Mitofusin 2 maintains haematopoietic stem cells with extensive lymphoid potential.

Haematopoietic stem cells (HSCs), which sustain production of all blood cell lineages, rely on glycolysis for ATP production, yet little attention has been paid to the role of mitochondria. Here we show in mice that the short isoform of a critical regulator of HSCs, Prdm16 (refs 4, 5), induces mitofusin 2 (Mfn2), a protein involved in mitochondrial fusion and in tethering of mitochondria to the endoplasmic reticulum. Overexpression and deletion studies, including single-cell transplantation assays, revealed that Mfn2 is specifically required for the maintenance of HSCs with extensive lymphoid potential, but not, or less so, for the maintenance of myeloid-dominant HSCs. Mfn2 increased buffering of intracellular Ca(2+), an effect mediated through its endoplasmic reticulum-mitochondria tethering activity, thereby negatively regulating nuclear translocation and transcriptional activity of nuclear factor of activated T cells (Nfat). Nfat inhibition rescued the effects of Mfn2 deletion in HSCs, demonstrating that negative regulation of Nfat is the prime downstream mechanism of Mfn2 in the maintenance of HSCs with extensive lymphoid potential. Mitochondria therefore have an important role in HSCs. These findings provide a mechanism underlying clonal heterogeneity among HSCs and may lead to the design of approaches to bias HSC differentiation into desired lineages after transplantation.

A composite biomarker of neutrophil-lymphocyte ratio and hemoglobin level correlates with clinical response to PD-1 and PD-L1 inhibitors in advanced non-small cell lung cancers.

BACKGROUND: Immune checkpoint inhibitors (ICIs) have been incorporated into various clinical oncology guidelines for systemic treatment of advanced non-small cell lung cancers (aNSCLC). However, less than 50% (and 20%) of the patients responded to the therapy as a first (or second) line of therapy. PD-L1 immunohistochemistry (IHC) is an extensively studied biomarker of response to ICI, but results from this test have equivocal predictive power. In order to identify other biomarkers that support clinical decision-making around whether to treat with ICIs or not, we performed a retrospective study of patients with aNSCLC who underwent ICI-based therapy in the Mount Sinai Health System between 2014 and 2019. METHODS: We analyzed data from standard laboratory tests performed in patients as a part of the routine clinical workup during treatment, including complete blood counts (CBC) and a comprehensive metabolic panel (CMP), to correlate test results with clinical response and survival. RESULTS: Of 11,138 NSCLC patients identified, 249 had been treated with ICIs. We found associations between high neutrophil-to-lymphocyte ratio (NLR ≥ 5) and poor survival in ICI-treated NSCLC. We further observed that sustained high NLR after initiation of treatment had a more profound impact on survival than baseline NLR, regardless of PD-L1 status. Hazard ratios when comparing patients with NLR ≥ 5 vs. NLR < 5 are 1.7 (p = 0.02), 3.4 (p = 4.2 × 10- 8), and 3.9 (p = 1.4 × 10- 6) at baseline, 2-8 weeks, and 8-14 weeks after treatment start, respectively. Mild anemia, defined as hemoglobin (HGB) less than 12 g/dL was correlated with survival independently of NLR. Finally, we developed a composite NLR and HGB biomarker. Patients with pretreatment NLR ≥ 5 and HGB < 12 g/dL had a median overall survival (OS) of 8.0 months (95% CI 4.5-11.5) compared to the rest of the cohort with a median OS not reached (95% CI 15.9-NE, p = 1.8 × 10- 5), and a hazard ratio of 2.6 (95% CI 1.7-4.1, p = 3.5 × 10- 5). CONCLUSIONS: We developed a novel composite biomarker for ICI-based therapy in NSCLC based on routine CBC tests, which may provide meaningful clinical utility to guide treatment decision. The results suggest that treatment of anemia to elevate HGB before initiation of ICI therapy may improve patient outcomes or the use of alternative non-chemotherapy containing regimens.

An algorithm to identify patients aged 0-3 with rare genetic disorders.

BACKGROUND: With over 7000 Mendelian disorders, identifying children with a specific rare genetic disorder diagnosis through structured electronic medical record data is challenging given incompleteness of records, inaccurate medical diagnosis coding, as well as heterogeneity in clinical symptoms and procedures for specific disorders. We sought to develop a digital phenotyping algorithm (PheIndex) using electronic medical records to identify children aged 0-3 diagnosed with genetic disorders or who present with illness with an increased risk for genetic disorders. RESULTS: Through expert opinion, we established 13 criteria for the algorithm and derived a score and a classification. The performance of each criterion and the classification were validated by chart review. PheIndex identified 1,088 children out of 93,154 live births who may be at an increased risk for genetic disorders. Chart review demonstrated that the algorithm achieved 90% sensitivity, 97% specificity, and 94% accuracy. CONCLUSIONS: The PheIndex algorithm can help identify when a rare genetic disorder may be present, alerting providers to consider ordering a diagnostic genetic test and/or referring a patient to a medical geneticist.

PRDM16 isoforms differentially regulate normal and leukemic hematopoiesis and inflammatory gene signature.

