Muscle fibre morphometric analysis (MusMA) correlates with muscle function and cardiovascular risk prognosis.

Morphometry of striated muscle fibres is critical for monitoring muscle health and function. Here, we evaluated functional parameters of skeletal and cardiac striated muscle in two experimental models using the Morphometric Analysis of Muscle Fibre tool (MusMA). The collagen-induced arthritis model was used to evaluate the function of skeletal striated muscle and the non-alcoholic fatty liver disease model was used for cardiac striated muscle analysis. After euthanasia, we used haeamatoxylin and eosin stained sections of skeletal and cardiac muscle to perform muscle fibre segmentation and morphometric analysis. Morphometric analysis classified muscle fibres into six subpopulations: normal, regular hypertrophic, irregular hypertrophic, irregular, irregular atrophic and regular atrophic. The percentage of atrophic fibres was associated with lower walking speed (p = 0.009) and lower body weight (p = 0.026), respectively. Fibres categorized as normal were associated with maximum grip strength (p < 0.001) and higher march speed (p < 0.001). In the evaluation of cardiac striated muscle fibres, the percentage of normal cardiomyocytes negatively correlated with cardiovascular risk markers such as the presence of abdominal adipose tissue (p = .003), miR-33a expression (p = .001) and the expression of miR-126 (p = .042) Furthermore, the percentage of atrophic cardiomyocytes correlated significantly with the Castelli risk index II (p = .014). MusMA is a simple and objective tool that allows the screening of striated muscle fibre morphometry, which can complement the diagnosis of muscle diseases while providing functional and prognostic information in basic and clinical research.

Modulation of tumor plasticity by senescent cells: Deciphering basic mechanisms and survival pathways to unravel therapeutic options.

Senescence is a cellular state in which the cell loses its proliferative capacity, often irreversibly. Physiologically, it occurs due to a limited capacity of cell division associated with telomere shortening, the so-called replicative senescence. It can also be induced early due to DNA damage, oncogenic activation, oxidative stress, or damage to other cellular components (collectively named induced senescence). Tumor cells acquire the ability to bypass replicative senescence, thus ensuring the replicative immortality, a hallmark of cancer. Many anti-cancer therapies, however, can lead tumor cells to induced senescence. Initially, this response leads to a slowdown in tumor growth. However, the longstanding accumulation of senescent cells (SnCs) in tumors can promote neoplastic progression due to the enrichment of numerous molecules and extracellular vesicles that constitutes the senescence-associated secretory phenotype (SASP). Among other effects, SASP can potentiate or unlock the tumor plasticity and phenotypic transitions, another hallmark of cancer. This review discusses how SnCs can fuel mechanisms that underlie cancer plasticity, like cell differentiation, stemness, reprogramming, and epithelial-mesenchymal transition. We also discuss the main molecular mechanisms that make SnCs resistant to cell death, and potential strategies to target SnCs. At the end, we raise open questions and clinically relevant perspectives in the field.

Subcellular structure, heterogeneity, and plasticity of senescent cells.

Cellular senescence is a state of permanent growth arrest. It can be triggered by telomere shortening (replicative senescence) or prematurely induced by stresses such as DNA damage, oncogene overactivation, loss of tumor suppressor genes, oxidative stress, tissue factors, and others. Advances in techniques and experimental designs have provided new evidence about the biology of senescent cells (SnCs) and their importance in human health and disease. This review aims to describe the main aspects of SnCs phenotype focusing on alterations in subcellular compartments like plasma membrane, cytoskeleton, organelles, and nuclei. We also discuss the heterogeneity, dynamics, and plasticity of SnCs' phenotype, including the SASP, and pro-survival mechanisms. We advance on the multiple layers of phenotypic heterogeneity of SnCs, such as the heterogeneity between inducers, tissues and within a population of SnCs, discussing the relevance of these aspects to human health and disease. We also raise the main challenges as well alternatives to overcome them. Ultimately, we present open questions and perspectives in understanding the phenotype of SnCs from the perspective of basic and applied questions.

