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Accumulating evidence demonstrates that the "Warburg effect" that glycolysis is enhanced even in the presence of oxygen existed in hematopoietic malignancies, contributing to extracellular acidosis. G-protein coupled receptor 68 (GPR68), as a proton sensing GPCR responding to extracellular acidosis, is expected to play a critical role in hematopoietic malignancies. In the present study, we found that GPR68 was overexpressed in acute myeloid leukemia (AML) cells, and GPR68 deficiency impaired AML cell survival in vitro and cell engraftment in vivo. Mechanistic studies revealed that unlike GPR68 regulates Calpain1 in myelodysplastic syndromes (MDS) cells, GPR68 deficiency reduced cytosolic Ca 2+ levels and calcineurin (CaN) activity in AML cells through an NFAT-independent mechanism. Moreover, the decreased Ca 2+ levels disturbed cellular respiration (i.e., oxidative phosphorylation, OxPhos) by inhibiting isocitrate dehydrogenase (IDH) activity; this was more pronounced when BCL2 was inhibited simultaneously. Interestingly, GPR68 inhibition also decreased aerobic glycolysis in AML cells in a Ca 2+ -independent manner, suggesting that GPR68 mediated glucose metabolic symbiosis. As glucose metabolic symbiosis and the heterogeneous dependencies on aerobic glycolysis and cellular respiration tremendously impact chemosensitivity, the inhibition of GPR68 potentiated the tumoricidal effect of first-line chemotherapeutic agents, including BCL-2 inhibitors targeting OxPhos and cytarabine (Ara-C) targeting glycolysis. Consistent with these in vitro observations, higher levels of GPR68 were associated with inferior clinical outcomes in AML patients who received chemotherapies. In short, GPR68 drives the Ca 2+ /CaN pro-survival pathway and mediates glucose metabolic pathways in AML cells. Targeting GPR68 eradicates AML cells and alleviates chemoresistance, which could be exploited as a therapeutic target., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

We investigated immune cytopenia in multiple myeloma (MM) patients with concurrent acquired aplastic anemia (AA), focusing on three clinical cases treated with plasma cell-directed therapy. All three patients achieved partial response in MM and one patient experienced complete resolution of AA. Two patients had partial improvement in transfusion requirement but continued to suffer from severe AA, leading to immunosuppressive therapy (IST) with improvement of transfusion requirement in both patients. In vitro serum testing of these patients demonstrated platelet mitochondrial dysfunction and platelet apoptosis but did not show sera-specific inhibition of erythroid colony formation in progenitor cells. The levels of IL8 and IL15 were elevated in MM patients with AA, implicating their potential roles in this co-occurrence. Response to IST points to the possibility of myeloma-dysregulated immune system leading to autoreactive T-cell destruction of hematopoietic stem and progenitor cells, offering insights for developing new treatment for cytopenia in MM.

Competing Interests: This study was supported by the National Center for Advancing Translational Science (Clinical and Translational Science Awards grant UL1 TR001863) a component of the National Institutes of Health to Dr Kwan. The authors have reported that they have no relationships relevant to the contents of this paper to disclose.

How to develop highly informative serology assays to evaluate the quality of immune protection against coronavirus disease-19 (COVID-19) has been a global pursuit over the past years. Here, a microfluidic high-plex immuno-serolomic assay is developed to simultaneously measure50 plasma or serum samples for50 soluble markers including 35proteins, 11 anti-spike/receptor binding domian (RBD) IgG antibodies spanningmajor variants, and controls. This assay demonstrates the quintuplicate test in a single run with high throughput, low sample volume, high reproducibilityand accuracy. It is applied to the measurement of 1012 blood samples including in-depth analysis of sera from 127 patients and 21 healthy donors over multiple time points, either with acute COVID infection or vaccination. The protein analysis reveals distinct immune mediator modules that exhibit a reduced degree of diversity in protein-protein cooperation in patients with hematologic malignancies or receiving B cell depletion therapy. Serological analysis identifies that COVID-infected patients with hematologic malignancies display impaired anti-RBD antibody response despite high level of anti-spike IgG, which can be associated with limited clonotype diversity and functional deficiency in B cells. These findings underscore the importance to individualize immunization strategies for these high-risk patients and provide an informative tool to monitor their responses at the systems level., (© 2023 The Authors. Small Methods published by Wiley-VCH GmbH.)

