SMARCB1 regulates the hypoxic stress response in sickle cell trait.

Renal medullary carcinoma (RMC) is an aggressive kidney cancer that almost exclusively develops in individuals with sickle cell trait (SCT) and is always characterized by loss of the tumor suppressor SMARCB1. Because renal ischemia induced by red blood cell sickling exacerbates chronic renal medullary hypoxia in vivo, we investigated whether the loss of SMARCB1 confers a survival advantage under the setting of SCT. Hypoxic stress, which naturally occurs within the renal medulla, is elevated under the setting of SCT. Our findings showed that hypoxia-induced SMARCB1 degradation protected renal cells from hypoxic stress. SMARCB1 wild-type renal tumors exhibited lower levels of SMARCB1 and more aggressive growth in mice harboring the SCT mutation in human hemoglobin A (HbA) than in control mice harboring wild-type human HbA. Consistent with established clinical observations, SMARCB1-null renal tumors were refractory to hypoxia-inducing therapeutic inhibition of angiogenesis. Further, reconstitution of SMARCB1 restored renal tumor sensitivity to hypoxic stress in vitro and in vivo. Together, our results demonstrate a physiological role for SMARCB1 degradation in response to hypoxic stress, connect the renal medullary hypoxia induced by SCT with an increased risk of SMARCB1-negative RMC, and shed light into the mechanisms mediating the resistance of SMARCB1-null renal tumors against angiogenesis inhibition therapies.

Syndecan 1 is a critical mediator of macropinocytosis in pancreatic cancer.

Pancreatic ductal adenocarcinoma (PDAC) remains recalcitrant to all forms of cancer treatment and carries a five-year survival rate of only 8%1. Inhibition of oncogenic KRAS (hereafter KRAS*), the earliest lesion in disease development that is present in more than 90% of PDACs, and its signalling surrogates has yielded encouraging preclinical results with experimental agents2-4. However, KRAS*-independent disease recurrence following genetic extinction of Kras* in mouse models anticipates the need for co-extinction strategies5,6. Multiple oncogenic processes are initiated at the cell surface, where KRAS* physically and functionally interacts to direct signalling that is essential for malignant transformation and tumour maintenance. Insights into the complexity of the functional cell-surface-protein repertoire (surfaceome) have been technologically limited until recently and-in the case of PDAC-the genetic control of the function and composition of the PDAC surfaceome in the context of KRAS* signalling remains largely unknown. Here we develop an unbiased, functional target-discovery platform to query KRAS*-dependent changes of the PDAC surfaceome, which reveals syndecan 1 (SDC1, also known as CD138) as a protein that is upregulated at the cell surface by KRAS*. Localization of SDC1 at the cell surface-where it regulates macropinocytosis, an essential metabolic pathway that fuels PDAC cell growth-is essential for disease maintenance and progression. Thus, our study forges a mechanistic link between KRAS* signalling and a targetable molecule driving nutrient salvage pathways in PDAC and validates oncogene-driven surfaceome annotation as a strategy to identify cancer-specific vulnerabilities.

Divergent expression patterns of IL-4 and IL-13 define unique functions in allergic immunity.

Interleukin 4 (IL-4) and IL-13 are critical for responses to parasitic helminthes. We used genetically engineered reporter mice to assess the temporal and spatial production of these cytokines in vivo. In lymph nodes, IL-4, but not IL-13, was made by follicular helper T cells (T(FH) cells). In contrast, tissue type 2 helper T cells (T(H)2 cells) produced both cytokines. There was also divergent production of IL-4 and IL-13 among cells of the innate immune system, whereby basophils produced IL-4, whereas innate helper type 2 cells (Ih2 cells) produced IL-13. IL-13 production by T(H)2 and Ih2 cells was dependent on the transcription factor GATA-3, which was present in large amounts in these cells, and in contrast to the small amount of GATA-3 in T(FH) cells and basophils. The distinct localization and cellular expression of IL-4 and IL-13 explains their unique roles during allergic immunity.

Suppression of alias and replica noises in phase holograms using fractal topologies.

Two-dimensional fractal topologies featuring (scaling) self-similarity, dense set of Bragg (diffraction) peaks, and inherent rotation symmetry, which are not achievable with regular grid-matrix geometries, exhibit optical robustness against structural damage and noise immunity of optical transmission paths. In this work, we numerically and experimentally demonstrate phase holograms using fractal plane-divisions. By taking advantage of the symmetries of the fractal topology, we propose numerical algorithms to design the fractal holograms. This algorithm solves the inapplicability of the conventional iterative Fourier transform algorithm (IFTA) method and enables efficient optimizations of millions of adjustable parameters in the optical element. Experimental samples show that the alias and replica noises in the image plane of fractal holograms are clearly suppressed, facilitating applications for high-accuracy and compact requirements.

