RESUMO
Since late 2019, the world has been devastated by the coronavirus disease 2019 (COVID-19) induced by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), with more than 760 million people affected and â¼seven million deaths reported. Although effective treatments for COVID-19 are currently limited, there has been a strong focus on developing new therapeutic approaches to address the morbidity and mortality linked to this disease. An approach that is currently being investigated is the use of exosome-based therapies. Exosomes are small, extracellular vesicles that play a role in many clinical diseases, including viral infections, infected cells release exosomes that can transmit viral components, such as miRNAs and proteins, and can also include receptors for viruses that facilitate viral entry into recipient cells. SARS-CoV-2 has the ability to impact the formation, secretion, and release of exosomes, thereby potentially facilitating or intensifying the transmission of the virus among cells, tissues and individuals. Therefore, designing synthetic exosomes that carry immunomodulatory cargo and antiviral compounds are proposed to be a promising strategy for the treatment of COVID-19 and other viral diseases. Moreover, exosomes generated from mesenchymal stem cells (MSC) might be employed as cell-free therapeutic agents, as MSC-derived exosomes can diminish the cytokine storm and reverse the suppression of host anti-viral defences associated with COVID-19, and boost the repair of lung damage linked to mitochondrial activity. The present article discusses the significance and roles of exosomes in COVID-19, and explores potential future applications of exosomes in combating this disease. Despite the challenges posed by COVID-19, exosome-based therapies could represent a promising avenue for improving patient outcomes and reducing the impact of this disease.
Assuntos
COVID-19 , Exossomos , SARS-CoV-2 , Exossomos/metabolismo , Humanos , COVID-19/terapia , COVID-19/virologia , SARS-CoV-2/fisiologia , Tratamento Farmacológico da COVID-19 , Células-Tronco Mesenquimais/virologia , Células-Tronco Mesenquimais/metabolismo , Antivirais/uso terapêutico , Antivirais/farmacologia , AnimaisRESUMO
PROPOSE: Human epidermal growth factor receptor 2 (HER2) is overexpressed on the surface of some kinds of cancer cells including breast cancer. In this study, we designed and produced a novel immunotoxin consisting anti-HER2 single-chain Fv (scFv) from pertuzumab and a modified form of Pseudomonas exotoxin (PE35KDEL). METHODS: The three-dimensional (3D) structure of the fusion protein (anti-HER IT) was predicted by MODELLER 9.23 and its interaction with HER2 receptor was assessed using HADDOCK web server. Anti-HER2 IT, anti-HER2 scFv, and PE35KDEL proteins were expressed by Escherichia coli BL21 (DE3). After purification of the proteins using Ni2+ affinity chromatography and refolding through dialysis, the cytotoxicity of proteins against breast cancer cell lines was examined by MTT assay. RESULTS: In-silico studies showed that (EAAAK)2 linker can efficiently prevent the formation of salt bridges between two functional domains and the constructed fusion protein has a high affinity to HER2 receptor. The optimum condition of anti-HER2 IT expression was 25 °C and 1 mM IPTG. The protein was successfully purified and refolded by dialysis with a final yield of 45.7 mg per 1 L of bacterial culture. The cytotoxicity results showed that anti-HER2 IT was much more toxic on HER2-overexpressing cells, BT-474 (IC50 ~ 95 nM) compared with HER2-negative cells, MDA-MB-23 (IC50 Ë 200 nM). CONCLUSION: This novel immunotoxin has the potential to be applied as a therapeutic candidate for HER2-targeted cancer therapy. However further in vitro and in vivo evaluations are still required to confirm the efficacy and safety of this protein.
