolide rhamnoside 2 exhibited potent cell proliferation inhibitory activity against SBC-3 cells after 24–48 h of treatment and apoptosis-inducing activity in SBC-3 cells via an intrinsic pathway after 72 h of treatment. The JFCR39 panel screening of 2 suggests that the molecular target of 2 is Na+,K+-ATPase.

Increased RNA and protein degradation is required for counteracting transcriptional burden and proteotoxic stress in human aneuploid cells.

Aneuploidy results in a stoichiometric imbalance of protein complexes that jeopardizes cellular fitness. Aneuploid cells thus need to compensate for the imbalanced DNA levels by regulating their RNA and protein levels, but the underlying molecular mechanisms remain unknown. Here, we dissected multiple diploid vs. aneuploid cell models. We found that aneuploid cells cope with transcriptional burden by increasing several RNA degradation pathways, and are consequently more sensitive to the perturbation of RNA degradation. At the protein level, aneuploid cells mitigate proteotoxic stress by reducing protein translation and increasing protein degradation, rendering them more sensitive to proteasome inhibition. These findings were recapitulated across hundreds of human cancer cell lines and primary tumors, and aneuploidy levels were significantly associated with the response of multiple myeloma patients to proteasome inhibitors. Aneuploid cells are therefore preferentially dependent on several key nodes along the gene expression process, creating clinically-actionable vulnerabilities in aneuploid cells.

Developmental analysis of Spalt function in the Drosophila prothoracic gland.

The Spalt transcriptional regulators participate in a variety of cell fate specification processes during development, regulating transcription through interactions with DNA AT-rich regions. Spalt proteins also bind to heterochromatic regions, and some of their effects require interactions with the NuRD chromatin remodeling and deacetylase complex. Most of the biological roles of Spalt proteins have been characterized in diploid cells engaged in cell proliferation. Here, we address the function of Drosophila Spalt genes in the development of a larval tissue formed by polyploid cells, the prothoracic gland, the cells of which undergo several rounds of DNA replication without mitosis during larval development. We show that prothoracic glands depleted of Spalt expression display severe changes in the size of the nucleolus, the morphology of the nuclear envelope and the disposition of the chromatin within the nucleus, leading to a failure in the synthesis of ecdysone. We propose that loss of ecdysone production in the prothoracic gland of Spalt mutants is primarily caused by defects in nuclear pore complex function that occur as a consequence of faulty interactions between heterochromatic regions and the nuclear envelope.

Effect of abiotic and biotic elicitors on vincristine accumulation in endosperm derived in vitro cultures in Catharanthus roseous (L.) G. Don

Catharanthus roseus (L.) G. Don (Apocynaceae) is a well-studied herb renowned for its in vitro culture as a source of the anti-cancer alkaloid, vincristine. However, despite the recognized advantages of triploid cells over diploid cells in terms of productivity, the triploid endosperm tissue of this important medicinal plant has not been utilized for in vitro culture initiation. In this investigation, zygotic embryos and endosperm tissues were cultured on Murashige and Skoog (MS) medium supplemented with various combinations of auxins and cytokinins. The medium containing 2.50 µM 6-Benzyladenine (BA) and 1.25 µM 2,4-Dichlorophenoxyacetic acid (2,4-D) proved to be the most effective for callus and cell culture formation. Ploidy analysis using ploidy analyzer confirmed that the endosperm-derived callus exhibited mixoploid, while the embryo-derived callus remained diploid. Liquid Chromatography Mass Spectrometry (LC–MS) analysis of callus and cell cultures grown on MS media with different combinations of auxins, cytokinins, elicitors, and precursors (both biotic and abiotic) revealed the accumulation of vincristine. Notably, treatment with a biotic elicitor derived from Aspergillus niger (300 mg/l) demonstrated superior efficacy in promoting the maximum accumulation of vincristine in endosperm-derived callus and cell biomass. These findings hold promise for the sustainable production of the anti-cancer alkaloid vincristine from endosperm-derived callus and cell cultures of Catharanthus roseus.

