Variations in heterochromatin content reveal important polymorphisms for studies of genetic improvement in garlic (Allium sativum L.)

Allium sativum L. is an herb of the Alliaceae family with a specific taste and aroma and medicinal and nutraceutical properties that are widely marketed in several countries. Brazil is one of the largest importers of garlic in the world, despite of its production is restricted and limited to internal consumption. Thus, explore the genetic diversity of commercial garlic conserved at germplasm banks is essential to generate additional genetic information about its economically important crop. A suitable tool for this purpose is the cytogenetic characterisation of these accessions. This study aimed to characterise the cytogenetic diversity among seven accessions of garlic from a Germplasm Bank in Brazil. The karyotypes were obtained by conventional staining and with chromomycin A3 (CMA) and 4,6-diamidino-2-phenylindole (DAPI) fluorochromes. All accessions analysed showed chromosome number 2n = 16, karyotype formula 6M+2SM, symmetrical karyotypes, reticulate interphase nuclei, and chromosomes with uniform chromatin condensation from prophase to metaphase. The fluorochromes staining showed differences in the amount and distribution of heterochromatin along the chromosomes and between accessions studied. Based on the distribution pattern of these small polymorphisms, it was possible to separate the seven accessions into three groups. It was also possible to differentiate some of the accessions individually. One of the results obtained showed a heteromorphic distension of the nucleolar organiser region observed on the chromosome pairs 6 or 7 with peculiar characteristics. It was suggested for example, that the heteromorphic block of heterochromatin (CMA+++/DAPI-) on chromosome 6 of the “Branco Mineiro Piauí” accession can be used as a marker to identify this genotype or may be associated with some character of economic interest.

Multiple parameters shape the 3D chromatin structure of single nuclei

The spatial organization of chromatin at the scale of topologically associating domains (TADs) and below displays large cell-to-cell variations. Up until now, how this heterogeneity in chromatin conformation is shaped by chromatin condensation, TAD insulation, and transcription has remained mostly elusive. Here, we used Hi-M, a multiplexed DNA-FISH imaging technique providing developmental timing and transcriptional status, to show that the emergence of TADs at the ensemble level partially segregates the conformational space explored by single nuclei during the early development of Drosophila embryos. Surprisingly, a substantial fraction of nuclei displayed strong insulation even before TADs emerged. Moreover, active transcription within a TAD led to minor changes to the local inter- and intra-TAD chromatin conformation in single nuclei and only weakly affected insulation to the neighboring TAD. Overall, our results indicate that multiple parameters contribute to shaping the chromatin architecture of single nuclei at the TAD scale.

High fat diet-induced obesity prolongs critical stages of the spermatogenic cycle in a Ldlr−/−.Leiden mouse model

Obesity can disturb spermatogenesis and subsequently affect male fertility and reproduction. In our study, we aim to elucidate at which cellular level of adult spermatogenesis the detrimental effects of obesity manifest. We induced high fat diet (HFD) obesity in low-density lipoprotein receptor knock-out Leiden (Ldlr−/−.Leiden) mice, and studied the morphological structure of the testes and histologically examined the proportion of Sertoli cells, spermatocytes and spermatids in the seminiferous tubules. We examined sperm DNA damage and chromatin condensation and measured plasma levels of leptin, testosterone, cholesterol and triglycerides. HFD-induced obesity caused high plasma leptin and abnormal testosterone levels and induced an aberrant intra-tubular organisation (ITO) which is associated with an altered spermatids/spermatocytes ratio (2:1 instead of 3:1). Mice fed a HFD had a higher level of tubules in stages VII + VIII in the spermatogenic cycle. The stages VII + VII indicate crucial processes in spermatogenic development like initiation of meiosis, initiation of spermatid elongation, and release of fully matured spermatids. In conclusion, HFD-induced obese Ldlr−/−.Leiden mice develop an aberrant ITO and alterations in the spermatogenic cycle in crucial stages (stages VII and VII). Thereby, our findings stress the importance of lifestyle guidelines in infertility treatments.

Characterization of cisplatin-loaded chitosan nanoparticles and rituximab-linked surfaces as target-specific injectable nano-formulations for combating cancer

