Establishment of closed 35S ribosomal RNA gene chromatin in stationary Saccharomyces cerevisiae cells.

As a first step in eukaryotic ribosome biogenesis RNA polymerase (Pol) I synthesizes a large ribosomal RNA (rRNA) precursor from multicopy rRNA gene loci. This process is essential for cellular growth and regulated in response to the cell's physiological state. rRNA gene transcription is downregulated upon growth to stationary phase in the yeast Saccharomyces cerevisiae.

Immune responses and clinical outcomes after COVID-19 vaccination in patients with liver disease and liver transplant recipients.

Background & Aims: Comparative assessments of immunogenicity following different COVID-19 vaccines in patients with distinct liver diseases are lacking. SARS-CoV-2-specific T-cell and antibody responses were evaluated longitudinally after one to three vaccine doses, with long-term follow-up for COVID-19-related clinical outcomes.

Methods: A total of 849 participants (355 with cirrhosis, 74 with autoimmune hepatitis [AIH], 36 with vascular liver disease [VLD], 257 liver transplant recipients [LTRs] and 127 healthy controls [HCs]) were recruited from four countries.

N-nitrosamine Mitigation with Nitrite Scavengers in Oral Pharmaceutical Drug Products.

N-nitrosamines are likely human carcinogens. After N-nitrosamine contaminants were detected in pharmaceutical products in 2018, regulatory authorities set a framework for the risk assessment, testing and mitigation of N-nitrosamines in drug products. One strategy to inhibit the formation of N-nitrosamines during the manufacture and storage of drug products involves the incorporation of nitrite scavengers in the formulation.

In-depth virological and immunological characterization of HIV-1 cure after CCR5Δ32/Δ32 allogeneic hematopoietic stem cell transplantation.

Despite scientific evidence originating from two patients published to date that CCR5Δ32/Δ32 hematopoietic stem cell transplantation (HSCT) can cure human immunodeficiency virus type 1 (HIV-1), the knowledge of immunological and virological correlates of cure is limited. Here we characterize a case of long-term HIV-1 remission of a 53-year-old male who was carefully monitored for more than 9 years after allogeneic CCR5Δ32/Δ32 HSCT performed for acute myeloid leukemia. Despite sporadic traces of HIV-1 DNA detected by droplet digital PCR and in situ hybridization assays in peripheral T cell subsets and tissue-derived samples, repeated ex vivo quantitative and in vivo outgrowth assays in humanized mice did not reveal replication-competent virus.

High-resolution analysis of individual spike peptide-specific CD4 T-cell responses in vaccine recipients and COVID-19 patients.

Objectives: Potential differences in the breadth, distribution and magnitude of CD4 T-cell responses directed against the SARS-CoV-2 spike glycoprotein between vaccinees, COVID-19 patients and subjects who experienced both ways of immunisation have not been comprehensively compared on a peptide level.

Methods: Following virus-specific cultivation, we determined the T-cell responses directed against 253 individual overlapping 15-mer peptides covering the entire SARS-CoV-2 spike glycoprotein using IFN-γ ELISpot and intracellular cytokine staining. HLA binding was determined for selected peptides.

High and Sustained Ex Vivo Frequency but Altered Phenotype of SARS-CoV-2-Specific CD4 T-Cells in an Anti-CD20-Treated Patient with Prolonged COVID-19.

Here, we longitudinally assessed the ex vivo frequency and phenotype of SARS-CoV-2 membrane protein (aa145-164) epitope-specific CD4 T-cells of an anti-CD20-treated patient with prolonged viral positivity in direct comparison to an immunocompetent patient through an MHC class II DRB1*11:01 Tetramer analysis. We detected a high and stable SARS-CoV-2 membrane-specific CD4 T-cell response in both patients, with higher frequencies of virus-specific CD4 T-cells in the B-cell-depleted patient. However, we found an altered virus-specific CD4 T-cell memory phenotype in the B-cell-depleted patient that was skewed towards late differentiated memory T-cells, as well as reduced frequencies of SARS-CoV-2-specific CD4 T-cells with CD45RA CXCR5 PD-1 circulating T follicular helper cell (cT) phenotype.

Inversed Ratio of CD39/CD73 Expression on γδ T Cells in HIV Versus Healthy Controls Correlates With Immune Activation and Disease Progression.