Craniofacial disorders arise in early pregnancy and are one of the most common congenital defects. To fully understand how craniofacial disorders arise, it is essential to characterize gene expression during the patterning of the craniofacial region. To address this, we performed bulk and single-cell RNA-seq on human craniofacial tissue from 4-8 weeks post conception. Comparisons to dozens of other human tissues revealed 239 genes most strongly expressed during craniofacial development. Craniofacial-biased developmental enhancers were enriched +/- 400 kb surrounding these craniofacial-biased genes. Gene co-expression analysis revealed that regulatory hubs are enriched for known disease causing genes and are resistant to mutation in the normal healthy population. Combining transcriptomic and epigenomic data we identified 539 genes likely to contribute to craniofacial disorders. While most have not been previously implicated in craniofacial disorders, we demonstrate this set of genes has increased levels of de novo mutations in orofacial clefting patients warranting further study., (© 2023. The Author(s).)

Purpose of Review: The aim of this study was to provide insight into how novel next-generation sequencing (NGS) techniques are set to revolutionize clinical practice., Recent Findings: Advances in sequencing technologies have focused on improved capture of mutations and reads and cellular resolution. Both short and long read DNA sequencing technology are being refined and combined in novel ways with other multiomic approaches to gain unprecedented biological insight into disease. Single-cell (sc)DNA-seq and integrated scDNA-seq with immunophenotyping provide granular information on disease composition such as clonal hierarchy, co-mutation status, zygosity, clonal diversity and genotype phenotype correlations. These and other techniques can identify rare cell populations providing the opportunity for increased sensitivity in measurable residual disease monitoring and precise characterization of residual clones permitting distinction of leukemic from pre/nonmalignant clones., Summary: Increasing genetics-based mechanistic insights and classification of myeloid diseases along with a decrease in the cost of high-throughput NGS mean novel sequencing technologies are closer to being a reality in standard clinical practice. These technologies are poised to improve diagnostics, our ability to monitor treatment response and minimal residual disease and allow the study of premalignant conditions such as clonal haematopoiesis., (Copyright © 2022 Wolters Kluwer Health, Inc. All rights reserved.)

The cellular and genetic mechanisms that coordinate formation of facial sensory structures with surrounding skeletal and soft tissue elements remain poorly understood. Alx1, a homeobox transcription factor, is a key regulator of midfacial morphogenesis. ALX1 mutations in humans are linked to severe congenital anomalies of the facial skeleton (frontonasal dysplasia, FND) with malformation or absence of eyes and orbital contents (micro- and anophthalmia). Zebrafish with loss-of-function alx1 mutations develop with craniofacial and ocular defects of variable penetrance, likely due to compensatory upregulation in expression of a paralogous gene, alx3. Here we show that zebrafish alx1;alx3 mutants develop with highly penetrant cranial and ocular defects that resemble human ALX1-linked FND. alx1 and alx3 are expressed in anterior cranial neural crest (aCNC), which gives rise to the anterior neurocranium (ANC), anterior segment structures of the eye and vascular pericytes. Consistent with a functional requirement for alx genes in aCNC, alx1; alx3 mutants develop with nearly absent ANC and grossly aberrant hyaloid vasculature and ocular anterior segment, but normal retina. In vivo lineage labeling identified a requirement for alx1 and alx3 during aCNC migration, and transcriptomic analysis suggested oxidative stress response as a key target mechanism of this function. Oxidative stress is a hallmark of fetal alcohol toxicity, and we found increased penetrance of facial and ocular malformations in alx1 mutants exposed to ethanol, consistent with a protective role for alx1 against ethanol toxicity. Collectively, these data demonstrate a conserved role for zebrafish alx genes in controlling ocular and facial development, and a novel role in protecting these key midfacial structures from ethanol toxicity during embryogenesis. These data also reveal novel roles for alx genes in ocular anterior segment formation and vascular development and suggest that retinal deficits in alx mutants may be secondary to aberrant ocular vascularization and anterior segment defects. This study establishes robust zebrafish models for interrogating conserved genetic mechanisms that coordinate facial and ocular development, and for exploring gene--environment interactions relevant to fetal alcohol syndrome., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2022. Published by The Company of Biologists Ltd.)