Combination of explainable machine learning and conceptual density functional theory: applications for the study of key solvation mechanisms.

We present explainable machine learning approaches for the accurate prediction and understanding of solvation free energies, enthalpies, and entropies for different salts in various protic and aprotic solvents. As key input features, we use fundamental contributions from the conceptual density functional theory (DFT) of solutions. The most accurate models with the highest prediction accuracy for the experimental validation data set are decision tree-based approaches such as extreme gradient boosting and extra trees, which highlight the non-linear influence of feature values on target predictions. The detailed assessment of the importance of features in terms of Gini importance criteria as well as Shapley Additive Explanations (SHAP) and permutation and reduction approaches underlines the prominent role of anion and cation solvation effects in combination with fundamental electronic properties of the solvents. These results are reasonably consistent with previous assumptions and provide a solid rationale for more recent theoretical approaches.

The costimulatory molecule ICOS regulates the expression of c-Maf and IL-21 in the development of follicular T helper cells and TH-17 cells.

The inducible costimulatory molecule ICOS has been suggested to be important in the development of interleukin 17 (IL-17)-producing helper T cells (T(H)-17 cells) and of follicular helper T cells (T(FH) cells). Here we show that ICOS-deficient mice had no defect in T(H)-17 differentiation but had fewer T(H)-17 cells after IL-23 stimulation and fewer T(FH) cells. We also show that T(FH) cells produced IL-17 and that T(FH) cells in ICOS-deficient mice were defective in IL-17 production. Both T(H)-17 and T(FH) cells had higher expression of the transcription factor c-Maf. Genetic loss of c-Maf resulted in a defect in IL-21 production and fewer T(H)-17 and T(FH) cells. Thus our data suggest that ICOS-induced c-Maf regulates IL-21 production that in turn regulates the expansion of T(H)-17 and T(FH) cells.

K-series approximation of vectorial optical fields for designing diffractive optical elements with subwavelength feature sizes.

Diffractive optical elements (DOEs) are widely applied as compact solutions for desired light manipulations via wavefront shaping. Recent advanced chip applications further require their feature sizes to move down to the subwavelength, which inevitably brings forth vectorial effects of optical fields and makes the typical scalar-based theory invalid. However, simulating and optimizing their vectorial fields, which are associated with billions of adjustable parameters in the optical element, are difficult to do, because of the issues of numerical stability and the highly-demanding computational cost. To address this problem, this research proposes an applicable algorithm by means of a wave-vector (k) series approximation of vectorial optical fields. On the basis of the semi-analytical rigorous coupled wave analysis (RCWA), an adequate selection scheme on k-series enables computationally efficient yet still predictive calculations for DOEs. The performance estimations for exemplary designs by the finite difference time domain (FDTD) method show that the predicted intensity profiles by the proposed algorithm agree with the target by just a fractional error. Together with optimizing the geometrical degrees of freedom (e.g., DOE depth h) as compensation for errors from the truncation of k-series, the algorithm demonstrates its outperformance by one or two orders of magnitude in accuracy versus the scalar-based model, and demands only a reasonable computational resource.

Ets1 Promotes the Differentiation of Post-Selected iNKT Cells through Regulation of the Expression of Vα14Jα18 T Cell Receptor and PLZF.

The transcription factor Ets1 is essential for the development/differentiation of invariant Natural Killer T (iNKT) cells at multiple stages. However, its mechanisms of action and target genes in iNKT cells are still elusive. Here, we show that Ets1 is required for the optimal expression of the Vα14Jα18 T cell receptor (TCR) in post-selected thymic iNKT cells and their immediate differentiation. Ets1 is also critical for maintaining the peripheral homeostasis of iNKT cells, which is a role independent of the expression of the Vα14Jα18 TCR. Genome-wide transcriptomic analyses of post-selected iNKT cells further reveal that Ets1 controls leukocytes activation, proliferation differentiation, and leukocyte-mediated immunity. In addition, Ets1 regulates the expression of ICOS and PLZF in iNKT cells. More importantly, restoring the expression of PLZF and the Vα14Jα18 TCR partially rescues the differentiation of iNKT cells in the absence of Ets1. Taken together, our results establish a detailed molecular picture of how Ets1 regulates the stepwise differentiation of iNKT cells.