Background: γδ T cells are unconventional T cells that have been demonstrated to be crucial for the pathogenesis and potentially for the cure of HIV-1 infection. The ectonucleotidase CD39 is part of the purinergic pathway that regulates immune responses by degradation of pro-inflammatory ATP in concert with CD73. Few studies on the expression of the ectoenzymes CD73 and CD39 on human γδ T cells in HIV have been performed to date.

Towards maximum acceleration of monoclonal antibody development: Leveraging transposase-mediated cell line generation to enable GMP manufacturing within 3 months using a stable pool.

In recent years, acceleration of development timelines has become a major focus within the biopharmaceutical industry to bring innovative therapies faster to patients. However, in order to address a high unmet medical need even faster further acceleration potential has to be identified to transform "speed-to-clinic" concepts into "warp-speed" development programs. Recombinant Chinese hamster ovary (CHO) cell lines are the predominant expression system for monoclonal antibodies (mAbs) and are routinely generated by random transgene integration (RTI) of the genetic information into the host cell genome.

Dual role of EZH2 in megakaryocyte differentiation.

EZH2, the enzymatic component of PRC2, has been identified as a key factor in hematopoiesis. EZH2 loss-of-function mutations have been found in myeloproliferative neoplasms, particularly in myelofibrosis, but the precise function of EZH2 in megakaryopoiesis is not fully delineated. Here, we show that EZH2 inhibition by small molecules and short hairpin RNA induces megakaryocyte (MK) commitment by accelerating lineage marker acquisition without change in proliferation.

Decreased Frequency of Intestinal CD39 γδ T Cells With Tissue-Resident Memory Phenotype in Inflammatory Bowel Disease.

The ectoenzymes CD39 and CD73 play a major role in controlling tissue inflammation by regulating the balance between adenosine triphosphate (ATP) and adenosine. Still, little is known about the role of these two enzymes and ATP and its metabolites in the pathophysiology of inflammatory bowel disease (IBD). We isolated mononuclear cells from peripheral blood and lamina propria of the large intestine of patients diagnosed with IBD and of healthy volunteers.

Analysis of Co-inhibitory Receptor Expression in COVID-19 Infection Compared to Acute Malaria: LAG-3 and TIM-3 Correlate With T Cell Activation and Course of Disease.

Coronavirus disease 2019 (COVID-19) which is caused by the novel SARS-CoV-2 virus is a severe flu-like illness which is associated with hyperinflammation and immune dysfunction. The virus induces a strong T and B cell response but little is known about the immune pathology of this viral infection. Acute malaria also causes acute clinical illness and is characterized by hyperinflammation due to the strong production of pro-inflammatory cytokines and a massive activation of T cells.

Detection of EXP1-Specific CD4+ T Cell Responses Directed Against a Broad Range of Epitopes Including Two Promiscuous MHC Class II Binders During Acute Malaria.

T cells are thought to play a major role in conferring immunity against malaria. This study aimed to comprehensively define the breadth and specificity of the -specific CD4+ T cell response directed against the exported protein 1 (EXP1) in a cohort of patients diagnosed with acute malaria. Peripheral blood mononuclear cells of 44 patients acutely infected with , and of one patient infected with , were stimulated and cultured with an overlapping set of 31 -specific 13-17-mer peptides covering the entire EXP1 sequence.

CABLES1 Deficiency Impairs Quiescence and Stress Responses of Hematopoietic Stem Cells in Intrinsic and Extrinsic Manners.

Bone marrow (BM) niche cells help to keep adult hematopoietic stem cells (HSCs) in a quiescent state via secreted factors and induction of cell-cycle inhibitors. Here, we demonstrate that the adapter protein CABLES1 is a key regulator of long-term hematopoietic homeostasis during stress and aging. Young mice lacking Cables1 displayed hyperproliferation of hematopoietic progenitor cells.

Remodeling of Bone Marrow Hematopoietic Stem Cell Niches Promotes Myeloid Cell Expansion during Premature or Physiological Aging.

Hematopoietic stem cells (HSCs) residing in the bone marrow (BM) accumulate during aging but are functionally impaired. However, the role of HSC-intrinsic and -extrinsic aging mechanisms remains debated. Megakaryocytes promote quiescence of neighboring HSCs.

In vitro and in vivo efficacy of an anti-CD203c conjugated antibody (AGS-16C3F) in mouse models of advanced systemic mastocytosis.

