Pan-inhibition of the three H2S synthesizing enzymes restrains tumor progression and immunosuppression in breast cancer.

BACKGROUND: Hydrogen sulfide (H2S) is a significant endogenous mediator that has been implicated in the progression of various forms of cancer including breast cancer (BC). Cystathionine-ß-synthase (CBS), cystathionine-γ-lyase (CSE), and 3-mercaptopyruvate sulfurtransferase (3MST) are the three principal mammalian enzymes responsible for H2S production. Overexpression of CBS, CSE and 3MST was found to be associated with poor prognosis of BC patients. Moreover, H2S was linked to an immune-suppressive tumor microenvironment in BC. Recently it was observed that BC cells, in response to single or dual inhibition of H2S synthesizing enzymes, develop an escape mechanism by overexpressing alternative sources of H2S generation. Thus, the aim of this work is to escape the H2S compensatory mechanism by pan repressing the three enzymes using microRNAs (miRNAs) and to investigate their impact on the oncogenic and immunogenic profile of BC cells. METHODS: BC female patients (n = 25) were recruited. In-silico analysis was used to identify miRNAs targeting CBS, CSE, and 3MST. MDA-MB-231 cells were cultured and transfected using oligonucleotides. Total RNA was extracted using Biazol, reverse transcribed and quantified using qRT-PCR. H2S levels were measured using AzMc assay. BC hallmarks were assessed using trans-well migration, wound healing, MTT, and colony forming assays. RESULTS: miR-193a and miR-548c were validated by eight different bioinformatics software to simultaneously target CBS, CSE and 3MST. MiR-193a and miR-548c were significantly downregulated in BC tissues compared to their non-cancerous counterparts. Ectopic expression of miR-193a and miR-548c in MDA-MB-231 TNBC cells resulted in a marked repression of CBS, CSE, and 3MST transcript and protein levels, a significant decrease in H2S levels, reduction in cellular viability, inhibition of migration and colony forming ability, repression of immune-suppressor proteins GAL3 GAL9, and CD155 and upregulation of the immunostimulatory MICA and MICB proteins. CONCLUSION: This study sheds the light onto miR-193a and miR-548c as potential pan-repressors of the H2S synthesizing enzymes. and identifies them as novel tumor suppressor and immunomodulatory miRNAs in TNBC.

Role of Hydrogen Sulfide in Oncological and Non-Oncological Disorders and Its Regulation by Non-Coding RNAs: A Comprehensive Review.

Recently, myriad studies have defined the versatile abilities of gasotransmitters and their synthesizing enzymes to play a "Maestro" role in orchestrating several oncological and non-oncological circuits and, thus, nominated them as possible therapeutic targets. Although a significant amount of work has been conducted on the role of nitric oxide (NO) and carbon monoxide (CO) and their inter-relationship in the field of oncology, research about hydrogen sulfide (H2S) remains in its infancy. Recently, non-coding RNAs (ncRNAs) have been reported to play a dominating role in the regulation of the endogenous machinery system of H2S in several pathological contexts. A growing list of microRNAs (miRNAs) and long non-coding RNAs (lncRNAs) are leading the way as upstream regulators for H2S biosynthesis in different mammalian cells during the development and progression of human diseases; therefore, their targeting can be of great therapeutic benefit. In the current review, the authors shed the light onto the biosynthetic pathways of H2S and their regulation by miRNAs and lncRNAs in various oncological and non-oncological disorders.

Dual targeting of H2S synthesizing enzymes; cystathionine ß-synthase and cystathionine γ-lyase by miR-939-5p effectively curbs triple negative breast cancer.

