Oncolytic Adenoviral Vector-Mediated Expression of an Anti-PD-L1-scFv Improves Anti-Tumoral Efficacy in a Melanoma Mouse Model

Oncolytic virotherapy is an emerging therapeutic approach based on replication-competent viruses able to selectively infect and destroy cancer cells, inducing the release of tumor-associated antigens and thereby recruiting immune cells with a subsequent increase in antitumoral immune response. To increase the anticancer activity, we engineered a specific oncolytic adenovirus expressing a single-chain variable fragment of an antibody against PD-L1 to combine blockage of PD-1/PD-L1 interaction with the antitumoral activity of Onc.Ad5. To assess its efficacy, we infected B16.OVA cells, a murine model of melanoma, with Ad5 Delta 24 -anti-PD-L1-scFv and then co-cultured them with C57BL/6J naive splenocytes. We observed that the combinatorial treatments were significantly more effective in inducing cancer cell death. Furthermore, we assessed the efficacy of intratumoral administrations of Ad5 Delta 24-anti-PD-L1-scFv in C57BL/6J mice engrafted with B16.OVA and compared this treatment to that of the parental Ad5 Delta 24 or placebo. Treatment with the scFv-expressing Onc.Ad induced a marked reduction of tumor growth concerning the parental Onc.Ad. Additionally, the evaluation of the lymphocytic population infiltrating the treated tumor reveals a favorable immune profile with an enhancement of the CD8(+) population. These data suggest that Onc.Ad-mediated expression of immune checkpoint inhibitors increases oncolytic virotherapy efficacy and could be an effective and promising tool for cancer treatments, opening a new way into cancer therapy.Peer reviewe

Essential Physiological Differences Characterize Short- and Long-Lived Strains of Drosophila melanogaster

Aging is a multifactorial process which affects all animals. Aging as a result of damage accumulation is the most accepted explanation but the proximal causes remain to be elucidated. There is also evidence indicating that aging has an important genetic component. Animal species age at different rates and specific signaling pathways, such as insulin/insulin-like growth factor, can regulate life span of individuals within a species by reprogramming cells in response to environmental changes. Here, we use an unbiased approach to identify novel factors that regulate life span in Drosophila melanogaster. We compare the transcriptome and metabolome of two wild-type strains used widely in aging research: short-lived Dahomey and long-lived Oregon R flies. We found that Dahomey flies carry several traits associated with short-lived individuals and species such as increased lipoxidative stress, decreased mitochondrial gene expression, and increased Target of Rapamycin signaling. Dahomey flies also have upregulated octopamine signaling known to stimulate foraging behavior. Accordingly, we present evidence that increased foraging behavior, under laboratory conditions where nutrients are in excess increases damage generation and accelerates aging. In summary, we have identified several new pathways, which influence longevity highlighting the contribution and importance of the genetic component of aging.This work was supported by the European Research Council (260632 - ComplexI&Aging to A.S.); the Academy of Finland (252048 to A.S); the Biotechnology and Biological Sciences Research Council ( BB/M023311/1 to A.S.); the Centre for International Mobility (CIMO) (TM-12- 8391 and TM-13-8919 to N.G.); the Spanish Ministry of Economy and Competitiveness, Institute of Health Carlos III (PI14/00328 to R.P. and PI17/01286 to P.N.); the Autonomous Government of Catalonia (2017SGR696 and SLT002/16/00250 to R.P.); the Ministry of Education and Science of Ukraine (grant number 0117U006426 to O.L.); FEDER funds from the European Union (“A way to build Europe” to R.P.); and the Doctoral Programme in Medicine and Life Sciences of University of Tampere (to T.R). R.S is a Sir Henry Wellcome Postdoctoral Fellow funded by Wellcome (204715/Z/16/Z

