MicroRNA-204 Regulates Angiogenesis and Vasculogenic Mimicry in CD44+/CD24- Breast Cancer Stem-like Cells.

Tumors have high requirements in terms of nutrients and oxygen. Angiogenesis is the classical mechanism for vessel formation. Tumoral vascularization has the function of nourishing the cancer cells to support tumor growth. Vasculogenic mimicry, a novel intratumoral microcirculation system, alludes to the ability of cancer cells to organize in three-dimensional (3D) channel-like architectures. It also supplies the tumors with nutrients and oxygen. Both mechanisms operate in a coordinated way; however, their functions in breast cancer stem-like cells and their regulation by microRNAs remain elusive. In the present study, we investigated the functional role of microRNA-204 (miR-204) on angiogenesis and vasculogenic mimicry in breast cancer stem-like cells. Using flow cytometry assays, we found that 86.1% of MDA-MB-231 and 92% of Hs-578t breast cancer cells showed the CD44+/CD24- immunophenotype representative of cancer stem-like cells (CSCs). The MDA-MB-231 subpopulation of CSCs exhibited the ability to form mammospheres, as expected. Interestingly, we found that the restoration of miR-204 expression in CSCs significantly inhibited the number and size of the mammospheres. Moreover, we found that MDA-MB-231 and Hs-578t CSCs efficiently undergo angiogenesis and hypoxia-induced vasculogenic mimicry in vitro. The transfection of precursor miR-204 in both CSCs was able to impair the angiogenesis in the HUVEC cell model, which was observed as a diminution in the number of polygons and sprouting cells. Remarkably, miR-204 mimics also resulted in the inhibition of vasculogenic mimicry formation in MDA-MB-231 and Hs-578t CSCs, with a significant reduction in the number of channel-like structures and branch points. Mechanistically, the effects of miR-204 were associated with a diminution of pro-angiogenic VEGFA and ß-catenin protein levels. In conclusion, our findings indicated that miR-204 abrogates the angiogenesis and vasculogenic mimicry development in breast cancer stem-like cells, suggesting that it could be a potential tool for breast cancer intervention based on microRNA replacement therapies.

Assessing the In Vitro and In Vivo Effect of Supplementation with a Garlic (Allium sativum) and Oregano (Origanum vulgare) Essential Oil Mixture on Digestibility in West African Sheep.

This study assessed the impact of a mixture of garlic (Allium sativum) and oregano (Origanum vulgare) essential oils (EOGOs) on in vitro dry matter digestibility (IVDMD) and in vivo apparent nutrient digestibility. Different EOGO inclusion levels were evaluated to assess the dose response and potential effects of the mixture. Three EOGO inclusion levels (0.5, 0.75, and 1 mL/kg of incubated dry matter) were evaluated in vitro, while four treatments (0.5, 0.75, and 1 mL/day of EOGO and a control group) were tested in vivo on 12 West African sheep. A randomized controlled trial was conducted using a 4 × 4 design. Blood parameters (glucose, blood urea nitrogen, and ß-hydroxybutyrate) were measured to observe the effect of EOGO on the metabolism. The results showed that the inclusion of EOGO significantly enhanced IVDMD at low levels (p < 0.052) compared with the highest levels in treatments containing 0.5 and 0.75 mL/kg of EOGO dry matter. A higher intake of dry matter (DM), crude protein (CP), and neutral detergent fiber (NDF) (p < 0.05) was observed in the in vivo diets with the inclusion of EOGO. In terms of in vivo apparent digestibility, significant differences were found among treatments in the digestibility coefficients of DM, CP, and NDF. EOGO inclusion increased the digestibility of DM. CP digestibility displayed a cubic effect (p < 0.038), with the lowest values of digestibility observed at 1 mL EOGO inclusion. Additionally, NDF digestibility showed a cubic effect (p < 0.012), with the highest value obtained at 0.75 mL of EOGO inclusion. The inclusion levels above 0.75 mL EOGO showed a cubic effect, which indicates that higher concentrations of EOGO may not be beneficial for the digestibility of CP and NDF. Although no significant difference was observed in total digestible nutrients, a linear trend was observed (p < 0.059). EOGO improved the intake of DM, CP, and NDF. EOGO supplementation improved the digestibility of DM and NDF, with optimal levels observed at 0.5 mL/day. No significant effects were observed in the blood parameters. These results suggest that EOGO has the potential as an additive in ruminal nutrition to improve food digestibility and serve as an alternative to antibiotic additives. The use of EOGO potentially improves fiber digestion and may reduce the use of antibiotics in livestock production. Garlic (A. sativum) and oregano (O. vulgare) essential oils effectively modulated fiber digestibility at 0.75 mL/day. Garlic (A. sativum) and oregano (O. vulgare) essential oils have the potential to improve digestibility at low inclusion levels and serve as an alternative to antibiotic additives. The effectiveness of essential oils is greater in a mixture and at lower doses.

