Combining metaphase cytogenetics with single nucleotide polymorphism arrays can improve the diagnostic yield and identify prognosis more precisely in myelodysplastic syndromes.

BACKGROUND: Myelodysplastic syndromes (MDS) encompass a group of heterogeneous haematopoietic stem cell malignancies characterised by ineffective haematopoiesis, cytological aberrations, and a propensity for progression to acute myeloid leukaemia. Diagnosis and disease prognostic stratification are much based on genomic abnormalities. The traditional metaphase cytogenetics analysis (MC) can detect about 40-60% aberrations. Single-nucleotide polymorphism arrays (SNP-A) karyotyping can detect copy number variations with a higher resolution and has a unique advantage in detection of copy number neutral loss of heterozygosity (CN-LOH). Combining these two methods may improve the diagnostic efficiency and accuracy for MDS. METHODS: We retrospectively analysed the data of 110 MDS patients diagnosed from January 2012 to December 2019 to compare the detection yield of chromosomal abnormalities by MC with by SNP-A, and the relationship between chromosomal abnormalities and prognosis. RESULTS: Our results showed that SNP-A improved the detection yield of chromosomal aberrations compared with MC (74.5 vs. 55.5%, p < .001). In addition, the positive yield could be further improved by combining MC with SNP-A to 77.3%, compared with MC alone. Univariate analysis showed that age >65 years, bone marrow blasts ≥5%, with acquired CN-LOH, new aberrations detected by SNP-A, TGA value > the median (81.435 Mb), higher risk by IPSS-R-MC, higher risk by IPSS-R-SNP-A all had poorer prognosis. More critically, multivariable analysis showed that age >65 years and higher risk by IPSS-R-SNP-A were independent predictors of inferior OS in MDS patients. CONCLUSION: The combination of MC and SNP-A based karyotyping can further improve the diagnostic yield and provide more precise prognostic stratification in MDS patients. However, SNP-A may not completely replace MC because of its inability to detect balanced translocation and to detect different clones. From a practical point of view, we recommend the concurrent use of SNP-A and MC in the initial karyotypic evaluation for MDS patients on diagnosis and prognosis stratification.KEY MESSAGESSNP-A based karyotyping can further improve the MDS diagnostic yield and provide more precise prognostic stratification in MDS patients.Acquired CN-LOH is a characteristic chromosomal aberration of MDS, which should be integrated to the diagnostic project of MDS.The concurrent use of SNP-A and MC in the initial karyotypic evaluation for MDS patients can be recommended.

Stochastic chromatin packing of 3D mitotic chromosomes revealed by coherent X-rays.

DNA molecules are atomic-scale information storage molecules that promote reliable information transfer via fault-free repetitions of replications and transcriptions. Remarkable accuracy of compacting a few-meters-long DNA into a micrometer-scale object, and the reverse, makes the chromosome one of the most intriguing structures from both physical and biological viewpoints. However, its three-dimensional (3D) structure remains elusive with challenges in observing native structures of specimens at tens-of-nanometers resolution. Here, using cryogenic coherent X-ray diffraction imaging, we succeeded in obtaining nanoscale 3D structures of metaphase chromosomes that exhibited a random distribution of electron density without characteristics of high-order folding structures. Scaling analysis of the chromosomes, compared with a model structure having the same density profile as the experimental results, has discovered the fractal nature of density distributions. Quantitative 3D density maps, corroborated by molecular dynamics simulations, reveal that internal structures of chromosomes conform to diffusion-limited aggregation behavior, which indicates that 3D chromatin packing occurs via stochastic processes.

New Data on Organization and Spatial Localization of B-Chromosomes in Cell Nuclei of the Yellow-Necked Mouse Apodemus flavicollis.