Coenzyme Q redox signalling and longevity

Mitochondria are the powerhouses of the cell. They produce a significant amount of the energy we need to grow, survive and reproduce. The same system that generates energy in the form of ATP also produces Reactive Oxygen Species (ROS). Mitochondrial Reactive Oxygen Species (mtROS) were considered for many years toxic by-products of metabolism, responsible for ageing and many degenerative diseases. Today, we know that mtROS are essential redox messengers required to determine cell fate and maintain cellular homeostasis. Most mtROS are produced by respiratory complex I (CI) and complex III (CIII). How and when CI and CIII produce ROS is determined by the redox state of the Coenzyme Q (CoQ) pool and the proton motive force (pmf) generated during respiration. During ageing, there is an accumulation of defective mitochondria that generate high levels of mtROS. This causes oxidative stress and disrupts redox signalling. Here, we review how mtROS are generated in young and old mitochondria and how CI and CIII derived ROS control physiological and pathological processes. Finally, we discuss why damaged mitochondria amass during ageing as well as methods to preserve mitochondrial redox signalling with age

The interplay between mitochondrial protein and iron homeostasis and its possible role in ageing

Free (labile or chelatable) iron is extremely redox-active and only represents a small fraction of the total mitochondrial iron population. Several studies have shown that the proportion of free iron increases with age, leading to increased Fenton chemistry in later life. It is not clear why free iron accumulates in mitochondria, but it does so in parallel with an inability to degrade and recycle damaged proteins that causes loss of mitochondrial protein homeostasis (proteostasis). The increase in oxidative damage that has been shown to occur with age might be explained by these two processes. While this accumulation of oxidative damage has often been cited as causative to ageing there are examples of model organisms that possess high levels of oxidative damage throughout their lives with no effect on lifespan. Interestingly, these same animals are characterised by an outstanding ability to maintain correct proteostasis during their entire life. ROS can damage critical components of the iron homeostasis machinery, while the efficacy of mitochondrial quality control mechanisms will determine how detrimental that damage is. Here we review the interplay between iron and organellar quality control in mitochondrial dysfunction and we suggest that a decline in mitochondrial proteostasis with age leaves iron homeostasis (where several key stages are thought to be dependent on proteostasis machinery) vulnerable to oxidative damage and other age-related stress factors. This will have severe consequences for the electron transport chain and TCA cycle (among other processes) where several components are acutely dependent on correct assembly, insertion and maintenance of iron–sulphur clusters, leading to energetic crisis and death

Practical Recommendations for the Use of the GeneSwitch Gal4 System to Knock-Down Genes in <i>Drosophila melanogaster</i>

<div><p><i>Drosophila melanogaster</i> is a popular research model organism thanks to its’ powerful genetic tools that allow spatial and temporal control of gene expression. The inducible GeneSwitch Gal4 system (GS) system is a modified version of the classic UAS/GAL4 system which allows inducible regulation of gene expression and eliminates background effects. It is widely acknowledged that the GS system is leaky, with low level expression of UAS transgenes in absence of the inducer RU-486 (the progesterone analog that activates the modified GAL4 protein). However, in the course of our experiments, we have observed that the extent of this leak depends on the nature of the transgene being expressed. In the absence of RU-486, when strong drivers are used to express protein coding transgenes, leaky expression is low or negligible, however expression of RNA interference (RNAi) transgenes results in complete depletion of protein levels. The majority of published studies, using the GS system and RNAi transgenes validate knock-down efficiency by comparing target gene mRNA levels between induced and non-induced groups. Here, we demonstrate that this approach is lacking and that both additional control groups and further validation is required at the protein level. Unfortunately, this experimental limitation of the GS system eliminates “the background advantage”, but does offer the possibility of performing more complex experiments (e.g. studying depletion and overexpression of different proteins in the same genetic background). The limitations and new possible applications of the GS system are discussed in detail.</p></div

Practical recommendations for the use of the GeneSwitch Gal4 system to knock-down genes in Drosophila melanogaster