IL-4 inhibits TGF-beta-induced Foxp3+ T cells and, together with TGF-beta, generates IL-9+ IL-10+ Foxp3(-) effector T cells.

Transcription factor Foxp3 is critical for generating regulatory T cells (T(reg) cells). Transforming growth factor-beta (TGF-beta) induces Foxp3 and suppressive T(reg) cells from naive T cells, whereas interleukin 6 (IL-6) inhibits the generation of inducible T(reg) cells. Here we show that IL-4 blocked the generation of TGF-beta-induced Foxp3(+) T(reg) cells and instead induced a population of T helper cells that produced IL-9 and IL-10. The IL-9(+)IL-10(+) T cells demonstrated no regulatory properties despite producing abundant IL-10. Adoptive transfer of IL-9(+)IL-10(+) T cells into recombination-activating gene 1-deficient mice induced colitis and peripheral neuritis, the severity of which was aggravated if the IL-9(+)IL-10(+) T cells were transferred with CD45RB(hi) CD4(+) effector T cells. Thus IL-9(+)IL-10(+) T cells lack suppressive function and constitute a distinct population of helper-effector T cells that promote tissue inflammation.

Utilizing a PTPN22 gene signature to predict response to targeted therapies in rheumatoid arthritis.

Despite the development of several targeted therapies for rheumatoid arthritis (RA), there is still no reliable drug-specific predictor to assist rheumatologists in selecting the most effective targeted therapy for each patient. Recently, a gene signature caused by impaired induction of PTPN22 in anti-CD3 stimulated peripheral blood mononuclear cells (PBMC) was observed in healthy at-risk individuals. However, the downstream target genes of PTPN22 and the molecular mechanisms regulating its expression are still poorly understood. Here we report that the PTPN22 gene signature is also present in PBMC from patients with active RA and can be reversed after effective treatment. The expression of PTPN22 correlates with that of more than 1000 genes in Th cells of anti-CD3 stimulated PBMC of healthy donors and is inhibited by TNFα or CD28 signals, but not IL-6, through distinct mechanisms. In addition, the impaired induction of PTPN22 in PBMC of patients with active RA can be normalized in vitro by several targeted therapies. More importantly, the in vitro normalization of PTPN22 expression correlates with clinical response to the targeted therapies in a longitudinal RA cohort. Thus, in vitro normalization of PTPN22 expression by targeted therapies can potentially be used to predict clinical response.

Rectal Delivery of a DNAzyme That Specifically Blocks the Transcription Factor GATA3 and Reduces Colitis in Mice.

BACKGROUND & AIMS: GATA3 is a transcription factor that regulates T-cell production of cytokines. We investigated the role of GATA3 in development of colitis in mice. METHODS: We performed quantitative polymerase chain reaction and immunofluorescence analyses of colon tissues from patients with Crohn's disease (n = 61) or ulcerative colitis (UC, n = 74) or from patients without inflammatory bowel diseases (n = 22), to measure levels of GATA3. Colitis was induced by administration of oxazolone or 2,4,6-trinitrobenzenesulfonic acid to control mice, mice with T-cell-specific deletion of GATA3, and mice with deletion of tumor necrosis factor receptor (TNFR) 1 and TNFR2 (TNFR double knockouts); some mice were given a GATA3-specific DNAzyme (hgd40) or a control DNAzyme via intrarectal administration, or systemic injections of an antibody to TNF before or during sensitization and challenge phase of colitis induction. Colon tissues were collected and immunofluorescence and histochemical analyses were performed. Lamina propria mononuclear cells and T cells were isolated and analyzed by flow cytometry or cytokine assays. Colonic distribution of labeled DNAzyme and inflammation were monitored by in vivo imaging (endoscopy) of mice. RESULTS: Levels of GATA3 messenger RNA were higher in colon tissues from patients with UC, but not ileal Crohn's disease, than control tissues; levels of GATA3 correlated with levels of inflammatory cytokines (interleukin [IL] 9, IL17A, IL6, IL5, IL4, IL13, and TNF). We observed increased expression of GATA3 by lamina propria T cells from mice with colitis compared with controls. Mice with T-cell-specific deletion of GATA3 did not develop colitis and their colonic tissues did not produce inflammatory cytokines (IL6, IL9, or IL13). The DNAzyme hgd40 inhibited expression of GATA3 messenger RNA by unstimulated and stimulated T cells, and distributed throughout the inflamed colons of mice with colitis. Colon tissues from mice given hgd40 had reduced expression of GATA3 messenger RNA, compared with mice given a control DNAzyme. Mice given hgd40 did not develop colitis after administration of oxazolone or 2,4,6-trinitrobenzenesulfonic acid; lamina propria cells from these mice expressed lower levels of IL6, IL9, and IL13 than cells from mice given the control DNAzyme. Mini-endoscopic images revealed that hgd40 and anti-TNF reduced colon inflammation over 3 days; hgd40 reduced colitis in TNFR double-knockout mice. CONCLUSIONS: Levels of GATA3 are increased in patients with UC and correlate with production of inflammatory cytokines in mice and humans. A DNAzyme that prevents expression of GATA3 reduces colitis in mice, independently of TNF, and reduces levels of cytokines in the colon. This DNAzyme might be developed for treatment of patients with UC.