The gene composition, function and evolution of B-chromosomes (Bs) have been actively discussed in recent years. However, the additional genomic elements are still enigmatic. One of Bs mysteries is their spatial organization in the interphase nucleus. It is known that heterochromatic compartments are not randomly localized in a nucleus. The purpose of this work was to study the organization and three-dimensional spatial arrangement of Bs in the interphase nucleus. Using microdissection of Bs and autosome centromeric heterochromatic regions of the yellow-necked mouse (Apodemus flavicollis) we obtained DNA probes for further two-dimensional (2D)- and three-dimensional (3D)- fluorescence in situ hybridization (FISH) studies. Simultaneous in situ hybridization of obtained here B-specific DNA probes and autosomal C-positive pericentromeric region-specific probes further corroborated the previously stated hypothesis about the pseudoautosomal origin of the additional chromosomes of this species. Analysis of the spatial organization of the Bs demonstrated the peripheral location of B-specific chromatin within the interphase nucleus and feasible contact with the nuclear envelope (similarly to pericentromeric regions of autosomes and sex chromosomes). It is assumed that such interaction is essential for the regulation of nuclear architecture. It also points out that Bs may follow the same mechanism as sex chromosomes to avoid a meiotic checkpoint.

GAS6 ameliorates advanced age-associated meiotic defects in mouse oocytes by modulating mitochondrial function.

Previously, we reported that the silencing of growth arrest-specific gene 6 (Gas6) expression in oocytes impairs cytoplasmic maturation by suppressing mitophagy and inducing mitochondrial dysfunction, resulting in fertilization failure. Here, we show that oocyte aging is accompanied by an increase in meiotic defects associated with chromosome misalignment and abnormal spindle organization. Intriguingly, decreased Gas6 mRNA and protein expression were observed in aged oocytes from older females. We further explored the effect of GAS6 on the quality and fertility of aged mouse oocytes using a GAS6 rescue analysis. After treatment with the GAS6 protein, aged oocytes matured normally to the meiosis II (MII) stage. Additionally, maternal age-related meiotic defects were reduced by GAS6 protein microinjection. Restoring GAS6 ameliorated the mitochondrial dysfunction induced by maternal aging. Ultimately, GAS6-rescued MII oocytes exhibited increased ATP levels, reduced ROS levels and elevated glutathione (GSH) levels, collectively indicating improved mitochondrial function in aged oocytes. Thus, the age-associated decrease in oocyte quality was prevented by restoring GAS6. Importantly, GAS6 protein microinjection in aged oocytes also rescued fertility. We conclude that GAS6 improves mitochondrial function to achieve sufficient cytoplasmic maturation and attenuates maternal age-related meiotic errors, thereby efficiently safeguarding oocyte quality and fertility.

The value of serum progesterone level on day of human chorionic gonadotrophin administration / metaphase II oocyte ratio in predicting IVF/ICSI outcome in patients with normal ovarian reserve.

BACKGROUND: The clinical implication of the increased serum progesterone level on the day of HCG administration in assisted reproduction treatment (ART) is still controversial. The current study aimed to compare the predictive value of serum progesterone on day of HCG administration / metaphase II oocyte (P/MII) ratio on IVF/ ICSI outcome to serum progesterone (P) level alone and the ratio of serum progesterone/estradiol level (P/E2) ratio in prediction of pregnancy rates after ART. MATERIAL & METHODS: Two hundred patients admitted to the IVF/ICSI program at Minia IVF center in Egypt in the period from October 2016 to May 2018 were included in this study. Serum Progesterone (P) and Estradiol (E2) levels were estimated on the day of HCG administration. The ratio between serum P and the number of MII oocytes (P/MII ratio) was calculated and the predictive values of the three parameters (P, P/E2 ratio and P/MII ratio) in prediction of cycle outcomes were measured. RESULTS: P/ MII oocyte ratio was significantly lower in patients who attained clinical pregnancy (n = 97) as compared with those who couldn't whilst there was no significant difference in P and P/E2 ratio between the two groups. Using a cut off value of 0.125, the sensitivity and specificity of progesterone/ MII ratio in prediction of no pregnancy in IVF/ICSI were 75.7 and 77.1% respectively with the area under The Receiver operating curve (ROC-AUC) = 0.808. The respective values of the ROC-AUC for the P and P/E2 ratio were 0.651 and 0.712 with sensitivity and specificity of 71.2 and 73.5%for P level and of 72.5 and 75.3% for P/E2 ratio. Implantation or clinical pregnancy rates were significantly different between patients with high and low P/MII ratio irrespective of day of embryo transfer (day 3 or 5). CONCLUSIONS: In patients with normal ovarian response, serum progesterone on day of HCG / MII oocyte ratio can be a useful predictor of pregnancy outcomes and in deciding on freezing of all embryos for later transfer instead of high progesterone level alone.