Background: Global efforts are needed to elucidate the epidemiology of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the underlying cause of coronavirus disease 2019 (COVID-19), including seroprevalence, risk factors, and long-term sequelae, as well as immune responses after vaccination across populations and the social dimensions of prevention and treatment strategies., Methods: In the United States, the National Cancer Institute in partnership with the National Institute of Allergy and Infectious Diseases, established the SARS-CoV-2 Serological Sciences Network (SeroNet) as the nation's largest coordinated effort to study coronavirus disease 2019. The network comprises multidisciplinary researchers bridging gaps and fostering collaborations among immunologists, epidemiologists, virologists, clinicians and clinical laboratories, social and behavioral scientists, policymakers, data scientists, and community members. In total, 49 institutions form the SeroNet consortium to study individuals with cancer, autoimmune disease, inflammatory bowel diseases, cardiovascular diseases, human immunodeficiency virus, transplant recipients, as well as otherwise healthy pregnant women, children, college students, and high-risk occupational workers (including healthcare workers and first responders)., Results: Several studies focus on underrepresented populations, including ethnic minorities and rural communities. To support integrative data analyses across SeroNet studies, efforts are underway to define common data elements for standardized serology measurements, cellular and molecular assays, self-reported data, treatment, and clinical outcomes., Conclusions: In this paper, we discuss the overarching framework for SeroNet epidemiology studies, critical research questions under investigation, and data accessibility for the worldwide scientific community. Lessons learned will help inform preparedness and responsiveness to future emerging diseases., (© The Author(s) 2022.. Published by Oxford University Press on behalf of Infectious Diseases Society of America.)

Human cardiac regeneration is limited by low cardiomyocyte replicative rates and progressive polyploidization by unclear mechanisms. To study this process, we engineer a human cardiomyocyte model to track replication and polyploidization using fluorescently tagged cyclin B1 and cardiac troponin T. Using time-lapse imaging, in vitro cardiomyocyte replication patterns recapitulate the progressive mononuclear polyploidization and replicative arrest observed in vivo. Single-cell transcriptomics and chromatin state analyses reveal that polyploidization is preceded by sarcomere assembly, enhanced oxidative metabolism, a DNA damage response, and p53 activation. CRISPR knockout screening reveals p53 as a driver of cell-cycle arrest and polyploidization. Inhibiting sarcomere function, or scavenging ROS, inhibits cell-cycle arrest and polyploidization. Finally, we show that cardiomyocyte engraftment in infarcted rat hearts is enhanced 4-fold by the increased proliferation of troponin-knockout cardiomyocytes. Thus, the sarcomere inhibits cell division through a DNA damage response that can be targeted to improve cardiomyocyte replacement strategies., Competing Interests: Declaration of interests C.E.M. is a scientific founder, employee, and equity holder in Sana Biotechnology. C.E.M. has multiple issued and pending patents pertaining to stem cell biology and heart regeneration., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Rationale: There is growing evidence that common variants and rare sequence alterations in regulatory sequences can result in birth defects or predisposition to disease. Congenital heart defects are the most common birth defect and have a clear genetic component, yet only a third of cases can be attributed to structural variation in the genome or a mutation in a gene. The remaining unknown cases could be caused by alterations in regulatory sequences., Objective: Identify regulatory sequences and gene expression networks that are active during organogenesis of the human heart. Determine whether these sites and networks are enriched for disease-relevant genes and associated genetic variation., Methods and Results: We characterized ChromHMM (chromatin state) and gene expression dynamics during human heart organogenesis. We profiled 7 histone modifications in embryonic hearts from each of 9 distinct Carnegie stages (13-14, 16-21, and 23), annotated chromatin states, and compared these maps to over 100 human tissues and cell types. We also generated RNA-sequencing data, performed differential expression, and constructed weighted gene coexpression networks. We identified 177 412 heart enhancers; 12 395 had not been previously annotated as strong enhancers. We identified 92% of all functionally validated heart-positive enhancers (n=281; 7.5× enrichment; P <2.2×10 -16 ). Integration of these data demonstrated novel heart enhancers are enriched near genes expressed more strongly in cardiac tissue and are enriched for variants associated with ECG measures and atrial fibrillation. Our gene expression network analysis identified gene modules strongly enriched for heart-related functions, regulatory control by heart-specific enhancers, and putative disease genes., Conclusions: Well-connected hub genes with heart-specific expression targeted by embryonic heart-specific enhancers are likely disease candidates. Our functional annotations will allow for better interpretation of whole genome sequencing data in the large number of patients affected by congenital heart defects.