A PTP1B-Cdk3 Signaling Axis Promotes Cell Cycle Progression of Human Glioblastoma Cells through an Rb-E2F Dependent Pathway.

PTP1B plays a key role in developing different types of cancer. However, the molecular mechanism underlying this effect is unclear. To identify molecular targets of PTP1B that mediate its role in tumorigenesis, we undertook a SILAC-based phosphoproteomic approach, which allowed us to identify Cdk3 as a novel PTP1B substrate. Substrate trapping experiments and docking studies revealed stable interactions between the PTP1B catalytic domain and Cdk3. In addition, we observed that PTP1B dephosphorylates Cdk3 at tyrosine residue 15 in vitro and interacts with it in human glioblastoma cells. Next, we found that pharmacological inhibition of PTP1B or its depletion with siRNA leads to cell cycle arrest with diminished activity of Cdk3, hypophosphorylation of Rb, and the downregulation of E2F target genes Cdk1, Cyclin A, and Cyclin E1. Finally, we observed that the expression of a constitutively active Cdk3 mutant bypasses the requirement of PTP1B for cell cycle progression and expression of E2F target genes. These data delineate a novel signaling pathway from PTP1B to Cdk3 required for efficient cell cycle progression in an Rb-E2F dependent manner in human GB cells.

Criminal reactions to drug-using offenders: A systematic review of the effect of treatment and/or punishment on reduction of drug use and/or criminal recidivism.

The association between substance use and crime is very common, but complex. Several countries have found strategies to face drug abuse and criminality that may exist associated to it, seeking to reduce overcrowded prisons and to promote the reductions of criminal recidivism and/or substance use. Through the guidelines of PRISMA, a systematic review was conducted with the aim to explore the different criminal reactions to individuals who use substances and are involved in the criminal justice system, namely the role of treatment and/or punishment in the reduction of crime recidivism and/or drug (ab)use. After gathering the following criteria of inclusion (individuals who use substances and are involved in the criminal justice system, between 18 and 65 years old, regardless of gender; consumers of licit/illicit psychoactive substances; without psychopathology not related with use/abuse of drugs; treatment programs; judicial interventions) the database found 155 articles between 1971 and 2022 from which 110 were selected for analysis (57 are from Academic Search Complete, 28 from PsycInfo, 10 from Academic Search Ultimate, seven from Sociology Source Ultimate, four from Business Source Complete, two from Criminal Justice Abstracts, and two from PsycArticles); additional records were included trough manual search. From these studies, 23 articles were included, as they answered the research question, and therefore, constitute the final sample of this revision. The results indicate treatment as an effective response of the criminal justice system in the reduction of criminal recidivism and/or drug use, addressing the criminogenic effect of reclusion/imprisonment. Therefore, interventions that privilege treatment should be chosen, although there are still gaps in terms of evaluation, monitoring and scientific publications regarding the effectiveness of treatment in this population.

Mexican Colorectal Cancer Research Consortium (MEX-CCRC): Etiology, Diagnosis/Prognosis, and Innovative Therapies.