Introduction: Hydrogen sulfide (H2S) has been recently scrutinized for its critical role in aggravating breast cancer (BC) tumorigenicity. Several cancers aberrantly express H2S synthesizing enzymes; Cystathionine-ß-synthase (CBS) and cystathionine-γ-lyase (CSE). However, their levels and interdependence in BC require further studies. Objectives: Firstly, this study aimed to demonstrate a comparative expression profile of H2S synthesizing enzymes in BC vs normal tissue. Moreover, to investigate the reciprocal relationship between CBS and CSE and highlight the importance of dual targeting. Finally, to search for a valid dual repressor of the H2S synthesizing enzymes that could cease H2S production and reduce TNBC pathogenicity. Methods: Pairwise analysis of tumor vs. normal tissues of 40 BC patients was carried out. The TNBC cell line MDA-MB-231 was transfected with oligonucleotides to study the H2S mediated molecular mechanisms. In silico screening was performed to identify dual regulator(s) for CBS and CSE. Gene expression analysis was performed using qRT-PCR and was confirmed on protein level using Western blot. TNBC hallmarks were evaluated using MTT, migration, and clonogenicity assays. H2S levels were detected using a AzMc fluorescent probe. Results: BC tissues exhibited elevated levels of both CBS and CSE. Interestingly, upon CBS knockdown, CSE levels increased compensating for H2S production in TNBC cells, underlining the importance of dually targeting both enzymes in TNBC. In silico screening suggested miR-939-5p as a regulator of both CBS and CSE with high binding scores. Low expression levels of miR-939-5p were found in BC tissues, especially the aggressive subtypes. Ectopic expression of miR-939-5p significantly repressed CBS and CSE transcript and protein levels, diminished H2S production and attenuated TNBC hallmarks. Moreover, it improved the immune surveillance potency of TNBC cells through up regulating the NKG2D ligands, MICB and ULBP2 and reducing the immune suppressive cytokine IL-10. Conclusion: This study sheds light on the reciprocal relationship between CBS and CSE and on the importance of their dual targeting, particularly in TNBC. It also postulates miR-939-5p as a potent dual repressor for CBS and CSE overcoming their redundancy in H2S production, a mechanism that can potentially attenuate TNBC oncogenicity and improves the immunogenic response.

Isolation, screening and characterization of phage.

Bacterial resistance threatens public health due to a lack of novel antibacterial classes since the 21st century. Bacteriophages, the most ubiquitous microorganism on Earth and natural predators of bacteria, have the potential to save the world from the post-antibiotic era. Therefore, phage isolation and characterization are in high demand to find suitable phages for therapeutic and bacterial control applications. The chapter presents brief guidance supported by recommendations on the isolation of phages, and initial screening of phage antimicrobial efficacy, in addition to, conducting comprehensive characterization addressing morphological, biological, genomic, and taxonomic features.

Decoding hepatocarcinogenesis from a noncoding RNAs perspective.

Being a leading lethal malignancy worldwide, the pathophysiology of hepatocellular carcinoma (HCC) has gained a lot of interest. Yet, underlying mechanistic basis of the liver tumorigenesis is poorly understood. The role of some coding genes and their respective translated proteins, then later on, some noncoding RNAs (ncRNAs) such as microRNAs have been extensively studied in context of HCC pathophysiology; however, the implication of long noncoding RNAs (lncRNAs) and circular RNAs (circRNAs) in HCC is indeed less investigated. As a subclass of the ncRNAs which has been elusive for long time ago, lncRNAs was found to be involved in plentiful cellular functions such as DNA, RNA, and proteins regulation. Hence, it is undisputed that lncRNAs dysregulation profoundly contributes to HCC via diverse etiologies. Accordingly, lncRNAs represent a hot research topic that requires prime focus in HCC. In this review, the authors discuss breakthrough discoveries involving lncRNAs and circRNAs dysregulation that have contributed to the contemporary concepts of HCC pathophysiology and how these concepts could be leveraged as potential novel diagnostic and prognostic HCC biomarkers. Further, this review article sheds light on future trends, thereby discussing the pathological roles of lncRNAs and circRNAs in HCC proliferation, migration, and epithelial-to-mesenchymal transition. Along this line of reasoning, future recommendations of how these targets could be exploited to achieve effective HCC-related drug development is highlighted.

Translational molecular imaging and drug development in Parkinson's disease.