Destabilization of mRNAs enhances competence to initiate meiosis in mouse spermatogenic cells.

The specialized cell cycle of meiosis transforms diploid germ cells into haploid gametes. In mammals, diploid spermatogenic cells acquire the competence to initiate meiosis in response to retinoic acid. Previous mouse studies revealed that MEIOC interacts with RNA-binding proteins YTHDC2 and RBM46 to repress mitotic genes and to promote robust meiotic gene expression in spermatogenic cells that have initiated meiosis. Here, we have used the enhanced resolution of scRNA-seq and bulk RNA-seq of developmentally synchronized spermatogenesis to define how MEIOC molecularly supports early meiosis in spermatogenic cells. We demonstrate that MEIOC mediates transcriptomic changes before meiotic initiation, earlier than previously appreciated. MEIOC, acting with YTHDC2 and RBM46, destabilizes its mRNA targets, including the transcriptional repressors E2f6 and Mga, in mitotic spermatogonia. MEIOC thereby derepresses E2F6- and MGA-repressed genes, including Meiosin and other meiosis-associated genes. This confers on spermatogenic cells the molecular competence to, in response to retinoic acid, fully activate the transcriptional regulator STRA8-MEIOSIN, which is required for the meiotic G1/S phase transition and for meiotic gene expression. We conclude that, in mice, mRNA decay mediated by MEIOC-YTHDC2-RBM46 enhances the competence of spermatogenic cells to initiate meiosis.

DSB profiles in human spermatozoa highlight the role of TMEJ in the male germline.

The male mammalian germline is characterized by substantial chromatin remodeling associated with the transition from histones to protamines during spermatogenesis, followed by the reversal to nucleohistones in the male pronucleus preceding the zygotic genome activation. Both transitions are associated with the extensive formation of DNA double-strand breaks (DSBs), requiring an estimated 5 to 10 million transient DSBs per spermatozoa. Additionally, the high transcription rate in early stages of spermatogenesis leads to transcription-coupled damage preceding meiotic homologous recombination, potentially further contributing to the DSB landscape in mature spermatozoa. Once meiosis is completed, spermatozoa remain haploid and therefore cannot rely on error-free homologous recombination, but instead depend on error-prone classical non-homologous end joining (cNHEJ). This DNA damage/repair-scenario is proposed to be one of the main causes of the observed paternal mutation propensity in human evolution. Recent studies have shown that DSBs in the male pronucleus are repaired by maternally provided Polθ in Caenorhabditis elegans through Polθ-mediated end joining (TMEJ). Additionally, population genetic datasets have revealed a preponderance of TMEJ signatures associated with human variation. Since these signatures are the result of the combined effect of TMEJ and DSB formation in spermatozoa and male pronuclei, we used a BLISS-based protocol to analyze recurrent DSBs in mature human sperm heads as a proxy of the male pronucleus before zygotic chromatin remodeling. The DSBs were found to be enriched in (YR)n short tandem repeats and in evolutionarily young SINEs, reminiscent to patterns observed in murine spermatids, indicating evolutionary hotspots of recurrent DSB formation in mammalian spermatozoa. Additionally, we detected a similar DSB pattern in diploid human IMR90 cells when cNHEJ was selectively inhibited, indicating the significant impact of absent cNHEJ on the sperm DSB landscape. Strikingly, regions associated with most retained histones, and therefore less condensed chromatin, were not strongly enriched with recurrent DSBs. In contrast, the fraction of retained H3K27me3 in the mature spermatozoa displayed a strong association with recurrent DSBs. DSBs in H3K27me3 are associated with a preference for TMEJ over cNHEJ during repair. We hypothesize that the retained H3K27me3 may trigger transgenerational DNA repair by priming maternal Polθ to these regions.

