(University of Science and Technology of China) Researchers from University of science and technology of China has shed new lights on the topic of solid-phase ion migration. Researchers demonstrated a unique in-situ strategy for visualizing the dynamic solid-phase ion migration between nanostructures with nanogap at the atomic scale. The research article entitled "Real-Time Visualization of Solid-Phase Ion Migration Kinetics on Nanowire Monolayer" was published in Journal of the American Chemical Society on April 29th.

The transcription factor forkhead box P3 (FOXP3) is a biomarker for regulatory T cells and can also be expressed in cancer cells, but its function in cancer appears to be divergent. The role of hepatocyte-expressed FOXP3 in hepatocellular carcinoma (HCC) is unknown. Here, we collected tumor samples and clinical information from 115 HCC patients and used five human cancer cell lines. We examined FOXP3 mRNA sequences for mutations, used a luciferase assay to assess promoter activities of FOXP3's target genes, and employed mouse tumor models to confirm in vitro results. We detected mutations in the FKH domain of FOXP3 mRNAs in 33% of the HCC tumor tissues, but in none of the adjacent nontumor tissues. None of the mutations occurred at high frequency, indicating that they occurred randomly. Notably, the mutations were not detected in the corresponding regions of FOXP3 genomic DNA, and many of them resulted in amino acid substitutions in the FKH region, altering FOXP3's subcellular localization. FOXP3 delocalization from the nucleus to the cytoplasm caused loss of transcriptional regulation of its target genes, inactivated its tumor-inhibitory capability, and changed cellular responses to histone deacetylase (HDAC) inhibitors. More complex FKH mutations appeared to be associated with worse prognosis in HCC patients. We conclude that mutations in the FKH domain of FOXP3 mRNA frequently occur in HCC and that these mutations are caused by errors in transcription and are not derived from genomic DNA mutations. Our results suggest that transcriptional mutagenesis of FOXP3 plays a role in HCC.

Prevention of aberrant cutaneous wound repair and appropriate regeneration of an intact and functional integument require the coordinated timing of fibroblast and keratinocyte migration. Here, we identified a mechanism whereby opposing cell-specific motogenic functions of a multifunctional intracellular and extracellular protein, the receptor for hyaluronan-mediated motility (RHAMM), coordinates fibroblast and keratinocyte migration speed and ensures appropriate timing of excisional wound closure. We found that, unlike in WT mice, in Rhamm-null mice, keratinocyte migration initiates prematurely in the excisional wounds, resulting in wounds that have re-surfaced before the formation of normal granulation tissue, leading to a defective epidermal architecture. We also noted aberrant keratinocyte and fibroblast migration in the Rhamm-null mice, indicating that RHAMM suppresses keratinocyte motility but increases fibroblast motility. This cell context–dependent effect resulted from cell-specific regulation of extracellular signal-regulated kinase 1/2 (ERK1/2) activation and expression of a RHAMM target gene encoding matrix metalloprotease 9 (MMP-9). In fibroblasts, RHAMM promoted ERK1/2 activation and MMP-9 expression, whereas in keratinocytes, RHAMM suppressed these activities. In keratinocytes, loss of RHAMM function or expression promoted epidermal growth factor receptor–regulated MMP-9 expression via ERK1/2, which resulted in cleavage of the ectodomain of the RHAMM partner protein CD44 and thereby increased keratinocyte motility. These results identify RHAMM as a key factor that integrates the timing of wound repair by controlling cell migration.