Parkinson's disease (PD) is a progressive neurodegenerative disorder that primarily affects elderly people and constitutes a major source of disability worldwide. Notably, the neuropathological hallmarks of PD include nigrostriatal loss and the formation of intracellular inclusion bodies containing misfolded α-synuclein protein aggregates. Cardinal motor symptoms, which include tremor, rigidity and bradykinesia, can effectively be managed with dopaminergic therapy for years following symptom onset. Nonetheless, patients ultimately develop symptoms that no longer fully respond to dopaminergic treatment. Attempts to discover disease-modifying agents have increasingly been supported by translational molecular imaging concepts, targeting the most prominent pathological hallmark of PD, α-synuclein accumulation, as well as other molecular pathways that contribute to the pathophysiology of PD. Indeed, molecular imaging modalities such as positron emission tomography (PET) and single-photon emission computed tomography (SPECT) can be leveraged to study parkinsonism not only in animal models but also in living patients. For instance, mitochondrial dysfunction can be assessed with probes that target the mitochondrial complex I (MC-I), while nigrostriatal degeneration is typically evaluated with probes designed to non-invasively quantify dopaminergic nerve loss. In addition to dopaminergic imaging, serotonin transporter and N-methyl-D-aspartate (NMDA) receptor probes are increasingly used as research tools to better understand the complexity of neurotransmitter dysregulation in PD. Non-invasive quantification of neuroinflammatory processes is mainly conducted by targeting the translocator protein 18 kDa (TSPO) on activated microglia using established imaging agents. Despite the overwhelming involvement of the brain and brainstem, the pathophysiology of PD is not restricted to the central nervous system (CNS). In fact, PD also affects various peripheral organs such as the heart and gastrointestinal tract - primarily via autonomic dysfunction. As such, research into peripheral biomarkers has taken advantage of cardiac autonomic denervation in PD, allowing the differential diagnosis between PD and multiple system atrophy with probes that visualize sympathetic nerve terminals in the myocardium. Further, α-synuclein has recently gained attention as a potential peripheral biomarker in PD. This review discusses breakthrough discoveries that have led to the contemporary molecular concepts of PD pathophysiology and how they can be harnessed to develop effective imaging probes and therapeutic agents. Further, we will shed light on potential future trends, thereby focusing on potential novel diagnostic tracers and disease-modifying therapeutic interventions.

Circular RNAs: New layer of complexity evading breast cancer heterogeneity.

Advances in high-throughput sequencing techniques and bioinformatic analysis have refuted the "junk" RNA hypothesis that was claimed against non-coding RNAs (ncRNAs). Circular RNAs (circRNAs); a class of single-stranded covalently closed loop RNA molecules have recently emerged as stable epigenetic regulators. Although the exact regulatory role of circRNAs is still to be clarified, it has been proven that circRNAs could exert their functions by interacting with other ncRNAs or proteins in their own physiologically authentic environment, regulating multiple cellular signaling pathways and other classes of ncRNAs. CircRNAs have also been reported to exhibit a tissue-specific expression and have been associated with the malignant transformation process of several hematological and solid malignancies. Along this line of reasoning, this review aims to highlight the importance of circRNAs in Breast Cancer (BC), which is ranked as the most prevalent malignancy among females. Notwithstanding the substantial efforts to develop a suitable anticancer therapeutic regimen against the heterogenous BC, inter- and intra-tumoral heterogeneity have resulted in an arduous challenge for drug development research, which in turn necessitates the investigation of other markers to be therapeutically targeted. Herein, the potential of circRNAs as possible diagnostic and prognostic biomarkers have been highlighted together with their possible application as novel therapeutic targets.

Molecular Engines, Therapeutic Targets, and Challenges in Pediatric Brain Tumors: A Special Emphasis on Hydrogen Sulfide and RNA-Based Nano-Delivery.