Application prospects of the 2BS cell-adapted China fixed rabies virus vaccine strain 2aG4-B40

BackgroundRabies is a fatal zoonotic disease whose pathogenesis has not been fully elucidated, and vaccination is the only effective method for protecting against rabies virus infection. Most inactivated vaccines are produced using Vero cells, which are African green monkey kidney cells, to achieve large-scale production. However, there is a potential carcinogenic risk due to nonhuman DNA contamination. Thus, replacing Vero cells with human diploid cells may be a safer strategy. In this study, we developed a novel 2BS cell-adapted rabies virus strain and analysed its sequence, virulence and immunogenicity to determine its application potential as a human diploid cell inactivated vaccine.Methods and resultsThe 2BS cell-adapted rabies virus strain 2aG4-B40 was established by passage for 40 generations and selection of plaques in 2BS cells. RNA sequence analysis revealed that mutations in 2BS cell-adapted strains were not located at key sites that regulate the production of neutralizing antibodies or virulence in the aG strain (GQ412744.1). The gradual increase in virulence (remaining above 7.0 logLD50/ml from the 40th to 55th generation) and antigen further indicated that these mutations may increase the affinity of the adapted strains for human diploid cells. Identification tests revealed that the 2BS cell-adapted virus strain was neutralized by anti-rabies serum, with a neutralization index of 19,952. PrEP and PEP vaccination and the NIH test further indicated that the vaccine prepared with the 2aG4-B40 strain had high neutralizing antibody levels (2.24 to 46.67 IU/ml), immunogenicity (protection index 270) and potency (average 11.6 IU/ml).ConclusionsIn this study, a 2BS cell-adapted strain of the 2aG4 rabies virus was obtained by passage for 40 generations. The results of sequencing analysis and titre determination of the adapted strain showed that the mutations in the adaptive process are not located at key sequence regions of the virus, and these mutations may enhance the affinity of the adapted strain for human diploid cells. Moreover, vaccines made from the adapted strain 2aG4-B40 had high potency and immunogenicity and could be an ideal candidate rabies virus strain for inactivated vaccine preparation.

P-806 Heritable genome editing in haploid mouse embryonic stem cells

Abstract Study question Can we edit a specific gene by CRISPR-Cas9 in haploid mouse embryonic stem cells? Summary answer We successfully carried out heritable genome editing of a coat pigmentation gene in haploid mouse embryonic stem cells via electroporation. What is known already Embryonic stem cells have been widely used as a substrate to carry out genetic studies due to their availability, low cost and ease of handling. CRISPR-Cas9 has been successful in editing genes in the human model. Gene editing occurred by homology directed repair or non-homologous end joining. The utilization of a haploid genome would confirm the feasibility of non-homologous end joining repair mechanism. Study design, size, duration In the past 3 months, diploid as positive control and experimental haploid embryonic stem cells were independently cultured for genome editing experiments. Electroporation was used as a means to introduce CRISPR-Cas9 into cells. The negative control cohort consisted of embryonic stem cells electroporated without delivering any CRISPR solution. CRISPR-Cas9 was designed to knock out the Tyr gene to create an albino phenotype. Participants/materials, setting, methods Diploid embryonic stem cells were derived from C57BL/6 mice. The haploid counterpart instead was obtained from haploid androgenetic embryos using spermatozoa from Oct4-EGFP mice. CRISPR solution was prepared by combining Tyr gRNA and Cas9 protein. For each experiment, one million cells were suspended in CRISPR solution and an electroporation buffer. Then, electric pulses were applied for electroporation with 4 different parameters. Gene modification at the target site was confirmed using T7E1 cleavage assay. Main results and the role of chance A 584 base pairs region around the CRISPR target site was amplified in all the diploid and haploid mouse embryonic stem cells. After performing T7E1 cleavage assay, editing efficiency was calculated following the manufacturer protocol. Precisely, after testing out different electroporation conditions on diploid mESCs, 1350V/20ms/2 pulses together with the combination of three sgRNAs (exon 1 and exon 2) and Cas9 protein, provided the best genome editing efficiency at 36.8% with a viability of 80%. Interestingly, when delivering only one type of gRNA (exon 2) together with Cas9 protein, we saw an efficiency rising to 77.6%. We then used the same condition to edit the Tyr gene in the genome of haploid androgenetic stem cells and confirmed that also in these cells, we were able to obtain a comparable genome editing efficiency at 68.4%. Limitations, reasons for caution While these experimental observations are limited, it is encouraging that it is possible to target and edit a haploid genome with a single allele. Wider implications of the findings The utilization of a haploid cell culture model paves the way to utilize CRISPR-Cas9 for gamete genome editing. This will provide a consistent and reliable method to correct inheritable genetic conditions on gametes allowing uniform gene editing of all cell progeny. Trial registration number Not applicable

