The Effect of Dysfunctional Ubiquitin Enzymes in the Pathogenesis of Most Common Diseases.

Ubiquitination is a multi-step enzymatic process that involves the marking of a substrate protein by bonding a ubiquitin and protein for proteolytic degradation mainly via the ubiquitin-proteasome system (UPS). The process is regulated by three main types of enzymes, namely ubiquitin-activating enzymes (E1), ubiquitin-conjugating enzymes (E2), and ubiquitin ligases (E3). Under physiological conditions, ubiquitination is highly reversible reaction, and deubiquitinases or deubiquitinating enzymes (DUBs) can reverse the effect of E3 ligases by the removal of ubiquitin from substrate proteins, thus maintaining the protein quality control and homeostasis in the cell. The dysfunction or dysregulation of these multi-step reactions is closely related to pathogenic conditions; therefore, understanding the role of ubiquitination in diseases is highly valuable for therapeutic approaches. In this review, we first provide an overview of the molecular mechanism of ubiquitination and UPS; then, we attempt to summarize the most common diseases affecting the dysfunction or dysregulation of these mechanisms.

Therapeutic Nanoparticles and Their Targeted Delivery Applications.

Nanotechnology offers many advantages in various fields of science. In this regard, nanoparticles are the essential building blocks of nanotechnology. Recent advances in nanotechnology have proven that nanoparticles acquire a great potential in medical applications. Formation of stable interactions with ligands, variability in size and shape, high carrier capacity, and convenience of binding of both hydrophilic and hydrophobic substances make nanoparticles favorable platforms for the target-specific and controlled delivery of micro- and macromolecules in disease therapy. Nanoparticles combined with the therapeutic agents overcome problems associated with conventional therapy; however, some issues like side effects and toxicity are still debated and should be well concerned before their utilization in biological systems. It is therefore important to understand the specific properties of therapeutic nanoparticles and their delivery strategies. Here, we provide an overview on the unique features of nanoparticles in the biological systems. We emphasize on the type of clinically used nanoparticles and their specificity for therapeutic applications, as well as on their current delivery strategies for specific diseases such as cancer, infectious, autoimmune, cardiovascular, neurodegenerative, ocular, and pulmonary diseases. Understanding of the characteristics of nanoparticles and their interactions with the biological environment will enable us to establish novel strategies for the treatment, prevention, and diagnosis in many diseases, particularly untreatable ones.

Lipid Droplets in Health and Disease.

Lipids are essential building blocks synthesized by complex molecular pathways and deposited as lipid droplets (LDs) in cells. LDs are evolutionary conserved organelles found in almost all organisms, from bacteria to mammals. They are composed of a hydrophobic neutral lipid core surrounding by a phospholipid monolayer membrane with various decorating proteins. Degradation of LDs provide metabolic energy for divergent cellular processes such as membrane synthesis and molecular signaling. Lipolysis and autophagy are two main catabolic pathways of LDs, which regulate lipid metabolism and, thereby, closely engaged in many pathological conditons. In this review, we first provide an overview of the current knowledge on the structural properties and the biogenesis of LDs. We further focus on the recent findings of their catabolic mechanism by lipolysis and autophagy as well as their connection ragarding the regulation and function. Moreover, we discuss the relevance of LDs and their catabolism-dependent pathophysiological conditions.

Hyaluronan-Sphingosine Polymersomes for Treatment of Ocular Neovascularization: Synthesis and Evaluation.

