MLIP and Its Potential Influence on Key Oncogenic Pathways.

Muscle-enriched A-type lamin-interacting protein (MLIP) is an emerging protein involved in cellular homeostasis and stress adaptation. Eukaryotic cells regulate various cellular processes, including metabolism, DNA repair, and cell cycle progression, to maintain cellular homeostasis. Disruptions in this homeostasis can lead to diseases such as cancer, characterized by uncontrolled cell growth and division. This review aims to explore for the first time the unique role MLIP may play in cancer development and progression, given its interactions with the PI3K/Akt/mTOR pathway, p53, MAPK9, and FOXO transcription factors, all critical regulators of cellular homeostasis and tumor suppression. We discuss the current understanding of MLIP's involvement in pro-survival pathways and its potential implications in cancer cells' metabolic remodeling and dysregulated homeostasis. Additionally, we examine the potential of MLIP as a novel therapeutic target for cancer treatment. This review aims to shed light on MLIP's potential impact on cancer biology and contribute to developing innovative therapeutic strategies.

Impact of Obesity-Related Endoplasmic Reticulum Stress on Cancer and Associated Molecular Targets.

Obesity, characterized by excessive body fat, is closely linked to endoplasmic reticulum (ER) stress, leading to insulin resistance and type 2 diabetes. Inflammatory pathways like c-Jun N-terminal kinase (JNK) worsen insulin resistance, impacting insulin signaling. Moreover, ER stress plays a substantial role in cancer, influencing tumor cell survival and growth by releasing factors like vascular endothelial growth factor (VEGF). The unfolded protein response (UPR) is pivotal in this process, offering both pro-survival and apoptotic pathways. This review offers an extensive exploration of the sophisticated connection between ER stress provoked by obesity and its role in both the onset and advancement of cancer. It delves into the intricate interplay between oncogenic signaling and the pathways associated with ER stress in individuals who are obese. Furthermore, this review sheds light on potential therapeutic strategies aimed at managing ER stress induced by obesity, with a focus on addressing cancer initiation and progression. The potential to alleviate ER stress through therapeutic interventions, which may encompass the use of small molecules, FDA-approved medications, and gene therapy, holds great promise. A more in-depth examination of pathways such as UPR, ER-associated protein degradation (ERAD), autophagy, and epigenetic regulation has the potential to uncover innovative therapeutic approaches and the identification of predictive biomarkers.

The Association between Lifestyle Factors and COVID-19: Findings from Qatar Biobank.

Coronavirus Disease 2019 (COVID-19) manifestations range from mild to severe life-threatening symptoms, including death. COVID-19 susceptibility has been associated with various factors, but studies in Qatar are limited. The objective of this study was to investigate the correlation between COVID-19 susceptibility and various sociodemographic and lifestyle factors, including age, gender, body mass index, smoking status, education level, dietary patterns, supplement usage, physical activity, a history of bariatric surgery, diabetes, and hypertension. We utilized logistic regression to analyze these associations, using the data of 10,000 adult participants, aged from 18 to 79, from Qatar Biobank. In total, 10.5% (n = 1045) of the participants had COVID-19. Compared to non-smokers, current and ex-smokers had lower odds of having COVID-19 (odds ratio [OR] = 0.55; 95% CI: 0.44-0.68 and OR = 0.70; 95% CI: 0.57-0.86, respectively). Vitamin D supplement use was associated with an 18% reduction in the likelihood of contracting COVID-19 (OR = 0.82; 95% CI: 0.69-0.97). Obesity (BMI ≥ 30 kg/m2), a history of bariatric surgery, and higher adherence to the modern dietary pattern-characterized by the consumption of foods high in saturated fat and refined carbohydrates-were positively associated with COVID-19. Our findings indicate that adopting a healthy lifestyle may be helpful in the prevention of COVID-19 infection.

Comparing Methods for Induction of Insulin Resistance in Mouse 3T3-L1 Cells.