In 2013, recognizing that Colorectal Cancer (CRC) is the second leading cause of death by cancer worldwide and that it was a neglected disease increasing rapidly in Mexico, the community of researchers at the Biomedicine Research Unit of the Facultad de Estudios Superiores Iztacala from the Universidad Nacional Autónoma de México (UNAM) established an intramural consortium that involves a multidisciplinary group of researchers, technicians, and postgraduate students to contribute to the understanding of this pathology in Mexico. This article is about the work developed by the Mexican Colorectal Cancer Research Consortium (MEX-CCRC): how the Consortium was created, its members, and its short- and long-term goals. Moreover, it is a narrative of the accomplishments of this project. Finally, we reflect on possible strategies against CRC in Mexico and contrast all the data presented with another international strategy to prevent and treat CRC. We believe that the Consortium's characteristics must be maintained to initiate a national strategy, and the reported data could be useful to establish future collaborations with other countries in Latin America and the world.

Maladaptive Beliefs of Young Adults in Interpersonal Relationships: A Systematic Literature Review.

Beliefs that are too rigid and misadjusted may legitimize interpersonal relationships marked by verbal, physical, or sexual violence. A systematic literature review was performed to identify the existence of maladaptive beliefs (MBs) in interpersonal relationships of young adults and their association with problematic behaviors. Following the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines, studies were obtained through a search in multiple databases: EBSCOhost, PubMed, and Web of Science. Of the 1,280 articles retrieved, 42 were retained for further analysis and 15 were considered eligible for inclusion. In addition, seven studies were added through manual search, leading to a final sample of 15 articles, published between 1964 and 2021. Only empirical studies with quantitative methodologies were included. Objectives, sample (age in years), sample type, country of origin of studies, instruments, control group, and results and main conclusions were extracted from each study. Results suggest the existence of MBs that may legitimize antisocial behavior and violence, whether physical, sexual, gender, domestic, or in intimate relationships. Some intervention programs were also identified, with different approaches with a reasonable degree of efficacy in changing these beliefs or in reducing the rates of recidivism by the aggressors.

Epidemiological Situation of Monkeypox Transmission by Possible Sexual Contact: A Systematic Review.

Monkeypox (MPX), a zoonotic infection caused by the monkeypox virus (MPXV), has re-emerged worldwide with numerous confirmed cases with person-to-person transmission through close contacts, including in sexual networks. Therefore, this study aimed to determine the epidemiological situation of monkeypox transmission by possible sexual contact. A systematic literature review was conducted using PubMed, Scopus, Web of Science, and Embase databases until 18 August 2022. The key search terms used were "monkeypox", "sexual contact", "sexual intercourse" and "sexual transmission". A total of 1291 articles were retrieved using the search strategy. After eliminating duplicates (n = 738) and examining by title, abstract, and full text, 28 studies reporting case reports of monkeypox with a detailed description of clinical features, sexually transmitted diseases, method of diagnosis, location and course of skin lesions, and treatment were included. A total of 4222 confirmed cases of monkeypox have been reported, of which 3876 monkeypox cases are the result of transmission by sexual contact distributed in twelve countries: 4152 cases were male with a mean age of 36 years. All confirmed cases of monkeypox were diagnosed by reverse transcriptase-polymerase chain reaction (RT-PCR). The most frequent clinical manifestations were fever, lymphadenopathy, headache, malaise, and painful perianal and genital lesions. The most frequent locations of the lesions were perianal, genital, oral, trunk, upper and lower extremities. Patients were in good clinical condition, with treatment based on analgesics and antipyretics to relieve some symptoms of monkeypox. A high proportion of STIs and frequent anogenital symptoms were found, suggesting transmissibility through local inoculation during close skin-to-skin or mucosal contact during sexual activity. The highest risk of monkeypox transmission occurs in men who have sex with men, and MPXV DNA could be recovered in seminal fluid. It is essential to establish health policies for the early detection and management of patients with monkeypox.

MicroRNA-204/CREB5 axis regulates vasculogenic mimicry in breast cancer cells.

