Target-seq: single workflow for detection of genome integration site, DNA translocation and off-target events.

Designed donor DNA delivery through viral or nonviral systems to target loci in the host genome is a critical step for gene therapy. Adeno-associated virus and lentivirus are leading vehicles for in vivo and ex vivo delivery of therapeutic genes due to their high delivery and editing efficiency. Nonviral editing tools, such as CRISPR/Cas9, are getting more attention for gene modification. However, there are safety concerns; for example, tumorigenesis due to off-target effects and DNA rearrangement. Analysis tools to detect and characterize on-target and off-target genome modification post editing in the host genome are pivotal for evaluating the success and safety of gene therapy. We developed Target-seq combined with different analysis tools to detect the genome integration site, DNA translocation and off-target events.

Protein kinase B (AKT) upregulation and Thy-1-αvß3 integrin-induced phosphorylation of Connexin43 by activated AKT in astrogliosis.

BACKGROUND: In response to brain injury or inflammation, astrocytes undergo hypertrophy, proliferate, and migrate to the damaged zone. These changes, collectively known as "astrogliosis", initially protect the brain; however, astrogliosis can also cause neuronal dysfunction. Additionally, these astrocytes undergo intracellular changes involving alterations in the expression and localization of many proteins, including αvß3 integrin. Our previous reports indicate that Thy-1, a neuronal glycoprotein, binds to this integrin inducing Connexin43 (Cx43) hemichannel (HC) opening, ATP release, and astrocyte migration. Despite such insight, important links and molecular events leading to astrogliosis remain to be defined. METHODS: Using bioinformatics approaches, we analyzed different Gene Expression Omnibus datasets to identify changes occurring in reactive astrocytes as compared to astrocytes from the normal mouse brain. In silico analysis was validated by both qRT-PCR and immunoblotting using reactive astrocyte cultures from the normal rat brain treated with TNF and from the brain of a hSOD1G93A transgenic mouse model. We evaluated the phosphorylation of Cx43 serine residue 373 (S373) by AKT and ATP release as a functional assay for HC opening. In vivo experiments were also performed with an AKT inhibitor (AKTi). RESULTS: The bioinformatics analysis revealed that genes of the PI3K/AKT signaling pathway were among the most significantly altered in reactive astrocytes. mRNA and protein levels of PI3K, AKT, as well as Cx43, were elevated in reactive astrocytes from normal rats and from hSOD1G93A transgenic mice, as compared to controls. In vitro, reactive astrocytes stimulated with Thy-1 responded by activating AKT, which phosphorylated S373Cx43. Increased pS373Cx43 augmented the release of ATP to the extracellular medium and AKTi inhibited these Thy-1-induced responses. Furthermore, in an in vivo model of inflammation (brain damage), AKTi decreased the levels of astrocyte reactivity markers and S373Cx43 phosphorylation. CONCLUSIONS: Here, we identify changes in the PI3K/AKT molecular signaling network and show how they participate in astrogliosis by regulating the HC protein Cx43. Moreover, because HC opening and ATP release are important in astrocyte reactivity, the phosphorylation of Cx43 by AKT and the associated increase in ATP release identify a potential therapeutic window of opportunity to limit the adverse effects of astrogliosis.

Extracapsular dissection and minimally invasive procedures of primary benign parotid tumours: Case series

Extracapsular dissection is an old technique use for the removal of benign parotid tumours, which is not generally chosen as the first treatment option due to the association of recurrences in the past but is currently considered again accord to the aesthetic requirements of the patients. The general trend in the last decade is to return to minimally invasive procedures for this type of lesions, which are mainly conditioned by the pleomorphic adenoma and its positive margins in its capsule. By this, the purpose of this case series study is to analyze those patients diagnosed with benign parotid tumors and treated by extracapsular dissection in a tertiary hospital in Chile between 2018-2020.

CRISPR-mediated TGFBR2 knockout renders human ovarian cancer tumor-infiltrating lymphocytes resistant to TGF-ß signaling.