Assuntos
Neoplasias da Mama , Imunotoxinas , Anticorpos de Cadeia Única , Humanos , Feminino , Anticorpos de Cadeia Única/genética , Anticorpos de Cadeia Única/química , Imunotoxinas/genética , Imunotoxinas/farmacologia , Receptor ErbB-2/genética , Neoplasias da Mama/metabolismo , Linhagem Celular Tumoral , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/farmacologia , Proteínas Recombinantes de Fusão/uso terapêuticoRESUMO
Background: The initiator of cytokine storm in Coronavirus disease (COVID-19) is still unknown. We recently suggested a complex interaction of matrix metalloproteinases (MMPs), Fas ligand (FasL), and viral entry factors could be responsible for the cytokine outrage In COVID-19. We explored the molecular dynamics of FasL/MMP7-9 in COVID-19 conditions in silico and provide neuroimmune insights for future. Methods: We enrolled and analyzed a clinical cohort of COVID-19 patients, and recorded their blood Na + levels and temperature at admission. A blood-like molecular dynamics simulation (MDS) box was then built. Four conditions were studied; MMP7/FasL (healthy), MMP7/FasL (COVID-19), MMP9-FasL (healthy), and MMP9/FasL (COVID-19). MDS was performed by GROningen MAchine for Chemical Simulation (GROMACS). We analyzed bonds, short-range energies, and free binding energies to draw conclusions on the interaction of MMP7/MMP9 and FasL to gain insights into COVID-19 immunopathology. Genevestigator was used study RNA-seq/microarray expression data of MMPs in the cells of immune and nervous systems. Finally, epitopes of MMP/FasL complexes were identified as drug targets by machine learning (ML) tools. Results: MMP7-FasL (Healthy), MMP7-FasL (COVID-19), MMP9-FasL (Healthy), and MMP9-FasL (COVID-19) systems showed 0, 1, 4, and 2 salt bridges, indicating MMP9 had more salt bridges. Moreover, in both COVID-19 and normal conditions, the number of interacting residues and surface area was higher for MMP9 compared to MMP7 group. The COVID-19 MMP9-FasL group had more H-bonds compared to MMP7-FasL group (12 vs. 7). 15 epitopes for FasL-MMP9 and 10 epitopes for FasL-MMP7 were detected. Extended MD simulation for 100 ns confirmed stronger binding of MMP9 based on Molecular Mechanics Generalized Borne Surface analysis (MM-GBSA) and Coul and Leonard-Jones (LJ) short-range energies. Conclusions: MMP9 interacts stronger than MMP7 with FasL, however, both molecules maintained strong interaction through the MDS. We suggested epitopes for MMP-FasL complexes as valuable therapeutic targets in COVID-19. These data could be utilized in future immune drug and protein design and repurposing efforts.
RESUMO
Background: The arterial stiffness measured by pulsed wave velocity (PWV) is associated with heart failure (HF). However, the effectiveness of arterial stiffness and PWV as prognostic indicators in patients with HFpEF and HFrEF is still unclear. In this systematic review and meta-analysis, we synthesized the prognostic value of PWV and arterial stiffness in HF patients. Methods: Four databases, including Embase, PubMed, Scopus, and Web of Science, were systematically searched for published studies assessing the relationship between PWV and HF from inception up to August 31, 2023. The Newcastle-Ottawa Scale (NOS) was used to assess the quality of the included studies. The standardized mean difference (SMD) and their corresponding 95% confidence intervals (CI) were used to compare PWV in HF (HFrEF and HFpEF) and controls. Meta-regressions based on age, year of publication, sample size, and gender (male percentage) were also conducted. Results: The systematic search yielded 5,977 results, of which 58 met our inclusion criteria and 24 were analyzed quantitatively. Studies included 64,687 patients with a mean age of 53.7 years, and 41,803 (67.3%) were male. Meta-analysis of 19 studies showed that PWV was significantly higher in HF patients compared to the controls (SMD 1.04, 95% CI 0.43-1.66, P < 0.001, I 2 = 93%). Moreover, nine studies have measured PWV among HFrEF and HFpEF patients and found no significant difference (SMD -0.51, 95% CI -1.03 to 0.02, P = 0.057, I2 = 95%). Moreover, increased PWV was linked to an increased chance of developing new-onset HF in individuals with cardiovascular risk factors. Conclusions: Patients with HF exhibit significantly higher arterial stiffness, as indicated by PWV, compared to the normal population. However, this association was not significant between HFrEF and HFpEF patients. Future research is warranted to establish the potential prognostic role of PWV in HF. Systematic Review Registration: https://www.crd.york.ac.uk/prospero/display_record.php?ID=CRD42023479683, PROSPERO (CRD42023479683).
