Keloid Disease Can Be Inhibited by Antagonizing Excessive mTOR Signaling With a Novel Dual TORC1/2 Inhibitor

Keloid disease (KD) is a fibroproliferative lesion of unknown etiopathogenesis that possibly targets the PI3K/Akt/mTOR pathway. We investigated whether PI3K/Akt/mTOR inhibitor, Palomid 529 (P529), which targets both mammalian target of rapamycin complex 1 (mTORC-1) and mTORC-2 signaling, could exert anti-KD effects in a novel KD organ culture assay and in keloid fibroblasts (KF). Treatment of KF with P529 significantly (P < 0.05) inhibited cell spreading, attachment, proliferation, migration, and invasive properties at a low concentration (5 ng/mL) and induced substantial KF apoptosis when compared with normal dermal fibroblasts. P529 also inhibited hypoxia-inducible factor-1α expression and completely suppressed Akt, GSK3β, mTOR, eukaryotic initiation factor 4E-binding protein 1, and S6 phosphorylation. P529 significantly (P < 0.05) inhibited proliferating cell nuclear antigen and cyclin D and caused considerable apoptosis. Compared with rapamycin and wortmannin, P529 also significantly (P < 0.05) reduced keloid-associated phenotypic markers in KF. P529 caused tissue shrinkage, growth arrest, and apoptosis in keloid organ cultures and substantially inhibited angiogenesis. pS6, pAkt-Ser473, and mTOR phosphorylation were also suppressed in situ. P529 reduced cellularity and expression of collagen, fibronectin, and α-smooth muscle actin (substantially more than rapamycin). These pre-clinical in vitro and ex vivo observations are evidence that the mTOR pathway is a promising target for future KD therapy and that the dual PI3K/Akt/mTOR inhibitor P529 deserves systematic exploration as a candidate agent for the future treatment of KD

Design a framework for IoT- Identification, Authentication and Anomaly detection using Deep Learning: A Review

The Internet of Things (IoT) connects billions of smart gadgets so that they may communicate with one another without the need for human intervention. With an expected 50 billion devices by the end of 2020, it is one of the fastest-growing industries in computer history. On the one hand, IoT technologies are critical in increasing a variety of real-world smart applications that can help people live better lives. The cross-cutting nature of IoT systems, on the other hand, has presented new security concerns due to the diverse components involved in their deployment. For IoT devices and their inherent weaknesses, security techniques such as encryption, authentication, permissions, network monitoring, \& application security are ineffective. To properly protect the IoT ecosystem, existing security solutions need to be strengthened. Machine learning and deep learning (ML/DL) have come a long way in recent years, and machine intelligence has gone from being a laboratory curiosity to being used in a variety of significant applications. The ability to intelligently monitor IoT devices is an important defense against new or negligible assaults. ML/DL are effective data exploration techniques for learning about 'normal' and 'bad' behavior in IoT devices and systems. Following a comprehensive literature analysis on Machine Learning methods as well as the importance of IoT security within the framework of different sorts of potential attacks, multiple DL algorithms have been evaluated in terms of detecting attacks as well as anomaly detection in this work. We propose a taxonomy of authorization and authentication systems in the Internet of Things based on the review, with a focus on DL-based schemes. The authentication security threats and problems for IoT are thoroughly examined using the taxonomy supplied. This article provides an overview of projects that involve the use of deep learning to efficiently and automatically provide IoT applications

Phase 1 clinical trial of CRISPR-engineered CAR19 universal T cells for treatment of children with refractory B cell leukemia

Genome editing of allogeneic T cells can provide “off-the-shelf” alternatives to autologous chimeric antigen receptor (CAR) T cell therapies. Disruption of T cell receptor α chain (TRAC) to prevent graft-versus-host disease (GVHD) and removal of CD52 (cluster of differentiation 52) for a survival advantage in the presence of alemtuzumab have previously been investigated using transcription activator–like effector nuclease (TALEN)-mediated knockout. Here, we deployed next-generation CRISPR-Cas9 editing and linked CAR expression to multiplexed DNA editing of TRAC and CD52 through incorporation of self-duplicating CRISPR guide RNA expression cassettes within the 3’ long terminal repeat of a CAR19 lentiviral vector. Three cell banks of TT52CAR19 T cells were generated and cryopreserved. A phase 1, open-label, non-randomized clinical trial was conducted and treated six children with relapsed/refractory CD19-positive B cell acute lymphoblastic leukemia (B-ALL) (NCT04557436). Lymphodepletion included fludarabine, cyclophosphamide, and alemtuzumab and was followed by a single infusion of 0.8 × 10^{6} to 2.0 × 10^{6} CAR19 T cells per kilogram with no immediate toxicities. Four of six patients infused with TT52CAR19 T cells exhibited cell expansion, achieved flow cytometric remission, and then proceeded to receive allogeneic stem cell transplantation. Two patients required biological intervention for grade II cytokine release syndrome, one patient developed transient grade IV neurotoxicity, and one patient developed skin GVHD, which resolved after transplant conditioning. Other complications were within expectations, and primary safety objectives were met. This study provides a demonstration of the feasibility, safety, and therapeutic potential of CRISPR-engineered immunotherapy