Cell culture plays a crucial role in addressing fundamental research questions, particularly in studying insulin resistance (IR) mechanisms. Multiple in vitro models are utilized for this purpose, but their technical distinctions and relevance to in vivo conditions remain unclear. This study aims to assess the effectiveness of existing in vitro models in inducing IR and their ability to replicate in vivo IR conditions. BACKGROUND: Insulin resistance (IR) is a cellular condition linked to metabolic disorders. Despite the utility of cell culture in IR research, questions persist regarding the suitability of various models. This study seeks to evaluate these models' efficiency in inducing IR and their ability to mimic in vivo conditions. Insights gained from this research could enhance our understanding of model strengths and limitations, potentially advancing strategies to combat IR and related disorders. OBJECTIVE: 1- Investigate the technical differences between existing cell culture models used to study molecular mediators of insulin resistance (IR). 2- Compare the effectiveness of present in vitro models in inducing insulin resistance (IR). 3- Assess the relevance of the existing cell culture models in simulating the in vivo conditions and environment that provoke the induction of insulin resistance (IR). METHODS AND MATERIAL: In vitro, eight sets of 3T3-L1 cells were cultured until they reached 90% confluence. Subsequently, adipogenic differentiation was induced using a differentiation cocktail (media). These cells were then divided into four groups, with four subjected to normal conditions and the other four to hypoxic conditions. Throughout the differentiation process, each cell group was exposed to specific factors known to induce insulin resistance (IR). These factors included 2.5nM tumor necrosis factor-alpha (TNFα), 20 ng/ml interleukin-6 (IL-6), 10 micromole 4-hydroxynonenal (4HNE), and high insulin (HI) at a concentration of 100nM. To assess cell proliferation, DAPI staining was employed, and the expression of genes associated with various metabolic pathways affected by insulin resistance was investigated using Real-Time PCR. Additionally, insulin signaling was examined using the Bio-plex Pro cell signaling Akt panel. RESULTS: We induced insulin resistance in 3T3-L1 cells using IL-6, TNFα, 4HNE, and high insulin in both hypoxic and normoxic conditions. Hypoxia increased HIF1a gene expression by approximately 30% (P<0.01). TNFα reduced cell proliferation by 10-20%, and chronic TNFα treatment significantly decreased mature adipocytes due to its cytotoxicity. We assessed the impact of insulin resistance (IR) on metabolic pathways, focusing on genes linked to branched-chain amino acid metabolism, detoxification, and chemotaxis. Notably, ALDH6A1 and MCCC1 genes, related to amino acid metabolism, were significantly affected under hypoxic conditions. TNFα treatment notably influenced MCP-1 and MCP-2 genes linked to chemotaxis, with remarkable increases in MCP-1 levels and MCP-2 expression primarily under hypoxia. Detoxification-related genes showed minimal impact, except for a significant increase in MAOA expression under acute hypoxic conditions with TNFα treatment. Additional genes displayed varying effects, warranting further investigation. To investigate insulin signaling's influence in vitro by IRinducing factors, we assessed phospho-protein levels. Our results reveal a significant p-Akt induction with chronic high insulin (10%) and acute TNFα (12%) treatment under hypoxia (both P<0.05). Other insulin resistance-related phospho-proteins (GSK3B, mTOR, PTEN) increased with IL-6, 4HNE, TNFα, and high insulin under hypoxia, while p-IRS1 levels remained unaffected. CONCLUSION: In summary, different in vitro models using inflammatory, oxidative stress, and high insulin conditions under hypoxic conditions can capture various aspects of in vivo adipose tissue insulin resistance (IR). Among these models, acute TNFα treatment may offer the most robust approach for inducing IR in 3T3-L1 cells.

The Role of Apolipoproteins in the Commonest Cancers: A Review.