Inter-α-inhibitor is a proteoglycan essential for mammalian reproduction and also plays a less well-characterized role in inflammation. It comprises two homologous “heavy chains” (HC1 and HC2) covalently attached to chondroitin sulfate on the bikunin core protein. Before ovulation, HCs are transferred onto the polysaccharide hyaluronan (HA) to form covalent HC·HA complexes, thereby stabilizing an extracellular matrix around the oocyte required for fertilization. Additionally, such complexes form during inflammatory processes and mediate leukocyte adhesion in the synovial fluids of arthritis patients and protect against sepsis. Here using X-ray crystallography, we show that human HC1 has a structure similar to integrin β-chains, with a von Willebrand factor A domain containing a functional metal ion-dependent adhesion site (MIDAS) and an associated hybrid domain. A comparison of the WT protein and a variant with an impaired MIDAS (but otherwise structurally identical) by small-angle X-ray scattering and analytical ultracentrifugation revealed that HC1 self-associates in a cation-dependent manner, providing a mechanism for HC·HA cross-linking and matrix stabilization. Surprisingly, unlike integrins, HC1 interacted with RGD-containing ligands, such as fibronectin, vitronectin, and the latency-associated peptides of transforming growth factor β, in a MIDAS/cation-independent manner. However, HC1 utilizes its MIDAS motif to bind to and inhibit the cleavage of complement C3, and small-angle X-ray scattering–based modeling indicates that this occurs through the inhibition of the alternative pathway C3 convertase. These findings provide detailed structural and functional insights into HC1 as a regulator of innate immunity and further elucidate the role of HC·HA complexes in inflammation and ovulation.

Antibodies are widely used as cancer therapeutics, but their current use is limited by the low number of antigens restricted to cancer cells. A receptor tyrosine kinase, receptor tyrosine kinase-like orphan receptor 2 (ROR2), is normally expressed only during embryogenesis and is tightly down-regulated in postnatal healthy tissues. However, it is up-regulated in a diverse set of hematologic and solid malignancies, thus ROR2 represents a candidate antigen for antibody-based cancer therapy. Here we describe the affinity maturation and humanization of a rabbit mAb that binds human and mouse ROR2 but not human ROR1 or other human cell-surface antigens. Co-crystallization of the parental rabbit mAb in complex with the human ROR2 kringle domain (hROR2-Kr) guided affinity maturation by heavy-chain complementarity-determining region 3 (HCDR3)-focused mutagenesis and selection. The affinity-matured rabbit mAb was then humanized by complementarity-determining region (CDR) grafting and framework fine tuning and again co-crystallized with hROR2-Kr. We show that the affinity-matured and humanized mAb retains strong affinity and specificity to ROR2 and, following conversion to a T cell–engaging bispecific antibody, has potent cytotoxicity toward ROR2-expressing cells. We anticipate that this humanized affinity-matured mAb will find application for antibody-based cancer therapy of ROR2-expressing neoplasms.

Patrick Herr
Apr 1, 2020; 19:608-623
Research

2 December 2019

The fallout from disinformation and online manipulation strategies have alerted Western democracies to the novel, nuanced vulnerabilities of our information society. This paper outlines the implications of the adoption of AI by the the legacy media, as well as by the new media, focusing on personalization.

Research Event

Event participants

Osamah Al Rawhani, Deputy Director, Sana’a Center for Strategic Studies
Moderator: Nadim Houry, Executive Director, Arab Reform Initiative