Drosophila melanogaster is a popular research model organism thanks to its’ powerful genetic tools that allow spatial and temporal control of gene expression. The inducible GeneSwitch Gal4 system (GS) system is a modified version of the classic UAS/GAL4 system which allows inducible regulation of gene expression and eliminates background effects. It is widely acknowledged that the GS system is leaky, with low level expression of UAS transgenes in absence of the inducer RU-486 (the progesterone analog that activates the modified GAL4 protein). However, in the course of our experiments, we have observed that the extent of this leak depends on the nature of the transgene being expressed. In the absence of RU-486, when strong drivers are used to express protein coding transgenes, leaky expression is low or negligible, however expression of RNA interference (RNAi) transgenes results in complete depletion of protein levels. The majority of published studies, using the GS system and RNAi transgenes validate knock-down efficiency by comparing target gene mRNA levels between induced and non-induced groups. Here, we demonstrate that this approach is lacking and that both additional control groups and further validation is required at the protein level. Unfortunately, this experimental limitation of the GS system eliminates “the background advantage”, but does offer the possibility of performing more complex experiments (e.g. studying depletion and overexpression of different proteins in the same genetic background). The limitations and new possible applications of the GS system are discussed in detail

No difference in protein levels between induced and non-induced groups using a strong GS driver.

<p>(A) Western blot analysis of ND-19 levels in control (without the RNAi transgene), induced and non-induced groups. (B) Quantification of A (n = 3). (C) Western blot analysis of ND-39 levels in control (without the RNAi transgene), induced and non-induced groups. (D) Quantification of C (n = 3). (E) Western blot analysis of Sod2 levels in control (without the RNAi transgene), induced and non-induced groups. (D) Quantification of E (n = 3). Tubulin is shown as loading control. <i>daGS</i>, <i>daughterless</i>-GeneSwitch GAL4; <i>tubGS</i>, <i>tubulin</i>-GeneSwitch GAL4; +/- indicate presence/absence of the transgene or 500 μM RU-486 (inducer).</p

No significant leak is detected when expressing protein coding transgenes using GS.

<p><b>(A</b>) qPCR expression data of NDI1 in induced vs. non-induced flies. Controls without the RNAi transgene are also included (n = 3). (B) Western blot analysis of NDI1 levels in induced vs non-induced flies. (C) Quantification of B (n = 2). (D) Western blot analysis of AOX expression driven by two (++) or one (+) copy of <i>daughterless</i>-GeneSwitch GAL4 (<i>daGS</i>) in flies carrying two (++) or one copy (+) of the AOX transgene in induced vs non-induced groups. (E) Quantification of D (n = 3). (F) Western blot analysis of NDI1 expression driven by two (++) or one (+) copy of <i>daughterless</i>-GeneSwitch GAL4 (<i>daGS</i>) in flies carrying two (++) or one copy (+) of the NDI1 gene in induced vs non-induced groups. (G) Quantification of F (n = 3). (H) Western blot analysis of AOX expression driven by two (++) or one (+) copy of <i>tubulin</i>-GeneSwitch GAL4 (<i>tubGS</i>) in flies carrying two (++) or one copy (+) of the AOX gene in induced vs non-induced groups. (I) Quantification of H (n = 3). (J) Western blot analysis of NDI1 expression driven by two (++) or one (+) copy of <i>tubulin</i>-GeneSwitch GAL4 (<i>tubGS</i>) in flies carrying two (++) or one copy (+) of the NDI1 gene in induced vs non-induced groups. (K) Quantification of J (n = 3). GAPDH or Tubulin is shown as loading control. +/- indicate presence/absence of the transgene or 500 μM RU-486 (inducer).</p

Expression of RNAi transgenes does not correlate with the presence of RU-486 in the fly food.

<p>(A) Western blot analysis of ND-39, NDI1 and ATP5A levels in control, induced and non-induced groups. (B) Quantification of A (n = 2). (C) Respirometry in <i>tubGS</i> flies with and without an ND-39 RNAi transgene (n = 3–8). (D) Western blot analysis of ND-39 and NDI1 levels in control, induced and non-induced groups. (E) Quantification of D (n = 2–3). (F) Western blot analysis of ND-39 levels in controls and experimental flies fed with RU-486 during development (1 μM), development and adulthood (1 μM and 500 μM respectively) or exclusively during adulthood (500 μM) (n = 2–3). All flies were 5 days old when proteins were extracted. Flies that were exclusively fed during development spent 5 days in food without RU-486. GAPDH is shown as loading control. <i>daGS</i>, <i>daughterless</i>-GeneSwitch GAL4; <i>tubGS</i>, <i>tubulin</i>-GeneSwitch GAL4; +/- indicate presence/absence of the transgene or 500 μM RU-486 (inducer).</p