Cellular senescence in reproduction: a two-edged sword†.

Cellular senescence (CS) is the state when cells are no longer capable to divide even after stimulation with grown factors. Cells that begin to undergo CS stop in the cell cycle and enter a suspended state without committing to programmed cell death. These cells assume a specific phenotype and influence their microenvironment by secreting molecules and extracellular vesicles that are part of the so-called senescent cell-associated secretory phenotype (SASP). Cellular senescence is intertwined with physiological and pathological conditions in the human organism. In terms of reproduction, senescent cells are present from reproductive tissues and germ cells to gestational tissues, and participate from fertilization to delivery, going through adverse reproductive outcomes such as pregnancy losses. Furthermore, various SASP molecules are enriched in gestational tissues throughout pregnancy. Thus, the aim of this review is to provide a basis about the features and potential roles played by CS throughout the reproductive process, encompassing its implication in each step of it and proposing a way to manage it in adverse reproductive contexts.

Rifaximin on epigenetics and autophagy in animal model of hepatocellular carcinoma secondary to metabolic-dysfunction associated steatotic liver disease.

BACKGROUND: Prevalence of hepatocellular carcinoma (HCC) is increasing, especially in patients with metabolic dysfunction-associated steatotic liver disease (MASLD). AIM: To investigate rifaximin (RIF) effects on epigenetic/autophagy markers in animals. METHODS: Adult Sprague-Dawley rats were randomly assigned (n = 8, each) and treated from 5-16 wk: Control [standard diet, water plus gavage with vehicle (Veh)], HCC [high-fat choline deficient diet (HFCD), diethylnitrosamine (DEN) in drinking water and Veh gavage], and RIF [HFCD, DEN and RIF (50 mg/kg/d) gavage]. Gene expression of epigenetic/autophagy markers and circulating miRNAs were obtained. RESULTS: All HCC and RIF animals developed metabolic-dysfunction associated steatohepatitis fibrosis, and cirrhosis, but three RIF-group did not develop HCC. Comparing animals who developed HCC with those who did not, miR-122, miR-34a, tubulin alpha-1c (Tuba-1c), metalloproteinases-2 (Mmp2), and metalloproteinases-9 (Mmp9) were significantly higher in the HCC-group. The opposite occurred with Becn1, coactivator associated arginine methyltransferase-1 (Carm1), enhancer of zeste homolog-2 (Ezh2), autophagy-related factor LC3A/B (Map1 Lc3b), and p62/sequestosome-1 (p62/SQSTM1)-protein. Comparing with controls, Map1 Lc3b, Becn1 and Ezh2 were lower in HCC and RIF-groups (P < 0.05). Carm1 was lower in HCC compared to RIF (P < 0.05). Hepatic expression of Mmp9 was higher in HCC in relation to the control; the opposite was observed for p62/Sqstm1 (P < 0.05). Expression of p62/SQSTM1 protein was lower in the RIF-group compared to the control (P = 0.024). There was no difference among groups for Tuba-1c, Aldolase-B, alpha-fetoprotein, and Mmp2 (P > 0.05). miR-122 was higher in HCC, and miR-34a in RIF compared to controls (P < 0.05). miR-26b was lower in HCC compared to RIF, and the inverse was observed for miR-224 (P < 0.05). There was no difference among groups regarding miR-33a, miR-143, miR-155, miR-375 and miR-21 (P > 0.05). CONCLUSION: RIF might have a possible beneficial effect on preventing/delaying liver carcinogenesis through epigenetic modulation in a rat model of MASLD-HCC.

The rational modulation of autophagy sensitizes colorectal cancer cells to 5-fluouracil and oxaliplatin.