Defects in patterning during human embryonic development frequently result in craniofacial abnormalities. The gene regulatory programs that build the craniofacial complex are likely controlled by information located between genes and within intronic sequences. However, systematic identification of regulatory sequences important for forming the human face has not been performed. Here, we describe comprehensive epigenomic annotations from human embryonic craniofacial tissues and systematic comparisons with multiple tissues and cell types. We identified thousands of tissue-specific craniofacial regulatory sequences and likely causal regions for rare craniofacial abnormalities. We demonstrate significant enrichment of common variants associated with orofacial clefting in enhancers active early in embryonic development, while those associated with normal facial variation are enriched near the end of the embryonic period. These data are provided in easily accessible formats for both craniofacial researchers and clinicians to aid future experimental design and interpretation of noncoding variation in those affected by craniofacial abnormalities., (Copyright © 2018 The Author(s). Published by Elsevier Inc. All rights reserved.)

Background: Prostate-specific antigen (PSA) is a pivotal downstream target gene of the androgen receptor (AR), and a serum biomarker to monitor prostate cancer (PrCa) progression. It has been reported that PSA transactivates AR, but the mechanistic requirements of this response have not been investigated., Methods: We studied the localization of PSA, AR, and Src in intracellular compartments of synthetic androgen (R1881)-stimulated LNCaP and C4-2B PrCa cells, using immunofluorescence and subcellular fractionation approaches. We also investigated the effect of downregulation of PSA on AR expression by immunoblotting and real-time PCR using short hairpin RNA (shRNA) and small interfering RNA (siRNA). Src activity was analyzed by immunoblotting., Results: R1881 stimulation induced nuclear localization of both PSA and AR in LNCaP and C4-2B PrCa cells as well as increased phosphorylation of Src. Stable shRNA or transient siRNA knockdown of PSA resulted in reduced AR protein levels as well as AR mRNA levels in C4-2B cells. Similar to C4-2B cells, ablation of AR levels upon silencing of PSA was also confirmed in VCaP cells, another androgen-independent cell line. Silencing of PSA did not cause significant changes in Src activation; besides, Src regulation by integrins did not appear to affect AR transcriptional activity., Conclusions: PSA localizes to nuclei of androgen-stimulated PrCa cells, and controls AR mRNA and protein levels. This regulatory loop is specific for PSA, does not involve known AR activators such as Src and AKT, and may contribute to AR signaling under conditions of increasing PSA levels in patients., (Copyright © 2011 Wiley Periodicals, Inc.)

Protein kinase R (PKR) is a key component of the interferon antiviral defense pathway. Upon binding double-stranded RNA, PKR undergoes autophosphorylation reactions that activate the kinase. PKR contains an N-terminal double-stranded RNA binding domain, which consists of two tandem double-stranded RNA binding motifs, and a C-terminal kinase domain. We have used small-angle X-ray scattering and small-angle neutron scattering to define the conformation of latent PKR in solution. Guinier analysis indicates a radius of gyration of about 35 A. The p(r) distance distribution function exhibits a peak near 30 A, with a broad shoulder extending to longer distances. Good fits to the scattering data require models that incorporate multiple compact and extended conformations of the two interdomain linker regions. Thus, PKR belongs to the growing family of proteins that contain intrinsically unstructured regions. We propose that the flexible linkers may allow PKR to productively dimerize upon interaction with RNA activators that have diverse structures.

Three cases, recently observed, of non-obstetrical gynaecological infections in adults caused by Haemophilus influenzae have prompted us to reconsider this rare pathology. Non-encapsulated strains of H. influenzae, particularly those belonging to biotype IV, might have special affinity and virulence for the genital tract. It seems, therefore, that such strains should systematically be looked for in samples collected from genital organs.