Apolipoproteins (APOs) are vital structural components of plasma lipoproteins that are involved in lipid metabolism and transport. Recent studies have reported an association between apolipoprotein dysregulation and the onset of a variety of human cancers; however, the role of certain APOs in cancer development remains unknown. Based on recent work, we hypothesize that APOs might be involved in the onset of cancer, with a focus on the most common cancers, including breast, lung, gynecological, colorectal, thyroid, gastric, pancreatic, hepatic, and prostate cancers. This review will focus on the evidence supporting this hypothesis, the mechanisms linking APOs to the onset of cancer, and the potential clinical relevance of its various inhibitors.

Evaluation of the Influence of Eggshell (ES) Concentration on the Degradation Behavior of Mg-2.5Zn Biodegradable Alloy in Simulated Body Fluid.

Currently, permanent vascular stents are fabricated using titanium and stainless steel implants that are nondegradable and offer high stability, but they have certain disadvantages. For example, the prolonged exposition of aggressive ions in the physiological media and the existence of defects in the oxide film create conditions for corrosion to occur, thus triggering unwanted biological events and compromising the mechanical integrity of the implants. Moreover, when the implant does not need to be permanent, there is the need to submit the patient for a second surgery for implant removal. As a solution for nonpermanent implants, biodegradable magnesium alloys have been deemed a promising substitute, for example, for cardiovascular-related applications and the construction of orthopedic devices. A biodegradable magnesium alloy (Mg-2.5Zn) reinforced by zinc and eggshell was employed in this study as an environment-conscious magnesium (eco) composite (Mg-2.5Zn-xES). Disintegrated melt deposition (DMD) was used to fabricate the composite. Experimental studies were conducted to investigate the biodegradation performance of Mg-Zn alloys containing 3 and 7 wt % eggshell (ES) in simulated body fluid (SBF) at 37 °C. Different corrosion techniques were used to study the corrosion behavior of the Mg-2.5Zn-xES composites, including weight loss measurements, hydrogen evolution, potentiodynamic polarization, electrochemical impedance spectroscopy (EIS), and scanning vibrating electrode technique (SVET). Scanning electron microscopy (SEM) coupled with energy-dispersive X-ray spectroscopy (EDS), X-ray photoelectron spectroscopy (XPS), and X-ray diffraction (XRD) were employed to scrutinize the corroded surfaces' morphology and composition. The outcomes indicated that Mg-2.5Zn-3ES possesses the lowest degradation activity.

Effect of sera from elite athletes on cytokine secretion and insulin signaling in preadipocytes and skeletal muscle cells.

Introduction: The immunomodulatory effect of physical activity can impact insulin signaling differentially in adipose tissues and skeletal muscle cells, depending on sport intensity. In this study, the effect of serum from elite athletes with varying endurance levels and playing different power sports on cytokine secretion and insulin signaling in preadipocyte and skeletal muscle cell lines was investigated. Methods: Preadipocytes (3T3-L1) and skeletal muscle cells (C2C12) were cultured in media containing pooled sera from elite athletes who play high-endurance (HE), high-power (HP), or low-endurance/low-power (LE/LP) sports for 72 h. Secreted cytokines (IL-6 and TNF-alpha) were assessed in the supernatant, and insulin signaling phosphoproteins levels were measured in lysates following treatment using cells multiplex immunoassays. Results: Sera from LE/LP and HP induced TNF-α secretion in C2C12, while serum from HE reduced IL-6 secretion compared to non-athlete serum control. All elite athlete sera groups caused decreased insulin sensitivity in 3T3-L1 cells, whereas in C2C12 cells, only HE athlete serum reduced insulin signaling, while LE/LP and HP caused increased insulin sensitivity. Conclusion: Sera from elite athletes of different sport disciplines can affect the inflammatory status and insulin signaling of preadipocytes and myoblasts differently, with risk of developing insulin resistance. Furthermore, investigation of the functional relevance of these effects on exercise physiology and pathophysiology is warranted.

In Vitro and In Vivo Validation of GATA-3 Suppression for Induction of Adipogenesis and Improving Insulin Sensitivity.

