Highly efficient reprogrammable mouse lines with integrated reporters to track the route to pluripotency.

Revealing the molecular events associated with reprogramming different somatic cell types to pluripotency is critical for understanding the characteristics of induced pluripotent stem cell (iPSC) therapeutic derivatives. Inducible reprogramming factor transgenic cells or animals-designated as secondary (2°) reprogramming systems-not only provide excellent experimental tools for such studies but also offer a strategy to study the variances in cellular reprogramming outcomes due to different in vitro and in vivo environments. To make such studies less cumbersome, it is desirable to have a variety of efficient reprogrammable mouse systems to induce successful mass reprogramming in somatic cell types. Here, we report the development of two transgenic mouse lines from which 2° cells reprogram with unprecedented efficiency. These systems were derived by exposing primary reprogramming cells containing doxycycline-inducible Yamanaka factor expression to a transient interruption in transgene expression, resulting in selection for a subset of clones with robust transgene response. These systems also include reporter genes enabling easy readout of endogenous Oct4 activation (GFP), indicative of pluripotency, and reprogramming transgene expression (mCherry). Notably, somatic cells derived from various fetal and adult tissues from these 2° mouse lines gave rise to highly efficient and rapid reprogramming, with transgene-independent iPSC colonies emerging as early as 1 wk after induction. These mouse lines serve as a powerful tool to explore sources of variability in reprogramming and the mechanistic underpinnings of efficient reprogramming systems.

Multicountry Spread of Influenza A(H1N1)pdm09 Viruses with Reduced Oseltamivir Inhibition, May 2023-February 2024.

Since May 2023, a novel combination of neuraminidase mutations, I223V + S247N, has been detected in influenza A(H1N1)pdm09 viruses collected in countries spanning 5 continents, mostly in Europe (67/101). The viruses belong to 2 phylogenetically distinct groups and display ≈13-fold reduced inhibition by oseltamivir while retaining normal susceptibility to other antiviral drugs.

Whole genome sequencing in (recurrent) glioblastoma: challenges related to informed consent procedures and data sharing.

Increased use of whole genome sequencing (WGS) in neuro-oncology for diagnostics and research purposes necessitates a renewed conversation about informed consent procedures and governance structures for sharing personal health data. There is currently no consensus on how to obtain informed consent for WGS in this population. In this narrative review, we analyze the formats and contents of frameworks suggested in literature for WGS in oncology and assess their benefits and limitations. We discuss applicability, specific challenges, and legal context for patients with (recurrent) glioblastoma. This population is characterized by the rarity of the disease, extremely limited prognosis, and the correlation of the stage of the disease with cognitive abilities. Since this has implications for the informed consent procedure for WGS, we suggest that the content of informed consent should be tailor-made for (recurrent) glioblastoma patients.

Interferon stimulation creates chromatin marks and establishes transcriptional memory.

Epigenetic memory for signal-dependent transcription has remained elusive. So far, the concept of epigenetic memory has been largely limited to cell-autonomous, preprogrammed processes such as development and metabolism. Here we show that IFNß stimulation creates transcriptional memory in fibroblasts, conferring faster and greater transcription upon restimulation. The memory was inherited through multiple cell divisions and led to improved antiviral protection. Of ∼2,000 IFNß-stimulated genes (ISGs), about half exhibited memory, which we define as memory ISGs. The rest, designated nonmemory ISGs, did not show memory. Surprisingly, mechanistic analysis showed that IFN memory was not due to enhanced IFN signaling or retention of transcription factors on the ISGs. We demonstrated that this memory was attributed to accelerated recruitment of RNA polymerase II and transcription/chromatin factors, which coincided with acquisition of the histone H3.3 and H3K36me3 chromatin marks on memory ISGs. Similar memory was observed in bone marrow macrophages after IFNγ stimulation, suggesting that IFN stimulation modifies the shape of the innate immune response. Together, external signals can establish epigenetic memory in mammalian cells that imparts lasting adaptive performance upon various somatic cells.

Replicative Fitness of Seasonal Influenza A Viruses With Decreased Susceptibility to Baloxavir.

Susceptibility of influenza A viruses to baloxavir can be affected by changes at amino acid residue 38 in the polymerase acidic (PA) protein. Information on replicative fitness of PA-I38-substituted viruses remains sparse. We demonstrated that substitutions I38L/M/S/T not only had a differential effect on baloxavir susceptibility (9- to 116-fold) but also on in vitro replicative fitness. Although I38L conferred undiminished growth, other substitutions led to mild attenuation. In a ferret model, control viruses outcompeted those carrying I38M or I38T substitutions, although their advantage was limited. These findings offer insights into the attributes of baloxavir-resistant viruses needed for informed risk assessment.