Colorectal cancer (CRC) is the third most common and deadliest cancer globally. Regimens using 5-fluorouracil (5FU) and Oxaliplatin (OXA) are the first-line treatment for CRC, but tumor recurrence is frequent. It is plausible to hypothesize that differential cellular responses are triggered after treatments depending on the genetic background of CRC cells and that the rational modulation of cell tolerance mechanisms like autophagy may reduce the regrowth of CRC cells. This study proposes investigating the cellular mechanisms triggered by CRC cells exposed to 5FU and OXA using a preclinical experimental design mimicking one cycle of the clinical regimen (i.e., 48 h of treatment repeated every 2 weeks). To test this, we treated CRC human cell lines HCT116 and HT29 with the 5FU and OXA, combined or not, for 48 h, followed by analysis for two additional weeks. Compared to single-drug treatments, the co-treatment reduced tumor cell regrowth, clonogenicity and stemness, phenotypes associated with tumor aggressiveness and poor prognosis in clinics. This effect was exerted by the induction of apoptosis and senescence only in the co-treatment. However, a week after treatment, cells that tolerated the treatment had high levels of autophagy features and restored the proliferative phenotype, resembling tumor recurrence. The pharmacologic suppression of early autophagy during its peak of occurrence, but not concomitant with chemotherapeutics, strongly reduced cell regrowth. Overall, our experimental model provides new insights into the cellular mechanisms that underlie the response and tolerance of CRC cells to 5FU and OXA, suggesting optimized, time-specific autophagy inhibition as a new avenue for improving the efficacy of current treatments.

Autophagy and nuclear morphometry are associated with histopathologic features in esophageal squamous cell carcinoma.

Less than 15% of patients with esophageal squamous cell carcinoma (ESCC) survive 5 years after diagnosis. A better understanding of the biology of these tumors and the development of clinical biomarkers is needed. Autophagy is a physiological mechanism involved in the turnover of cellular components that plays a key role in cancer. This study evaluated the differential levels of three key regulators of autophagy (SQSTM1, MAP1LC3B, and BECN1) in patients with ESCC, associating autophagy with histopathologic features, including the grade of differentiation, mitotic rate, inflammation score, and the intensity of tumor-infiltrating lymphocytes. Nuclear morphometry of the tumor parenchyma was also assessed, associating it with autophagy and histopathology. All three markers significantly increased in patients with ESCC compared to the control group. Based on the mean expression of each protein in the control group, 57% of patients with ESCC had high levels of all three markers compared to control patients (14%). The most frequent profiles found in ESCC were BECNhigh/MAP1LC3high and BECNhigh/SQSTM1high. According to the TCGA database, we found that the main autophagy genes were upregulated in ESCC. Moreover, high levels of autophagy markers were associated with a poor prognosis. Considering nuclear morphometry, ESCC samples showed a significant reduction in nuclear area, which was strongly negatively correlated with autophagy. Finally, the percentage of normal nuclei was associated with tumor differentiation, while poorly differentiated tumors showed lower SQSTM1 levels. ESCC progression may involve increased autophagy and changes in nuclear structure, associated with clinically relevant histopathological features. KEY MESSAGES: Autophagy markers are co-increased in primary ESCC. Autophagy negatively correlates with nuclear morphometry in ESCC parenchyma. Autophagy and nuclear morphometry are associated with histopathological features. Autophagy is increased in ESCC-TCGA database and associated with poor prognosis.

Mitochondrial response of glioma cells to temozolomide.

Metabolic adaptations are central for carcinogenesis and response to therapy, but little is known about the contribution of mitochondrial dynamics to the response of glioma cells to the standard treatment with temozolomide (TMZ). Glioma cells responded to TMZ with mitochondrial mass increased and the production of round structures of dysfunctional mitochondria. At single-cell level, asymmetric mitosis contributed to the heterogeneity of mitochondrial levels. It affected the fitness of cells in control and treated condition, indicating that the mitochondrial levels are relevant for glioma cell fitness in the presence of TMZ.