Impaired adipogenesis is associated with the development of insulin resistance and an increased risk of type 2 diabetes (T2D). GATA Binding Protein 3 (GATA3) is implicated in impaired adipogenesis and the onset of insulin resistance. Therefore, we hypothesize that inhibition of GATA3 could promote adipogenesis, restore healthy fat distribution, and enhance insulin signaling. Primary human preadipocytes were treated with GATA3 inhibitor (DNAzyme hgd40). Cell proliferation, adipogenic capacity, gene expression, and insulin signaling were measured following well-established protocols. BALB/c mice were treated with DNAzyme hgd40 over a period of 2 weeks. Liposomes loaded with DNAzyme hgd40, pioglitazone (positive), or vehicle (negative) controls were administered subcutaneously every 2 days at the right thigh. At the end of the study, adipose tissues were collected and weighed from the site of injection, the opposite side, and the omental depot. Antioxidant enzyme (superoxide dismutase and catalase) activities were assessed in animals' sera, and gene expression was measured using well-established protocols. In vitro GATA3 inhibition induced the adipogenesis of primary human preadipocytes and enhanced insulin signaling through the reduced expression of p70S6K. In vivo GATA3 inhibition promoted adipogenesis at the site of injection and reduced MCP-1 expression. GATA3 inhibition also reduced omental tissue size and PPARγ expression. These findings suggest that modulating GATA3 expression offers a potential therapeutic benefit by correcting impaired adipogenesis, promoting healthy fat distribution, improving insulin sensitivity, and potentially lowering the risk of T2D.

Role of Inflammatory Cytokines, Growth Factors and Adipokines in Adipogenesis and Insulin Resistance.

Obesity, manifested by increased adiposity, represents a main cause of morbidity in the developed countries, causing increased risk of insulin resistance and type 2 diabetes mellitus. Recruitment of macrophages and activation of innate immunity represent the initial insult, which can be further exacerbated through secretion of chemokines and adipocytokines from activated macrophages and other cells within the adipose tissue. These events can impact adipogenesis, causing dysfunction of the adipose tissue and increased risk of insulin resistance. Various factors mediate adiposity and related insulin resistance including inflammatory and non-inflammatory factors such as pro and anti-inflammatory cytokines, adipokines and growth factors. In this review we will discuss the role of these factors in adipogenesis and development of insulin resistance and type 2 diabetes mellitus in the context of obesity. Understanding the molecular mechanisms that mediate adipogenesis and insulin resistance could help the development of novel therapeutic strategies for individuals at higher risk of insulin resistance and type 2 diabetes mellitus.

Production of Long-Acting CNGRC-CPG2 Fusion Proteins: New Derivatives to Overcome Drug Immunogenicity of Ligand-Directed Enzyme Prodrug Therapy for Targeted Cancer Treatment.

Objectives: Aminopeptidase N (APN) is an enzyme highly expressed in metastatic cancers and could be used in targeted cancer therapy. Our previous work showed the successful construction of CNGRC-carboxypeptidase G2 (CPG2) and CNGRC-CPG2-CNGRC fusion proteins. Our conjugates and prodrugs were effective in targeting high APN-expressing cancer cells. In the present study, we aim to produce long-acting fusion proteins to overcome 2 of the main drawbacks of antibody-directed enzyme prodrug therapy. Methods: N-terminal and N-, C-terminal fusion CPG2, CNGRC-CPG2, and CNGRC-CPG2-CNGRC, respectively, were PEGylated using polyethylene glycol (PEG) maleimide (40K). We examined the effect of PEGylation on the therapeutic efficacy of the new products. The resulting PEGylated fusion proteins were tested for their stability, ex vivo immunotoxicity, binding capacity to their target on high HT1080, and low A549 APN-expressing cells. The catalytic activity of the resulting PEGylated fusion CPG2 proteins was investigated. Pro-drug "ZD2767P" cytotoxic effect in association with PEG CPG2-CNGRC fusion proteins on cancer cells was studied. Results: Our work demonstrated that the properties of the PEGylated single-fused proteins were significantly improved over that of un-PEGylated fused CPG2, and its kinetic activity and APN-binding affinity were not negatively affected by the PEGylation. Significantly, The PEGylated single-fused CPG2 had lower immunogenicity than the un-PEGylated CPG2. Our results, however, were different in the case of the PEGylated double-fused CPG2. Although its stability in human serum under physiological conditions was not significantly affected, the kinetic activity and its binding affinity to their cellular marker (APN) were substantially reduced. When the study was performed with high and low APN-expressing cancer cell lines, using the prodrug ZD2767p, the PEGylated fusion CPG2 demonstrated cancer cell killing effects. Conclusion: We have successfully produced PEGylated-CNGRC-CPG2, which is bioactive and with lower immunogenicity in ligand-directed enzyme prodrug therapy for cancer treatment.