A new four-way complex translocation variant involving the t(8;5;21;4)(q21;q13,q22,q31) and the relocalization of AML1/ETO fusion gene.

In acute myeloid leukemia, t(8;21) detected with a frequency of 10% is associated with good prognosis. However, variant t(8;21) is observed in 4% of these cases, and although the prognostic effects of these variant translocations have not been clearly revealed, there are findings that they affect the prognosis poorly. Here, we report on a 39 years old man, detected 4-way varyant t(8;21) which include relocalization of RUNX1/RUNX1T1 fusion gene, and loss of Y chromosome. RT-PCR also confirmed RUNX1/RUNX1T1 fusion transcript. Additionally, D820G and N822K mutations on KIT gene and mut B on NMP1 gene were detected. A complete remission could not achieved after first chemotherapy treatment. Due to primary resistance and variant of t(8;21), stem cell transplantation was performed. The variant translocation we have reported is unique and also the case is the second case that was reported in the literature in terms of the relocation of the AML1/ETO fusion gene. Since c-KIT mutations and LOY were also observed, it is not possible to predict the prognosis. To highlight the importance of variant translocations and relocalization of fusion gene, more cytogenetic and molecular data are needed.

Fluorescence in Situ Hybridization (FISH) Utility for Risk Score Assessment in Patients With MDS With Normal Metaphase Karyotype.

BACKGROUND: Cytogenetic profile is an essential parameter in myelodysplastic syndromes (MDS) risk stratification by both International Prognostic Symptom Score (IPSS) and Revised (R)-IPSS. Almost one-half of patients with MDS have normal cytogenetics by metaphase karyotype. Here we report the yield of MDS fluorescence in situ hybridization (FISH) panel detecting cytogenetic abnormalities in these patients and its impact on risk stratification. PATIENTS AND METHODS: Among patients with normal metaphase karyotype, we assessed those patients who had cytogenetic abnormalities detected by an MDS FISH panel, which included probes for del (5), del (7), del (20), trisomy 8, and del (17p). Risk stratification was calculated by both IPSS and R-IPSS. RESULTS: Of 1600 patients with MDS with normal metaphase karyotype, 53 (3%) patients had cytogenetic abnormality detected by MDS FISH panel. Integrating the MDS FISH panel cytogenetics (IPSS + FISH restaging) resulted in upstaging the score, where 53% of low-risk IPSS were upstaged to intermediate (int)-1, 56% of int-1 were upstaged to int-2, and 78% of int-2 were upstaged to high risk. Based on the R-IPSS, 61% of very low-risk patients, all low-risk patients, 92% of intermediate-risk patients, and 50% of high-risk patients with FISH abnormalities were upstaged, respectively. CONCLUSION: The yield of MDS FISH panel detecting cytogenetic abnormalities in patients with normal karyotype by G-banding is low and may not warrant ordering the panel in all patients. Among the 3% of patients with normal karyotype who had cytogenetic abnormality detected by FISH, the risk score assignment by IPSS and R-IPSS was upstaged.

Antisense Oligonucleotides Used to Identify Telomeric G-Quadruplexes in Metaphase Chromosomes and Fixed Cells by Fluorescence Lifetime Imaging Microscopy of o-BMVC Foci.

Identification of the existence of G-quadruplex (G4) structure, from a specific G-rich sequence in cells, is critical to the studies of structural biology and drug development. Accumulating evidence supports the existence of G4 structure in vivo. Particularly, time-gated fluorescence lifetime imaging microscopy (FLIM) of a G4 fluorescent probe, 3,6-bis(1-methyl-2-vinylpyridinium) carbazole diiodide (o-BMVC), was used to quantitatively measure the number of G4 foci, not only in different cell lines, but also in tissue biopsy. Here, circular dichroism spectra and polyacrylamide gel electrophoresis assays show that the use of antisense oligonucleotides unfolds their G4 structures in different percentages. Using antisense oligonucleotides, quantitative measurement of the number of o-BMVC foci in time-gated FLIM images provides a method for identifying which G4 motifs form G4 structures in fixed cells. Here, the decrease of the o-BMVC foci number, upon the pretreatment of antisense sequences, (CCCTAA)3CCCTA, in fixed cells and at the end of metaphase chromosomes, allows us to identify the formation of telomeric G4 structures from TTAGGG repeats in fixed cells.

