Antioxidant functions of DHHC3 suppress anti-cancer drug activities.

Ablation of protein acyltransferase DHHC3 selectively enhanced the anti-cancer cell activities of several chemotherapeutic agents, but not kinase inhibitors. To understand why this occurs, we used comparative mass spectrometry-based palmitoyl-proteomic analysis of breast and prostate cancer cell lines, ± DHHC3 ablation, to obtain the first comprehensive lists of candidate protein substrates palmitoylated by DHHC3. Putative substrates included 22-28 antioxidant/redox-regulatory proteins, thus predicting that DHHC3 should have antioxidant functions. Consistent with this, DHHC3 ablation elevated oxidative stress. Furthermore, DHHC3 ablation, together with chemotherapeutic drug treatment, (a) elevated oxidative stress, with a greater than additive effect, and (b) enhanced the anti-growth effects of the chemotherapeutic agents. These results suggest that DHHC3 ablation enhances chemotherapeutic drug potency by disabling the antioxidant protections that contribute to drug resistance. Affirming this concept, DHHC3 ablation synergized with another anti-cancer drug, PARP inhibitor PJ-34, to decrease cell proliferation and increase oxidative stress. Hence, DHHC3 targeting can be a useful strategy for selectively enhancing potency of oxidative stress-inducing anti-cancer drugs. Also, comprehensive identification of DHHC3 substrates provides insight into other DHHC3 functions, relevant to in vivo tumor growth modulation.

Microstructural Characterization of GaN Grown on SiC.

GaN films have been grown on SiC substrates with an AlN nucleation layer by using a metal organic chemical vapor deposition technique. Micro-cracking of the GaN films has been observed in some of the grown samples. In order to investigate the micro-cracking and microstructure, the samples have been studied using various characterization techniques such as optical microscopy, atomic force microscopy, Raman spectroscopy, scanning electron microscopy and transmission electron microscopy (TEM). The surface morphology of the AlN nucleation layer is related to the stress evolution in subsequent overgrown GaN epilayers. It is determined via TEM evidence that, if the AlN nucleation layer has a rough surface morphology, this leads to tensile stresses in the GaN films, which finally results in cracking. Raman spectroscopy results also suggest this, by showing the existence of considerable tensile residual stress in the AlN nucleation layer. Based on these various observations and results, conclusions or propositions relating to the microstructure are presented.

Tetraspanin TSPAN12 regulates tumor growth and metastasis and inhibits ß-catenin degradation.

Ablation of tetraspanin protein TSPAN12 from human MDA-MB-231 cells significantly decreased primary tumor xenograft growth, while increasing tumor apoptosis. Furthermore, TSPAN12 removal markedly enhanced tumor-endothelial interactions and increased metastasis to mouse lungs. TSPAN12 removal from human MDA-MB-231 cells also caused diminished association between FZD4 (a key canonical Wnt pathway receptor) and its co-receptor LRP5. The result likely explains substantially enhanced proteosomal degradation of ß-catenin, a key effecter of canonical Wnt signaling. Consistent with disrupted canonical Wnt signaling, TSPAN12 ablation altered expression of LRP5, Naked 1 and 2, DVL2, DVL3, Axin 1, and GSKß3 proteins. TSPAN12 ablation also altered expression of several genes regulated by ß-catenin (e.g. CCNA1, CCNE2, WISP1, ID4, SFN, ME1) that may help to explain altered tumor growth and metastasis. In conclusion, these results provide the first evidence for TSPAN12 playing a role in supporting primary tumor growth and suppressing metastasis. TSPAN12 appears to function by stabilizing FZD4-LRP5 association, in support of canonical Wnt-pathway signaling, leading to enhanced ß-catenin expression and function.

Palmitoylation by DHHC3 is critical for the function, expression, and stability of integrin α6ß4.