Bio-vehicles of cytotoxic drugs for delivery to tumor specific targets for cancer precision therapy.

Abnormal structural and molecular changes in malignant tissues were thoroughly investigated and utilized to target tumor cells, hence rescuing normal healthy tissues and lowering the unwanted side effects as non-specific cytotoxicity. Various ligands for cancer cell specific markers have been uncovered and inspected for directional delivery of the anti-cancer drug to the tumor site, in addition to diagnostic applications. Over the past few decades research related to the ligand targeted therapy (LTT) increased tremendously aiming to treat various pathologies, mainly cancers with well exclusive markers. Malignant tumors are known to induce elevated levels of a variety of proteins and peptides known as cancer "markers" as certain antigens (e.g., Prostate specific membrane antigen "PSMA", carcinoembryonic antigen "CEA"), receptors (folate receptor, somatostatin receptor), integrins (Integrin αvß3) and cluster of differentiation molecules (CD13). The choice of an appropriate marker to be targeted and the design of effective ligand-drug conjugate all has to be carefully selected to generate the required therapeutic effect. Moreover, since some tumors express aberrantly high levels of more than one marker, some approaches investigated targeting cancer cells with more than one ligand (dual or multi targeting). We aim in this review to report an update on the cancer-specific receptors and the vehicles to deliver cytotoxic drugs, including recent advancements on nano delivery systems and their implementation in targeted cancer therapy. We will discuss the advantages and limitations facing this approach and possible solutions to mitigate these obstacles. To achieve the said aim a literature search in electronic data bases (PubMed and others) using keywords "Cancer specific receptors, cancer specific antibody, tumor specific peptide carriers, cancer overexpressed proteins, gold nanotechnology and gold nanoparticles in cancer treatment" was carried out.

Age and Sport Intensity-Dependent Changes in Cytokines and Telomere Length in Elite Athletes.

BACKGROUND: Exercise-associated immune response plays a crucial role in the aging process. The aim of this study is to investigate the effect of sport intensity on cytokine levels, oxidative stress markers and telomere length in aging elite athletes. METHODS: In this study, 80 blood samples from consenting elite athletes were collected for anti-doping analysis at an anti-doping laboratory in Italy (FMSI). Participants were divided into three groups according to their sport intensity: low-intensity skills and power sports (LI, n = 18); moderate-intensity mixed soccer players (MI, n = 31); and high-intensity endurance sports (HI, n = 31). Participants were also divided into two age groups: less than 25 (n = 45) and above 25 years old (n = 35). Serum levels of 10 pro and anti-inflammatory cytokines and two antioxidant enzymes were compared in age and sport intensity groups and telomere lengths were measured in their respective blood samples. RESULTS: Tumor necrosis factor-alpha (TNF-α) was the only cytokine showing significantly higher concentration in older athletes, regardless of sport intensity. Interleukin (IL)-10 increased significantly in HI regardless of age group, whereas IL-6 concentration was higher in the older HI athletes. IL-8 showed a significant interaction with sport intensity in different age groups. Overall, significant positive correlations among levels of IL-6, IL-10, IL-8 and TNF-α were identified. The antioxidant catalase activity was positively correlated with levels of TNF-α. Telomere length increased significantly with sport intensity, especially in the younger group. CONCLUSION: HI had longer telomeres and higher levels of pro- and anti-inflammatory cytokines, suggesting less aging in HI compared to low and moderate counterparts in association with heightened immune response. Investigation of the functional significance of these associations on the health and performance of elite athletes is warranted.