Antibody-drug conjugates (ADCs) are a new class of therapeutics that use antibodies to deliver potent cytotoxic drugs selectively to cancer cells. CD203c, an ecto-nucleotide pyrophosphatase-phosphodiesterase 3, is overexpressed on neoplastic mast cells (MCs) in systemic mastocytosis (SM), thus representing a promising target for antibody-mediated therapy. In this study, we have found that human neoplastic MC lines (ROSA and ROSA), which express high levels of CD203c, are highly and specifically sensitive to the antiproliferative effects of an ADC against CD203c (AGS-16C3F).

CD32 Expression of Different Memory T Cell Subpopulations in the Blood and Lymph Nodal Tissue of HIV Patients and Healthy Controls Correlates With Immune Activation.

Background: Recently, CD32 has been described to be a specific surface marker of latently HIV-infected CD4 T cells, but little is known about the frequency and distribution of CD32 expression on naive and memory CD8 and CD4 T cell populations in HIV patients and healthy individuals.

Methods: We studied peripheral blood samples of 36 HIV-1-infected patients [23 viremic patients / 13 antiretroviral therapy(ART)-treated] and healthy individuals (n = 14) as well as cells from lymph nodes (8 HIV infected, 5 controls) using a multiparametric flow cytometry panel determining surface expression of CD3, CD8, CD4, CD45RA, CCR7, CD27, CD25, CD127, CCR5, CCR6, CXCR4, CD38, HLA-DR, TIGIT, and PD-1.

Results: Overall, expression of CD32 on total peripheral CD4 T cells between viremic HIV patients, ART-treated and healthy individuals only slightly differed (mean values 1.

Lymphoid differentiation of hematopoietic stem cells requires efficient Cxcr4 desensitization.

The CXCL12/CXCR4 signaling exerts a dominant role in promoting hematopoietic stem and progenitor cell (HSPC) retention and quiescence in bone marrow. Gain-of-function mutations that affect homologous desensitization of the receptor have been reported in the WHIM Syndrome (WS), a rare immunodeficiency characterized by lymphopenia. The mechanisms underpinning this remain obscure.

CXCR4/CXCL12 axis counteracts hematopoietic stem cell exhaustion through selective protection against oxidative stress.

Hematopoietic stem cells (HSCs) undergo self-renewal to maintain hematopoietic homeostasis for lifetime, which is regulated by the bone marrow (BM) microenvironment. The chemokine receptor CXCR4 and its ligand CXCL12 are critical factors supporting quiescence and BM retention of HSCs. Here, we report an unknown function of CXCR4/CXCL12 axis in the protection of HSCs against oxidative stress.

CXCL12/CXCR4 pathway is activated by oncogenic JAK2 in a PI3K-dependent manner.

JAK2 activation is the driver mechanism in negative myeloproliferative neoplasms (MPN). These diseases are characterized by an abnormal retention of hematopoietic stem cells within the bone marrow microenvironment and their increased trafficking to extramedullary sites. The CXCL12/CXCR4 axis plays a central role in hematopoietic stem cell/ progenitor trafficking and retention in hematopoietic sites.

Involvement of the L-Type Amino Acid Transporter Lat2 in the Transport of 3,3'-Diiodothyronine across the Plasma Membrane.

Thyroid hormones are transported across cell membranes by transmembrane transporter proteins, for example by members of the monocarboxylate transporter (MCT) and the L-type amino acid transporter (LAT) families. LATs consist of a light chain (e.g.

Purification of specific chromatin domains from single-copy gene loci in Saccharomyces cerevisiae.

Most methods currently available for the analysis of chromatin in vivo rely on a priori knowledge of putative chromatin components or their posttranslational modification state. The isolation of defined native chromosomal regions provides an attractive alternative to obtain a largely unbiased molecular description of chromatin. Here, we describe a strategy combining site-specific recombination at the chromosome with an efficient tandem affinity purification protocol to isolate a single-copy gene locus from the yeast Saccharomyces cerevisiae.

Compositional and structural analysis of selected chromosomal domains from Saccharomyces cerevisiae.

Chromatin is the template for replication and transcription in the eukaryotic nucleus, which needs to be defined in composition and structure before these processes can be fully understood. We report an isolation protocol for the targeted purification of specific genomic regions in their native chromatin context from Saccharomyces cerevisiae. Subdomains of the multicopy ribosomal DNA locus containing transcription units of RNA polymerases I, II or III or an autonomous replication sequence were independently purified in sufficient amounts and purity to analyze protein composition and histone modifications by mass spectrometry.