The present study was carried out to develop cisplatin-loaded chitosan nanoparticles (CCNP) and cisplatin-loaded chitosan nanoparticle surface linked to rituximab (mAbCCNP) as targeted delivery formulations. The two formulations (CCNP and mAbCCNP) exhibited significant physicochemical properties. The zetapotential (ZP) values of CCNP and mAbCCNP were 30.50 ± 5.64 and 26.90 ± 9.09 mV, respectively; while their particle sizes were 308.10 ± 1.10 and 349.40 ± 3.20 z.d.nm, respectively. The poly dispersity index (PDI) of CCNP was 0.257 ± 0.030 (66.6% PDI), while that of mAbCCNP was 0.444 ± 0.007 (57.60% PDI). Differential scanning calorimetry (DSC) revealed that CCNP had endothermic peaks at temperatures ranging from 135.50 to 157.69 °C. A sharp exothermic peak was observed at 95.79 °C, and an endothermic peak was observed at 166.60 °C. The XRD study on CCNP and mAbCCNP revealed distinct peaks at 2θ. Four peaks at 35.38°, 37.47°, 49.29°, and 59.94° corresponded to CCNP, while three distinct peaks at 36.6°, 49.12°, and 55.08° corresponded to mAbCCNP. The in vitro release of cisplatin from nanoparticles followed zero order kinetics in both CCNP and mAbCCNP. The profile for CCNP showed 43.80% release of cisplatin in 6 h (R2 = 0.9322), indicating linearity of release with minimal deviation. However, the release profile of mAbCCNP showed 22.52% release in 4 h (R2 = 0.9416), indicating linearity with sustained release. In vitro cytotoxicity studies on MCF-7 ATCC human breast cancer cell line showed that CCNP exerted good cytotoxicity, with IC50 of 4.085 ± 0.065 µg/mL. However, mAbCCNP did not elicit any cytotoxic effect. At a dose of 4.00 µg/mL cisplatin induced early apoptosis and late apoptosis, chromatin condensation, while it produced secondary necrosis at a dose of 8.00 µg/mL. Potential delivery system for cisplatin CCNP and mAbCCNP were successfully formulated. The results indicated that CCNP was a more successful formulation than mAbCCNP due to lack of specificity of rituximab against MCF-7 ATCC human breast cancer cells.

Knockdown of NRMT enhances sensitivity of retinoblastoma cells to cisplatin through upregulation of the CENPA/Myc/Bcl2 axis

Chemotherapy resistance of tumor cells causes failure in anti-tumor therapies. Recently, N-terminal regulator of chromatin condensation 1 methyltransferase (NRMT) is abnormally expressed in different cancers. Hence, we speculate that NRMT may pay a crucial role in the development of chemosensitivity in retinoblastoma. We characterized the upregulation of NRMT in the developed cisplatin (CDDP)-resistant retinoblastoma cell line relative to parental cells. Loss-of-function experiments demonstrated that NRMT silencing enhanced chemosensitivity of retinoblastoma cells to CDDP. Next, NRMT was identified to enrich histone-H3 lysine 4 trimethylation in the promoter of centromere protein A (CENPA) by chromatin immunoprecipitation assay. Rescue experiments suggested that CENPA reduced chemosensitivity by increasing the viability and proliferation and reducing apoptosis of CDDP-resistant retinoblastoma cells, which was reversed by NRMT. Subsequently, CENPA was witnessed to induce the transcription of Myc and to elevate the expression of B cell lymphoma-2. At last, in vivo experiments confirmed the promotive effect of NRMT knockdown on chemosensitivity of retinoblastoma cells to CDDP in tumor-bearing mice. Taken together, NRMT is an inhibitor of chemosensitivity in retinoblastoma. Those findings shed new light on NRMT-targeted therapies for retinoblastoma.

Histone deacetylase inhibitor overrides the effect of soft hydrogel on the mechanoresponse of human mesenchymal stem cells

Human Mesenchymal cells (hMSCs) are promising in regenerative medicine for their multi-lineage differentiation capability. It has been demonstrated that lineage specification is governed by both chemical and mechanical cues. Among all the different mechanical cues known to control hMSCs fate, substrate stiffness is the most well-studied. It has been shown that the naive mesenchymal stem cells when cultured on soft gel, they commit towards adipogenic lineage while when cultured on stiff gel they become osteogenic. Soft substrates also cause less cell spreading, less traction, less focal adhesion assembly and stress fibre formation. Furthermore, chromatin condensation increases when cells are cultured on soft substrates. As the nucleus has been postulated to be mechanosensor and mechanotransducer, in this paper we asked the question how mechanosensing and mechanoresponse process will be influenced if we change the chromatin condensation by using an external chemical stimulus. To address this question, we treated hMSCs cultured on soft polyacrylamide (PA) gels with a histone deacetylase inhibitor (HDACi) called Valproic Acid (VA) which decondense the chromatin by hyperacetylation of histone proteins. We found that the treatment with VA overrides the effect of soft substrates on hMSCs morphology, cellular traction, nuclear localization of mechnosensory protein YAP, and differentiation. VA treated cells behaved as if they are on stiff substrates in all aspects tested here. Furthermore, we have shown that VA controls hMSCs differentiation via activation of ERK/MAPK pathway by increasing the p-ERK expression which inhibits adipogenic differentiation potential of mesenchymal stem cells. Collectively, these findings for the first time demonstrate that inhibiting histone acetylation can override the mechanoresponse of hMSCs. This work will help us to fundamentally understand the mechanosignalling process and to control the hMSCs differentiation in tissue engineering and regenerative medicine.