GATA-3 as a Potential Therapeutic Target for Insulin Resistance and Type 2 Diabetes Mellitus.

Impaired adipogenesis plays an important role in the development of obesity-associated insulin resistance and type 2 diabetes as it leads to ectopic fat deposition. The anti-adipogenic transcription factor GATA-3 was identified as one of the potential molecular targets responsible for the impairment of adipogenesis. The expression of GATA-3 is higher in insulinresistant obese individuals compared to BMI-matched insulin-sensitive counterparts. Adipose tissue inflammation is a crucial mediator of this process. Hyperglycemia mediates the activation of the immune system, partially through upregulation of GATA- 3, causing exacerbation of the inflammatory state associated with obesity. This review discusses the evidence supporting the inhibition of GATA-3 as a useful therapeutic strategy in obesity-associated insulin resistance and type 2 diabetes, through up-regulation adipogenesis and amelioration of the immune response.

Metabolomics of Lean/Overweight Insulin-Resistant Females Reveals Alterations in Steroids and Fatty Acids.

BACKGROUND: The global diabetes epidemic is largely attributed to obesity-triggered metabolic syndrome. However, the impact of insulin resistance (IR) prior to obesity on the high prevalence of diabetes and the molecular mediators remain largely unknown. This study aims to compare the metabolic profiling of apparently healthy lean/overweight participants with IR and insulin sensitivity (IS), and identify the metabolic pathways underlying IR. METHODS: In this cross-sectional study, clinical and metabolic data for 200 seemingly healthy young female participants (100 IR and 100 IS) was collected from Qatar Biobank. Orthogonal partial least square analysis was performed to assess the extent of separation between individuals from the 2 groups based on measured metabolites. Classical linear models were used to identify the metabolic signature of IR, followed by elastic-net-regularized generalized linear model (GLMNET) and receiver operating characteristic (ROC) analysis to determine top metabolites associated with IR. RESULTS: Compared to lean/overweight participants with IS, those with IR showed increased androgenic steroids, including androsterone glucuronide, in addition to various microbiota byproducts, such as the phenylalanine derivative carboxyethylphenylalanine. On the other hand, participants with IS had elevated levels of long-chain fatty acids. A ROC analysis suggested better discriminatory performance using 20 metabolites selected by GLMNET in comparison to the classical clinical traits (area under curve: 0.93 vs 0.73, respectively). CONCLUSION: Our data confirm the multifactorial mechanism of IR with a diverse spectrum of emerging potential biomarkers, including steroids, long-chain fatty acids, and microbiota metabolites. Further studies are warranted to validate these markers for diagnostic and therapeutic applications.

Assessment of Serum Cytokines and Oxidative Stress Markers in Elite Athletes Reveals Unique Profiles Associated With Different Sport Disciplines.