The 3D Topography of Mitotic Chromosomes.

A long-standing conundrum is how mitotic chromosomes can compact, as required for clean separation to daughter cells, while maintaining close parallel alignment of sister chromatids. Pursuit of this question, by high resolution 3D fluorescence imaging of living and fixed mammalian cells, has led to three discoveries. First, we show that the structural axes of separated sister chromatids are linked by evenly spaced "mini-axis" bridges. Second, when chromosomes first emerge as discrete units, at prophase, they are organized as co-oriented sister linear loop arrays emanating from a conjoined axis. We show that this same basic organization persists throughout mitosis, without helical coiling. Third, from prophase onward, chromosomes are deformed into sequential arrays of half-helical segments of alternating handedness (perversions), accompanied by correlated kinks. These arrays fluctuate dynamically over <15 s timescales. Together these discoveries redefine the foundation for thinking about the evolution of mitotic chromosomes as they prepare for anaphase segregation.

A prognostic model based on cell-cycle control predicts outcome of breast cancer patients.

BACKGROUND: A prognostic model combining biomarkers of metaphase-anaphase transition of the cell cycle was developed for invasive breast cancer. The prognostic value and clinical applicability of the model was evaluated in comparison with the routine prognosticators of invasive breast carcinoma. METHODS: The study comprised 1135 breast cancer patients with complete clinical data and up to 22-year follow-up. Regulators of metaphase-anaphase transition were detected immunohistochemically and the biomarkers with the strongest prognostic impacts were combined into a prognostic model. The prognostic value of the model was tested and evaluated in separate patient materials originating from two Finnish breast cancer centers. RESULTS: The designed model comprising immunoexpressions of Securin, Separase and Cdk1 identified 8.4-fold increased risk of breast cancer mortality (p < 0.0001). A survival difference exceeding 15 years was observed between the majority (> 75%) of patients resulting with favorable as opposed to unfavorable outcome of the model. Along with nodal status, the model showed independent prognostic impact for all breast carcinomas and for subgroups of luminal, N+ and N- disease. CONCLUSIONS: The impact of the proposed prognostic model in predicting breast cancer survival was comparable to nodal status. However, the model provided additional information in N- breast carcinoma in identifying patients with aggressive course of disease, potentially in need of adjuvant treatments. Concerning N+, in turn, the model could provide evidence for withholding chemotherapy from patients with favorable outcome.

Telomere and Centromere Staining Followed by M-FISH Improves Diagnosis of Chromosomal Instability and Its Clinical Utility.

Dicentric chromosomes are a relevant marker of chromosomal instability. Their appearance is associated with telomere dysfunction, leading to cancer progression and a poor clinical outcome. Here, we present Telomere and Centromere staining followed by M-FISH (TC+M-FISH) for improved detection of telomere dysfunction and the identification of dicentric chromosomes in cancer patients and various genetic syndromes. Significant telomere length shortening and significantly higher frequencies of telomere loss and deletion were found in the peripheral lymphocytes of patients with cancer and genetic syndromes relative to similar age-matched healthy donors. We assessed our technique against conventional cytogenetics for the detection of dicentric chromosomes by subjecting metaphase preparations to both approaches. We identified dicentric chromosomes in 28/50 cancer patients and 21/44 genetic syndrome patients using our approach, but only 7/50 and 12/44, respectively, using standard cytogenetics. We ascribe this discrepancy to the identification of the unique configuration of dicentric chromosomes. We observed significantly higher frequencies of telomere loss and deletion in patients with dicentric chromosomes (p < 10-4). TC+M-FISH analysis is superior to classical cytogenetics for the detection of chromosomal instability. Our approach is a relatively simple but useful tool for documenting telomere dysfunction and chromosomal instability with the potential to become a standard additional diagnostic tool in medical genetics and the clinic.

SKP1 drives the prophase I to metaphase I transition during male meiosis.