Laterality influence on gene expression of DNA damage repair in colorectal cancer.

Colorectal carcinoma (CRC) is the third most common malignancy worldwide, and second in number of deaths in the world. The molecular pathogenesis of CRC is heterogeneous and can affect several genes. Moreover, genomic instability is recognized as an important part of CRC carcinogenesis and is tightly connected to DNA damage response. DNA damage repair (DDR) pathways are intrinsically associated with cancer development and establishment. Traditionally, CRC is considered as one coherent disease, however, new evidence shows that left and right-sided CRC present differences observed in clinical settings, as well as in pre-clinical studies. Therefore, this study aimed to investigate the impact of DDR transcriptional profiles on survival in different sublocations of the colon and rectum using Cox regression, survival analysis and differential gene expression. Right side colon (RSC) has DDR genes' expression associated only with higher risk of death, while left side colon (LSC) and Rectum have most genes' expression associated with lower risk. The pattern is the same with survival analysis. All significant DDR genes had lower expression associated with better survival in RSC, as opposed to LSC and Rectum. Our results demonstrate that RSC is distinctively different from LSC and Rectum. LSC and Rectum have similar DDR expression profiles.

The Application of Static Magnetic Stimulation Reduces Survival of SH-SY5Y Neuroblastoma Cells.

BACKGROUND/AIM: Central nervous system cancer is still a major public health issue. The effectiveness of treatments is limited and varies depending on the severity of disease. Therefore, there is a demand for the development of novel therapies. Static magnetic stimulation (SMS) emerges as a new therapeutic option. The aim of this study was to evaluate the SMS effects on neuroblastoma cells in culture. MATERIALS AND METHODS: SH-SY5Y neuroblastoma cells were exposed to 0.3T SMS for 6, 12, 24, 36, 72 h, and 6 days. Cell viability (MTT), cell death (annexin-V/PI staining) and cell cycle (DNA content), cell proliferation (CFSE), autophagy (acridine orange), and total mitochondrial mass (MitoTracker™ Red) were analyzed to establish the cellular response to SMS. RESULTS: The viability of SH-SY5Y cells was reduced after exposure to SMS for 24 h and 6 days (p<0.05), without differences for the other times (p>0.05); however, this effect was not related to cell death or cell cycle arrest (p>0.05). In contrast, the viability of human malignant melanoma (HMV-II) cells, used as a tumoral control, was not affected. In addition, stimulated SH-SY5Y cells presented a decrease in mitochondrial mass at both exposure times and a reduction in autophagy and cell proliferation after 6 days (p<0.05). CONCLUSION: SMS application appears to be a promising adjuvant therapy for the treatment of neuroblastoma since it decreases the survival of SH-SY5Y neuroblastoma cells.

Comparison of Nonsequencing Techniques for Identification of NPM1 Mutations and Associated Blast Morphology in Patients With Acute Myeloid Leukemia.

CONTEXT.­: Nucleophosmin 1 (NPM1) mutations affect 20% to 30% of all acute myeloid leukemia (AML) patients; several methods are employed to analyze NPM1 mutations, each of them with its advantages and limitations. OBJECTIVE.­: To compare 3 nonsequencing protocols capable of detecting the main NPM1 mutations and to evaluate nuclear morphometric analysis (NMA) as an alternative to cuplike blast detection. DESIGN.­: We selected multiparameter flow cytometry (MFC), amplification refractory mutation system-polymerase chain reaction (ARMS-PCR), and a quantitative PCR (qPCR) kit to identify NPM1 mutations in AML patients at diagnosis. We also evaluated the presence of cuplike blasts and assessed nuclear morphometry using NMA. RESULTS.­: MFC appears as a screening method for NPM1 mutations because of its lower specificity. ARMS-PCR demonstrated specificity similar to that of the qPCR kit, although it was more laborious. qPCR testing, conversely, is relatively fast and easy to standardize. Of these methods, qPCR was the only one capable of identifying the type of NPM1 mutation. With regard to morphology, NMA could be used as an alternative for the evaluation of cuplike blasts in AML smears. CONCLUSIONS.­: qPCR appears to be the best option to identify NPM1 mutations, with ARMS-PCR representing a cheaper alternative. MFC may be used as a screening method, in which results falling within and above the gray zone should be confirmed by molecular testing.