Divergent reprogramming routes lead to alternative stem-cell states.

Pluripotency is defined by the ability of a cell to differentiate to the derivatives of all the three embryonic germ layers: ectoderm, mesoderm and endoderm. Pluripotent cells can be captured via the archetypal derivation of embryonic stem cells or via somatic cell reprogramming. Somatic cells are induced to acquire a pluripotent stem cell (iPSC) state through the forced expression of key transcription factors, and in the mouse these cells can fulfil the strictest of all developmental assays for pluripotent cells by generating completely iPSC-derived embryos and mice. However, it is not known whether there are additional classes of pluripotent cells, or what the spectrum of reprogrammed phenotypes encompasses. Here we explore alternative outcomes of somatic reprogramming by fully characterizing reprogrammed cells independent of preconceived definitions of iPSC states. We demonstrate that by maintaining elevated reprogramming factor expression levels, mouse embryonic fibroblasts go through unique epigenetic modifications to arrive at a stable, Nanog-positive, alternative pluripotent state. In doing so, we prove that the pluripotent spectrum can encompass multiple, unique cell states.

Genome-wide characterization of the routes to pluripotency.

Somatic cell reprogramming to a pluripotent state continues to challenge many of our assumptions about cellular specification, and despite major efforts, we lack a complete molecular characterization of the reprograming process. To address this gap in knowledge, we generated extensive transcriptomic, epigenomic and proteomic data sets describing the reprogramming routes leading from mouse embryonic fibroblasts to induced pluripotency. Through integrative analysis, we reveal that cells transition through distinct gene expression and epigenetic signatures and bifurcate towards reprogramming transgene-dependent and -independent stable pluripotent states. Early transcriptional events, driven by high levels of reprogramming transcription factor expression, are associated with widespread loss of histone H3 lysine 27 (H3K27me3) trimethylation, representing a general opening of the chromatin state. Maintenance of high transgene levels leads to re-acquisition of H3K27me3 and a stable pluripotent state that is alternative to the embryonic stem cell (ESC)-like fate. Lowering transgene levels at an intermediate phase, however, guides the process to the acquisition of ESC-like chromatin and DNA methylation signature. Our data provide a comprehensive molecular description of the reprogramming routes and is accessible through the Project Grandiose portal at http://www.stemformatics.org.

Susceptibility of Influenza A, B, C, and D Viruses to Baloxavir1.

Baloxavir showed broad-spectrum in vitro replication inhibition of 4 types of influenza viruses (90% effective concentration range 1.2-98.3 nmol/L); susceptibility pattern was influenza A ˃ B ˃ C ˃ D. This drug also inhibited influenza A viruses of avian and swine origin, including viruses that have pandemic potential and those resistant to neuraminidase inhibitors.

UV light perception is modulated by the odour element of an olfactory-visual compound in restrained honeybees.

Honeybees use visual and olfactory cues to detect flowers during foraging trips. Hence, the reward association of a nectar source is a multimodal construct which has at least two major components - olfactory and visual cues. How both sensory modalities are integrated to form a common reward association and whether and how they may interfere, is an open question. The present study used stimulation with UV, blue and green light to evoke distinct photoreceptor activities in the compound eye and two odour components (geraniol, citronellol). To test if a compound of both modalities is perceived as the sum of its elements (elemental processing) or as a unique cue (configural processing), we combined monochromatic light with single odour components in positive (PP) and negative patterning (NP) experiments. During PP, the compound of two modalities was rewarded, whereas the single elements were not. For NP, stimuli comprising a single modality were rewarded, whereas the olfactory-visual compound was not. Furthermore, we compared the differentiation abilities between two light stimuli that were or were not part of an olfactory-visual compound. Interestingly, the behavioural performances revealed a prominent case of configural processing, but only in those cases when UV light was an element of an olfactory-visual compound. Instead, learning with green- and blue-containing compounds rather supports elemental processing theory.

Assessing baloxavir susceptibility of influenza viruses circulating in the United States during the 2016/17 and 2017/18 seasons.

The anti-influenza therapeutic baloxavir targets cap-dependent endonuclease activity of polymerase acidic (PA) protein. We monitored baloxavir susceptibility in the United States with next generation sequencing analysis supplemented by phenotypic one-cycle infection assay. Analysis of PA sequences of 6,891 influenza A and B viruses collected during 2016/17 and 2017/18 seasons showed amino acid substitutions: I38L (two A(H1N1)pdm09 viruses), E23G (two A(H1N1)pdm09 viruses) and I38M (one A(H3N2) virus); conferring 4-10-fold reduced susceptibility to baloxavir.

WHSC1 links transcription elongation to HIRA-mediated histone H3.3 deposition.