Ocular neovascularization is a hallmark of several sight-threatening diseases, including diabetic retinopathy and age-related macular degeneration. Currently, available treatments are limited and often associated with side effects. Therefore, a novel approach to ocular neovascularization treatment through utilization of polymersomes from self-assembled sphingosine-grafted hyaluronic acid (HA-Sph) amphiphilic polymers is presented. The polymersomes are generated in spherical morphologies and sizes between 97.95 - 161.9 nm with homogenous size distributions. Experiments reveal that HA-Sph polymersomes, with concentrations ≥150 µg mL-1, significantly inhibit the proliferation of human umbilical vein endothelial cells (HUVECs), while concurrently promoting the proliferation of retinal pigment epithelial cells. The polymersomes demonstrate gradual disintegration in vitro, leading to sustained release of sphingosine, which prolongs the inhibition of HUVEC proliferation (from 87.5% at 24 h to 35.2% viability at 96 h). The efficacy of polymersomes in inhibiting angiogenesis is confirmed through tube formation assay, revealing a substantial reduction in tube length compared to the control group. The findings also validate the ocular penetration capability of polymersomes through ex vivo whole porcine eye ocular penetration study, indicating their suitability for topical administration. Potentially, HA-Sph polymersomes can be harnessed to develop intricate drug delivery systems that protect the retina and effectively treat ocular diseases.

On the application of hydrodynamic cavitation on a chip in cellular injury and drug delivery.

Hydrodynamic cavitation (HC) is a phase change phenomenon, where energy release in a fluid occurs upon the collapse of bubbles, which form due to the low local pressures. During recent years, due to advances in lab-on-a-chip technologies, HC-on-a-chip (HCOC) and its potential applications have attracted considerable interest. Microfluidic devices enable the performance of controlled experiments by enabling spatial control over the cavitation process and by precisely monitoring its evolution. In this study, we propose the adjunctive use of HC to induce distinct zones of cellular injury and enhance the anticancer efficacy of Doxorubicin (DOX). HC caused different regions (lysis, necrosis, permeabilization, and unaffected regions) upon exposure of different cancer and normal cells to HC. Moreover, HC was also applied to the confluent cell monolayer following the DOX treatment. Here, it was shown that the combination of DOX and HC exhibited a more pronounced anticancer activity on cancer cells than DOX alone. The effect of HC on cell permeabilization was also proven by using carbon dots (CDs). Finally, the cell stiffness parameter, which was associated with cell proliferation, migration and metastasis, was investigated with the use of cancer cells and normal cells under HC exposure. The HCOC offers the advantage of creating well-defined zones of bio-responses upon HC exposure simultaneously within minutes, achieving cell lysis and molecular delivery through permeabilization by providing spatial control. In conclusion, micro scale hydrodynamic cavitation proposes a promising alternative to be used to increase the therapeutic efficacy of anticancer drugs.

A feasibility study of different commercially available serum-free mediums to enhance lentivirus and adeno-associated virus production in HEK 293 suspension cells.

Lentivirus and adeno-associated viruses are invaluable tools for biotechnology applications due to their genetic material delivery abilities both in vitro and in vivo. However, their large-scale productions with Good Manufacturing Practices yield low efficiency when adherent and serum dependent HEK293 (Human Embryonic Kidney) cells are used as the host. To increase production efficiency, HEK293 cells are adapted to grow in suspension using commercially available and chemically defined serum-free mediums. Suspended cells can be transiently transfected for viral vector production; however, significant improvements are still needed to increase yield and thereby cost effectiveness. Here, we evaluated four most preferred commercially available mediums that are IVY, FreeStyle293, LV-MAX, and BalanCD HEK293 for the transient transfection feasibility of lentiviral (LV) and adeno-associated virus serotype 2 (AAV2) production in FlorabioHEK293 suspension cells. The highest transfection efficiency was over 90% and obtained by using polyethyleneimine (PEI) 25 K and by media adaptation in IVY without using any transfection enhancer. For the first time the feasibility of HEK293 cells, which were adapted to grow in suspension culture by Florabio and IVY media, were tested for virus production. This study demonstrates the best transfection medium for scalable and optimized production of Lentivirus and Adeno-Associated Virus in suspended HEK293 cell culture. Supplementary Information: The online version contains supplementary material available at 10.1007/s10616-022-00551-1.

Hydrodynamic Cavitation on a Chip: A Tool to Detect Circulating Tumor Cells.