The meiotic prophase I to metaphase I (PI/MI) transition requires chromosome desynapsis and metaphase competence acquisition. However, control of these major meiotic events is poorly understood. Here, we identify an essential role for SKP1, a core subunit of the SKP1-Cullin-F-box (SCF) ubiquitin E3 ligase, in the PI/MI transition. SKP1 localizes to synapsed chromosome axes and evicts HORMAD proteins from these regions in meiotic spermatocytes. SKP1-deficient spermatocytes display premature desynapsis, precocious pachytene exit, loss of PLK1 and BUB1 at centromeres, but persistence of HORMAD, γH2AX, RPA2, and MLH1 in diplonema. Strikingly, SKP1-deficient spermatocytes show sharply reduced MPF activity and fail to enter MI despite treatment with okadaic acid. SKP1-deficient oocytes exhibit desynapsis, chromosome misalignment, and progressive postnatal loss. Therefore, SKP1 maintains synapsis in meiosis of both sexes. Furthermore, our results support a model where SKP1 functions as the long-sought intrinsic metaphase competence factor to orchestrate MI entry during male meiosis.

Impaired condensin complex and Aurora B kinase underlie mitotic and chromosomal defects in hyperdiploid B-cell ALL.

B-cell acute lymphoblastic leukemia (ALL; B-ALL) is the most common pediatric cancer, and high hyperdiploidy (HyperD) identifies the most common subtype of pediatric B-ALL. Despite HyperD being an initiating oncogenic event affiliated with childhood B-ALL, the mitotic and chromosomal defects associated with HyperD B-ALL (HyperD-ALL) remain poorly characterized. Here, we have used 54 primary pediatric B-ALL samples to characterize the cellular-molecular mechanisms underlying the mitotic/chromosome defects predicated to be early pathogenic contributors in HyperD-ALL. We report that HyperD-ALL blasts are low proliferative and show a delay in early mitosis at prometaphase, associated with chromosome-alignment defects at the metaphase plate leading to robust chromosome-segregation defects and nonmodal karyotypes. Mechanistically, biochemical, functional, and mass-spectrometry assays revealed that condensin complex is impaired in HyperD-ALL cells, leading to chromosome hypocondensation, loss of centromere stiffness, and mislocalization of the chromosome passenger complex proteins Aurora B kinase (AURKB) and Survivin in early mitosis. HyperD-ALL cells show chromatid cohesion defects and an impaired spindle assembly checkpoint (SAC), thus undergoing mitotic slippage due to defective AURKB and impaired SAC activity, downstream of condensin complex defects. Chromosome structure/condensation defects and hyperdiploidy were reproduced in healthy CD34+ stem/progenitor cells upon inhibition of AURKB and/or SAC. Collectively, hyperdiploid B-ALL is associated with a defective condensin complex, AURKB, and SAC.

Probing lasting cryoinjuries to oocyte-embryo transcriptome.

Clinical applications of oocytes cryopreservation include preservation of future fertility of young cancer patients, substitution of embryo freezing to avoid associated legal and ethical issues, and delaying childbearing years. While the outcome of oocyte cryopreservation has recently been improved, currently used vitrification method still suffer from increased biosafety risk and handling issues while slow freezing techniques yield overall low success. Understanding better the mechanism of cryopreservation-induced injuries may lead to development of more reliable and safe methods for oocyte cryopreservation. Using the mouse model, a microarray study was conducted on oocyte cryopreservation to identify cryoinjuries to transcriptionally active genome. To this end, metaphase II (MII) oocytes were subjected to standard slow freezing, and then analyzed at the four-cell stage after embryonic genome activation. Non-frozen four-cell embryos served as controls. Differentially expressed genes were identified and validated using RT-PCR. Embryos produced from the cryopreserved oocytes displayed 200 upregulated and 105 downregulated genes, associated with the regulation of mitochondrial function, protein ubiquitination and maintenance, cellular response to stress and oxidative states, fatty acid and lipid regulation/metabolism, and cell cycle maintenance. These findings reveal previously unrecognized effects of standard slow oocyte freezing on embryonic gene expression, which can be used to guide improvement of oocyte cryopreservation methods.

Identification and characterization of a new stripe rust resistance gene Yr83 on rye chromosome 6R in wheat.