Pediatric primary brain tumors represent a real challenge in the oncology arena. Besides the psychosocial burden, brain tumors are considered one of the most difficult-to-treat malignancies due to their sophisticated cellular and molecular pathophysiology. Notwithstanding the advances in research and the substantial efforts to develop a suitable therapy, a full understanding of the molecular pathways involved in primary brain tumors is still demanded. On the other hand, the physiological nature of the blood-brain barrier (BBB) limits the efficiency of many available treatments, including molecular therapeutic approaches. Hydrogen Sulfide (H2S), as a member of the gasotransmitters family, and its synthesizing machinery have represented promising molecular targets for plentiful cancer types. However, its role in primary brain tumors, generally, and pediatric types, particularly, is barely investigated. In this review, the authors shed the light on the novel role of hydrogen sulfide (H2S) as a prominent player in pediatric brain tumor pathophysiology and its potential as a therapeutic avenue for brain tumors. In addition, the review also focuses on the challenges and opportunities of several molecular targeting approaches and proposes promising brain-delivery strategies for the sake of achieving better therapeutic results for brain tumor patients.

Assessment of cholesterol homeostasis in the living human brain.

Alterations in brain cholesterol homeostasis have been broadly implicated in neurological disorders. Notwithstanding the complexity by which cholesterol biology is governed in the mammalian brain, excess neuronal cholesterol is primarily eliminated by metabolic clearance via cytochrome P450 46A1 (CYP46A1). No methods are currently available for visualizing cholesterol metabolism in the living human brain; therefore, a noninvasive technology that quantitatively measures the extent of brain cholesterol metabolism via CYP46A1 could broadly affect disease diagnosis and treatment options using targeted therapies. Here, we describe the development and testing of a CYP46A1-targeted positron emission tomography (PET) tracer, 18F-CHL-2205 (18F-Cholestify). Our data show that PET imaging readouts correlate with CYP46A1 protein expression and with the extent to which cholesterol is metabolized in the brain, as assessed by cross-species postmortem analyses of specimens from rodents, nonhuman primates, and humans. Proof of concept of in vivo efficacy is provided in the well-established 3xTg-AD murine model of Alzheimer's disease (AD), where we show that the probe is sensitive to differences in brain cholesterol metabolism between 3xTg-AD mice and control animals. Furthermore, our clinical observations point toward a considerably higher baseline brain cholesterol clearance via CYP46A1 in women, as compared to age-matched men. These findings illustrate the vast potential of assessing brain cholesterol metabolism using PET and establish PET as a sensitive tool for noninvasive assessment of brain cholesterol homeostasis in the clinic.

Fat-soluble vitamins: updated review of their role and orchestration in human nutrition throughout life cycle with sex differences.

Age and Gender are vital determinants for the micronutrient demands of normal indviduals. Among these micronutrients are vitamins that are required in small amounts for optimum metabolism, homeostasis, and a healthy lifestyle, acting as coenzymes in several biochemical reactions. The majority of previous studies have examined such issues that relates to a specific vitamin or life stage, with the majority merely reporting the effect of either excess or deficiency. Vitamins are classified into water-soluble and fat-soluble components. The fat-soluble vitamins include vitamins (A, D, E, and K). Fat-soluble vitamins were found to have an indisputable role in an array of physiological processes such as immune regulation, vision, bone and mental health. Nonetheless, the fat-soluble vitamins are now considered a prophylactic measurement for a multitude of diseases such as autism, rickets disease, gestational diabetes, and asthma. Herein, in this review, a deep insight into the orchestration of the four different fat-soluble vitamins requirements is presented for the first time across the human life cycle beginning from fertility, pregnancy, adulthood, and senility with an extensive assessment ofthe interactions among them and their underlying mechanistic actions. The influence of sex for each vitamin is also presented at each life stage to highlight the different daily requirements and effects.