Lentiviral Engineered Fibroblasts Expressing Codon-Optimized COL7A1 Restore Anchoring Fibrils in RDEB

Cells therapies, engineered to secrete replacement proteins, are being developed to ameliorate otherwise debilitating diseases. Recessive dystrophic epidermolysis bullosa (RDEB) is caused by defects of type VII collagen, a protein essential for anchoring fibril formation at the dermal-epidermal junction. Whereas allogeneic fibroblasts injected directly into the dermis can mediate transient disease modulation, autologous gene-modified fibroblasts should evade immunological rejection and support sustained delivery of type VII collagen at the dermal-epidermal junction. We demonstrate the feasibility of such an approach using a therapeutic grade, self-inactivating-lentiviral vector, encoding codon-optimized COL7A1, to transduce RDEB fibroblasts under conditions suitable for clinical application. Expression and secretion of type VII collagen was confirmed with transduced cells exhibiting supranormal levels of protein expression, and ex vivo migration of fibroblasts was restored in functional assays. Gene-modified RDEB fibroblasts also deposited type VII collagen at the dermal-epidermal junction of human RDEB skin xenografts placed on NOD-scid IL2Rgammanull recipients, with reconstruction of human epidermal structure and regeneration of anchoring fibrils at the dermal-epidermal junction. Fibroblast-mediated restoration of protein and structural defects in this RDEB model strongly supports proposed therapeutic applications in man

Integrin‐Targeted, Short Interfering RNA Nanocomplexes for Neuroblastoma Tumor‐Specific Delivery Achieve MYCN Silencing with Improved Survival

The authors aim to develop siRNA therapeutics for cancer that can be administered systemically to target tumors and retard their growth. The efficacy of systemic delivery of siRNA to tumors with nanoparticles based on lipids or polymers is often compromised by their rapid clearance from the circulation by the liver. Here, multifunctional cationic and anionic siRNA nanoparticle formulations are described, termed receptor‐targeted nanocomplexes (RTNs), that comprise peptides for siRNA packaging into nanoparticles and receptor‐mediated cell uptake, together with lipids that confer nanoparticles with stealth properties to enhance stability in the circulation, and fusogenic properties to enhance endosomal release within the cell. Intravenous administration of RTNs in mice leads to predominant accumulation in xenograft tumors, with very little detected in the liver, lung, or spleen. Although non‐targeted RTNs also enter the tumor, cell uptake appears to be RGD peptide‐dependent indicating integrin‐mediated uptake. RTNs with siRNA against MYCN (a member of the Myc family of transcription factors) in mice with MYCN‐amplified neuroblastoma tumors show significant retardation of xenograft tumor growth and enhanced survival. This study shows that RTN formulations can achieve specific tumor‐targeting, with minimal clearance by the liver and so enable delivery of tumor‐targeted siRNA therapeutics

Interactions in vivo between the Vif protein of HIV-1 and the precursor (Pr55GAG) of the virion nucleocapsid proteins

The abnormality of viral core structure seen in vif-defective HIV-1 grown in PBMCs has suggested a role for Vif in viral morphogenesis. Using an in vivo mammalian two-hybrid assay, the interaction between Vif and the precursor (Pr55GAG) of the virion nucleocapsid proteins has been analysed. This revealed the amino-terminal (aa 1–22) and central (aa 70–100) regions of Vif to be essential for its interaction with Pr55GAG, but deletion of the carboxy-terminal (aa 158–192) region of the protein had only a minor effect on its interaction. Initial deletion studies carried out on Pr55GAG showed that a 35-amino-acid region of the protein bridging the MA(p17)–CA(p24) junction was essential for its ability to interact with Vif. Site-directed mutagenesis of a conserved tryptophan (Trp21) near the amino terminus of Vif showed it to be important for the interaction with Pr55GAG. By contrast, mutagenesis of the highly conserved YLAL residues forming part of the BC-box motif, shown to be important in Vif promoting degradation of APOBEC3G/3F, had little or no effect on the Vif–Pr55GAG interaction