Objectives: The detection of cancer micrometastasis for early diagnosis and treatment poses a great challenge for conventional imaging techniques. The aim of study is to evaluate the performance of photoacoustic imaging (PAI) in detecting hepatic micrometastases from melanoma in a very early stage and perform tumor resection by intraoperative photoacoustic image-guidance. Methods: In vivo studies were performed by following protocols approved by the Ethical Committee for Animal Research at Xiamen University. First, a B16 melanoma hepatic metastasis mouse model (n = 10) was established to study the development of micrometastases in vivo. Next, the hepatic metastasis mice models were imaged by scalable PAI instrument, ultrasound, 9.4 T high-resolution magnetic resonance imaging (MRI), positron emission tomography/computed tomography (PET/CT), and bioluminescence imaging. Photoacoustic images acquired with optical wavelengths spanning from 680 to 850 nm were spectrally unmixed by using a linear least-squares method to differentiate various components. Differences in the signal-to-background ratios among different modalities were determined with the two-tailed paired t test. The diagnosis results were assessed with histologic examinations. Excised liver samples from patients diagnosed with hepatic cancer were also examined to identify tumor boundary. In vivo metastatic melanoma removal in surgery was precisely guided by the portable PAI system. Results: PAI achieved as small as ~400 µm hepatic melanoma detection at a depth up to 7 mm in vivo, which could early detect small melanoma compared with ultrasound and MRI in mouse models. The signal ratio of tumor-to-liver acquired with PAI in micrometastases at 8 days (4.2 ± 0.2, n = 6) and 14 days (9.2 ± 0.4, n = 5) were significantly higher than those obtained with PET/CT (1.8 ± 0.1, n = 5 and 4.5 ± 0.2, n = 5, P <0.001 for both). Functional PAI provided dynamic oxygen saturation changes during tumor growth. The limit of detection was measured to be approximately 219 cells per microliter in vitro. We successfully performed intraoperative photoacoustic image-guided surgery in vivo using the rapid portable PAI system. Conclusion: Our findings offer a rapid and effective tool to noninvasively detect micrometastases and guide intraoperative resection as a complementary clinical imaging application.

2-Deoxy-2-[18F]fluoro-D-glucose (2-FDG) with positron emission tomography (2-FDG-PET) is undeniably useful in the clinic, among other uses, to monitor change over time using the 2-FDG standardized uptake values (SUV) metric. This report suggests some potentially serious caveats for this and related roles for 2-FDG PET. Most critical is the assumption that there is an exact proportionality between glucose metabolism and 2-FDG metabolism, called the lumped constant, LC. This report describes that LC is not constant for a specific tissue and may be variable before and after disease treatment. The purpose of this work is not to deny the clinical value of 2-FDG PET; it is a reminder that when one extends the use of an appropriately qualified imaging method, new observations may arise and further validation would be necessary. Current understanding of glucose-based energetics in vivo is based on the quantification of glucose metabolic rates with 2-FDG PET, a method that permits the non-invasive assessment in various human disorders. However, 2-FDG is only a good substrate for facilitated-glucose transporters (GLUTs) but not for sodium-dependent glucose co-transporters (SGLTs), which have recently been shown to be distributed in multiple human tissues. Thus, the GLUT-mediated in vivo glucose utilization measured by 2-FDG PET would be blinded to the potentially substantial role of functional SGLTs in glucose transport and utilization. Therefore, in these circumstances the 2-FDG LC used to quantify in vivo glucose utilization should not be expected to remain constant. 2-FDG LC variations have been especially significant in tumors, particularly at different stages of cancer development, affecting the accuracy of quantitative glucose measures and potentially limiting the prognostic value of 2-FDG, as well as its accuracy in monitoring treatments. SGLT-mediated glucose transport can be estimated using α-methyl-4-deoxy-4-[18F]fluoro-D-glucopyranoside (Me-4FDG). Utilizing both 2-FDG and Me-4FDG should provide a more complete picture of glucose utilization via both GLUT and SGLT transporters in health and disease stages. Given the widespread use of 2-FDG PET to infer glucose metabolism, appreciating the potential limitations of 2-FDG as a surrogate for glucose metabolic rate and the potential reasons for variability in LC is relevant. Even when the readout for the 2-FDG PET study is only an SUV parameter, variability in LC is important, particularly if it changes over the course of disease progression (e.g., an evolving tumor).

3 October 2019

The Chatham House Africa Programme designed the Sudan Stakeholder Dialogues series to help identify the factors that have led to the current economic crisis, the immediate steps that need to be taken to avert collapse and stabilize the economy, and the longer-term structural reforms required to set Sudan on the path to recovery. The project is funded by Humanity United.