The laminin-binding integrin α6ß4 plays key roles in both normal epithelial and endothelial cells and during tumor cell progression, metastasis, and angiogenesis. Previous cysteine mutagenesis studies have suggested that palmitoylation of α6ß4 protein supports a few integrin-dependent functions and molecular associations. Here we took another approach and obtained strikingly different results. We used overexpression and RNAi knockdown in multiple cell types to identify protein acyl transferase DHHC3 as the enzyme responsible for integrin ß4 and α6 palmitoylation. Ablation of DHHC3 markedly diminished integrin-dependent cellular cable formation on Matrigel, integrin signaling through Src, and ß4 phosphorylation on key diagnostic amino acids (S1356 and 1424). However, unexpectedly, and in sharp contrast to prior α6ß4 mutagenesis results, knockdown of DHHC3 accelerated the degradation of α6ß4, likely due to an increase in endosomal exposure to cathepsin D. When proteolytic degradation was inhibited (by Pepstatin A), rescued α6ß4 accumulated intracellularly, but was unable to reach the cell surface. DHHC3 ablation effects were strongly selective for α6ß4. Cell-surface levels of ~10 other proteins (including α3ß1) were not diminished, and the appearance of hundreds of other palmitoylated proteins was not altered. Results obtained here demonstrate a new substrate for the DHHC3 enzyme and provide novel opportunities for modulating α6ß4 expression, distribution, and function.

The great lockdown, uncertainty and trade: asymmetric impact on imports.

The world economy has recently navigated through the pandemic caused by the coronavirus. Almost all the affected countries have responded with stringency measures to control the pandemic. However, these restrictions appear to have critically impacted the global supply chain and cross-border movement of goods. In this regard, we attempt to investigate the impact of pandemic-related stringency measures on India's import demand. For this purpose, we use bilateral monthly import data of India with its major trading counterparts. Our findings suggest that stringency measures have a positive impact on imports, indicating that the economy relies more on imported items when its domestic production and supply chain are disrupted by the pandemic-related restrictions. Conversely, the import origin countries' restrictions have a negative impact on Indian imports, indicating that these restrictions have adversely affected the production and supply chain in origin countries, thereby reducing the overall flow of imports to India. We also find that economic policy uncertainty of home and product origin countries has a negative impact on Indian imports. Our results also confirm that the pandemic-related restrictions and different types of uncertainty have an asymmetric effect on imports.

Fabrication and Synthesis of Thiococlchicoside loaded Matrix type Transdermal Patch.

BACKGROUND: The goal of this work was to synthesize and fabricate matrix type transdermal patches based on a combination of polymers (Eudragit L100, HPMC and PVP K30), plasticizer and crosslinking agents (propylene glycol and triethyl citrate) and adhesives (Dura Tak 87-6908) to increase Thiocolchicoside (THC) absorption via topical route. This method allows avoidance of first-pass metabolism along with a consistent and extended duration of therapeutic activity. METHODS: Fabrication and casting of polymeric solutions containing THC was done either in petri plates or through lab coater to get transdermal patches. Finally, the formulated patches were studied for their physicochemical and biological evaluation using scanning electron microscopy, FTIR, DSC, XRD and ex-vivo permeation studies using pig ear skin. RESULTS: FTIR studies confirm that the THC characteristics peaks (carbonyl (Amide I) at 1525.5 cm-1, C=O stretching (tropane ring) at 1664.4 cm-1, Amide II band (N-H stretching) at 3325.9cm-1, thioether band at 2360.7cm-1, and OH group stretching band at 3400.2 cm-1) are still present in the polymer mixture even after formulation as a transdermal patch, indicating compatibility among all excipients. While on the other hand, DSC studies confirm endothermic peaks for all the polymers along with THC with the highest enthalpy of 65.979 J/g, which is an indicator of sharp endothermic peak at 198℃, leading to the melting of THC. The percentage drug content and percentage moisture uptake of all the formulation was found in the range of 96±2.04 to 98.56±1.34% and 4.13±1.16 to 8.23±0.90%, respectively. Drug release and release kinetics studies confirm that it is dependent on the composition of individual formulation. CONCLUSION: All these findings support the possibility of using suitable polymeric composition, as well as proper formulation and manufacturing circumstances, to create a one-of-a-kind technology platform for transdermal drug administration.

Antioxidant and Anticancer Functions of Protein Acyltransferase DHHC3.