Circulating tumor cells (CTCs) are essential biomarkers for cancer diagnosis. Although various devices have been designed to detect, enumerate, and isolate CTCs from blood, some of these devices could have some drawbacks, such as the requirement of labeling, long process time, and high cost. Here, we present a microfluidic device based on the concept of "hydrodynamic cavitation-on-chip (HCOC)", which can detect CTCs in the order of minutes. The working principle relies on the difference of the required inlet pressure for cavitation inception of working fluids when they pass through the microfluidic device. The interface among the solid/floating particles, liquid, and vapor phases plays an important role in the strength of the fluid to withstand the rupture and cavitation formation. To this end, four experimental groups, including the "cell culture medium", "medium + Jurkat cells", "medium + Jurkat cells + CTCs", and "medium + CTCs", were tested as a proof of concept with two sets of fabricated microfluidic chips with the same geometrical dimensions, in which one set contained structural sidewall roughness elements. Jurkat cells were used to mimic white blood cells, and MDA-MB-231 cells were spiked into the medium as CTCs. Accordingly, the group with CTCs led to detectable earlier cavitation inception. Additionally, the effect of the CTC concentration on cavitation inception and the effect of the presence of sidewall roughness elements on the earlier inception were evaluated. Furthermore, CTC detection tests were performed with cancer cell lines spiked in blood samples from healthy donors. The results showed that this approach, HCOC, could be a potential approach to detect the presence of CTCs based on cavitation phenomenon and offer a cheap, user-friendly, and rapid tool with no requirement for any biomarker or extensive films acting as a biosensor. This approach also possesses straightforward application procedures to be employed for detection of CTCs.

A Flexible Cystoscope Based on Hydrodynamic Cavitation for Tumor Tissue Ablation.

OBJECTIVE: Hydrodynamic cavitation is characterized by the formation of bubbles inside a flow due to local reduction of pressure below the saturation vapor pressure. The resulting growth and violent collapse of bubbles lead to a huge amount of released energy. This energy can be implemented in different fields such as heat transfer enhancement, wastewater treatment and chemical reactions. In this study, a cystoscope based on small scale hydrodynamic cavitation was designed and fabricated to exploit the destructive energy of cavitation bubbles for treatment of tumor tissues. The developed device is equipped with a control system, which regulates the movement of the cystoscope in different directions. According to our experiments, the fabricated cystoscope was able to locate the target and expose cavitating flow to the target continuously and accurately. The designed cavitation probe embedded into the cystoscope caused a significant damage to prostate cancer and bladder cancer tissues within less than 15 minutes. The results of our experiments showed that the cavitation probe could be easily coupled with endoscopic devices because of its small diameter. We successfully integrated a biomedical camera, a suction tube, tendon cables, and the cavitation probe into a 6.7 mm diameter cystoscope, which could be controlled smoothly and accurately via a control system. The developed device is considered as a mechanical ablation therapy, can be a solid alternative for minimally invasive tissue ablation methods such as radiofrequency (RF) and laser ablation, and could have lower side effects compared to ultrasound therapy and cryoablation.

The influence of RS738409 I148M polymorphism of patatin-like phospholipase domain containing 3 gene on the susceptibility of non-alcoholic fatty liver disease.

ABSTRACT: We aimed to elucidate the frequency of polymorphic genotypes and alleles of patatin-like phospholipase domain containing 3 rs738409 polymorphism and its possible associations with non-alcoholic fatty liver disease (NAFLD) and non-alcoholic steatohepatitis in a cohort from Turkey.We enrolled 200 patients diagnosed with NAFLD and genotyped for rs738409 I148M polymorphism by real-time polymerase chain reaction, particularly by melting curve analysis. SPSS analysis software was used for statistical significance. Continuous variable values were expressed as mean ±â€Šstandard deviation. Significant statistical level was chosen as p  = 0.05.Our results demonstrate in a cohort from Turkey that rs738409 C > G polymorphism (I148M) of patatin-like phospholipase domain containing 3 gene is significantly able to affect individuals to have NAFLD in unadjusted regression model.Consistent with the previous studies in other populations, our study group showed a significantly higher risk of having NAFLD in unadjusted regression model but not in the adjusted model indicating that non-genetic factors such as age and sex may be responsible for the association. However, independent studies need to validate our findings with a larger group of NAFLD patients, as well as in different ethnic cohorts.