Characterization and arbovirus susceptibility of cultured CERNI cells derived from sika deer (Cervus nippon).

Cervus nippon (sika deer) are widely distributed throughout eastern Asia. Deer possess a variety of antibodies against several zoonotic pathogens, indicating that they act as reservoir of zoonoses. In this study, we reported the characterization of cultured cells derived from sika deer and evaluated their susceptibility to arthropod-borne viruses to clarify their usefulness in virological studies. Cells derived from testicular tissue in Dulbecco's modified eagle medium with 16% fetal bovine serum started growing as primary cultured cells. The diploid cells consisted of 68 chromosomes, consistent with those of Japanese sika deer previously reported. The phylogenetic analysis showed the cells formed a robust clade with Japanese population of C. nippon, indicating that the cultured cells established in this study were originated from the Japanese sika deer. The cells immortalized by the simian virus 40 T-antigen were predominantly spindle-shaped cells exhibiting adhesive properties, and cultivated at 37°C and 5% CO2, which are common culture conditions for many mammalian cell lines. Western blotting analysis indicated that the cultured cells were multiple types of cells that coexist, including at least epithelial, fibroblast, and also Leydig cells. We confirmed that the cells have susceptibility to several arboviruses distributed in Japan: Getah virus, Japanese encephalitis virus, Oz virus, and severe fever with thrombocytopenia syndrome virus, but not to Tarumiz tick virus. From these results, the cells contribute to clarify the role of sika deer as a reservoir of zoonoses in nature and deer-associated experimental research at the cellular and molecular levels.

P-796 A novel method of somatic cell nuclear transfer to generate artificial oocytes