RESUMO
BACKGROUND: An essential relationship between insulin resistance (IR) and atrial fibrillation (AF) has been demonstrated. Among the methods used to assess IR, the triglyceride-glucose (TyG) index is the more straightforward, dimensionless, and low-cost tool. However, the possible usage of this index in clinical practice to predict and diagnose AF has yet to be determined and consolidated. OBJECTIVE AND RATIONALE: Herein, we performed a systematic review and meta-analysis to assess the association between the TyG index and AF. METHODS: Databases (PubMed, Embase, Scopus, and Web of Science) were systematically searched for studies evaluating the TyG index in AF. The inclusion criteria were observational studies investigating AF and TyG index correlation in individuals older than 18 years, while preclinical studies and those without the relevant data were excluded. Random effect meta-analyses comparing TyG levels between AF and non-AF cases, AF recurrence after radiofrequency ablation, and post-procedural AF were performed using standardized mean differences (SMD) with their matching 95% confidence intervals (CIs). RESULTS: Our screening identified nine studies to be analyzed, including 6,171 participants including 886 with AF. The meta-analysis demonstrated that the TyG index resulted higher in patients with AF than non-AF counterparts (SMD 1.23, 95% CI 0.71 to 1.75, I2 98%, P < 0.001). Subgroup analysis showed the same results for post-procedure AF (SMD 0.99, 95% CI 0.78 to 1.20, I2 10%, P < 0.001) and post-ablation AF (SMD 1.25, 95% CI 1.07 to 1.43, I2 46%, P < 0.001), while no difference was found in population-based cohorts (SMD 1.45, 95% CI - 0.41 to 3.31, I2 100%, P = 0.13). Publication year (P = 0.036) and sample size (P = 0.003) showed significant associations with the effect size, using multivariable meta-regression. CONCLUSION: The TyG index is an easy-to-measure surrogate marker of IR in patients with AF. Further clinical studies are warranted to demonstrate its ability for routine clinical use and as a screening tool.
Assuntos
Fibrilação Atrial , Ablação por Cateter , Resistência à Insulina , Humanos , Fibrilação Atrial/diagnóstico , Glucose , Triglicerídeos , Biomarcadores , Ablação por Cateter/métodosRESUMO
Tinnitus is a conscious attended awareness perception of sourceless sound. Widespread theoretical and evidence-based neurofunctional and psychological models have tried to explain tinnitus-related distress considering the influence of psychological and cognitive factors. However, tinnitus models seem to be less focused on causality, thereby easily misleading interpretations. Also, they may be incapable of individualization. This study proposes a Conceptual Cognitive Framework (CCF) providing insight into cognitive mechanisms involved in the predisposition, precipitation, and perpetuation of tinnitus and consequent cognitive-emotional disturbances. The current CCF for tinnitus relies on evaluative conditional learning and appraisal, generating negative valence (emotional value) and arousal (cognitive value) to annoyance, distress, and distorted perception. The suggested methodology is well-defined, reproducible, and accessible, which can help foster future high-quality clinical databases. Perceived tinnitus through the perpetual-learning process can always lead to annoyance, but only in the clinical stage directly cause annoyance. In the clinical stage, tinnitus perception can lead indirectly to distress only with experiencing annoyance either with (" I n d - 1 C " = 1.87; 95% CI 1.18-2.72)["1st indirect path in the Clinical stage model": Tinnitus Loudness â Attention Bias â Cognitive-Emotional Value â Annoyance â Clinical Distress]or without (" I n d - 2 C "= 2.03; 95% CI 1.02-3.32)[ "2nd indirect path in the Clinical stage model": Tinnitus Loudness â Annoyance â Clinical Distress] the perpetual-learning process. Further real-life testing of the CCF is expected to express a meticulous, decision-supporting platform for cognitive rehabilitation and clinical interventions. Furthermore, the suggested methodology offers a reliable platform for CCF development in other cognitive impairments and supports the causal clinical data models. It may also enhance our knowledge of psychological disorders and complicated comorbidities by supporting the design of different rehabilitation interventions and comprehensive frameworks in line with the "preventive medicine" policy.