KEY MESSAGE: A physical map of Secale cereale chromosome 6R was constructed using deletion mapping, and a new stripe rust resistance gene Yr83 was mapped to the deletion bin of FL 0.73-1.00 of 6RL. Rye (Secale cereale L., RR) possesses valuable genes for wheat improvement. In the current study, we report a resistance gene conferring stripe rust resistance effective from seedling to adult plant stages located on chromosome 6R. This chromosome was derived from triticale line T-701 and also carries highly effective resistance to the cereal cyst nematode species Heterodera avenae Woll. A wheat-rye 6R(6D) disomic substitution line exhibited high levels of seedling resistance to Australian pathotypes of the stripe rust (Puccinia striiformis f. sp. tritici; Pst) pathogen and showed an even greater resistance to the Chinese Pst pathotypes in the field. Ten chromosome 6R deletion lines and five wheat-rye 6R translocation lines were developed earlier in the attempt to transfer the nematode resistance gene to wheat and used herein to map the stripe rust resistance gene. These lines were subsequently characterized by sequential multicolor fluorescence in situ hybridization (mc-FISH), genomic in situ hybridization (GISH), mc-GISH, PCR-based landmark unique gene (PLUG), and chromosome 6R-specific length amplified fragment sequencing (SLAF-Seq) marker analyses to physically map the stripe rust resistance gene. The new stripe rust resistance locus was located in a chromosomal bin with fraction length (FL) 0.73-1.00 on 6RL and was named Yr83. A wheat-rye translocation line T6RL (#5) carrying the stripe rust resistance gene will be useful as a new germplasm in breeding for resistance.

Characterization of a Saccharum spontaneum with a basic chromosome number of x = 10 provides new insights on genome evolution in genus Saccharum.

KEY MESSAGE: A novel tetraploid S. spontaneum with basic chromosome x = 10 was discovered, providing us insights in the origin and evolution in Saccharum species. Sugarcane (Saccharum spp., Poaceae) is a leading crop for sugar production providing 80% of the world's sugar. However, the genetic and genomic complexities of this crop such as its high polyploidy level and highly variable chromosome numbers have significantly hindered the studies in deciphering the genomic structure and evolution of sugarcane. Here, we developed the first set of oligonucleotide (oligo)-based probes based on the S. spontaneum genome (x = 8), which can be used to simultaneously distinguish each of the 64 chromosomes of octaploid S. spontaneum SES208 (2n = 8x = 64) through fluorescence in situ hybridization (FISH). By comparative FISH assay, we confirmed the chromosomal rearrangements of S. spontaneum (x = 8) and S. officinarum (2n = 8x = 80), the main contributors of modern sugarcane cultivars. In addition, we examined a S. spontaneum accession, Np-X, with 2n = 40 chromosomes, and we found that it was a tetraploid with the unusual basic chromosome number of x = 10. Assays at the cytological and DNA levels demonstrated its close relationship with S. spontaneum with basic chromosome number x = 8 (the most common accessions in S. spontaneum), confirming its S. spontaneum identity. Population genetic structure and phylogenetic relationship analyses between Np-X and 64 S. spontaneum accessions revealed that Np-X belongs to the ancient Pan-Malaysia group, indicating a close relationship to S. spontaneum with basic chromosome number of x = 8. This finding of a tetraploid S. spontaneum with basic chromosome number of x = 10 suggested a parallel evolution path of genomes and polyploid series in S. spontaneum with different basic chromosome numbers.

Avian Expression Patterns and Genomic Mapping Implicate Leptin in Digestion and TNF in Immunity, Suggesting That Their Interacting Adipokine Role Has Been Acquired Only in Mammals

In mammals, leptin and tumor-necrosis factor (TNF) are prominent interacting adipokines mediating appetite control and insulin sensitivity. While TNF pleiotropically functions in immune defense and cell survival, leptin is largely confined to signaling energy stores in adipocytes. Knowledge about the function of avian leptin and TNF is limited and they are absent or lowly expressed in adipose, respectively. Employing radiation-hybrid mapping and FISH-TSA, we mapped TNF and its syntenic genes to chicken chromosome 16 within the major histocompatibility complex (MHC) region. This mapping position suggests that avian TNF has a role in regulating immune response. To test its possible interaction with leptin within the immune system and beyond, we compared the transcription patterns of TNF, leptin and their cognate receptors obtained by meta-analysis of GenBank RNA-seq data. While expression of leptin and its receptor (LEPR) were detected in the brain and digestive tract, TNF and its receptor mRNAs were primarily found in viral-infected and LPS-treated leukocytes. We confirmed leptin expression in the duodenum by immunohistochemistry staining. Altogether, we suggest that whereas leptin and TNF interact as adipokines in mammals, in birds, they have distinct roles. Thus, the interaction between leptin and TNF may be unique to mammals.