Offspring production of haploid spermatid-like cells derived from mouse female germline stem cells with chromatin condensation

Background During male meiosis, the Y chromosome can form perfect pairing with the X chromosome. However, it is unclear whether mammalian Female germline stem cells (FGSCs) without a Y chromosome can transdifferentiate into functional haploid spermatid-like cells (SLCs). Results We found that spermatogenesis was restarted by transplanting FGSCs into Kitw/wv mutant testes. Complete meiosis and formation of SLCs was induced in vitro by testicular cells of Kitw/wv mutant mice, cytokines and retinoic acid. Healthy offspring were produced by sperm and SLCs derived from the in vivo and in vitro transdifferentiation of FGSCs, respectively. Furthermore, high-throughput chromosome conformation capture sequencing(Hi-C-seq) and “bivalent” (H3K4me3-H3K27me3) micro chromatin immunoprecipitation sequencing (μChIP-seq) experiments showed that stimulated by retinoic acid gene 8 (STRA8)/protamine 1 (PRM1)-positive transdifferentiated germ cells (tGCs) and male germ cells (mGCs) display similar chromatin dynamics and chromatin condensation during in vitro spermatogenesis. Conclusion This study demonstrates that sperm can be produced from FGSCs without a Y chromosome. This suggests a strategy for dairy cattle breeding to produce only female offspring with a high-quality genetic background.

Transient nuclear lamin A/C accretion aids in recovery from vapor nanobubble-induced permeabilisation of the plasma membrane

Vapor nanobubble (VNB) photoporation is a physical method for intracellular delivery that has gained significant interest in the past decade. It has successfully been used to introduce molecular cargo of diverse nature into different cell types with high throughput and minimal cytotoxicity. For translational purposes, it is important to understand whether and how photoporation affects cell homeostasis. To obtain a comprehensive view on the transcriptional rewiring that takes place after VNB photoporation, we performed a longitudinal shotgun RNA-sequencing experiment. Six hours after photoporation, we found a marked upregulation of LMNA transcripts as well as their protein products, the A-type lamins. At the same time point, we observed a significant increase in several heterochromatin marks, suggesting a global stiffening of the nucleus. These molecular features vanished 24 h after photoporation. Since VNB-induced chromatin condensation was prolonged in LMNA knockout cells, A-type lamins may be required for restoring the nucleus to its original state. Selective depletion of A-type lamins reduced cell viability after VNB photoporation, while pharmacological stimulation of LMNA transcription increased the percentage of successfully transfected cells that survived after photoporation. Therefore, our results suggest that cells respond to VNB photoporation by temporary upregulation of A-type lamins to facilitate their recovery.

Relevance of AIF/CypA Lethal Pathway in SH-SY5Y Cells Treated with Staurosporine

The AIF/CypA complex exerts a lethal activity in several rodent models of acute brain injury. Upon formation, it translocates into the nucleus of cells receiving apoptotic stimuli, inducing chromatin condensation, DNA fragmentation, and cell death by a caspase-independent mechanism. Inhibition of this complex in a model of glutamate-induced cell death in HT-22 neuronal cells by an AIF peptide (AIF(370-394)) mimicking the binding site on CypA, restores cell survival and prevents brain injury in neonatal mice undergoing hypoxia-ischemia without apparent toxicity. Here, we explore the effects of the peptide on SH-SY5Y neuroblastoma cells stimulated with staurosporine (STS), a cellular model widely used to study Parkinson’s disease (PD). This will pave the way to understanding the role of the complex and the potential therapeutic efficacy of inhibitors in PD. We find that AIF(370-394) confers resistance to STS-induced apoptosis in SH-SY5Y cells similar to that observed with CypA silencing and that the peptide works on the AIF/CypA translocation pathway and not on caspases activation. These findings suggest that the AIF/CypA complex is a promising target for developing novel therapeutic strategies against PD.

FRET-FISH probes chromatin compaction at individual genomic loci in single cells

The density or compaction of chromatin throughout the cell nucleus is a key biophysical property that influences DNA replication, transcription, and repair. Chromatin accessibility is often used as a proxy for chromatin compaction or density, however it is not clear how these two properties relate to each other, given the lack of tools for directly probing compaction at defined genomic loci. To fill in this gap, here we developed FRET-FISH, a microscopy-based method combining fluorescence resonance energy transference (FRET) with DNA fluorescence in situ hybridization (FISH) to probe chromatin compaction at selected loci in single cells. We optimized FRET-FISH by testing different probe designs in situ in fixed cells, readily detecting FRET generated by DNA FISH probes. To validate FRET-FISH, we compared it with ATAC-seq and Hi-C, demonstrating that local chromatin compaction and accessibility are strongly correlated and that the frequency of intra-genic contacts measured by Hi-C may be an even better proxy for local chromatin density. To further validate FRET-FISH, we showed that it can detect expected differences in chromatin compaction along the nuclear radius, with peripheral loci being more compacted and central ones less compacted. Lastly, we assessed the sensitivity of FRET-FISH, demonstrating its ability to reproducibly detect differences in chromatin density (i) upon treatment of cells with drugs that perturb global chromatin condensation; (ii) during prolonged cell culture; and (iii) in different phases of the cell cycle. We conclude that FRET-FISH is a robust tool for probing chromatin compaction at selected loci in single cells and for studying inter-allelic and cell-to-cell variability in chromatin density.