Abstract Study question Can we identify an alternative method to Sendai-Virus fusion for somatic cell nuclear transfer (SCNT) aiming at generating artificial oocyte? Summary answer The utilization lipofectamine proved itself as a valid alternative method to virus-mediated SCNT, allowing successful membrane fusion and supporting proper embryo development. What is known already SCNT involves transferring a diploid cell’s nucleus into an enucleated oocyte. Early attempts of SCNT involves direct injection and electrofusion but both approaches can affect oolemma integrity. To avoid mechanical damage to oolemma, fusogenic agents have been employed for SCNT including polyethylene glycol (PEG) and more efficient Hemagglutinating Virus of Japan Envelope (HVJ-E). However, despite its high efficiency, HVJ-E is difficult to be used in human because it is a virus derivative, and a recent shortage of this compound made it not readily available. On the other hand, cationic phospholipid is fusogenic and is widely utilized in transfection experiments. Study design, size, duration In the past 4 months, SCNT was carried out through PEG-mediated, HVJ-E-mediated, or novel lipofectamine-mediated fusion using cumulus cells as nuclear donor. Post-SCNT oocytes were inseminated and cultured up to the blastocyst stage. Embryo development and morphokinetics were assessed and compared among the three SCNT experimental cohorts. In some cases, blastocysts were transferred into pseudo-pregnant mice, post-natal development were monitored. Fertilization, embryo development, and offspring wellbeing were monitored. Participants/materials, setting, methods Oocytes from B6D2F1 mice were enucleated by micromanipulation. The ooplasts were allocated to 3 cohorts: 1) exposure to 45% PEG, 2) subzonal transfer of donor cell with HVJ-E, or 3) subzonal transfer the donor cell exposed to 1.75% lipofectamine. Reconstructed oocytes were monitored for fusion 30 minutes after exposure to fusogens and inseminated by spermatozoa from B6-EGFP mice to confirm paternal genome provenance. Fertilization and embryo development were monitored in a time-lapse system. Main results and the role of chance A total of 165 oocytes were enucleated and 160 (97.0%) survived and allocated to PEG-fusion (n = 50), Sendai-virus-fusion (n = 41) and lipofecamine-fusion (n = 80) cohorts. Fusion rate was 56.0% (28/50) for the PEG-fusion cohort, while Sendai-virus and lipofectamine-fusion cohorts yielded higher fusion rate at 92.7%(38/41) and 86.3%(69/80), respectively (P<0.0001). Control oocytes yielded 96.7% (145/150) fertilization rate and developed to 2-cell stage at 93.8% (136/145), and yielded an eventual blastocyst rate at 80.7% (117/145). Despite a lower oocyte reconstruction rate, PEG-fusion cohort fertilized at 89.3% (25/28), but cleavage rate to 2-cell stage was lower at 48.0% (12/25, P<0.0001) and did not yield any blastocyst. The HVJ-E cohort yielded a fertilization rate of 94.7% (36/38) and cleavage rate at 97.2% (35/36) similar to control. The blastocyst rate was observed at 22.9% (8/35, P<0.0001) when comparing to control. Lipofectamine-fusion cohort yielded 91.3% (63/69) fertilization rate and subsequent cleavage rate at 95.2% (60/63). Albeit at a lower blastocyst development rate comparing to control, the lipofectamine cohort yielded similar full-preimplantation rate at 19.0% (12/63) when comparing to HVJ-E cohort. All blastocysts (n = 20) were transferred into 2 pseudo-pregnant mice and 3 healthy female offspring were yielded to date. Limitations, reasons for caution HVJ-E mediated membrane fusion has been well established with proven safety record; however, its recent shortage has led to a temporary cessation of its utilization. Our lipofectamine-mediated cell fusion technique yielded satisfactory fusion, fertilization, blastocyst formation and some live offspring, however the safety and efficacy still remain to be validated. Wider implications of the findings This novel lipofectamine-mediated cell fusion has broader implications for advancing cloning technologies. Lipofection is a non-viral approach, overcomes shortage of HVJ-E and therefore propose itself as a more appealing cell fusion method for human SCNT. Trial registration number N/A

Immunogenicity of 4-dose Essen intramuscular regimen for rabies post-exposure prophylaxis: A multi-center cross-sectional study in China

BackgroundThe 4-dose Essen intramuscular (IM) regimen for rabies post-exposure prophylaxis (PEP) has been recommended by Advisory Committee on Immunization Practices (ACIP) and World Health Organization (WHO), but the large-sample clinical evidence is still limited. MethodRabies virus neutralizing antibodies of 11,752 patients were detected from 409 rabies prevention clinics in 27 provinces in China. Patients with serum collected before or no later than 1 h after injection on the day of the fifth dose (day 28) of 5-dose Essen regimen were included in Group A to observe the immune efficacy of 4-dose Essen IM regimen, and patients with serum collected 14–28 days after injection of the fifth dose were included in Group B to observe the immune efficacy of 5-dose Essen IM regimen. ResultsFinally, 2351 cases met the inclusion and exclusion criteria, including 2244 cases in Group A and 107 cases in Group B. The antibody titer of Group A was higher than that of Group B [12.21 (4.15, 32.10) IU/ml vs. 9.41 (3.87, 27.38) IU/ml] (P = 0.002). In Group A, the median antibody titers were 4.01IU/ml, 11.63IU/ml and 29.46IU/ml in patients vaccinated with purified hamster kidney cell vaccine (PHKCV), purified Vero cell vaccine (PVRV), and human diploid cell rabies vaccine (HDCV), respectively, with statistical significance (P < 0.001). ConclusionsThe 4-dose Essen IM regimen could provide satisfactory immune effect, and HDCV induced higher antibody titer than PHKCV or PVRV.