Silencing of DHHC3, an acyltransferase enzyme in the DHHC family, extensively upregulates oxidative stress (OS). Substrates for DHHC3-mediated palmitoylation include several antioxidant proteins and many other redox regulatory proteins. This helps to explain why DHHC3 ablation upregulates OS. DHHC3 also plays a key role in cancer. DHHC3 ablation leads to diminished xenograft growth of multiple cancer cell types, along with diminished metastasis. Furthermore, DHHC3 protein is upregulated on malignant/metastatic cancer samples, and upregulated gene expression correlates with diminished patient survival in several human cancers. Decreased primary tumor growth due to DHHC3 ablation may be partly explained by an elevated OS → senescence → innate immune cell recruitment mechanism. Elevated OS due to DHHC3 ablation may also contribute to adaptive anticancer immunity and impair tumor metastasis. In addition, DHHC3 ablation disrupts antioxidant protection mechanisms, thus enhancing the efficacy of OS-inducing anticancer drugs. A major focus has thus far been on OS regulation by DHHC3. However, remaining to be studied are multiple DHHC3 substrates that may affect tumor behavior independent of OS. Nonetheless, the currently established properties of DHHC3 make it an attractive candidate for therapeutic targeting in situations in which antioxidant protections need to be downmodulated, and also in cancer.

A Comprehensive Analysis of Myocarditis in Formerly Healthy Individuals Following SARS-CoV-2 Vaccination (COVID-19 Immunization).

Due to the rapid development of the coronavirus disease 2019 (COVID-19) pandemic, the Food and Drug Administration (FDA) expedited the authorization of immunizations to counteract life-threatening COVID-19 effects. COVID-19 immunization was seen as an essential component of surviving endemically with COVID-19. Although there were no major adverse event reports that mandated an early authorization of the mass vaccination approval in initial studies, a few significant adverse events were reported after real-world usage. The most prevalent adverse events are regional reactions, such as discomfort at the injection site. Anaphylactic shock and acute responses were quite infrequent. Current evidence strongly convince the community that the advantages of immunization outweigh the risks. The review investigates the potential adverse reaction in the form of myocarditis caused by the COVID-19 vaccine. Age, sexuality, vaccination type, clinical manifestations, and diagnostic modalities were among the confounding factors associated with vaccine-induced myocarditis. This picture depicts COVID-19 immunization-induced myocarditis and the treatment options available to practitioners. Further evaluation is needed to establish the underlying cause of this association. We compiled the most recent data on SARS-CoV-2 vaccine-induced myocarditis after reviewing available research. Information sources including PubMed and Google Scholar were evaluated retrospectively.

Immune Thrombocytopenia in Previously Healthy Individuals Following SARS-CoV-2 Vaccination (COVID-19 Immunization): A Descriptive Research of 70 Instances With a Focus on Biomarkers, Predictive Outcomes, and Consequences.

The coronavirus disease-2019 (COVID-19) pandemic is exacerbating the worldwide healthcare crisis. The pandemic has had an impact on nearly every system of our body. The Food and Drug Administration (FDA) gave immediate authorization of several vaccines to avoid critical COVID-19 outcomes following the rapid spread of the COVID-19. There have only been a few cases of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccination-induced immune thrombocytopenia (ITP) so far. There should be enough information to identify whether some vaccination adverse effects, such as ITP, are caused by the vaccine. This study aims to determine how common ITP occurs after receiving the SARS-CoV-2 vaccine, as well as gender, age, symptoms, biomarkers, predicted outcomes, and sequelae. We looked at a number of research and compiled the best evidence of SARS-CoV-2 vaccine-induced thrombocytopenia currently available. To find the recommended reporting items, the search technique included keywords like "Immune thrombocytopenia," "COVID-19," "SARS-CoV-2," and "Vaccination." The search results were grouped using Boolean operators ("OR," "AND").

Tetraspanin protein contributions to cancer.