m(en)TOR(ing) differentiating T helper cells.

The decision of naive Th cells to assume the fate of effector or regulator Th cells heavily influences the outcome of adaptive immune responses. In this issue of Immunity, Delgoffe et al. (2009) use genetic approaches and demonstrate that mTOR kinase plays a critical role in this decision-making process.

Study of wide-spectrum and high-resolution diffraction optical elements by stacks of binary phase gratings.

This work theoretically investigates wide-spectrum and high-resolution diffraction optical elements that are made of stacks of low-resolution binary phase gratings, whereby the two-dimensional grids in different grating layers are arranged with specified displacements. We remodel the common kinoform algorithm for this multi-scale architecture. Numerical computations show that, by increasing the number of stacking layers, the resolution of the far-field image can be improved and also that the optical elements are more insensitive to variations of incident wavelengths at the cost of part accuracy of the image reconstructions. Practical concern focuses on largely increasing the number of grating layers and efficiency of the optical designs in theory and on the manufacture of stacks of ultra-thin grating films.

Metabolomics of Diabetic Retinopathy.

PURPOSE OF REVIEW: Metabolomics is the study of dysregulated metabolites in biological materials. We reviewed the use of the technique to elucidate the genetic and environmental factors that contribute to the development of diabetic retinopathy. RECENT FINDINGS: With regard to metabolomic studies of diabetic retinopathy, the field remains in its infancy with few studies published to date and little replication of results. Vitreous and serum samples are the main tissues examined, and dysregulation in pathways such as the pentose phosphate pathway, arginine to proline pathway, polyol pathway, and ascorbic acidic pathways have been reported. Few studies have examined the metabolomic underpinnings of diabetic retinopathy. Further research is required to replicate findings to date and determine longitudinal associations with disease.

Small Artery Elastin Distribution and Architecture-Focus on Three Dimensional Organization.

The distribution of ECM proteins within the walls of resistance vessels is complex both in variety of proteins and structural arrangement. In particular, elastin exists as discrete fibers varying in orientation across the adventitia and media as well as often resembling a sheet-like structure in the case of the IEL. Adding to the complexity is the tissue heterogeneity that exists in these structural arrangements. For example, small intracranial cerebral arteries lack adventitial elastin while similar sized arteries from skeletal muscle and intestinal mesentery exhibit a complex adventitial network of elastin fibers. With regard to the IEL, several vascular beds exhibit an elastin sheet with punctate holes/fenestrae while in others the IEL is discontinuous and fibrous in appearance. Importantly, these structural patterns likely sub-serve specific functional properties, including mechanosensing, control of external forces, mechanical properties of the vascular wall, cellular positioning, and communication between cells. Of further significance, these processes are altered in vascular disorders such as hypertension and diabetes mellitus where there is modification of ECM. This brief report focuses on the three-dimensional wall structure of small arteries and considers possible implications with regard to mechanosensing under physiological and pathophysiological conditions.

GATA-3 links tumor differentiation and dissemination in a luminal breast cancer model.

How breast cancers are able to disseminate and metastasize is poorly understood. Using a hyperplasia transplant system, we show that tumor dissemination and metastasis occur in discrete steps during tumor progression. Bioinformatic analysis revealed that loss of the transcription factor GATA-3 marked progression from adenoma to early carcinoma and onset of tumor dissemination. Restoration of GATA-3 in late carcinomas induced tumor differentiation and suppressed tumor dissemination. Targeted deletion of GATA-3 in early tumors led to apoptosis of differentiated cells, indicating that its loss is not sufficient for malignant conversion. Rather, malignant progression occurred with an expanding GATA-3-negative tumor cell population. These data indicate that GATA-3 regulates tumor differentiation and suppresses tumor dissemination in breast cancer.