BACKGROUND: Vasculogenic mimicry (VM) is characterized by formation of three-dimensional (3D) channels-like structures by tumor cells, supplying the nutrients needed for tumor growth. VM is stimulated by hypoxic tumor microenvironment, and it has been associated with increased metastasis and clinical poor outcome in cancer patients. cAMP responsive element (CRE)-binding protein 5 (CREB5) is a hypoxia-activated transcription factor involved in tumorigenesis. However, CREB5 functions in VM and if its regulated by microRNAs remains unknown in breast cancer. OBJECTIVE: We aim to study the functional relationships between VM, CREB5 and microRNA-204-5p (miR-204) in breast cancer cells. METHODS: CREB5 expression was evaluated by mining the public databases, and using RT-qPCR and Western blot assays. CREB5 expression was silenced using short-hairpin RNAs in MDA-MB-231 and MCF-7 breast cancer cells. VM formation was analyzed using matrigel-based cultures in hypoxic conditions. MiR-204 expression was restored in cancer cells by transfection of RNA mimics. Luciferase reporter assays were performed to evaluate the binding of miR-204 to 3'UTR of CREB5. RESULTS: Our data showed that CREB5 mRNA expression was upregulated in a set of breast cancer cell lines and clinical tumors, and it was positively associated with poor prognosis in lymph nodes positive and grade 3 basal breast cancer patients. Silencing of CREB5 impaired the hypoxia-induced formation of 3D channels-like structures representative of the early stages of VM in MDA-MB-231 cells. In contrast, VM formation was not observed in MCF-7 cells. Interestingly, we found that CREB5 expression was negatively regulated by miR-204 mimics in breast cancer cells. Functional analysis confirmed that miR-204 binds to CREB5 3'-UTR indicating that it's an ulterior effector. CONCLUSIONS: Our findings suggested that CREB5 could be a potential biomarker of disease progression in basal subtype of breast cancer, and that perturbations of the miR-204/CREB5 axis plays an important role in VM development in breast cancer cells.

Biological Adaptations of Tumor Cells to Radiation Therapy.

Radiation therapy has been used worldwide for many decades as a therapeutic regimen for the treatment of different types of cancer. Just over 50% of cancer patients are treated with radiotherapy alone or with other types of antitumor therapy. Radiation can induce different types of cell damage: directly, it can induce DNA single- and double-strand breaks; indirectly, it can induce the formation of free radicals, which can interact with different components of cells, including the genome, promoting structural alterations. During treatment, radiosensitive tumor cells decrease their rate of cell proliferation through cell cycle arrest stimulated by DNA damage. Then, DNA repair mechanisms are turned on to alleviate the damage, but cell death mechanisms are activated if damage persists and cannot be repaired. Interestingly, some cells can evade apoptosis because genome damage triggers the cellular overactivation of some DNA repair pathways. Additionally, some surviving cells exposed to radiation may have alterations in the expression of tumor suppressor genes and oncogenes, enhancing different hallmarks of cancer, such as migration, invasion, and metastasis. The activation of these genetic pathways and other epigenetic and structural cellular changes in the irradiated cells and extracellular factors, such as the tumor microenvironment, is crucial in developing tumor radioresistance. The tumor microenvironment is largely responsible for the poor efficacy of antitumor therapy, tumor relapse, and poor prognosis observed in some patients. In this review, we describe strategies that tumor cells use to respond to radiation stress, adapt, and proliferate after radiotherapy, promoting the appearance of tumor radioresistance. Also, we discuss the clinical impact of radioresistance in patient outcomes. Knowledge of such cellular strategies could help the development of new clinical interventions, increasing the radiosensitization of tumor cells, improving the effectiveness of these therapies, and increasing the survival of patients.

Recent advances in PTP1B signaling in metabolism and cancer.