Among the 33 human tetraspanin proteins, CD151, CD9 and Tspan12 play particularly important roles in cancer. Tetraspanin CD151, in partnership with integrins α6ß1 and α6ß4, modulates tumour cell growth, invasion, migration, metastasis, signalling and drug sensitivity. Tetraspanin CD9 has suppressor functions in multiple tumour cell types. Major CD9 partner proteins, such as EWI-2 and EWI-F, may modulate these tumour-suppressor functions. Tetraspanin Tspan12 mutations are linked to a human disease called familial exudative vitreoretinopathy. In addition, as a regulator of the metalloprotease ADAM10 (a disintegrin and metalloprotease 10) maturation and function, Tspan12 probably contributes to the pro-tumorigenic functions of ADAM10.

Biomass-Derived Activated Carbon-Supported Copper Catalyst: An Efficient Heterogeneous Magnetic Catalyst for Base-Free Chan-Lam Coupling and Oxidations.

Development of heterogeneous catalysts from biomass-derived activated carbon is a challenging task. Biomass-derived activated carbon possesses a large specific surface area, highly porous structure, and good thermal/chemical stability. Magnetic copper catalysts based on biomass-derived activated carbon exhibited good catalytic activity in base-free Chan-Lam coupling and oxidations. Herein, biomass-derived activated carbon was prepared by the carbonization of neem dead leaves (abundant waste biomass) followed by chemical activation with KOH. Such a porous carbon material was used as a low cost and highly efficient support material for the preparation of inexpensive and environmentally benign magnetic catalysts [Cu@KF-C/MFe2O4, M = Co, Cu, Ni, and Zn]. In addition, KF modification was done to impart basic character to the catalyst that can perform C-N coupling under base-free conditions. Initially, Brunauer-Emmett-Teller (BET) analysis of the synthesized catalysts was carried out, which indicated that Cu@KF-C/CoFe2O4 possess more surface area as well as pore volume, and so accounting for the highest activity among the other synthesized catalysts. Further, X-ray photoelectron spectroscopy (XPS) analysis was performed, which inferred that Cu@KF-C/CoFe2O4 contains most of the copper in reduced form, i.e., Cu(0), which is the active species responsible for better catalytic activity toward Chan-Lam coupling reactions as well as oxidation of alcohols and hydrocarbons. The physiochemical properties of the most active catalyst, Cu@KF-C/CoFe2O4, was examined by BET, XPS, Fourier transform infrared Spectroscopy (FTIR), thermogravimetric analysis (TGA), field emission gun scanning electron microscopy (FEG-SEM), high-resolution transmission electron microscopy (HR-TEM), energy dispersive X-ray (EDX) mapping, energy dispersive X-ray (EDX), inductively coupled plasma atomic emission spectroscopy (ICP-AES), powder X-ray diffraction (XRD), and vibrating sample magnetometry (VSM). Moreover, Cu@KF-C/CoFe2O4 shows excellent stability as well as reusability and could be easily separated with the help of an external magnet.

Tetraspanin12 regulates ADAM10-dependent cleavage of amyloid precursor protein.

Using mass spectrometry, we identified ADAM10 (a membrane-associated metalloproteinase) as a partner for TSPAN12, a tetraspanin protein. TSPAN12-ADAM10 interaction was confirmed by reciprocal coimmunoprecipitation in multiple tumor cell lines. TSPAN12, to a greater extent than other tetraspanins (CD81, CD151, CD9, and CD82), associated with ADAM10 but not with ADAM17. Overexpression of TSPAN12 enhanced ADAM10-dependent shedding of amyloid precursor protein (APP) in MCF7 (breast cancer) and SH-SY5Y (neuroblastoma) cell lines. Conversely, siRNA ablation of endogenous TSPAN12 markedly diminished APP proteolysis in both cell lines. Furthermore, TSPAN12 overexpression enhanced ADAM10 prodomain maturation, whereas TSPAN12 ablation diminished ADAM10 maturation. A palmitoylation-deficient TSPAN12 mutant failed to associate with ADAM10, inhibited ADAM10-dependent proteolysis of APP, and inhibited ADAM10 maturation, most likely by interfering with endogenous wild-type TSPAN12. In conclusion, TSPAN12 serves as a novel and robust partner for ADAM10 and promotes ADAM10 maturation, thereby facilitating ADAM10-dependent proteolysis of APP. This novel mode of regulating APP cleavage is of relevance to Alzheimer's disease therapy.