Waves of regulated protein expression and phosphorylation rewire the proteome to drive gametogenesis in budding yeast

Sexually reproducing eukaryotes employ a developmentally regulated cell division program-meiosis-to generate haploid gametes from diploid germ cells. To understand how gametes arise, we generated a proteomic census encompassing the entire meiotic program of budding yeast. We found that concerted waves of protein expression and phosphorylation modify nearly all cellular pathways to support meiotic entry, meiotic progression, and gamete morphogenesis. Leveraging this comprehensive resource, we pinpointed dynamic changes in mitochondrial components and showed that phosphorylation of the FoF1-ATP synthase complex is required for efficient gametogenesis. Furthermore, using cryoET as an orthogonal approach to visualize mitochondria, we uncovered highly ordered filament arrays of Ald4ALDH2, a conserved aldehyde dehydrogenase that is highly expressed and phosphorylated during meiosis. Notably, phosphorylation-resistant mutants failed to accumulate filaments, suggesting that phosphorylation regulates context-specific Ald4ALDH2 polymerization. Overall, this proteomic census constitutes a broad resource to guide the exploration of the unique sequence of events underpinning gametogenesis.

Multi-signal regulation of the GSK-3β homolog Rim11 controls meiosis entry in budding yeast

Starvation in diploid budding yeast cells triggers a cell-fate program culminating in meiosis and spore formation. Transcriptional activation of early meiotic genes (EMGs) hinges on the master regulator Ime1, its DNA-binding partner Ume6, and GSK-3β kinase Rim11. Phosphorylation of Ume6 by Rim11 is required for EMG activation. We report here that Rim11 functions as the central signal integrator for controlling Ume6 phosphorylation and EMG transcription. In nutrient-rich conditions, PKA suppresses Rim11 levels, while TORC1 retains Rim11 in the cytoplasm. Inhibition of PKA and TORC1 induces Rim11 expression and nuclear localization. Remarkably, nuclear Rim11 is required, but not sufficient, for Rim11-dependent Ume6 phosphorylation. In addition, Ime1 is an anchor protein enabling Ume6 phosphorylation by Rim11. Subsequently, Ume6-Ime1 coactivator complexes form and induce EMG transcription. Our results demonstrate how various signaling inputs (PKA/TORC1/Ime1) converge through Rim11 to regulate EMG expression and meiosis initiation. We posit that the signaling-regulatory network elucidated here generates robustness in cell-fate control.

New thiazole derivatives linked to pyridine, fused pyridine, pyrimidine and thiazolopyrimidine scaffolds with potential dual anticancer and antimicrobial activities: Design, synthesis and docking simulation