Research Event

Event participants

Mohamed Guleid, Chief Executive Officer, Frontier Counties Development Council
Nuradin Dirie, Chair, Puntland Presidential Advisory Council
Aden Abdi, Horn of Africa Programme Director, Conciliation Resources
Chair: Dr Zahbia Yousuf, Senior Research Advisor, Saferworld 

The cell cycle is a highly conserved process involving the coordinated separation of a single cell into two daughter cells. To relate transcriptional regulation across the cell cycle with oscillatory changes in protein abundance and activity, we carried out a proteome- and phospho-proteome-wide mass spectrometry profiling. We compared protein dynamics with gene transcription, revealing many transcriptionally regulated G2 mRNAs that only produce a protein shift after mitosis. Integration of CRISPR/Cas9 survivability studies further highlighted proteins essential for cell viability. Analyzing the dynamics of phosphorylation events and protein solubility dynamics over the cell cycle, we characterize predicted phospho-peptide motif distributions and predict cell cycle-dependent translocating proteins, as exemplified by the S-adenosylmethionine synthase MAT2A. Our study implicates this enzyme in translocating to the nucleus after the G1/S-checkpoint, which enables epigenetic histone methylation maintenance during DNA replication. Taken together, this data set provides a unique integrated resource with novel insights on cell cycle dynamics.

4 September 2019 , Volume 95, Number 5

Lipid droplets (LDs) are frequently increased when excessive lipid accumulation leads to cellular dysfunction. Distinct from mouse beta cells, LDs are prominent in human beta cells, however, the regulation of LD mobilization (lipolysis) in human beta cells remains unclear. We found that glucose increases lipolysis in non-diabetic human islets, but not in type 2 diabetic (T2D) islets, indicating dysregulation of lipolysis in T2D islets. Silencing adipose triglyceride lipase (ATGL) in human pseudoislets (shATGL) increased triglycerides, and the number and size of LDs indicating that ATGL is the principal lipase in human beta cells. In shATGL pseudoislets, biphasic glucose-stimulated insulin secretion (GSIS) and insulin secretion to IBMX and KCl were all reduced without altering oxygen consumption rate compared with scramble control. Like human islets, INS1 cells showed visible LDs, glucose responsive lipolysis, and impairment of GSIS after ATGL silencing. ATGL deficient INS1 cells and human pseudoislets showed reduced Stx1a, a key SNARE component. Proteasomal degradation of Stx1a was accelerated likely through reduced palmitoylation in ATGL deficient INS1 cells. Therefore, ATGL is responsible for LD mobilization in human beta cells and supports insulin secretion by stabilizing Stx1a. The dysregulated lipolysis may contribute to LD accumulation and beta cell dysfunction in T2D islets.

Lipid droplets (LDs) are frequently increased when excessive lipid accumulation leads to cellular dysfunction. Distinct from mouse beta cells, LDs are prominent in human beta cells, however, the regulation of LD mobilization (lipolysis) in human beta cells remains unclear. We found that glucose increases lipolysis in non-diabetic human islets, but not in type 2 diabetic (T2D) islets, indicating dysregulation of lipolysis in T2D islets. Silencing adipose triglyceride lipase (ATGL) in human pseudoislets (shATGL) increased triglycerides, and the number and size of LDs indicating that ATGL is the principal lipase in human beta cells. In shATGL pseudoislets, biphasic glucose-stimulated insulin secretion (GSIS) and insulin secretion to IBMX and KCl were all reduced without altering oxygen consumption rate compared with scramble control. Like human islets, INS1 cells showed visible LDs, glucose responsive lipolysis, and impairment of GSIS after ATGL silencing. ATGL deficient INS1 cells and human pseudoislets showed reduced Stx1a, a key SNARE component. Proteasomal degradation of Stx1a was accelerated likely through reduced palmitoylation in ATGL deficient INS1 cells. Therefore, ATGL is responsible for LD mobilization in human beta cells and supports insulin secretion by stabilizing Stx1a. The dysregulated lipolysis may contribute to LD accumulation and beta cell dysfunction in T2D islets.