Ornithine Aspartate and Vitamin-E Combination Has Beneficial Effects on Cardiovascular Risk Factors in an Animal Model of Nonalcoholic Fatty Liver Disease in Rats.

Cardiovascular (CV) disease is the main cause of death in nonalcoholic fatty liver disease (NAFLD), a clinical condition without any approved pharmacological therapy. Thus, we investigated the effects of ornithine aspartate (LOLA) and/or Vitamin E (VitE) on CV parameters in a steatohepatitis experimental model. Adult Sprague Dawley rats were randomly assigned (10 animals each) and treated from 16 to 28 weeks with gavage as follows: controls (standard diet plus distilled water (DW)), NAFLD (high-fat choline-deficient diet (HFCD) plus DW), NAFLD+LOLA (HFCD plus LOLA (200 mg/kg/day)), NAFLD+VitE (HFCD plus VitE (150 mg twice a week)) or NAFLD+LOLA+VitE in the same doses. Atherogenic ratios were higher in NAFLD when compared with NAFLD+LOLA+VitE and controls (p < 0.05). Serum concentration of IL-1ß, IL-6, TNF-α, MCP-1, e-selectin, ICAM-1, and PAI-1 were not different in intervention groups and controls (p > 0.05). NAFLD+LOLA decreased miR-122, miR-33a, and miR-186 (p < 0.05, for all) in relation to NAFLD. NAFLD+LOLA+VitE decreased miR-122, miR-33a and miR-186, and increased miR-126 (p < 0.05, for all) in comparison to NAFLD and NAFLD+VitE. NAFLD+LOLA and NAFLD+LOLA+VitE prevented liver collagen deposition (p = 0.006) in comparison to NAFLD. Normal cardiac fibers (size and shape) were lower in NAFLD in relation to the others; and the inverse was reported for the percentage of regular hypertrophic cardiomyocytes. NAFLD+LOLA+VitE promoted a significant improvement in atherogenic dyslipidemia, liver fibrosis, and paracrine signaling of lipid metabolism and endothelial dysfunction. This association should be further explored in the treatment of NAFLD-associated CV risk factors.

Vincristine promotes differential levels of apoptosis, mitotic catastrophe, and senescence depending on the genetic background of glioblastoma cells.

Vincristine (VCR) is a classical chemotherapeutic that has been revisited to treat refractory solid tumors producing encouraging results. VCR binds to tubulin and decreases the rate of microtubule dynamics, thus triggering many cellular responses and behaviors. However, the dynamics of these responses and fates are uncharacterized. This study combined systems biology approaches with acute and long-term in vitro experiments to predict key pathways and mechanisms associated with cell fates during and after VCR treatment. Glioblastoma (GBM) cells were treated with clinically relevant doses of VCR, and interconnected cell fates were explored. A correlation matrix based on experimental cell analysis reported strong negative correlations between cell number, nuclear irregularities, senescence, or apoptosis, depending on the cells' genetic makeup and treatment regimen. P53 would be essential in all analyzed processes according to topological network analysis. Furthermore, despite the high acute sensitivity, both cell lines re-growth in the long term after a single VCR treatment, especially in those populations with high levels of autophagy. These multiple responses may also be triggered in patients' exposed tumors, which should be considered to allow the rational design of VCR protocols, including modulators of the cell fates and pathways mentioned above.

P2Y12 receptor antagonism inhibits proliferation, migration and leads to autophagy of glioblastoma cells.