Actively transcribed genes are enriched with the histone variant H3.3. Although H3.3 deposition has been linked to transcription, mechanisms controlling this process remain elusive. We investigated the role of the histone methyltransferase Wolf-Hirschhorn syndrome candidate 1 (WHSC1) (NSD2/MMSET) in H3.3 deposition into interferon (IFN) response genes. IFN treatment triggered robust H3.3 incorporation into activated genes, which continued even after cessation of transcription. Likewise, UV radiation caused H3.3 deposition in UV-activated genes. However, in Whsc1(-/-) cells IFN- or UV-triggered H3.3 deposition was absent, along with a marked reduction in IFN- or UV-induced transcription. We found that WHSC1 interacted with the bromodomain protein 4 (BRD4) and the positive transcription elongation factor b (P-TEFb) and facilitated transcriptional elongation. WHSC1 also associated with HIRA, the H3.3-specific histone chaperone, independent of BRD4 and P-TEFb. WHSC1 and HIRA co-occupied IFN-stimulated genes and supported prolonged H3.3 incorporation, leaving a lasting transcriptional mark. Our results reveal a previously unrecognized role of WHSC1, which links transcriptional elongation and H3.3 deposition into activated genes through two molecularly distinct pathways.

Fluctuations in histone H4 isoforms during cellular reprogramming monitored by middle-down proteomics.

Cellular reprogramming remodels the gene expression program by re-setting the epigenome of somatic cells into an embryonic-like pluripotent state. Post-translational modifications of histones play an important role in this process. Previously, we found by ChIP-seq widespread changes of specific histone H3 marks in two divergent reprogramming routes leading to alternative pluripotent sates . Here, using an unbiased middle-down proteomics approach we have identified 72 unique isoforms of histone H4 and quantified 56 of them in the same set of samples. We found substantial differences between somatic and late-phase reprogramming cells. Also, ESCs and iPSCs displayed higher levels of H4 acetylation and tri-methylation concomitantly with lower levels of mono- and di-methylation when compared to cells undergoing reprogramming. Our data shows that the epigenetic remodeling induced by the reprogramming process goes beyond histone H3 and reveals the importance of H4 modifications as well. The presented data is a valuable resource to study the epigenetic mechanisms involved in the acquisition of induced pluripotency. All MS data have been deposited in the ProteomeXchange with identifier PXD002062 (http://proteomecentral.proteomexchange.org/dataset/PXD002062).

Loss of Siah2 does not impact angiogenic potential of murine endothelial cells.

Angiogenesis is triggered in response to hypoxia under many circumstances, from healthy cells and tissues during embryogenesis to pathological conditions like the formation of new blood vessels to supply tumours and promote invasive cancer. Siah2 has been shown to regulate the hypoxia pathway upstream of hypoxia-induced transcription factor subunit Hif-1alpha, and therefore may play an important role in angiogenesis in response to hypoxic stress in endothelial cells. This study aims to investigate the basic function of Siah2 in endothelial cells under hypoxia and to test the ability of Siah2 deficient cells to mount an angiogenic response when deprived of oxygen. We and others have previously shown that Siah2 is crucial for mediating the hypoxic response in many different cell types studied. In this study however, we describe that Siah2(-/-) endothelial cells have an intact hypoxic signalling pathway, including Hif-1alpha stabilisation and gene expression, the first report of a tissue or cell lineage in which the loss of Siah2 does not seem to impact hypoxic response signalling. In mice, the infiltration of Siah2(-/-) endothelial cells into a Matrigel plug containing a VEGF-A attractant was similar compared with wildtype endothelial cells. Ex vivo however, there was a reduced capacity of Siah2(-/-) aorta to form tubes or new vessels. Thus, we conclude that Siah2 is not essential for the hypoxic response of endothelial cells.

Differentiation of vascular smooth muscle cells from local precursors during embryonic and adult arteriogenesis requires Notch signaling.

Vascular smooth muscle cells (VSMC) have been suggested to arise from various developmental sources during embryogenesis, depending on the vascular bed. However, evidence also points to a common subpopulation of vascular progenitor cells predisposed to VSMC fate in the embryo. In the present study, we use binary transgenic reporter mice to identify a Tie1(+)CD31(dim)vascular endothelial (VE)-cadherin(-)CD45(-) precursor that gives rise to VSMC in vivo in all vascular beds examined. This precursor does not represent a mature endothelial cell, because a VE-cadherin promoter-driven reporter shows no expression in VSMC during murine development. Blockade of Notch signaling in the Tie1(+) precursor cell, but not the VE-cadherin(+) endothelial cell, decreases VSMC investment of developing arteries, leading to localized hemorrhage in the embryo at the time of vascular maturation. However, Notch signaling is not required in the Tie1(+) precursor after establishment of a stable artery. Thus, Notch activity is required in the differentiation of a Tie1(+) local precursor to VSMC in a spatiotemporal fashion across all vascular beds.