Mobilization of hematopoietic stem/progenitor cells (HSPCs) from the bone marrow (BM) is impaired in diabetes. Excess oncostatin M (OSM) produced by M1 macrophages in the diabetic BM signals through p66Shc to induce Cxcl12 in stromal cells and retain HSPCs. BM adipocytes are another source of CXCL12 that blunts mobilization. We tested a strategy of pharmacologic macrophage reprogramming to rescue HSPC mobilization. In vitro, PPAR- activation with pioglitazone switched macrophages from M1 to M2, reduced Osm expression, and prevented transcellular induction of Cxcl12. In diabetic mice, pioglitazone treatment downregulated Osm, p66Shc and Cxcl12 in the hematopoietic BM, restored the effects of granulocyte-colony stimulation factor (G-CSF), and partially rescued HSPC mobilization, but it increased BM adipocytes. Osm deletion recapitulated the effects of pioglitazone on adipogenesis, which was p66Shc-independent, and double knockout of Osm and p66Shc completely rescued HSPC mobilization. In the absence of OSM, BM adipocytes produced less CXCL12, being arguably devoid of HSPC-retaining activity, whereas pioglitazone failed to downregulate Cxcl12 in BM adipocytes. In diabetic patients under pioglitazone therapy, HSPC mobilization after G-CSF was partially rescued. In summary, pioglitazone reprogrammed BM macrophages and suppressed OSM signaling, but sustained Cxcl12 expression by BM adipocytes could limit full recovery of HSPC mobilization.

Cardiac glucose uptake and oxidation are reduced in diabetes despite hyperglycemia. Mitochondrial dysfunction contributes to heart failure in diabetes. It is unclear if these changes are adaptive or maladaptive. To directly evaluate the relationship between glucose delivery and mitochondrial dysfunction in diabetic cardiomyopathy we generated transgenic mice with inducible cardiomyocyte-specific expression of the glucose transporter (GLUT4). We examined mice rendered hyperglycemic following low-dose streptozotocin prior to increasing cardiomyocyte glucose uptake by transgene induction. Enhanced myocardial glucose in non-diabetic mice decreased mitochondrial ATP generation and was associated with echocardiographic evidence of diastolic dysfunction. Increasing myocardial glucose delivery after short-term diabetes onset, exacerbated mitochondrial oxidative dysfunction. Transcriptomic analysis revealed that the largest changes, driven by glucose and diabetes, were in genes involved in mitochondrial function. This glucose-dependent transcriptional repression was in part mediated by O-GlcNAcylation of the transcription factor Sp1. Increased glucose uptake induced direct O-GlcNAcylation of many electron transport chain subunits and other mitochondrial proteins. These findings identify mitochondria as a major target of glucotoxicity. They also suggest reduced glucose utilization in diabetic cardiomyopathy might defend against glucotoxicity and caution that restoring glucose delivery to the heart in the context of diabetes could accelerate mitochondrial dysfunction by disrupting protective metabolic adaptations.

The transcription factor forkhead box P3 (FOXP3) is a biomarker for regulatory T cells and can also be expressed in cancer cells, but its function in cancer appears to be divergent. The role of hepatocyte-expressed FOXP3 in hepatocellular carcinoma (HCC) is unknown. Here, we collected tumor samples and clinical information from 115 HCC patients and used five human cancer cell lines. We examined FOXP3 mRNA sequences for mutations, used a luciferase assay to assess promoter activities of FOXP3's target genes, and employed mouse tumor models to confirm in vitro results. We detected mutations in the FKH domain of FOXP3 mRNAs in 33% of the HCC tumor tissues, but in none of the adjacent nontumor tissues. None of the mutations occurred at high frequency, indicating that they occurred randomly. Notably, the mutations were not detected in the corresponding regions of FOXP3 genomic DNA, and many of them resulted in amino acid substitutions in the FKH region, altering FOXP3's subcellular localization. FOXP3 delocalization from the nucleus to the cytoplasm caused loss of transcriptional regulation of its target genes, inactivated its tumor-inhibitory capability, and changed cellular responses to histone deacetylase (HDAC) inhibitors. More complex FKH mutations appeared to be associated with worse prognosis in HCC patients. We conclude that mutations in the FKH domain of FOXP3 mRNA frequently occur in HCC and that these mutations are caused by errors in transcription and are not derived from genomic DNA mutations. Our results suggest that transcriptional mutagenesis of FOXP3 plays a role in HCC.