BACKGROUND: The correlation between elevated T-cell infiltration and improved survival of ovarian cancer (OvCa) patients suggests that endogenous tumor-infiltrating lymphocytes (TIL) possess some degree of antitumor activity that can be harnessed for OvCa immunotherapy. We previously optimized a protocol for ex vivo OvCa TIL expansion for adoptive cell therapy, which is now being tested in a clinical trial at our institution (NCT03610490). Building on this success, we embarked on genetic modification of OvCa TIL to overcome key immunosuppressive factors present in the tumor microenvironment. Here, we present the preclinical optimization of CRISPR/Cas9-mediated knockout of the TGF-ß receptor 2 (TGFBR2) in patient-derived OvCa TIL. METHODS: OvCa TILs were generated from four patients' tumor samples obtained at surgical resection and subjected to CRISPR/Cas9-mediated knockout of TGFBR2 before undergoing a rapid expansion protocol. TGFBR2-directed gRNAs were comprehensively evaluated for their TGFBR2 knockout efficiency and off-target activity. Furthermore, the impact of TGFBR2 knockout on TIL expansion, function, and downstream signaling was assayed. RESULTS: TGFBR2 knockout efficiencies ranging from 59±6% to 100%±0% were achieved using 5 gRNAs tested in four independent OvCa TIL samples. TGFBR2 knockout TIL were resistant to immunosuppressive TGF-ß signaling as evidenced by a lack of SMAD phosphorylation, a lack of global transcriptional changes in response to TGF-ß stimulation, equally strong secretion of proinflammatory cytokines in the presence and absence of TGF-ß, and improved cytotoxicity in the presence of TGF-ß. CRISPR-modification itself did not alter the ex vivo expansion efficiency, immunophenotype, nor the TCR clonal diversity of OvCa TIL. Importantly for clinical translation, comprehensive analysis of CRISPR off-target effects revealed no evidence of off-target activity for our top two TGFBR2-targeting gRNAs. CONCLUSIONS: CRISPR/Cas9-mediated gene knockout is feasible and efficient in patient-derived OvCa TIL using clinically-scalable methods. We achieved efficient and specific TGFBR2 knockout, yielding an expanded OvCa TIL product that was resistant to the immunosuppressive effects of TGF-ß. This study lays the groundwork for clinical translation of CRISPR-modified TIL, providing opportunities for engineering more potent TIL therapies not only for OvCa treatment, but for the treatment of other solid cancers as well.

Do total hip arthroplasty polyethylene liners without free radicals oxidize in vivo or ex vivo?

Crosslinking substantially reduces the wear of ultra-high molecular weight polyethylene (UHMWPE) used in total hip arthroplasty (THA) but some reports have indicated that first generation liners manufactured without antioxidants may be vulnerable to in vivo oxidation. This study evaluated maximum oxidation using Fourier transform infrared spectroscopy per ASTM F2102-06ε1 and linear head penetration using a coordinate measuring machine among 66 revision-retrieved THA components with in vivo durations ranging from 0.02 to 24.6 years. These included 30 liners crosslinked with 5 Mrad of gamma radiation and then melted, 13 non-crosslinked, never-irradiated liners sterilized with gas plasma and 23 non-crosslinked, never-irradiated liners sterilized with ethylene oxide. All liners were vacuum-sealed and stored at -20°C prior to analysis with the exception of three retrievals of each material type that were stored in air for 9.9 to 21.5 years. All 57 vacuum-sealed and frozen retrievals demonstrated good oxidative stability with maximum oxidation indices (OIs) less than 1.0 and 75% (43/57) of these liners had maximum OIs less than 0.1. Linear penetration measurements were lower in the crosslinked liners compared to non-crosslinked retrievals. Although instances of oxidation and embrittlement were found after ex vivo storage in air among liners that did not have free radicals at the time of implantation, in vivo oxidation does not appear to be a clinical concern through the first decade of service for crosslinked liners and at up to 25 years after surgery for non-crosslinked liners.

Characterizing environmental stress responses of aposymbiotic Astrangia poculata to divergent thermal challenges.