Modified graphene supported Ag-Cu NPs with enhanced bimetallic synergistic effect in oxidation and Chan-Lam coupling reactions.

Herein, well dispersed Ag-Cu NPs supported on modified graphene have been synthesized via a facile and rapid approach using sodium borohydride as a reducing agent under ambient conditions. Dicyandiamide is selected as an effective nitrogen source with TiO2 as an inorganic material to form two kinds of supports, labelled as TiO2-NGO and NTiO2-GO. Initially, the surface area analysis of these two support materials was carried out which indicated that N-doping of GO followed by anchoring with TiO2 has produced support material of larger surface area. Using both types of supports, ten nano-metal catalysts based on Ag and Cu were synthesized. Benefiting from the bimetallic synergistic effect and larger specific surface area of TiO2-NGO, Cu@Ag-TiO2-NGO is found to be a highly active and reusable catalyst out of other synthesized catalysts. It exhibits excellent catalytic activity for oxidation of alcohols and hydrocarbons as well as Chan-Lam coupling reactions. The nanocatalyst is intensively characterized by BET, SEM, HR-TEM, ICP-AES, EDX, CHN, FT-IR, TGA, XRD and XPS.

Pneumocystis jirovecii infection of bilateral adrenal glands in an immunocompetent adult: a case report.

Pneumocystis jirovecii (PJ) infection is one of the most common opportunistic infections occurring in patients with HIV/AIDS and other immunocompromised states. It is not known to cause clinically significant illness in immunocompetent hosts. We report a 48-year-old HIV-negative, diabetic male who presented with fever and adrenal insufficiency. Abdominal sonography and PET-CT revealed bilateral enlarged adrenal glands with peripheral enhancement and central necrosis. An endoscopic ultrasound-guided fine-needle aspiration cytology of the left adrenal gland demonstrated well-defined, round cysts of PJ. There was no evidence of pulmonary involvement. The response to first-line treatment was poor and the patient responded to second-line treatment for Pneumocystis infection.

Overexpression of fatty acid synthase is associated with palmitoylation of Wnt1 and cytoplasmic stabilization of beta-catenin in prostate cancer.

Fatty acid synthase (FASN), a key metabolic enzyme for liponeogenesis highly expressed in several human cancers, displays oncogenic properties such as resistance to apoptosis and induction of proliferation when overexpressed. To date, no mechanism has been identified to explain the oncogenicity of FASN in prostate cancer. We generated immortalized prostate epithelial cells (iPrECs) overexpressing FASN, and found that (14)C-acetate incorporation into palmitate synthesized de novo by FASN was significantly elevated in immunoprecipitated Wnt-1 when compared to isogenic cells not overexpressing FASN. Overexpression of FASN caused membranous and cytoplasmic beta-catenin protein accumulation and activation, whereas FASN knockdown by short-hairpin RNA resulted in a reduction in the extent of beta-catenin activation. Orthotopic transplantation of iPrECs overexpressing FASN in nude mice resulted in invasive tumors that overexpressed beta-catenin. A strong significant association between FASN and cytoplasmic (stabilized) beta-catenin immunostaining was found in 862 cases of human prostate cancer after computerized subtraction of the membranous beta-catenin signal (P<0.001, Spearman's rho=0.33). We propose that cytoplasmic stabilization of beta-catenin through palmitoylation of Wnt-1 and subsequent activation of the pathway is a potential mechanism of FASN oncogenicity in prostate cancer.

Protein Acyltransferase DHHC3 Regulates Breast Tumor Growth, Oxidative Stress, and Senescence.