Prevention of aberrant cutaneous wound repair and appropriate regeneration of an intact and functional integument require the coordinated timing of fibroblast and keratinocyte migration. Here, we identified a mechanism whereby opposing cell-specific motogenic functions of a multifunctional intracellular and extracellular protein, the receptor for hyaluronan-mediated motility (RHAMM), coordinates fibroblast and keratinocyte migration speed and ensures appropriate timing of excisional wound closure. We found that, unlike in WT mice, in Rhamm-null mice, keratinocyte migration initiates prematurely in the excisional wounds, resulting in wounds that have re-surfaced before the formation of normal granulation tissue, leading to a defective epidermal architecture. We also noted aberrant keratinocyte and fibroblast migration in the Rhamm-null mice, indicating that RHAMM suppresses keratinocyte motility but increases fibroblast motility. This cell context–dependent effect resulted from cell-specific regulation of extracellular signal-regulated kinase 1/2 (ERK1/2) activation and expression of a RHAMM target gene encoding matrix metalloprotease 9 (MMP-9). In fibroblasts, RHAMM promoted ERK1/2 activation and MMP-9 expression, whereas in keratinocytes, RHAMM suppressed these activities. In keratinocytes, loss of RHAMM function or expression promoted epidermal growth factor receptor–regulated MMP-9 expression via ERK1/2, which resulted in cleavage of the ectodomain of the RHAMM partner protein CD44 and thereby increased keratinocyte motility. These results identify RHAMM as a key factor that integrates the timing of wound repair by controlling cell migration.

Motor protein-based active transport is essential for mRNA localization and local translation in animal cells, yet how mRNA granules interact with motor proteins remains poorly understood. Using an unbiased yeast two–hybrid screen for interactions between murine RNA-binding proteins (RBPs) and motor proteins, here we identified protein interaction with APP tail-1 (PAT1) as a potential direct adapter between zipcode-binding protein 1 (ZBP1, a β-actin RBP) and the kinesin-I motor complex. The amino acid sequence of mouse PAT1 is similar to that of the kinesin light chain (KLC), and we found that PAT1 binds to KLC directly. Studying PAT1 in mouse primary hippocampal neuronal cultures from both sexes and using structured illumination microscopic imaging of these neurons, we observed that brain-derived neurotrophic factor (BDNF) enhances co-localization of dendritic ZBP1 and PAT1 within granules that also contain kinesin-I. PAT1 is essential for BDNF-stimulated neuronal growth cone development and dendritic protrusion formation, and we noted that ZBP1 and PAT1 co-locate along with β-actin mRNA in actively transported granules in living neurons. Acute disruption of the PAT1–ZBP1 interaction in neurons with PAT1 siRNA or a dominant-negative ZBP1 construct diminished localization of β-actin mRNA but not of Ca2+/calmodulin-dependent protein kinase IIα (CaMKIIα) mRNA in dendrites. The aberrant β-actin mRNA localization resulted in abnormal dendritic protrusions and growth cone dynamics. These results suggest a critical role for PAT1 in BDNF-induced β-actin mRNA transport during postnatal development and reveal a new molecular mechanism for mRNA localization in vertebrates.