ALFI: Cell cycle phenotype annotations of label-free time-lapse imaging data from cultured human cells.

Detecting and tracking multiple moving objects in a video is a challenging task. For living cells, the task becomes even more arduous as cells change their morphology over time, can partially overlap, and mitosis leads to new cells. Differently from fluorescence microscopy, label-free techniques can be easily applied to almost all cell lines, reducing sample preparation complexity and phototoxicity. In this study, we present ALFI, a dataset of images and annotations for label-free microscopy, made publicly available to the scientific community, that notably extends the current panorama of expertly labeled data for detection and tracking of cultured living nontransformed and cancer human cells. It consists of 29 time-lapse image sequences from HeLa, U2OS, and hTERT RPE-1 cells under different experimental conditions, acquired by differential interference contrast microscopy, for a total of 237.9 hours. It contains various annotations (pixel-wise segmentation masks, object-wise bounding boxes, tracking information). The dataset is useful for testing and comparing methods for identifying interphase and mitotic events and reconstructing their lineage, and for discriminating different cellular phenotypes.

MLL4-associated condensates counterbalance Polycomb-mediated nuclear mechanical stress in Kabuki syndrome.

The genetic elements required to tune gene expression are partitioned in active and repressive nuclear condensates. Chromatin compartments include transcriptional clusters whose dynamic establishment and functioning depend on multivalent interactions occurring among transcription factors, cofactors and basal transcriptional machinery. However, how chromatin players contribute to the assembly of transcriptional condensates is poorly understood. By interrogating the effect of KMT2D (also known as MLL4) haploinsufficiency in Kabuki syndrome, we found that mixed lineage leukemia 4 (MLL4) contributes to the assembly of transcriptional condensates through liquid-liquid phase separation. MLL4 loss of function impaired Polycomb-dependent chromatin compartmentalization, altering the nuclear architecture. By releasing the nuclear mechanical stress through inhibition of the mechanosensor ATR, we re-established the mechanosignaling of mesenchymal stem cells and their commitment towards chondrocytes both in vitro and in vivo. This study supports the notion that, in Kabuki syndrome, the haploinsufficiency of MLL4 causes an altered functional partitioning of chromatin, which determines the architecture and mechanical properties of the nucleus.

Alterations of redox and iron metabolism accompany the development of HIV latency.

HIV-1 persists in a latent form during antiretroviral therapy, mainly in CD4+ T cells, thus hampering efforts for a cure. HIV-1 infection is accompanied by metabolic alterations, such as oxidative stress, but the effect of cellular antioxidant responses on viral replication and latency is unknown. Here, we show that cells survive retroviral replication, both in vitro and in vivo in SIVmac-infected macaques, by upregulating antioxidant pathways and the intertwined iron import pathway. These changes are associated with remodeling of promyelocytic leukemia protein nuclear bodies (PML NBs), an important constituent of nuclear architecture and a marker of HIV-1 latency. We found that PML NBs are hyper-SUMOylated and that PML protein is degraded via the ubiquitin-proteasome pathway in productively infected cells, before latency establishment and after reactivation. Conversely, normal numbers of PML NBs were restored upon transition to latency or by decreasing oxidative stress or iron content. Our results highlight antioxidant and iron import pathways as determinants of HIV-1 latency and support their pharmacologic inhibition as tools to regulate PML stability and impair latency establishment.

Dysfunctional polycomb transcriptional repression contributes to lamin A/C-dependent muscular dystrophy.

Lamin A is a component of the inner nuclear membrane that, together with epigenetic factors, organizes the genome in higher order structures required for transcriptional control. Mutations in the lamin A/C gene cause several diseases belonging to the class of laminopathies, including muscular dystrophies. Nevertheless, molecular mechanisms involved in the pathogenesis of lamin A-dependent dystrophies are still largely unknown. The polycomb group (PcG) of proteins are epigenetic repressors and lamin A interactors, primarily involved in the maintenance of cell identity. Using a murine model of Emery-Dreifuss muscular dystrophy (EDMD), we show here that lamin A loss deregulated PcG positioning in muscle satellite stem cells, leading to derepression of non-muscle-specific genes and p16INK4a, a senescence driver encoded in the Cdkn2a locus. This aberrant transcriptional program caused impairment in self-renewal, loss of cell identity, and premature exhaustion of the quiescent satellite cell pool. Genetic ablation of the Cdkn2a locus restored muscle stem cell properties in lamin A/C-null dystrophic mice. Our findings establish a direct link between lamin A and PcG epigenetic silencing and indicate that lamin A-dependent muscular dystrophy can be ascribed to intrinsic epigenetic dysfunctions of muscle stem cells.

Thymidine kinase and deoxycytidine kinase activity in mononuclear cells from antiretroviral-naive HIV-infected patients.

OBJECTIVE: To evaluate whether an inter-individual variability in the activity of thymidine kinase (TK) and deoxycytidine kinase (dCK), which are involved in the first step of phosphorylation of some nucleoside analogues, exists in antiretroviral-naive, HIV-seropositive patients. DESIGN: Forty-five randomly selected antiretroviral-naive HIV-infected patients were recruited, together with 26 healthy volunteers with no concurrent infection and under no pharmacological treatment. METHODS: Peripheral blood mononuclear cells (PBMC) were isolated from venous blood and their TK and dCK activities evaluated. CD4 T cells and HIV-RNA were measured in HIV-infected patients, too. RESULTS: There was a broad range of variability in TK activity in HIV-infected individuals. Furthermore, the activity in PBMC was significantly higher in HIV-infected individuals than in healthy volunteers. dCK activity in seropositive patients was significantly lower than in healthy volunteers. A marked inter-individual variability in dCK levels was observed in the HIV-infected group. No correlations were found between TK or dCK activities and plasma viral load, CD4 cell count, sex or age of patients. CONCLUSIONS: A marked range of inter-individual variability of TK and dCK activities in PBMC exists in HIV-infected individuals but not in healthy volunteers, indicating that the activity of enzymes with key roles in drug activation could vary greatly from one patient to another. Furthermore, TK expression is greater in HIV-infected individuals than in healthy volunteers. Better understanding of the viral or cellular factors that contribute to this variability, as well as their effect on responses to antiretroviral treatment, may aid optimization of the management of HIV-infected patients.

Is human immunodeficiency virus RNA load composed of neutralized immune complexes?

During acute human immunodeficiency virus (HIV) infection, both virus load (HIV RNA) and infectivity are high (10(3)-10(7) RNA copies/mL or TCID(50)/mL) until antibody is produced, which may reduce the HIV infectivity. In HIV carriers, the HIV RNA load is elevated (10(3)-10(5) copies/mL), but infectivity is low (10(0)-10(2) TCID(50)/mL). The low infectivity in carriers could be due to neutralization by antibody in serum, resulting in immune complexes (ICs). We demonstrated that ICs in plasma, prepared with protein A beads, contained HIV RNA (80%-100%) in association with immunoglobulin G (IgG). In comparison, ICs from patients with acute HIV infection and little or no antibody contained virtually no HIV RNA. Moreover, ICs prepared by ultrafiltration contained IgG and specifically and irreversibly neutralized HIV, which indicates that the ICs contained neutralizing antibody. These findings indicate that the HIV RNA in the plasma of carriers is frequently composed of antibody-neutralized HIV as ICs.