Infrared nanospectroscopic mapping of a single metaphase chromosome.

The integrity of the chromatin structure is essential to every process occurring within eukaryotic nuclei. However, there are no reliable tools to decipher the molecular composition of metaphase chromosomes. Here, we have applied infrared nanospectroscopy (AFM-IR) to demonstrate molecular difference between eu- and heterochromatin and generate infrared maps of single metaphase chromosomes revealing detailed information on their molecular composition, with nanometric lateral spatial resolution. AFM-IR coupled with principal component analysis has confirmed that chromosome areas containing euchromatin and heterochromatin are distinguishable based on differences in the degree of methylation. AFM-IR distribution of eu- and heterochromatin was compared to standard fluorescent staining. We demonstrate the ability of our methodology to locate spatially the presence of anticancer drug sites in metaphase chromosomes and cellular nuclei. We show that the anticancer 'rule breaker' platinum compound [Pt[N(p-HC6F4)CH2]2py2] preferentially binds to heterochromatin, forming localized discrete foci due to condensation of DNA interacting with the drug. Given the importance of DNA methylation in the development of nearly all types of cancer, there is potential for infrared nanospectroscopy to be used to detect gene expression/suppression sites in the whole genome and to become an early screening tool for malignancy.

Gas6 is a reciprocal regulator of mitophagy during mammalian oocyte maturation.

Previously, we found that the silencing of growth arrest-specific gene 6 (Gas6) expression in oocytes impairs cytoplasmic maturation through mitochondrial overactivation with concurrent failure of pronuclear formation after fertilization. In this study, we report that Gas6 regulates mitophagy and safeguards mitochondrial activity by regulating mitophagy-related genes essential to the complete competency of oocytes. Based on RNA-Seq and RT-PCR analysis, in Gas6-silenced MII oocytes, expressions of mitophagy-related genes were decreased in Gas6-silenced MII oocytes, while mitochondrial proteins and Ptpn11, the downstream target of Gas6, was increased. Interestingly, GAS6 depletion induced remarkable MTOR activation. Gas6-depleted MII oocytes exhibited mitochondrial accumulation and aggregation caused by mitophagy inhibition. Gas6-depleted MII oocytes had a markedly lower mtDNA copy number. Rapamycin treatment rescued mitophagy, blocked the increase in MTOR and phosphorylated-MTOR, and increased the mitophagy-related gene expression in Gas6-depleted MII oocytes. After treatment with Mdivi-1, a mitochondrial division/mitophagy inhibitor, all oocytes matured and these MII oocytes showed mitochondrial accumulation but reduced Gas6 expression and failure of fertilization, showing phenomena very similar to the direct targeting of Gas6 by RNAi. Taken together, we conclude that the Gas6 signaling plays a crucial role in control of oocytes cytoplasmic maturation by modulating the dynamics and activity of oocyte mitochondria.

Clathrin heavy chain phosphorylated at T606 plays a role in proper cell division.

Clathrin regulates mitotic progression, in addition to membrane trafficking. However, the detailed regulatory mechanisms of clathrin during mitosis remain elusive. Here, we demonstrate novel regulation of clathrin during mitotic phase of the cell cycle. Clathrin heavy chain (CHC) was phosphorylated at T606 by its association partner cyclin G-associated kinase (GAK). This phosphorylation was required for proper cell proliferation and tumor growth of cells implanted into nude mice. Immunofluorescence analysis showed that the localization of CHC-pT606 signals changed during mitosis. CHC-pT606 signals localized in the nucleus and at the centrosome during interphase, whereas CHC signals were mostly cytoplasmic. Co-immunoprecipitation suggested that CHC formed a complex with GAK and polo-like kinase 1 (PLK1). Depletion of GAK using siRNA induced metaphase arrest and aberrant localization of CHC-pT606, which abolished Kiz-pT379 (as a phosphorylation target of PLK1) signals on chromatin at metaphase. Taken together, we propose that the GAK_CHC-pT606_PLK1_Kiz-pT379 axis plays a role in proliferation of cancer cells.