Anthropogenic climate change threatens corals globally and both high and low temperatures are known to induce coral bleaching. However, coral stress responses across wide thermal breadths remain understudied. Disentangling the role of symbiosis on the stress response in obligately symbiotic corals is challenging because this response is inherently coupled with nutritional stress. Here, we leverage aposymbiotic colonies of the facultatively symbiotic coral, Astrangia poculata, which lives naturally with and without its algal symbionts, to examine how broad thermal challenges influence coral hosts in the absence of symbiosis. A. poculata were collected from their northern range limit and thermally challenged in two independent 16-day common garden experiments (heat and cold challenge) and behavioural responses to food stimuli and genome-wide gene expression profiling (TagSeq) were performed. Both thermal challenges elicited significant reductions in polyp extension. However, there were five times as many differentially expressed genes (DEGs) under cold challenge compared to heat challenge. Despite an overall stronger response to cold challenge, there was significant overlap in DEGs between thermal challenges. We contrasted these responses to a previously identified module of genes associated with the environmental stress response (ESR) in tropical reef-building corals. Cold challenged corals exhibited a pattern consistent with more severe stressors while the heat challenge response was consistent with lower intensity stressors. Given that these responses were observed in aposymbiotic colonies, many genes previously implicated in ESRs in tropical symbiotic species may represent the coral host's stress response in or out of symbiosis.

Myoglobin induces mitochondrial fusion, thereby inhibiting breast cancer cell proliferation.

Myoglobin is a monomeric heme protein expressed ubiquitously in skeletal and cardiac muscle and is traditionally considered to function as an oxygen reservoir for mitochondria during hypoxia. It is now well established that low concentrations of myoglobin are aberrantly expressed in a significant proportion of breast cancer tumors. Despite being expressed only at low levels in these tumors, myoglobin is associated with attenuated tumor growth and a better prognosis in breast cancer patients, but the mechanism of this myoglobin-mediated protection against further cancer growth remains unclear. Herein, we report a signaling pathway by which myoglobin regulates mitochondrial dynamics and thereby decreases cell proliferation. We demonstrate in vitro that expression of human myoglobin in MDA-MB-231, MDA-MB-468, and MCF7 breast cancer cells induces mitochondrial hyperfusion by up-regulating mitofusins 1 and 2, the predominant catalysts of mitochondrial fusion. This hyperfusion causes cell cycle arrest and subsequent inhibition of cell proliferation. Mechanistically, increased mitofusin expression was due to myoglobin-dependent free-radical production, leading to the oxidation and degradation of the E3 ubiquitin ligase parkin. We recapitulated this pathway in a murine model in which myoglobin-expressing xenografts exhibited decreased tumor volume with increased mitofusin, markers of cell cycle arrest, and decreased parkin expression. Furthermore, in human triple-negative breast tumor tissues, mitofusin and myoglobin levels were positively correlated. Collectively, these results elucidate a new function for myoglobin as a modulator of mitochondrial dynamics and reveal a novel pathway by which myoglobin decreases breast cancer cell proliferation and tumor growth by up-regulating mitofusin levels.

Relevancia de profilaxis antibiótica ante procedimientos dentales generadores de bacteriemias transitorias / Antimicrobial prophylaxis for transient bacteremia during dental procedures

This review examines the evidence about the relationship between dental procedures and the incidence of transient bacteremia. One of the main obstacles was to define "invasive dental procedure" as an indication for antimicrobial prophylaxis for patients with high risk of bacteremia. A search in WorldWideScience and ScienceDirect was performed and 20 articles were selected for review. Two contradictions stood out. There is no concrete evidence that a transient bacteremia arising during a dental procedure may be a risk factor for the appearance of bacterial endocarditis. There is no certainty about the effectiveness of antimicrobial prophylaxis, due to the lack of clinical trials of good quality. There is a similitude between bacteremia resulting from invasive and non-invasive dental procedures. The importance of periodontal health as a preventive measure for bacterial endocarditis among high risk patients is highlighted.

Osteonecrosis mandibular relacionada con medicamentos: conceptos teóricos y prácticos actuales para el odontólogo general

La osteonecrosis mandibular relacionada con medicamentos (ONMRM), es un síndrome asociado al uso de fármacos antirresortivos (bifosfonatos), inhibidores de ligando RANK-L y de angiogénesis, administrados para el tratamiento de enfermedades como cáncer y osteoporosis. Objetivos: actualizar contenidos respecto a etiopatogenia y tratamientos de ONMRM, enfatizando el rol del factor "infección", y en la perspectiva de que el tratamiento, pueda ser implementado por el odontólogo general. Materiales y métodos: se realiza un scoping review mediante acceso a las bases de datos MEDLINE (Pubmed) y Cochrane library, de artículos publicados desde el año 2010 en adelante, en inglés y castellano, empleando palabras claves incluidas en los medical subject headings (MeSH). Conclusiones: La evidencia recogida demuestra que, teniendo en cuenta los factores de riesgo individuales, no existen en general contraindicaciones para llevar a cabo exodoncias, bajo específicos protocolos quirúrgicos, en pacientes con terapias antiresortivas o antiangiogénicas, ya que al parecer, el origen de la ONMRM se asocia a contaminación por biofilm, más que al uso o efecto de estos fármacos. En el caso del tratamiento de una ONMRM ya instalada, las terapias quirúrgicas resultan ser más eficaces que las paliativas en la remisión del cuadro.