Concise review: Embryonic stem cells versus induced pluripotent stem cells: the game is on.

Extraordinary advances in pluripotent stem cell research have initiated an era of hope for regenerative strategies to treat human disease. Besides embryonic stem cells, the discovery of induced pluripotent stem cells widened the possibility of patient-specific cell therapy, drug discovery, and disease modeling. Although similar, it has become clear that these two pluripotent cell types display significant differences. In this review, we explore current knowledge of the molecular and functional similarities and differences between these two cell types to emphasize the necessity for thorough characterization of their properties as well as their differentiation capabilities in the pluripotent state. Such comparative studies will be crucial for determining the more suitable cell type for future stem cell-based therapies for human degenerative diseases.

Siah2-deficient mice show impaired skin wound repair.

Hypoxia is associated with the dermal wound healing process and hypoxia signaling is presumed to be crucial for normal wound repair. The Siah2 ubiquitin ligase controls the abundance of hypoxia-inducible factor-1 alpha, and loss of Siah2 results in destabilization of hypoxia-inducible factor-1 alpha under hypoxia. Utilizing Siah2(-/-) mice we demonstrate that cutaneous wound healing is impaired in these mice. Wounds in Siah2(-/-) mice heal slower and are associated with delayed induction of myofibroblast infiltration and reduced collagen deposition. This coincides with delayed angiogenesis and reduced macrophage infiltration into the wounds of Siah2(-/-) mice. We furthermore demonstrate that primary Siah2(-/-) dermal fibroblasts have reduced migratory capacities and produce less collagen than wild-type fibroblasts. Additionally, Siah2(-/-) fibroblasts showed conserved responses to transforming growth factor-ß at the receptor level (pSmad 2C activation) but reduced responses downstream. Together, our data show, for the first time, that Siah2 is involved as a positive regulator in the wound healing response. Understanding the role of hypoxia signaling in tissue repair and fibrosis and interference with the hypoxia signaling pathway via regulation of Siah2 may provide new targets for clinical regulation of fibrosis and scarring.

Respiratory Syncytial Virus Infection Among Hospitalized Infants in Four Middle-Income Countries.

BACKGROUND: Understanding respiratory syncytial virus (RSV) global epidemiology is important to inform future prevention strategies. METHODS: Hospitalized infants <1-year-old with acute illness were enrolled prospectively in Albania, Jordan, Nicaragua, and Philippines during respiratory seasons in 2015-2017. Medical chart review, parental interview, and post-discharge follow up were conducted. Respiratory specimens were tested using real-time RT-PCR for RSV. Infant characteristics associated with very severe illness (intensive care unit [ICU] admission or receipt of supplemental oxygen) were assessed using logistic regression to adjust for potential confounders (age, sex, study site, and preterm birth). RESULTS: Of 3634 enrolled hospitalized infants, 1129 (31%) tested positive for RSV. The median age of RSV-positive infants was 2.7 (IQR: 1.4-6.1) months and 665 (59%) were male. Very severe illness in 583 (52%) RSV-positive infants was associated with younger age (aOR 4.1, 95% CI: 2.6-6.5 for 0-2 compared to 9-11-months; P < .01), low weight-for-age z-score (aOR 1.9, 95% CI: 1.2-2.8; P < .01), ICU care after birth (aOR 1.6, 95% CI: 1.0-2.5; P = .048), and cesarean delivery (aOR 1.4, 95% CI: 1.0-1.8; P = .03). RSV subgroups A and B co-circulated at all sites with alternating predominance by year; subgroup was not associated with severity (aOR 1.0, 95% CI: 0.8-1.4). Nine (0.8%) RSV-positive infants died during admission or within ≤30 days of discharge, of which 7 (78%) were <6-months-old. CONCLUSIONS: RSV was associated with nearly a third of infant acute illness hospitalizations in four middle-income countries during the respiratory season, where, in addition to young age, factors including low weight-for-age might be important predictors of severity. RSV prevention strategies targeting young infants could substantially reduce RSV-associated hospitalizations in middle-income countries.

Recurrent granulosa cell tumor of the retroperitoneum during the COVID-19 Pandemic.

Extraovarian granulosa cell tumors are rare with very few cases of isolated retroperitoneal granulosa cell tumors reported in the literature. Granulosa cell tumors are notorious for late recurrences and patients should have long term oncologic follow up. We describe a case of recurrent granulosa cell tumor of the retroperitoneum that originally presented as a renal mass, which has not been described before in the literature. Her management was delayed in part due to the COVID-19 Pandemic. Oncologists must be vigilant regarding the consequences of postponed care during this difficult time.