Assuntos
Zumbido , Humanos , Emoções , Cognição , Sintomas Afetivos , Nível de AlertaRESUMO
BACKGROUND: Unlike diabetes, the effect of prediabetes on outcomes in patients with acute coronary syndrome (ACS) who underwent percutaneous coronary intervention (PCI) is not much investigated. We investigated the association between fasting glycemic status and major adverse cardiovascular and cerebrovascular events (MACCE) in patients with ACS undergoing PCI and had mid to long-term follow-up after coronary stenting. METHODS: Registry-based retrospective cohort study included ACS patients who underwent PCI at the Tehran Heart Center from 2015 to 2021 with a median follow-up of 378 days. Patients were allocated into normoglycemic, prediabetic, and diabetic groups. The primary and secondary outcomes were MACCE and its components, respectively. Unadjusted and adjusted Cox models were used to evaluate the association between glycemic status and outcomes. RESULTS: Among 13 682 patients, 3151 (23%) were prediabetic, and 5834 (42.6%) were diabetic. MACCE risk was significantly higher for diabetic versus normoglycemic (adjusted hazard ratio [aHR]: 1.22, 95% confidence interval [CI]: 1.06-1.41), but nonsignificantly higher for prediabetic versus normoglycemic (aHR: 0.95, 95% CI: 0.78-1.10). All-cause mortality risk was significantly higher in diabetic versus normoglycemic (aHR: 1.42, 95% CI: 1.08-1.86), but nonsignificantly higher for prediabetic versus normoglycemic (aHR: 1.15, 95% CI: 0.84-1.59). Among other components of MACCE, only coronary artery bypass grafting was significantly higher in diabetic patients, and not prediabetic, compared with normoglycemic. CONCLUSIONS: Prediabetic ACS patients undergoing PCI, unlike diabetics, are not at increased risk of MACCE and all-cause mortality. While prediabetic patients could be regarded as having the same risk as nondiabetics, careful consideration to provide more intensive pre- and post-PCI care in diabetic patients is mandatory.
Assuntos
Síndrome Coronariana Aguda , Doença da Artéria Coronariana , Diabetes Mellitus , Intervenção Coronária Percutânea , Estado Pré-Diabético , Humanos , Estado Pré-Diabético/complicações , Estado Pré-Diabético/diagnóstico , Estado Pré-Diabético/epidemiologia , Síndrome Coronariana Aguda/complicações , Síndrome Coronariana Aguda/diagnóstico , Síndrome Coronariana Aguda/epidemiologia , Intervenção Coronária Percutânea/efeitos adversos , Estudos Retrospectivos , Resultado do Tratamento , Irã (Geográfico)/epidemiologia , Diabetes Mellitus/epidemiologia , Fatores de RiscoRESUMO
Loss of myelination is common among neurological diseases. It causes significant disability, even death, if it is not treated instantly. Different mechanisms involve the pathophysiology of demyelinating diseases, such as genetic background, infectious, and autoimmune inflammation. Recently, regenerative medicine and stem cell therapy have shown to be promising for the treatment of demyelinating disorders. Stem cells, including embryonic stem cells (ESCs), induced pluripotent stem cells (iPSCs), and adult stem cells (ASCs), can differentiate into oligodendrocyte progenitor cells (OPCs), which may convert to oligodendrocytes (OLs) and recover myelination. IPSCs provide an endless source for OPCs generation. However, the restricted capacity of proliferation, differentiation, migration, and myelination of iPSC-derived OPCs is a notable gap for future studies. In this article, we have first reviewed stem cell therapy in demyelinating diseases. Secondly, methods of different protocols have been discussed among in vitro and in vivo studies on iPSC-derived OPCs to contrast OPCs' transplantation efficacy. Lastly, we have reviewed the results of iPSCs-derived OLs production in each demyelination model.