Glioblastoma (GBM) is the most aggressive and lethal among the primary brain tumors, with a low survival rate and resistance to radio and chemotherapy. The P2Y12 is an adenosine diphosphate (ADP) purinergic chemoreceptor, found mainly in platelets. In cancer cells, its activation has been described to induce proliferation and metastasis. Bearing in mind the need to find new treatments for GBM, this study aimed to investigate the role of the P2Y12R in the proliferation and migration of GBM cells, as well as to evaluate the expression of this receptor in patients' data obtained from the TCGA data bank. Here, we used the P2Y12R antagonist, ticagrelor, which belongs to the antiplatelet agent's class. The different GBM cells (cell line and patient-derived cells) were treated with ticagrelor, with the agonist, ADP, or both, and the effects on cell proliferation, colony formation, ADP hydrolysis, cell cycle and death, migration, and cell adhesion were analyzed. The results showed that ticagrelor decreased the viability and the proliferation of GBM cells. P2Y12R antagonism also reduced colony formation and migration potentials, with alterations on the expression of metalloproteinases, and induced autophagy in GBM cells. Changes were observed at the cell cycle level, and only the U251 cell line showed a significant reduction in the ADP hydrolysis profile. TCGA data analysis showed a higher expression of P2Y12R in gliomas samples when compared to the other tumors. These data demonstrate the importance of the P2Y12 receptor in gliomas development and reinforce its potential as a pharmacological target for glioma treatment.

High P2X6 receptor expression in human bladder cancer predicts good survival prognosis.

As alterations in purinergic signaling have been observed in bladder diseases, we aimed to assess the potential prognostic role of purinergic receptors in bladder cancer in a translational approach based on clinical databases and in vitro data. The prognostic role of purinergic receptors in the survival of patients with bladder cancer and the expression profile of the altered P2 receptors in normal and in tumor samples were determined using The Cancer Genome Atlas databank. In T24 and RT4 human bladder cancer cell lines, the P2 purinergic receptors were characterized by RT-PCR and RT-qPCR analysis including radiotherapy exposure as treatment. The cell number and the cumulative population doubling were also assessed. The expression profile of P2X6 receptor in the cancer pathological stage and in the nodal metastasis status was in agreement with Kaplan-Meier analysis, indicating that high expression of this receptor was related to an increased survival rate in patients with bladder cancer. Of all the P2 receptors expressed on T24 cell line, P2X6 presented high expression after radiotherapy, while it was not altered in RT4 cells. In addition, irradiation promoted a decrease of T24 cell number, but did not change the cell number of RT4 after the same time and radiation dose. Along 7 days after irradiation exposure, both cells regrew. However, while P2X6 receptor was downregulated in T24 cells, it was upregulated in RT4 cells. Our findings indicated that high P2X6 receptor expression induced by radiation in T24 cell line may predict a good survival prognostic factor.

Bioimaging approaches for quantification of individual cell behavior during cell fate decisions.

Tracking individual cells has allowed a new understanding of cellular behavior in human health and disease by adding a dynamic component to the already complex heterogeneity of single cells. Technically, despite countless advances, numerous experimental variables can affect data collection and interpretation and need to be considered. In this review, we discuss the main technical aspects and biological findings in the analysis of the behavior of individual cells. We discuss the most relevant contributions provided by these approaches in clinically relevant human conditions like embryo development, stem cells biology, inflammation, cancer and microbiology, along with the cellular mechanisms and molecular pathways underlying these conditions. We also discuss the key technical aspects to be considered when planning and performing experiments involving the analysis of individual cells over long periods. Despite the challenges in automatic detection, features extraction and long-term tracking that need to be tackled, the potential impact of single-cell bioimaging is enormous in understanding the pathogenesis and development of new therapies in human pathophysiology.

Protocol to segment and characterize the morphometry of individual cross-sectioned striated muscle fibers from adult murine models.