Medication-related osteonecrosis of the jaw (MRONJ) is a syndrome associated to the use of antiresorptive therapy (bisphosphonates), RANK-ligand inhibitors and angiogenesis inhibitors drugs, used for the treatment of cancer and osteoporosis among other diseases. Purpose: Update the knowledge on its etiopathogenesis emphasizing the role of infection, and in doing so, look for treatments that can be carried out by general practitioners. Materials and methods: A scoping review is performed through Medline (Pubmed) and Cochrane databases, of articles published from to 2010, in English and Spanish, including MeSH keywords such as "osteonecrosis of the jaw; "antiresorptive drug"; "bisphosphonates"; "Denosumab"; "surgical wound infection"; "dental extraction". Conclusions: The current evidence demonstrate that, taking into account individual risk factors, teeth extractions can be harmless performed in patients under antiresorptive or antiangiogenic interetherapies because the origin of MRONJ appears to be more related to biofilm contamination than with the use or effects of any specific drug. Once MRONJ is installed, surgical therapies prove to be more effective in the treatment and remission of these lesions.

Cutting edge: FYCO1 recruitment to dectin-1 phagosomes is accelerated by light chain 3 protein and regulates phagosome maturation and reactive oxygen production.

L chain 3 (LC3)-associated phagocytosis is a process in which LC3, a protein canonically involved in engulfing intracellular materials (autophagy), is recruited to traditional phagosomes during internalization of extracellular payloads. LC3's association with phagosomes has been implicated in regulating microbial killing, Ag processing, and phagosome maturation; however, the mechanism by which LC3 influences these processes has not been clear. In this study, we report that FYVE and coiled-coil domain containing 1 (FYCO1), a protein previously implicated in autophagosome trafficking, is recruited directly by LC3 to Dectin-1 phagosomes. During LC3-associated phagocytosis, FYCO1 recruitment facilitates maturation of early p40phox(+) phagosomes into late LAMP1(+) phagosomes. When FYCO1 is lacking, phagosomes stay p40phox(+) longer and produce more reactive oxygen.

Phagosomal degradation increases TLR access to bacterial ligands and enhances macrophage sensitivity to bacteria.

Signaling by innate immune receptors initiates and orchestrates the overall immune responses to infection. Macrophage receptors recognizing pathogens can be broadly grouped into surface receptors and receptors restricted to intracellular compartments, such as phagosomes and the cytoplasm. There is an expectation that ingestion and degradation of microorganisms by phagocytes contributes to activation of intracellular innate receptors, although direct demonstrations of this are rare, and many model ligands are studied in soluble form, outside of their microbial context. By comparing a wild-type strain of Staphylococcus aureus and a lysozyme-sensitive mutant, we have been able directly to address the role of degradation of live bacteria by mouse macrophages in determining the overall innate cellular inflammatory response. Our investigations revealed a biphasic response to S. aureus that consisted of an initial signal resulting from the engagement of surface TLR2, followed by a later, second wave on inflammatory gene induction. This second wave of inflammatory signaling was dependent on and correlated with the timing of bacterial degradation in phagosomes. We found that TLR2 signaling followed by TLR2/TLR9 signaling enhanced sensitivity to small numbers of bacteria. We further found that treating wild-type bacteria with the peptidoglycan synthesis-inhibiting antibiotic vancomycin made S. aureus more susceptible to degradation and resulted in increased inflammatory responses, similar to those observed for mutant degradation-sensitive bacteria.

Activation of the innate immune receptor Dectin-1 upon formation of a 'phagocytic synapse'.