Antitumor Efficacy of Ceranib-2 with Nano-Formulation of PEG and Rosin Esters.

Ceranib-2 is a recently discovered, poorly water-soluble potent ceramidase inhibitor, with the ability to suppress cancer cell proliferation and delay tumor growth. However, its poor water solubility and weak cellular bioavailability hinder its use as a therapeutic agent for cancer. PEGylated rosin esters are an excellent platform as a natural polymer for drug delivery applications, especially for controlling drug release due to their degradability, biocompatibility, capability to improve solubility, and pharmacokinetics of potent drugs. In this study, stable aqueous amphiphilic submicron-sized PEG400-rosin ester-ceranib-2 (PREC-2) particles, ranging between 100 and 350 nm in a 1:1 mixture, were successfully synthesized by solvent evaporation mediated by sonication.Conclusion: Stable aqueous PEGylated rosin ester nanocarriers might present a significant solution to improve solubility, pharmacokinetic, and bioavailability of ceranib-2, and hold promises for use as an anticancer adjacent drug after further investigations.

Protein Kinase C Isozymes and Autophagy during Neurodegenerative Disease Progression.

Protein kinase C (PKC) isozymes are members of the Serine/Threonine kinase family regulating cellular events following activation of membrane bound phospholipids. The breakdown of the downstream signaling pathways of PKC relates to several disease pathogeneses particularly neurodegeneration. PKC isozymes play a critical role in cell death and survival mechanisms, as well as autophagy. Numerous studies have reported that neurodegenerative disease formation is caused by failure of the autophagy mechanism. This review outlines PKC signaling in autophagy and neurodegenerative disease development and introduces some polyphenols as effectors of PKC isozymes for disease therapy.

Niosomal Drug Delivery Systems for Ocular Disease-Recent Advances and Future Prospects.

The eye is a complex organ consisting of several protective barriers and particular defense mechanisms. Since this organ is exposed to various infections, genetic disorders, and visual impairments it is essential to provide necessary drugs through the appropriate delivery routes and vehicles. The topical route of administration, as the most commonly used approach, maybe inefficient due to low drug bioavailability. New generation safe, effective, and targeted drug delivery systems based on nanocarriers have the capability to circumvent limitations associated with the complex anatomy of the eye. Nanotechnology, through various nanoparticles like niosomes, liposomes, micelles, dendrimers, and different polymeric vesicles play an active role in ophthalmology and ocular drug delivery systems. Niosomes, which are nano-vesicles composed of non-ionic surfactants, are emerging nanocarriers in drug delivery applications due to their solution/storage stability and cost-effectiveness. Additionally, they are biocompatible, biodegradable, flexible in structure, and suitable for loading both hydrophobic and hydrophilic drugs. These characteristics make niosomes promising nanocarriers in the treatment of ocular diseases. Hereby, we review niosome based drug delivery approaches in ophthalmology starting with different preparation methods of niosomes, drug loading/release mechanisms, characterization techniques of niosome nanocarriers and eventually successful applications in the treatment of ocular disorders.

Autophagy as a molecular target for cancer treatment.

Autophagy is an evolutionarily conserved catabolic mechanism, by which eukaryotic cells recycle or degrades internal constituents through membrane-trafficking pathway. Thus, autophagy provides the cells with a sustainable source of biomolecules and energy for the maintenance of homeostasis under stressful conditions such as tumor microenvironment. Recent findings revealed a close relationship between autophagy and malignant transformation. However, due to the complex dual role of autophagy in tumor survival or cell death, efforts to develop efficient treatment strategies targeting the autophagy/cancer relation have largely been unsuccessful. Here we review the two-faced role of autophagy in cancer as a tumor suppressor or as a pro-oncogenic mechanism. In this sense, we also review the shared regulatory pathways that play a role in autophagy and malignant transformation. Finally, anti-cancer therapeutic agents used as either inhibitors or inducers of autophagy have been discussed.