Protein tyrosine phosphorylation is one of the major post-translational modifications in eukaryotic cells and represents a critical regulatory mechanism of a wide variety of signaling pathways. Aberrant protein tyrosine phosphorylation has been linked to various diseases, including metabolic disorders and cancer. Few years ago, protein tyrosine phosphatases (PTPs) were considered as tumor suppressors, able to block the signals emanating from receptor tyrosine kinases. However, recent evidence demonstrates that misregulation of PTPs activity plays a critical role in cancer development and progression. Here, we will focus on PTP1B, an enzyme that has been linked to the development of type 2 diabetes and obesity through the regulation of insulin and leptin signaling, and with a promoting role in the development of different types of cancer through the activation of several pro-survival signaling pathways. In this review, we discuss the molecular aspects that support the crucial role of PTP1B in different cellular processes underlying diabetes, obesity and cancer progression, and its visualization as a promising therapeutic target.

Evaluation of Acuros® XB accuracy for static small fields dose calculations based on the IAEA/AAPM TRS-483 recommendation.

PURPOSE: Evaluate Acuros® XB dose calculation accuracy following TRS-483 recommendations in small static fields for flattened and un-flattened 6 MV X-ray beams. METHODS: Field output factors were measured following TRS-483 recommendations using four radiation detectors. Two sets of field output factors were measured. One set was used to configure the beam model into Acuros® XB down to a jaw-defined field size of 1.0 cm × 1.0 cm. The second set was used to evaluate the differences between calculated and measured field output factors for MLC-fields down to a field size of 0.5 cm × 0.5 cm. RESULTS: Acuros® XB showed an accuracy within 1.5% down to an MLC-field of 1.0 cm × 1.0 cm, for a focal spot size of 1.0 and 0.0 mm in the cross and in-plane directions. For an MLC-field of 0.5 cm × 0.5 cm, an agreement was found within 3% between calculated and measured field output factors. These results were addressed by optimizing the focal spot size to minimize the differences between calculated and measured dose profiles. CONCLUSIONS: By optimizing the focal spot size, Acuros® XB showed an acceptable agreement within 3% down to an MLC-field of 0.5 cm × 0.5 cm. The results of this work suggest that if static and modulated delivery of very small targets is planned, then a field output factor table down to a field size of 1.0 cm is required in the beam configuration model.

Deciphering the Long Non-Coding RNAs and MicroRNAs Coregulation Networks in Ovarian Cancer Development: An Overview.

Non-coding RNAs are emergent elements from the genome, which do not encode for proteins but have relevant cellular functions impacting almost all the physiological processes occurring in eukaryotic cells. In particular, microRNAs and long non-coding RNAs (lncRNAs) are a new class of small RNAs transcribed from the genome, which modulate the expression of specific genes at transcriptional and posttranscriptional levels, thus adding a new regulatory layer in the flux of genetic information. In cancer cells, the miRNAs and lncRNAs interactions with its target genes and functional pathways are deregulated as a consequence of epigenetic and genetic alterations occurring during tumorigenesis. In this review, we summarize the actual knowledge on the interplay of lncRNAs with its cognate miRNAs and mRNAs pairs, which interact in coregulatory networks with a particular emphasis on the mechanisms underlying its oncogenic behavior in ovarian cancer. Specifically, we reviewed here the evidences unraveling the relevant roles of lncRNAs/miRNAs pairs in altered regulation of cell migration, angiogenesis, therapy resistance, and Warburg effect. Finally, we also discussed its potential clinical implications in ovarian cancer and related endocrine disease therapies.

20 years of Portuguese drug policy - developments, challenges and the quest for human rights.

Portugal decriminalized the public and private use, acquisition, and possession of all drugs in 2000; adopting an approach focused on public health rather than public-order priorities. Arguing that the Portuguese Drug Policy Model has not proven influential enough to emancipate drug use from the stigma that associates it either with crime or pathology, this article critically discusses the developments and current challenges the Portuguese drug policy confronts, namely the growing diversity of drug use patterns observed in Portugal as well as in Europe. To this end, international and national legal instruments concerning drugs and official local data were analysed. Despite encouraging results, conclusions indicate that these policies are marked by contradictions and ambiguities that have permeated its history since the very beginning, and modest ambitions, particularly regarding the implementation of harm reduction measures. Moreover, the polemical Supreme Court judgment that reestablished, in 2008, drug use as a crime when the quantities at play exceeded those required for an average individual's use for 10 days, might have impacted the landscape of drug use penalization. The last decade saw an increase of punitiveness targeted at drug users, including criminal sentences of jail terms. We finish with some suggestions that could be employed in the practical application of drug policy.