Regulation of IL-4 Expression in Immunity and Diseases.

IL-4 was first identified as a T cell-derived growth factor for B cells. Studies over the past several decades have markedly expanded our understanding of its cellular sources and function. In addition to T cells, IL-4 is produced by innate lymphocytes, such as NTK cells, and myeloid cells, such as basophils and mast cells. It is a signature cytokine of type 2 immune response but also has a nonimmune function. Its expression is tightly regulated at several levels, including signaling pathways, transcription factors, epigenetic modifications, microRNA, and long noncoding RNA. This chapter will review in detail the molecular mechanism regulating the cell type-specific expression of IL-4 in physiological and pathological type 2 immune responses.

Ets-1 facilitates nuclear entry of NFAT proteins and their recruitment to the IL-2 promoter.

E26 transformation-specific sequence 1 (Ets-1), the prototype of the ETS family of transcription factors, is critical for the expression of IL-2 by murine Th cells; however, its mechanism of action is still unclear. Here we show that Ets-1 is also essential for optimal production of IL-2 by primary human Th cells. Although Ets-1 negatively regulates the expression of Blimp1, a known suppressor of IL-2 expression, ablation of B lymphocyte-induced maturation protein 1 (Blimp1) does not rescue the expression of IL-2 by Ets-1-deficient Th cells. Instead, Ets-1 physically and functionally interacts with the nuclear factor of activated T-cells (NFAT) and is required for the recruitment of NFAT to the IL-2 promoter. In addition, Ets-1 is located in both the nucleus and cytoplasm of resting Th cells. Nuclear Ets-1 quickly exits the nucleus in response to calcium-dependent signals and competes with NFAT proteins for binding to protein components of noncoding RNA repressor of NFAT complex (NRON), which serves as a cytoplasmic trap for phosphorylated NFAT proteins. This nuclear exit of Ets-1 precedes rapid nuclear entry of NFAT and Ets-1 deficiency results in impaired nuclear entry, but not dephosphorylation, of NFAT proteins. Thus, Ets-1 promotes the expression of IL-2 by modulating the activity of NFAT.

Parts per billion detection of uranium with a porphyrinoid-containing nanoparticle and in vivo photoacoustic imaging.

Chemical tools that can report radioactive isotopes would be of interest to the defense community. Here we report ∼250 nm polymeric nanoparticles containing porphyrinoid macrocycles with and without pre-complexed depleted uranium and demonstrate that the latter species may be detected easily and with high sensitivity via photoacoustic imaging. The porphyrinoid macrocycles used in the present study are non-aromatic in the absence of the uranyl cation, but aromatic after cation complexation. We solubilized both the freebase and metalated forms of the macrocycles in poly(lactic-co-glycolic acid) and found a peak in the photoacoustic spectrum at 910 nm excitation in the case of the uranyl complex. The signal was stable for at least 15 minutes and allowed detection of uranium concentrations down to 6.2 ppb (5.7 nM) in vitro and 0.57 ppm (19 fCi; 0.52 µM) in vivo. To the best of our knowledge, this is the first report of a nanoparticle that detects an actinide cation via photoacoustic imaging.

GATA-3 regulates the homeostasis and activation of CD8+ T cells.

GATA-3, a C2C2-type zinc finger transcription factor, regulates many steps of T cell development and differentiation. It is also required for optimal production of type 2 cytokines by CD8(+) T cells. However, its role in the development and function of this subset of T cells is still poorly characterized. In this paper, we report that GATA-3 is required for MHC-mediated positive selection and final maturation of CD8 single-positive thymocytes. Deficiency of GATA-3 mediated by a CD4cre transgene led to age-dependent lymphadenopathy partly because of abnormal expansion of CD8(+) T cells driven by a cell-extrinsic mechanism. Paradoxically, GATA-3-deficient CD8(+) T cells were hyporesponsive to Ag stimulation due to a defect in the maintenance/progression, but not initiation, of activation signals. More importantly, GATA-3-deficient CD8(+) T cells were less efficient in killing Ag-bearing tumor cells in vivo. Taken together, our data further expand the role of GATA-3 in T cells.