The muscle fiber morphometric analysis (MusMA) is a protocol to segment and characterize the morphometry of individual cross-sectioned striated muscle fibers. Using a semi-automated Excel spreadsheet, the protocol allows the objective measurement of muscle fibers' subpopulations, aiming to characterize physiopathological conditions related to muscle tissue. The main limitation of MusMA is the need for high-quality tissue slides and images and control samples to set up the analyses.

Primary cells derived from Tuberous Sclerosis Complex patients show autophagy alteration in the haploinsufficiency state.

Tuberous sclerosis complex (TSC) is an autosomal dominant cancer predisposition disorder caused by heterozygous mutations in TSC1 or TSC2 genes and characterized by mTORC1 hyperactivation. TSC-associated tumors develop after loss of heterozygosity mutations and their treatment involves the use of mTORC1 inhibitors. We aimed to evaluate cellular processes regulated by mTORC1 in TSC cells with different mutations before tumor development. Flow cytometry analyses were performed to evaluate cell viability, cell cycle and autophagy in non-tumor primary TSC cells with different heterozygous mutations and in control cells without TSC mutations, before and after treatment with rapamycin (mTORC1 inhibitor). We did not observe differences in cell viability and cell cycle between the cell groups. However, autophagy was reduced in mutated cells. After rapamycin treatment, mutated cells showed a significant increase in the autophagy process (p=0.039). We did not observe differences between cells with distinct TSC mutations. Our main finding is the alteration of autophagy in non-tumor TSC cells. Previous studies in literature found autophagy alterations in tumor TSC cells or knock-out animal models. We showed that autophagy could be an important mechanism that leads to TSC tumor formation in the haploinsufficiency state. This result could guide future studies in this field.

In Situ Gene Therapy.

Gene therapy is a technique that aims at the delivery of nucleic acids to cells, to obtain a therapeutic effect. In situ gene therapy consists of the administration of the gene product to a specific site. It possesses several advantages, such as the reduction in potential side effects, the need for a lower vector dose, and, as a consequence, reduced costs, compared to intravenous administration. Different vectors, administration routes and doses involving in situ gene transfer have been tested both in animal models and humans, with in situ gene therapy drugs already approved in the market. In this review, we present applications of in situ gene therapy for different diseases, ranging from monogenic to multifactorial diseases, focusing mainly on therapies designed for the intra-articular and intraocular compartments, as well as gene therapies for the central nervous system (CNS) and for tumors. Gene therapy finally seems to blossom as a viable therapeutic approach. The growth in the number of clinical protocols shown here is evident, and the positive outcomes observed in several clinical trials indicate that more products based on in situ gene therapy should reach the market in the next years.

The paradox of autophagy in Tuberous Sclerosis Complex.

Tuberous sclerosis complex (TSC) is an autosomal dominant genetic disorder caused by germline mutations in TSC1 or TSC2 genes, which leads to the hyperactivation of the mTORC1 pathway, an important negative regulator of autophagy. This leads to the development of hamartomas in multiple organs. The variability in symptoms presents a challenge for the development of completely effective treatments for TSC. One option is the treatment with mTORC1 inhibitors, which are targeted to block cell growth and restore autophagy. However, the therapeutic effect of rapamycin seems to be more efficient in the early stages of hamartoma development, an effect that seems to be associated with the paradoxical role of autophagy in tumor establishment. Under normal conditions, autophagy is directly inhibited by mTORC1. In situations of bioenergetics stress, mTORC1 releases the Ulk1 complex and initiates the autophagy process. In this way, autophagy promotes the survival of established tumors by supplying metabolic precursors during nutrient deprivation; paradoxically, excessive autophagy has been associated with cell death in some situations. In spite of its paradoxical role, autophagy is an alternative therapeutic strategy that could be explored in TSC. This review compiles the findings related to autophagy and the new therapeutic strategies targeting this pathway in TSC.