Innate immune cells must be able to distinguish between direct binding to microbes and detection of components shed from the surface of microbes located at a distance. Dectin-1 (also known as CLEC7A) is a pattern-recognition receptor expressed by myeloid phagocytes (macrophages, dendritic cells and neutrophils) that detects ß-glucans in fungal cell walls and triggers direct cellular antimicrobial activity, including phagocytosis and production of reactive oxygen species (ROS). In contrast to inflammatory responses stimulated upon detection of soluble ligands by other pattern-recognition receptors, such as Toll-like receptors (TLRs), these responses are only useful when a cell comes into direct contact with a microbe and must not be spuriously activated by soluble stimuli. In this study we show that, despite its ability to bind both soluble and particulate ß-glucan polymers, Dectin-1 signalling is only activated by particulate ß-glucans, which cluster the receptor in synapse-like structures from which regulatory tyrosine phosphatases CD45 and CD148 (also known as PTPRC and PTPRJ, respectively) are excluded (Supplementary Fig. 1). The 'phagocytic synapse' now provides a model mechanism by which innate immune receptors can distinguish direct microbial contact from detection of microbes at a distance, thereby initiating direct cellular antimicrobial responses only when they are required.

Thermosensitive liposomes modified with poly(N-isopropylacrylamide-co-propylacrylic acid) copolymers for triggered release of doxorubicin.

A novel polymer-modified thermosensitive liposome (pTSL) was developed for the delivery of Doxorubicin (DOX) for cancer therapy. Copolymers containing temperature-responsive N-isopropylacrylamide (NIPAAm) and pH-responsive propylacrylic acid (PAA) were synthesized via reversible addition-fragmentation chain transfer (RAFT) polymerization, yielding copolymers with dual pH/temperature-dependent phase transition properties. When attached to liposomes, these copolymers were membrane-disruptive in a pH/temperature-dependent manner. pTSL demonstrated enhanced release profile and significantly lower thermal dose threshold when compared to traditional thermosensitive formulations and were stable in serum with minimal drug leakage over time. These liposomes thus have the potential to dramatically reduce the risk of damage to healthy tissues that is normally associated with liposomal cancer therapy.

Staphylococcus aureus evades lysozyme-based peptidoglycan digestion that links phagocytosis, inflammasome activation, and IL-1beta secretion.

IL-1beta produced by phagocytes is important for protection against the mucosal pathogen Staphylococcus aureus. Processing and maturation of this cytokine requires activation of the multiprotein inflammasome complex. We observed that the bacterial cell wall component peptidoglycan (PGN) must be particulate and internalized via phagocytosis to activate NLRP3 inflammasomes and IL-1beta secretion. In the context of S. aureus infection of macrophages, we find that phagocytosis and lysozyme-based bacterial cell wall degradation are necessary to induce IL-1beta secretion. Further, an S. aureus enzyme, PGN O-acetyltransferase A, previously demonstrated to make cell wall PGN resistant to lysozyme, strongly suppresses inflammasome activation and inflammation in vitro and in vivo. These observations demonstrate that phagocytosis and lysozyme-based cell wall degradation of S. aureus are functionally coupled to inflammasome activation and IL-1beta secretion and illustrate a case whereby a bacterium specifically subverts IL-1beta secretion through chemical modification of its cell wall PGN.

Americas Antibody Congress 2009. 21-23 September 2009, Washington DC, USA.

The Americas Antibody Congress 2009, held in Washington DC, included topics covering new state-of-the-art advances in antibody therapeutics. This conference report highlights selected presentations on enhanced antibody functionality, bispecific antibodies and antibody-drug conjugates. Technological approaches discussed include Potelligent technology from BioWa Inc, stability-engineered antibodies and BiTE antibodies.

Characterization of inhibitory anti-insulin-like growth factor receptor antibodies with different epitope specificity and ligand-blocking properties: implications for mechanism of action in vivo.