In Vivo and In Vitro Functional Studies on the HIV-1 Vif Protein

Human Immunodeficiency virus type-l (HIV-l), has a number of regulatory genes in addition to the gag, pol, and env that are common to all replication competent retrovimses. It expresses six auxilialY proteins, tat, rev, Vi/, vpu, vpx and nef. Vif (Viral Infectivity Factor) is a 23 kDa basic protein of 192 aa. Vif has been shown to be essential for the modulation of virion infectivity in nom1al host cells and is believed to function by interacting with both viral and cellular proteins. More recent studies have focused on its involvement in controlling the encapsidation of cellular proteins ~f the APOBEC3 family. However earlier work of Vif suggested an involvement in viral morphogenesis and this was the main focus of the present study. Vifhas been shown to be associated with the viral nucleocapsid and to be specifically packed into HIV patiicles, either by interaction with viral RNA and/or Gag and GagPol precursors. This study had as its primary aim definition of the molecular interactions of Vif with the Gag precursor (Pr55GAG ), the viral Protease (RR), and the antiviral cellular proteins APOBEC3G/3F. An in vivo mammalian two-hybrid assay was used to study the interactions between Vif and both Pr55GAG and the viral PR. This found that Vif interacts with Pr55GAG • To begin mapping the positions of this interaction, a series of mutations were made in both proteins. Complimenting previous studies on Vif done at Warwick, amino acid 21 was found to be cmcial for the interaction between Vif and Pr55GAG in the mammalian two-hybrid assay. Interaction between Vif and Pr55GAG was fmiher confirmed using an independent in vitro GST pull-down assay. No interactions were found between Vif and PR and between PR and Pr55GAG • The second objective of this study was to analyse the molecular interactions of HIV-1 Vif, Pr55GAG , and PR with the APOBEC3 family of cellular proteins using both an in vivo mammalian two-hybrid assay and an in vitro GST pull-down assay. In the mammalian two-hybrid assay a direct interaction between Pr55GAG and both APOBEC3G and 3F was identified. These interactions were further confirmed using the GST pull-down assay. A direct interaction between Vif and the two APOBEC proteins APOBEC3G and 3F was also seen in vitro in the GST pull-down assay; however, these interactions could not be seen in vivo using the mammalian two hybrid assay. A third are~ of work was concemed with the high level expression of Vifin a bacterial expression system and purification of the protein for structural studies. This experimental work was complemented by computer based model building using a comparative modeling method. The aim of this work being to produce an atomic level resolution model for Vifwhich could be tested against the experimental results achieved in interaction site mapping studies. Building on earlier work done at Warwick a fourth area of this study involved in vivo experiments aimed at understanding the role of HIV-1 Vif iil resistance to protease inhibitors (Adekale et al., 2005). This involved establishing the molecular reagents to allow the generation of infectious molecular clones carrying various variants of Vifand the HIV-1 protease. Plasmid constructs were generated to allow the inse11ion of different variants of the PRgene into an infectious molecular clone building on the previously available strategy which allowed similar exchanges with the Vifgene.EThOS - Electronic Theses Online ServiceGBUnited Kingdo

Influence of the human leukocyte antigen complex on the development of cutaneous fibrosis: An immunogenetic perspective

A number of aesthetically and physically distressing disorders of the skin come under the general term "cutaneous fibrosis", all sharing a common abnormal wound healing process These disorders are often incurable and effective treatments remain to be established and, as such, they present a significant burden for patients and a therapeutic challenge for clinicians The aim of this review is to investigate the evidence of either positive or negative associations of the human leukocyte antigen (HLA) system with various types of cutaneous fibrosis, focussing in particular on keloid scars, hypertrophic scars and scleroderma A standard systematic literature search was performed The strengths and limitations of studies were evaluated in terms of significance, methodology and reproducibility There is a clear association between specific HLA alleles and predilection or protection to cutaneous fibrosis Of these candidate HLA alleles, the class II loci seem to be the most promising in terms of a genetic biomarker, with the DQ and DR alleles having significant associations with abnormal wound healing and cutaneous fibrosis There is strong evidence of a significant immune component in the pathogenesis of each type of fibrotic disorder explored in this review However, the exact mechanisms remain to be elucidated, since the pathogenesis of cutaneous fibrosis and abnormal wound healing are not fully understoo