PRDM16 is a transcriptional coregulator involved in translocations in acute myeloblastic leukemia (AML), myelodysplastic syndromes, and T acute lymphoblastic leukemia that is highly expressed in and required for the maintenance of hematopoietic stem cells (HSCs), and can be aberrantly expressed in AML. Prdm16 is expressed as full-length (fPrdm16) and short (sPrdm16) isoforms, the latter lacking the N-terminal PR domain. The role of both isoforms in normal and malignant hematopoiesis is unclear. We show here that fPrdm16 was critical for HSC maintenance, induced multiple genes involved in GTPase signaling, and repressed inflammation, while sPrdm16 supported B cell development biased toward marginal zone B cells and induced an inflammatory signature. In a mouse model of human MLL-AF9 leukemia, fPrdm16 extended latency, while sPrdm16 shortened latency and induced a strong inflammatory signature, including several cytokines and chemokines that are associated with myelodysplasia and with a worse prognosis in human AML. Finally, in human NPM1-mutant and in MLL-translocated AML, high expression of PRDM16, which negatively impacts outcome, was associated with inflammatory gene expression, thus corroborating the mouse data. Our observations demonstrate distinct roles for Prdm16 isoforms in normal HSCs and AML, and identify sPrdm16 as one of the drivers of prognostically adverse inflammation in leukemia.

Dye-Independent Methods Reveal Elevated Mitochondrial Mass in Hematopoietic Stem Cells.

Hematopoietic stem cells (HSCs) produce most cellular energy through glycolysis rather than through mitochondrial respiration. Consistent with this notion, mitochondrial mass has been reported to be low in HSCs. However, we found that staining with MitoTracker Green, a commonly used dye to measure mitochondrial content, leads to artefactually low fluorescence specifically in HSCs because of dye efflux. Using mtDNA quantification, enumeration of mitochondrial nucleoids, and fluorescence intensity of a genetically encoded mitochondrial reporter, we unequivocally show here that HSCs and multipotential progenitors (MPPs) have higher mitochondrial mass than lineage-committed progenitors and mature cells. Despite similar mitochondrial mass, respiratory capacity of MPPs exceeds that of HSCs. Furthermore, although elevated mitophagy has been invoked to explain low mitochondrial mass in HSCs, we observed that mitochondrial turnover capacity is comparatively low in HSCs. We propose that the role of mitochondria in HSC biology may have to be revisited in light of these findings.

Double-Tip Artefact Removal from Atomic Force Microscopy Images.

The Atomic Force Microscope (AFM) allows the measurement of interactions at interfaces with nanoscale resolution. Imperfections in the shape of the tip often lead to the presence of imaging artefacts such as the blurring and repetition of objects within images. Generally, these artefacts can only be avoided by discarding data and replacing the probe. Under certain circumstances (e.g., rare, high value samples, or extensive chemical/physical tip modification) such an approach is not feasible. Here, we apply a novel deblurring technique, using a Bayesian framework, to yield a reliable estimation of the real surface topography without any prior knowledge of the tip geometry (blind reconstruction). A key contribution is to leverage the significant recently successful body of work in natural image deblurring to solve this problem. We focus specifically on the 'double-tip' effect, where two asperities 1 are present on the tip, each contributing to the image formation mechanism. Finally, we demonstrate that the proposed technique successfully removes the 'double-tip' effect from high resolution AFM images which demonstrate this artefact whilst preserving feature resolution.

Algorithms for the digital restoration of torn films.

This paper presents algorithms for the digital restoration of films damaged by tear. As well as causing local image data loss, a tear results in a noticeable relative shift in the frame between the regions at either side of the tear boundary. This paper describes a method for delineating the tear boundary and for correcting the displacement. This is achieved using a graph-cut segmentation framework that can be either automatic or interactive when automatic segmentation is not possible. Using temporal intensity differences to form the boundary conditions for the segmentation facilitates the robust division of the frame. The resulting segmentation map is used to calculate and correct the relative displacement using a global-motion estimation approach based on motion histograms. A high-quality restoration is obtained when a suitable missing-data treatment algorithm is used to recover any missing pixel intensities.

Analysis of hypoxia and hypoxia-like states through metabolite profiling.

BACKGROUND: In diverse organisms, adaptation to low oxygen (hypoxia) is mediated through complex gene expression changes that can, in part, be mimicked by exposure to metals such as cobalt. Although much is known about the transcriptional response to hypoxia and cobalt, little is known about the all-important cell metabolism effects that trigger these responses. METHODS AND FINDINGS: Herein we use a low molecular weight metabolome profiling approach to identify classes of metabolites in yeast cells that are altered as a consequence of hypoxia or cobalt exposures. Key findings on metabolites were followed-up by measuring expression of relevant proteins and enzyme activities. We find that both hypoxia and cobalt result in a loss of essential sterols and unsaturated fatty acids, but the basis for these changes are disparate. While hypoxia can affect a variety of enzymatic steps requiring oxygen and heme, cobalt specifically interferes with diiron-oxo enzymatic steps for sterol synthesis and fatty acid desaturation. In addition to diiron-oxo enzymes, cobalt but not hypoxia results in loss of labile 4Fe-4S dehydratases in the mitochondria, but has no effect on homologous 4Fe-4S dehydratases in the cytosol. Most striking, hypoxia but not cobalt affected cellular pools of amino acids. Amino acids such as aromatics were elevated whereas leucine and methionine, essential to the strain used here, dramatically decreased due to hypoxia induced down-regulation of amino acid permeases. CONCLUSIONS: These studies underscore the notion that cobalt targets a specific class of iron proteins and provide the first evidence for hypoxia effects on amino acid regulation. This research illustrates the power of metabolite profiling for uncovering new adaptations to environmental stress.