Supporting Future Cannabis Policy - Developing a Standard Joint Unit: A Brief Back-Casting Exercise.

The standardization of cannabis doses is a priority for research, policy-making, clinical and harm-reduction interventions and consumer security. Scientists have called for standard units of dosing for cannabis, similar to those used for alcohol. A Standard Joint Unit (SJU) would facilitate preventive and intervention models in ways similar to the Standard Drink (SD). Learning from the SD experiences allows researchers to tackle emerging barriers to the SJU by applying modern forecasting methods. During a workshop at the Lisbon Addictions Conference 2019, a back-casting foresight method was used to address challenges and achieve consensus in developing an SJU. Thirty-two professionals from 13 countries and 10 disciplines participated. Descriptive analysis of the workshop was carried out by the organizers and shared with the participants in order to suggest amendments. Several characteristics of the SJU were defined: (1) core values: easy-to use, universal, focused on THC, accurate, and accessible; (2) key challenges: sudden changes in patterns of use, heterogeneity of cannabis compounds as well as in administration routes, variations over time in THC concentrations, and of laws that regulate the legal status of recreational and medical cannabis use); and (3) facilitators: previous experience with standardized measurements, funding opportunities, multi-stakeholder support, high prevalence of cannabis users, and widespread changes in legislation. Participants also identified three initial steps for the implementation of a SJU by 2030: (1) Building a task-force to develop a consensus-based SJU; (2) Expanded available national-level data; (3) Linking SJU consumption to the concept of "risky use," based on evidence of harms.

LPA1 Receptor Promotes Progesterone Receptor Phosphorylation through PKCα in Human Glioblastoma Cells.

Lysophosphatidic acid (LPA) induces a wide range of cellular processes and its signaling is increased in several cancers including glioblastoma (GBM), a high-grade astrocytoma, which is the most common malignant brain tumor. LPA1 receptor is expressed in GBM cells and its signaling pathways activate protein kinases C (PKCs). A downstream target of PKC, involved in GBM progression, is the intracellular progesterone receptor (PR), which can be phosphorylated by this enzyme, increasing its transcriptional activity. Interestingly, in GBM cells, PKCα isotype translocates to the nucleus after LPA stimulation, resulting in an increase in PR phosphorylation. In this study, we determined that LPA1 receptor activation induces protein-protein interaction between PKCα and PR in human GBM cells; this interaction increased PR phosphorylation in serine400. Moreover, LPA treatment augmented VEGF transcription, a known PR target. This effect was blocked by the PR selective modulator RU486; also, the activation of LPA1/PR signaling promoted migration of GBM cells. Interestingly, using TCGA data base, we found that mRNA expression of LPAR1 increases according to tumor malignancy and correlates with a lower survival in grade III astrocytomas. These results suggest that LPA1/PR pathway regulates GBM progression.

Validation of a second-generation appropriateness classification system for total knee arthroplasty: a prospective cohort study.