DHHC-type protein acyltransferases may regulate the localization, stability, and/or activity of their substrates. In this study, we show that the protein palmitoyltransferase DHHC3 is upregulated in malignant and metastatic human breast cancer. Elevated expression of DHHC3 correlated with diminished patient survival in breast cancer and six other human cancer types. ZDHHC3 ablation in human MDA-MB-231 mammary tumor cell xenografts reduced the sizes of both the primary tumor and metastatic lung colonies. Gene array data and fluorescence dye assays documented increased oxidative stress and senescence in ZDHHC3-ablated cells. ZDHHC3-ablated tumors also showed enhanced recruitment of innate immune cells (antitumor macrophages, natural killer cells) associated with clearance of senescent tumors. These antitumor effects were reversed upon reconstitution with wild-type, but not enzyme-active site-deficient DHHC3. Concomitant ablation of the upregulated oxidative stress protein TXNIP substantially negated the effects of ZDHHC3 depletion on oxidative stress and senescence. Diminished DHHC3-dependent palmitoylation of ERGIC3 protein likely played a key role in TXNIP upregulation. In conclusion, DHHC3-mediated protein palmitoylation supports breast tumor growth by modulating cellular oxidative stress and senescence. Cancer Res; 77(24); 6880-90. ©2017 AACR.

Gastrin-mediated activation of cyclin D1 transcription involves beta-catenin and CREB pathways in gastric cancer cells.

Gastrin and its precursors promote proliferation in different gastrointestinal cells. Since mature, amidated gastrin (G-17) can induce cyclin D1, we determined whether G-17-mediated induction of cyclin D1 transcription involved Wnt signaling and CRE-binding protein (CREB) pathways. Our studies indicate that G-17 induces protein, mRNA expression and transcription of the G(1)-specific marker cyclin D1, in the gastric adenocarcinoma cell line AGSE (expressing the gastrin/cholecystokinin B receptor). This was associated with an increase in steady-state levels of total and nonphospho beta-catenin and its nuclear translocation, indicating the activation of the Wnt-signaling pathway. In addition, G-17-mediated increase in cyclin D1 transcription was significantly attenuated by axin or dominant-negative (dn) T-cell factor 4(TCF4), suggesting crosstalk of G-17 with the Wnt-signaling pathway. Mutational analysis indicated that this effect was mediated through the cyclic AMP response element (CRE) (predominantly) and the TCF sites in the cyclin D1 promoter, which was also inhibited by dnCREB. Furthermore, G-17 stimulation resulted in increased CRE-responsive reporter activity and CREB phosphorylation, indicating an activation of CREB. Chromatin immunoprecipitation studies revealed a G-17-mediated increase in the interaction of beta-catenin with cyclin D1 CRE, which was attenuated by dnTCF4 and dnCREB. These results indicate that G-17 induces cyclin D1 transcription, via the activation of beta-catenin and CREB pathways.

EWI-2 negatively regulates TGF-ß signaling leading to altered melanoma growth and metastasis.

In normal melanocytes, TGF-ß signaling has a cytostatic effect. However, in primary melanoma cells, TGF-ß-induced cytostasis is diminished, thus allowing melanoma growth. Later, a second phase of TGF-ß signaling supports melanoma EMT-like changes, invasion and metastasis. In parallel with these "present-absent-present" TGF-ß signaling phases, cell surface protein EWI motif-containing protein 2 (EWI-2 or IgSF8) is "absent-present-absent" in melanocytes, primary melanoma, and metastatic melanoma, respectively, suggesting that EWI-2 may serve as a negative regulator of TGF-ß signaling. Using melanoma cell lines and melanoma short-term cultures, we performed RNAi and overexpression experiments and found that EWI-2 negatively regulates TGF-ß signaling and its downstream events including cytostasis (in vitro and in vivo), EMT-like changes, cell migration, CD271-dependent invasion, and lung metastasis (in vivo). When EWI-2 is present, it associates with cell surface tetraspanin proteins CD9 and CD81 - molecules not previously linked to TGF-ß signaling. Indeed, when associated with EWI-2, CD9 and CD81 are sequestered and have no impact on TßR2-TßR1 association or TGF-ß signaling. However, when EWI-2 is knocked down, CD9 and CD81 become available to provide critical support for TßR2-TßR1 association, thus markedly elevating TGF-ß signaling. Consequently, all of those TGF-ß-dependent functions specifically arising due to EWI-2 depletion are reversed by blocking or depleting cell surface tetraspanin proteins CD9 or CD81. These results provide new insights into regulation of TGF-ß signaling in melanoma, uncover new roles for tetraspanins CD9 and CD81, and strongly suggest that EWI-2 could serve as a favorable prognosis indicator for melanoma patients.