Bacteriophages: from Isolation to Application

Bacteriophages are considered as a potential alternative to fight pathogenic bacteria during the antibiotic resistance era. With their high specificity, they are widely used in various applications: medicine, food industry, agriculture, animal farms, biotechnology, diagnosis, etc. Many techniques have been designed by different researchers for phage isolation, purification, and amplification, each of which has strengths and weaknesses. However, all aim at having a reasonably pure phage sample that can be further characterized. Phages can be characterized based on their physiological, morphological or inactivation tests. Microscopy, in particular, opened a wide gate, not only for visualizing phage morphological structure, but also for monitoring biochemistry and behavior. Meanwhile, computational analysis of phage genomes provides more details about phage history, lifestyle, and the potential for toxigenic or lysogenic conversion, which translate to safety in biocontrol and phage therapy applications. This review article summarizes phage application pipelines at different levels, and addresses specific restrictions and knowledge gaps in the field. Recently developed computational approaches, which are used in phage genome analysis, are critically assessed. We hope that this assessment provides researchers with useful insights for the selection of suitable approaches for phage-related research aims and applications.

The bacteriophage decides own tracks: When they are with or against the bacteria.

Bacteriophages, bacteria-infecting viruses, are considered by many researchers a promising solution for antimicrobial resistance. On the other hand, some phages have shown contribution to bacterial resistance phenomenon by transducing antimicrobial resistance genes to their bacterial hosts. Contradictory consequences of infections are correlated to different phage lifecycles. Out of four known lifecycles, lysogenic and lytic pathways have been riddles since the uncontrolled conversion between them could negatively affect the intended use of phages. However, phages still can be engineered for applications against bacterial and viral infections to ensure high efficiency. This review highlights two main aspects: (1) the different lifecycles as well as the different factors that affect lytic-lysogenic switch are discussed, including the intracellular and molecular factors control this decision. In addition, different models which describe the effect of phages on the ecosystem are compared, besides the approaches to study the switch. (2) An overview on the contribution of the phage in the evolution of the bacteria, instead of eating them, as a consequence of different mode of actions. As well, how phage display has helped in restricting phage cheating and how it could open new gates for immunization and pandemics control will be tacked.

Interaction of nanoparticles with biological macromolecules: a review of molecular docking studies.

The high frequency of using engineered nanoparticles in various medical applications entails a deep understanding of their interaction with biological macromolecules. Molecular docking simulation is now widely used to study the binding of different types of nanoparticles with proteins and nucleic acids. This helps not only in understanding the mechanism of their biological action but also in predicting any potential toxicity. In this review, the computational techniques used in studying the nanoparticles interaction with biological macromolecules are covered. Then, a comprehensive overview of the docking studies performed on various types of nanoparticles will be offered. The implication of these predicted interactions in the biological activity and/or toxicity is also discussed for each type of nanoparticles.

Encapsulation of E. coli phage ZCEC5 in chitosan-alginate beads as a delivery system in phage therapy.

Bacteriophages can be used successfully to treat pathogenic bacteria in the food chain including zoonotic pathogens that colonize the intestines of farm animals. However, harsh gastric conditions of low pH and digestive enzyme activities affect phage viability, and accordingly reduce their effectiveness. We report the development of a natural protective barrier suitable for oral administration to farm animals that confers acid stability before functional release of bead-encapsulated phages. Escherichia coli bacteriophage ZSEC5 is rendered inactive at pH 2.0 but encapsulation in chitosan-alginate bead with a honey and gelatin matrix limited titer reductions to 1 log10 PFU mL-1. The encapsulated phage titers were stable upon storage in water but achieved near complete release over 4-5 h in a simulated intestinal solution (0.1% bile salt, 0.4% pancreatin, 50 mM KH2PO4 pH 7.5) at 37 °C. Exposure of E. coli O157:H7 to the bead-encapsulated phage preparations produced a delayed response, reaching a maximal reductions of 4.2 to 4.8 log10 CFU mL-1 after 10 h at 37 °C under simulated intestinal conditions compared to a maximal reduction of 5.1 log10 CFU mL-1 at 3 h for free phage applied at MOI = 1. Bead-encapsulation is a promising reliable and cost-effective method for the functional delivery of bacteriophage targeting intestinal bacteria of farm animals.