Impairment of lipophagy by PNPLA1 mutations causes lipid droplet accumulation in primary fibroblasts of Autosomal Recessive Congenital Ichthyosis patients.

BACKGROUND: Autosomal Recessive Congenital Ichthyosis (ARCI) is a group of epidermal keratinization disorders. One of the disease-associated proteins, patatin-like phospholipase domain-containing protein-1 (PNPLA1), plays a key role in the epidermal omega-O-acylceramide synthesis and localizes on the surface of lipid droplets (LDs). OBJECTIVE: Previously, routine clinical test results showed abnormal LD accumulation in blood smear samples of our ARCI patients with PNPLA1 mutations. To investigate the abnormal accumulation of LDs, we analyzed primary fibroblast cells of ARCI patients with PNPLA1 mutations (p.Y245del and p.D172N). We hypothesized that PNPLA1 mutations might affect lipophagy-mediated regulation of LDs and cause intracellular lipid accumulation in ARCI patients. METHODS: LD accumulation was analyzed by fluorescence staining with BODIPY®493/503 in the fibroblasts of patient cells and PNPLA1 siRNA transfected control fibroblast cells. The expression of PNPLA1 and its effects on the lipophagy-mediated degradation of LDs were analyzed by immunocytochemistry and immunoblotting. RESULTS: Our results showed that mutant or downregulated PNPLA1 protein causes abnormal intracellular LD accumulation. We found that PNPLA1 mutations affect neither the cellular localization nor the expression levels of the protein in fibroblast cells. When we analyzed lipophagic degradation process, LC3 expression and the number of autophagosomes were significantly decreased in fibroblast cells of the patients. In addition, co-localization of LDs with autophagosomes and lysosomes were markedly less than that of the control group. CONCLUSION: PNPLA1 mutations caused disturbances in both autophagosome formation and fusion of autophagosomes with lysosomes. Our results indicate a possible role for PNPLA1 protein in LD regulation via lipophagy-mediated degradation.

Magnetofection of Green Fluorescent Protein Encoding DNA-Bearing Polyethyleneimine-Coated Superparamagnetic Iron Oxide Nanoparticles to Human Breast Cancer Cells.

Gene therapy is a developing method for the treatment of various diseases. For this purpose, the search for nonviral methods has recently accelerated to avoid toxic effects. A strong alternative method is magnetofection, which involves the use of superparamagnetic iron oxide nanoparticles (SPIONs) with a proper organic coating and external magnetic field to enhance the localization of SPIONs at the target site. In this study, a new magnetic actuation system consisting of four rare-earth magnets on a rotary table was designed and manufactured to obtain improved magnetofection. As a model, green fluorescent protein DNA-bearing polyethyleneimine-coated SPIONs were used. Magnetofection was tested on MCF7 cells. The system reduced the transfection time (down to 1 h) of the standard polyethyleneimine transfection protocol. As a result, we showed that the system could be effectively used for gene transfer.

Molecular Pathogenesis of Nonalcoholic Steatohepatitis- (NASH-) Related Hepatocellular Carcinoma.

The proportion of obese or diabetic population has been anticipated to increase in the upcoming decades, which rises the prevalence of nonalcoholic fatty liver disease (NAFLD) and its progression to nonalcoholic steatohepatitis (NASH). Recent evidence indicates that NASH is the main cause of chronic liver diseases and it is an important risk factor for development of hepatocellular carcinoma (HCC). Although the literature addressing NASH-HCC is growing rapidly, limited data is available about the etiology of NASH-related HCC. Experimental studies on the molecular mechanism of HCC development in NASH reveal that the carcinogenesis is relevant to complex changes in signaling pathways that mediate cell proliferation and energy metabolism. Genetic or epigenetic modifications and alterations in metabolic, immunologic, and endocrine pathways have been shown to be closely related to inflammation, liver injury, and fibrosis in NASH along with its subsequent progression to HCC. In this review, we provide an overview on the current knowledge of NASH-related HCC development and emphasize molecular signaling pathways regarding their mechanism of action in NASH-derived HCC.