BACKGROUND: To test the validity of a second-generation appropriateness system in a cohort of patients undergoing total knee arthroplasty (TKA). METHODS: We applied the RAND/UCLA Appropriateness Method to derive our second-generation system and conducted a prospective study of patients diagnosed with knee osteoarthritis in eight public hospitals in Spain. Main outcome questionnaires were the Western Ontario and McMaster Universities Osteoarthritis Index (WOMAC), Short-Form-12 (SF-12), and the Knee Society Score satisfaction scale (KSS), completed before and 6 months after TKA. Baseline, changes from baseline to 6 months (journey outcome), and 6-month scores (destination outcome) were compared according to appropriateness category. Percentage of patients attaining the minimal clinically important difference (MCID) and responders according to Outcome Measures in Rheumatology-Osteoarthritis Research Society (OMERACT-OARSI) criteria were also reported. RESULTS: A total of 282 patients completed baseline and 6-month questionnaires. Of these, 142 (50.4%) were classified as Appropriate, 90 (31.9%) as Uncertain, and 50 (17.7%) as Inappropriate. Patients classified as Appropriate had worse preoperative pain, function, and satisfaction (p < 0.001) and had greater improvements (i.e., journey scores) than those classified as Inappropriate (p < 0.001). At 6 months, destination scores for pain, function, or satisfaction were not significantly different across appropriateness categories. The percentage of patients meeting responder criteria (p < 0.001) and attaining MCID was statistically higher in Appropriate versus Inappropriate groups in pain (p = 0.04) and function (p = 0.004). CONCLUSIONS: The validity of our second-generation appropriateness system was generally supported. The findings highlight a critical issue in TKA healthcare: whether TKA appropriateness should be driven by the extent of improvement, by patient final state, or by both.

EsxA mainly contributes to the miR-155 overexpression in human monocyte-derived macrophages and potentially affect the immune mechanism of macrophages through miRNA dysregulation.

BACKGROUND/PURPOSE: Mycobacterium tuberculosis is a successful intracellular pathogen that uses multiple proteins to survive within macrophages, one of the most remarkable is the virulence factor EsxA. In this study, we evaluate the participation of EsxA in the miRNAs expression profile of human monocyte-derived macrophages (hMDM), to mapping out the contribution of this virulence factor in the miRNA profile and how these changes can influence and alter immune-related processes and pathways. METHODS: The cytotoxic effect of rEsxA on hMDM was evaluated by the neutral red assay. The evaluation of miRNA expression profile in infected and rEsxA-stimulated hMDM was done using TaqMan Low Density Assays, and in silico analyses was carried on to construct Protein-Protein Interaction network of miRNAs targets. RESULTS: miR-155 was the only miRNA upregulated consistently in hMDM infected with M. tuberculosis H37Rv or stimulated with rEsxA. In hMDM stimulated with rEsxA, we found 25 miRNA's dysregulated (8 up-regulated and 17 down-regulated). The most significant were the miR-155 and miR-622 that has been observed in the analysis carried out with two different endogenous controls (U6 snRNA and RNU44) for the normalization of expression analysis. This result suggests that rEsxA induces the deregulation of miRNAs that potentially target genes in key pathways for the infection control, like the MAPK signaling pathway, cytokines, and chemokine signaling pathways, and several connected pathways involved in mycobacterial uptake, vesicular traffic, and endosome maturation. CONCLUSION: Higher expression levels of miR-155 suggest potential roles of these miRNA in EsxA-dependent immune subversion.

p21-Activated Kinase 1 Promotes Breast Tumorigenesis via Phosphorylation and Activation of the Calcium/Calmodulin-Dependent Protein Kinase II.

p21-Activated kinase-1 (Pak1) is frequently overexpressed and/or amplified in human breast cancer and is necessary for transformation of mammary epithelial cells. Here, we show that Pak1 interacts with and phosphorylates the Calcium/Calmodulin-dependent Protein Kinase II (CaMKII), and that pharmacological inhibition or depletion of Pak1 leads to diminished activity of CaMKII. We found a strong correlation between Pak1 and CaMKII expression in human breast cancer samples, and combined inhibition of Pak1 and CaMKII with small-molecule inhibitors was synergistic and induced apoptosis more potently in Her2 positive and triple negative breast cancer (TNBC) cells. Co-adminstration of Pak and CaMKII small-molecule inhibitors resulted in a dramatic reduction of proliferation and an increase in apoptosis in a 3D cell culture setting, as well as an impairment in migration and invasion of TNBC cells. Finally, mice bearing xenografts of TNBC cells showed a significant delay in tumor growth when treated with small-molecule inhibitors of Pak and CaMKII. These data delineate a signaling pathway from Pak1 to CaMKII that is required for efficient proliferation, migration and invasion of mammary epithelial cells, and suggest new therapeutic strategies in breast cancer.