Assuntos
Doenças Desmielinizantes , Células-Tronco Pluripotentes Induzidas , Células Precursoras de Oligodendrócitos , Humanos , Oligodendroglia , Diferenciação Celular/genética , Doenças Desmielinizantes/terapiaRESUMO
Cancer and cardiovascular disorders are known as the two main leading causes of mortality worldwide. Cardiotoxicity is a critical and common adverse effect of cancer-related chemotherapy. Chemotherapy-induced cardiotoxicity has been associated with various cancer treatments, such as anthracyclines, immune checkpoint inhibitors, and kinase inhibitors. Different methods have been reported for the management of chemotherapy-induced cardiotoxicity. In this regard, sodium-glucose cotransporter-2 inhibitors (SGLT2i), a class of antidiabetic agents, have recently been applied to manage heart failure patients. Further, SGLT2i drugs such as EMPA exert protective cardiac and systemic effects. Moreover, it can reduce inflammation through the mediation of major inflammatory components, such as Nucleotide-binding domain-like receptor protein 3 (NLRP3) inflammasomes, Adenosine 5'-monophosphate-activated protein kinase (AMPK), and c-Jun N-terminal kinase (JNK) pathways, Signal transducer and activator of transcription (STAT), and overall decreasing transcription of proinflammatory cytokines. The clinical outcome of EMPA administration is related to improving cardiovascular risk factors, including body weight, lipid profile, blood pressure, and arterial stiffness. Intriguingly, SGLT2 suppressors can regulate microglia-driven hyperinflammation affecting neurological and cardiovascular disorders. In this review, we discuss the protective effects of EMPA in chemotherapy-induced cardiotoxicity from molecular, immunological, and neuroimmunological aspects to preclinical and clinical outcomes.
Assuntos
Antineoplásicos , Doenças Cardiovasculares , Diabetes Mellitus Tipo 2 , Inibidores do Transportador 2 de Sódio-Glicose , Humanos , Inibidores do Transportador 2 de Sódio-Glicose/uso terapêutico , Inibidores do Transportador 2 de Sódio-Glicose/farmacologia , Diabetes Mellitus Tipo 2/tratamento farmacológico , Cardiotoxicidade/tratamento farmacológico , Compostos Benzidrílicos/efeitos adversos , Doenças Cardiovasculares/tratamento farmacológico , Antineoplásicos/uso terapêuticoRESUMO
Our perception of physical distance to individuals and stimuli is influenced by our mental distance and relatedness. The present study aimed to investigate the role of the dorsolateral prefrontal cortex (dlPFC), ventromedial prefrontal cortex (vmPFC), and right temporoparietal junction (rTPJ) in interpersonal comfortable distance and approach behaviors towards emotional stimuli. Twenty healthy volunteers received brain stimulation in four separate sessions with a one-week interval, including anodal left dlPFC, anodal right vmPFC, anodal rTPJ, and sham condition, with an extracranial return electrode. Our results revealed an increase in interpersonal distance during anodal rTPJ stimulation and a decrease in distance to positive pictures during anodal vmPFC stimulation. These findings suggest that the rTPJ plays a role in the perceptual component of self-other distancing, while the vmPFC is involved in approaching positive emotions.
Assuntos
Estimulação Transcraniana por Corrente Contínua , Humanos , Estimulação Transcraniana por Corrente Contínua/métodos , Emoções/fisiologia , Córtex Pré-Frontal/fisiologiaRESUMO
Around 30% of the general population experience subjective tinnitus, characterized by conscious attended awareness perception of sound without an external source. Clinical distress tinnitus is more than just experiencing a phantom sound, as it can be highly disruptive and debilitating, leading those affected to seek clinical help. Effective tinnitus treatments are crucial for psychological well-being, but our limited understanding of the underlying neural mechanisms and a lack of a universal cure necessitate further treatment development. In light of the neurofunctional tinnitus model predictions and transcranial electrical stimulation, we conducted an open-label, single-arm, pilot study that utilized high-definition transcranial direct current stimulation (HD-tDCS) concurrent with positive emotion induction (PEI) techniques for ten consecutive sessions to down-regulate tinnitus negative valence in patients with clinical distress tinnitus. We acquired resting-state functional magnetic resonance imaging scans of 12 tinnitus patients (7 females, mean age = 51.25 ± 12.90 years) before and after the intervention to examine resting-state functional connectivity (rsFC) alterations in specific seed regions. The results showed reduced rsFC at post-intervention between the attention and emotion processing regions as follows: (1) bilateral amygdala and left superior parietal lobule (SPL), (2) left amygdala and right SPL, (3) bilateral dorsolateral prefrontal cortex (dlPFC) and bilateral pregenual anterior cingulate cortex (pgACC), and (4) left dlPFC and bilateral pgACC (FWE corrected p < 0.05). Furthermore, the post-intervention tinnitus handicap inventory scores were significantly lower than the pre-intervention scores (p < 0.05). We concluded that concurrent HD-tDCS and PEI might be effective in reducing tinnitus negative valence, thus alleviating tinnitus distress.