The present study involved the design and synthesis of a novel series of thiazole derivatives 3, 5, 7, 8, 10–13 having various heterocyclic scaffolds, including pyrazole, pyridine, and/or pyrimidine. Upon screening of their antiproliferative efficacy, pyrazolo[3,4-b]pyridine 5a,b and pyrimidine 10a,b, afforded excellent activity against cancerous HepG-2 and MCF-7 cell lines with safety borders against normal human diploid cell line WI-38 comparing with doxorubicin and erlotinib. Then, their impact upon the apoptotic indicators; caspase-3, Bax and Bcl-2 was assessed. Regarding to antimicrobial activities, all screened Gram-positive and Gram-negative bacteria and fungal strains except Candida albicans, were effectively inhibited by the thiazole moiety bearing pyrazolo[3,4-b]pyridine 5a,b and pyrimidine 10a,b. Additionally, thiazole-pyrazolo[3,4-b]pyridine 5b exhibited remarkable and superior inhibitory properties against EGFR and E. coli DNA gyrase (IC50 = 0.3‏5 ± 0.‏25 and ‏‏2‏‏‏.55‏ ± 0.03 µM, respectively) in comparison with references erlotinib and novobiocin (IC50 = 0.27 ± 0.02 and 3.78 ± 0.02 µM, respectively). In order to provide improved optimization and rationalization of effective novel anticancer and antimicrobial leads, in silico ADMET and docking investigations were ultimately constructed.

Abstract B003: Identification of novel genes that regulate aneuploidy tolerance by attenuating aneuploidy-induced stresses

Abstract Aneuploidy, an imbalanced number of chromosomes or chromosome arms, is the most common genetic aberration in cancer and is associated with a worse prognosis and advanced disease. However, in non-transformed cells, aneuploidy has a substantial fitness cost, leading to cell cycle arrest and cell death. This fitness cost is the result of several cellular stresses associated with aneuploidy, including proteotoxic, metabolic, mitotic and replication stress. Therefore, for aneuploidy to become beneficial, tolerance mechanisms that enable cancer cells to cope with aneuploidy-induced cellular stresses must come into play. Such mechanisms may uncover novel synthetic lethalities of aneuploid cancer cells. To this end, we systematically dissected the cellular mechanisms that facilitate aneuploidy tolerance. We combined large-scale analyses of gene expression, as well as genetic and pharmacological dependency datasets from human tumors, cancer cell lines and non-transformed cells, to identify genes and pathways associated with aneuploidy tolerance. We identified 55 genes associated (with high confidence) with aneuploidy tolerance. Gene set enrichment analysis (GSEA) revealed an enrichment for ribosome biogenesis and translation, DNA repair, replication stress and the p53 pathway. Interestingly, these pathways were all previously implicated with the aforementioned aneuploidy-induced cellular stresses. We plan to conduct a dependency mini-screen to validate the top ‘hits’ from our comprehensive bioinformatic analysis, and to explore the role of validated candidates in conferring aneuploidy tolerance. One of the top ‘hits’ of our exploratory analysis was a novel gene with an unclear cellular function. We experimentally confirmed that this gene was over-expressed in aneuploid cells, across several isogenic systems of matched diploid-aneuploid cell lines. Aneuploidy induction using MPS1 inhibitors led to its significant upregulation, demonstrating that its increased expression is a general feature of aneuploid cells. Mechanistically, we found that the novel gene had a role in regulating chromosome segregation, as its knockdown increased the rate of mitotic aberrations – as well as the sensitivity to mitotic checkpoint inhibition – resulting in increased chromosomal instability. We now study the molecular basis for the effect of this novel gene on aneuploidy tolerance. Specifically, we knocked it down in aneuploid cells, and over-expressed it in diploid cells, and we currently explore how these manipulations affect the above-mentioned aneuploidy tolerance pathways, namely replication stress, ribosome biogenesis, DNA damage repair and tp53 activation. In summary, we revealed a novel gene that is a key regulator of aneuploidy tolerance in cancer cells, likely due to its direct involvement in pathways required for the cellular coping with aneuploidy-induced stresses. Citation Format: Yonatan Eliezer, Adi V Tarrab, Tal Ben-Yishay, Hajime Okada, Tom Winkler, Uri Ben-David. Identification of novel genes that regulate aneuploidy tolerance by attenuating aneuploidy-induced stresses [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Expanding and Translating Cancer Synthetic Vulnerabilities; 2024 Jun 10-13; Montreal, Quebec, Canada. Philadelphia (PA): AACR; Mol Cancer Ther 2024;23(6 Suppl):Abstract nr B003.