Therapeutic antibodies directed against the type 1 insulin-like growth factor receptor (IGF-1R) have recently gained significant momentum in the clinic because of preliminary data generated in human patients with cancer. These antibodies inhibit ligand-mediated activation of IGF-1R and the resulting down-stream signaling cascade. Here we generated a panel of antibodies against IGF-1R and screened them for their ability to block the binding of both IGF-1 and IGF-2 at escalating ligand concentrations (>1 microm) to investigate allosteric versus competitive blocking mechanisms. Four distinct inhibitory classes were found as follows: 1) allosteric IGF-1 blockers, 2) allosteric IGF-2 blockers, 3) allosteric IGF-1 and IGF-2 blockers, and 4) competitive IGF-1 and IGF-2 blockers. The epitopes of representative antibodies from each of these classes were mapped using a purified IGF-1R library containing 64 mutations. Most of these antibodies bound overlapping surfaces on the cysteine-rich repeat and L2 domains. One class of allosteric IGF-1 and IGF-2 blocker was identified that bound a separate epitope on the outer surface of the FnIII-1 domain. Using various biophysical techniques, we show that the dual IGF blockers inhibit ligand binding using a spectrum of mechanisms ranging from highly allosteric to purely competitive. Binding of IGF-1 or the inhibitory antibodies was associated with conformational changes in IGF-1R, linked to the ordering of dynamic or unstructured regions of the receptor. These results suggest IGF-1R uses disorder/order within its polypeptide sequence to regulate its activity. Interestingly, the activity of representative allosteric and competitive inhibitors on H322M tumor cell growth in vitro was reflective of their individual ligand-blocking properties. Many of the antibodies in the clinic likely adopt one of the inhibitory mechanisms described here, and the outcome of future clinical studies may reveal whether a particular inhibitory mechanism leads to optimal clinical efficacy.

Flexibility in the ABC transporter MsbA: Alternating access with a twist.

ATP-binding cassette (ABC) transporters are integral membrane proteins that translocate a wide variety of substrates across cellular membranes and are conserved from bacteria to humans. Here we compare four x-ray structures of the bacterial ABC lipid flippase, MsbA, trapped in different conformations, two nucleotide-bound structures and two in the absence of nucleotide. Comparison of the nucleotide-free conformations of MsbA reveals a flexible hinge formed by extracellular loops 2 and 3. This hinge allows the nucleotide-binding domains to disassociate while the ATP-binding half sites remain facing each other. The binding of the nucleotide causes a packing rearrangement of the transmembrane helices and changes the accessibility of the transporter from cytoplasmic (inward) facing to extracellular (outward) facing. The inward and outward openings are mediated by two different sets of transmembrane helix interactions. Altogether, the conformational changes between these structures suggest that large ranges of motion may be required for substrate transport.

The structures of MsbA: Insight into ABC transporter-mediated multidrug efflux.

ATP-binding cassette (ABC) transporters are integral membrane proteins that couple ATP hydrolysis to the transport of various molecules across cellular membranes. Found in both prokaryotes and eukaryotes, a sub-group of these transporters are involved in the efflux of hydrophobic drugs and lipids, causing anti-microbial and chemotherapeutic multidrug resistance. In this review, we examine recent structural and functional analysis of the ABC transporter MsbA and implications on the mechanism of multidrug efflux.

Structure of the ABC transporter MsbA in complex with ADP.vanadate and lipopolysaccharide.

Select members of the adenosine triphosphate (ATP)-binding cassette (ABC) transporter family couple ATP binding and hydrolysis to substrate efflux and confer multidrug resistance. We have determined the x-ray structure of MsbA in complex with magnesium, adenosine diphosphate, and inorganic vanadate (Mg.ADP.Vi) and the rough-chemotype lipopolysaccharide, Ra LPS. The structure supports a model involving a rigid-body torque of the two transmembrane domains during ATP hydrolysis and suggests a mechanism by which the nucleotide-binding domain communicates with the transmembrane domain. We propose a lipid "flip-flop" mechanism in which the sugar groups are sequestered in the chamber while the hydrophobic tails are dragged through the lipid bilayer.

Lipopolysaccharide stabilizes the crystal packing of the ABC transporter MsbA.

The ABC transporter MsbA is an integral membrane protein involved in the transport of lipid A and lipopolysaccharides to the outer leaflet of the inner membrane in bacteria. Here, the critical role of the natural substrate lipopolysaccharide in the crystallization and diffraction quality of MsbA crystals is reported. Initial crystals grown in complex with ATP-vanadate alone diffracted to approximately 9 A. Screening of the natural substrate lipopolysaccharides led to the crystallization of MsbA in complex with ADP-vanadate and Ra lipopolysaccharide. The increased order within the crystal lattice allowed structure determination to 4.2 A.