RESUMO
The use of transcranial Electrical Stimulation (tES) in the modulation of cognitive brain functions to improve neuropsychiatric conditions has extensively increased over the decades. tES techniques have also raised new challenges associated with study design, stimulation protocol, functional specificity, and dose-response relationship. In this paper, we addressed challenges through the emerging methodology to investigate the dose-response relationship of High Definition-transcranial Direct Current Stimulation (HD tDCS), identifying the role of negative valence in tinnitus perception. In light of the neurofunctional testable framework and tES application, hypotheses were formulated to measure clinical and surrogate endpoints. We posited that conscious pairing adequately pleasant stimuli with tinnitus perception results in correction of the loudness misperception and would be reinforced by concurrent active HD-tDCS on the left Dorsolateral Prefrontal Cortex (dlPFC). The dose-response relationship between HD-tDCS specificity and the loudness perception is also modeled. We conducted a double-blind, randomized crossover pilot study with six recruited tinnitus patients. Accrued data was utilized to design a well-controlled adaptive seamless Bayesian dose-response study. The sample size (n = 47, for 90% power and 95% confidence) and optimum interims were anticipated for adaptive decision-making about efficacy, safety, and single session dose parameters. Furthermore, preliminary pilot study results were sufficient to show a significant difference (90% power, 99% confidence) within the longitudinally detected self-report tinnitus loudness between before and under positive emotion induction. This study demonstrated a research methodology used to improve emotion regulation in tinnitus patients. In the projected method, positive emotion induction is essential for promoting functional targeting under HD-tDCS anatomical specificity to indicate the efficacy and facilitate the dose-finding process. The continuous updating of prior knowledge about efficacy and dose during the exploratory stage adapts the anticipated dose-response model. Consequently, the effective dose range to make superiority neuromodulation in correcting loudness misperception of tinnitus will be redefined. Highly effective dose adapts the study to a standard randomized trial and transforms it into the confirmatory stage in which active HD-tDCS protocol is compared with a sham trial (placebo-like). Establishing the HD-tDCS intervention protocols relying on this novel method provides reliable evidence for regulatory agencies to approve or reject the efficacy and safety. Furthermore, this paper supports a technical report for designing multimodality data-driven complementary investigations in emotion regulation, including EEG-driven neuro markers, Stroop-driven attention biases, and neuroimaging-driven brain network dynamics.
RESUMO
Although severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection has caused many complications, the invention of coronavirus disease 2019 (COVID-19) vaccines has also brought about several adverse events, from common side effects to unexpected and rare ones. Common vaccine-related adverse reactions manifest locally or systematically following any vaccine, including COVID-19 vaccines. Specific side effects, known as adverse events of particular interest (AESI), are unusual and need more evaluation. Here, we discuss some of the most critical rare adverse events of COVID-19 vaccines.
RESUMO
Insomnia is a widespread neuropsychological sleep-related disorder known to result in various predicaments including cognitive impairments, emotional distress, negative thoughts, and perceived sleep insufficiency besides affecting the incidence and aggravation of other medical disorders. Despite the available insomnia-related theoretical cognitive models, clinical studies, and related guidelines, an evidence-based conceptual framework for a personalized approach to insomnia seems to be lacking. This study proposes a conceptual cognitive framework (CCF) providing insight into cognitive mechanisms involved in the predisposition, precipitation, and perpetuation of insomnia and consequent cognitive deficits. The current CCF for insomnia relies on evaluative conditional learning and appraisal which generates negative valence (emotional value) and arousal (cognitive value). Even with the limitations of this study, the suggested methodology is well-defined, reproducible, and accessible can help foster future high-quality clinical databases. During clinical insomnia but not the neutral one, negative mood (trait-anxiety) causes cognitive impairments only if mediating with a distorted perception of insomnia ( Ind-1 = 0.161, 95% CI 0.040-0.311). Further real-life testing of the CCF is intended to formulate a meticulous, decision-supporting platform for clinical interventions. Furthermore, the suggested methodology is expected to offer a reliable platform for CCF-development in other cognitive impairments and support the causal clinical data models. It may also improve our knowledge of psychological disturbances and complex comorbidities to help design rehabilitation interventions and comprehensive frameworks in line with the "preventive medicine" policies.