OBJECTIVES: Circulating cytokines and oxidative stress markers vary in response to different exercise regimens. This study aims to compare the immune-inflammatory and oxidative stress profiles of elite athletes from different sport disciplines as potential biomarkers of muscle damage, and cardiovascular demand. METHODS: Serum samples from 88 consented elite male athletes from different sports disciplines (aquatics, n = 11, athletics, n = 22, cycling, n = 19, football, n = 28 and weightlifting, n = 8) collected at the anti-doping lab in Italy were screened for 38 cytokines and oxidative stress markers. Comparisons were made between different level of power, cardiovascular demand (CD) and endurance, as well as among the sport types. RESULTS: The anti-inflammatory interleukin (IL)-10 was higher (p = 0.04) in moderate power compared with the high power group. Conversely, superoxide dismutase (SOD; p = 0.001) and malondialdehyde (MDA; p = 0.007) levels were greater in the higher power groups compared with the lower power counterpart. Among athletes who belong to different CD ranks, IL-1ß and monocyte chemoattractant protein-1(MCP1) levels were higher (p = 0.03) in the low CD-rank group compared with high CD counterpart, whereas, SOD levels were higher (p = 0.001) in high and moderate CD-rank groups compared to low counterpart. For endurance groups, IL-10 and macrophage inflammatory protein (MIP)-1beta were increased (p = 0.03) in low/moderate endurance compared with the high endurance group. Finally, MIP1-beta, SOD and catalase varied significantly among the sports groups. CONCLUSION: Specific markers of inflammation and oxidative stress are associated with different sports disciplines and could be utilized as potential biomarkers of athletes' health, performance, and recovery from injury.

Calreticulin regulates Src kinase in osteogenic differentiation from embryonic stem cells.

Calreticulin, the major Ca2+ buffer of the endoplasmic reticulum plays an important role in the choice of fate by embryonic stem cells. Using the embryoid body method of organogenesis, we showed impaired osteogenesis in crt-/- cells vis-à-vis calreticulin-containing osteogenic WT cells. In the non-osteogenic crt-/- cells, c-Src- a non-receptor tyrosine kinase- was activated and its inhibition rescued osteogenesis. Most importantly, we demonstrated that calreticulin-containing cells had lower c-Src kinase activity, and this was accomplished via the Ca2+-homeostatic function of calreticulin. Specifically, lowering cytosolic [Ca2+] in calreticulin-containing osteogenic WT cells with BAPTA-AM, activated c-Src and impaired osteogenic differentiation. Conversely, increasing cytosolic [Ca2+] in crt-/- cells with ionomycin deactivated c-Src kinase and restored osteogenesis. The immediate effector of calreticulin, the Ser/Thr phosphatase calcineurin, was less active in crt-/- cells, however, its activity was rescued upon inhibition of c-Src activity by small molecule inhibitors. Finally, we showed that higher activity of calcineurin correlated with increased level of nuclear Runx2, a transcription factor that is the master regulator of osteogenesis. Collectively, our work has identified a novel pathway involving calreticulin regulated Ca2+ signalling via c-Src in osteogenic differentiation of embryonic stem cells.

Suppression of GATA-3 increases adipogenesis, reduces inflammation and improves insulin sensitivity in 3T3L-1 preadipocytes.

Impaired adipogenesis plays an important role in the development of obesity-associated insulin resistance and type 2 diabetes. Adipose tissue inflammation is a crucial mediator of this process. GATA-3 plays important roles in adipogenesis and inflammation. The aim of this study is to investigate the impact of GATA-3 suppression on improving adipogenesis, lowering inflammation and reversing insulin resistance. GATA-3 levels were measured in subcutaneous (SC) and omental (OM) adipose tissues obtained from insulin sensitive (IS) and insulin resistant (IR) obese individuals during weight reduction surgeries. The effect of GATA-3 suppression on adipogenesis, expression of inflammatory cytokines and insulin resistance biomarkers was performed in 3T3L-1 mouse preadipocytes via transfection with GATA-3-specific DNAzyme. GATA-3 expression was higher in OM compared to SC adipose tissues and in stromal vascular fraction-derived differentiating preadipocytes from IR obese individuals compared to their IS counterparts. Suppression of GATA-3 expression in 3T3L-1 mouse preadipocytes with GATA-3 specific inhibitor reversed 4-hydroxynonenal-induced impaired adipogenesis and triggered changes in the expression of insulin signaling-related genes. GATA-3 inhibition also modulated the expression of IL-6 and IL-10 and lowered the expression of insulin resistance biomarkers (PAI-1 and resistin) and insulin resistance phosphoproteins (p-BAD, p-PTEN and p-GSK3ß). Inhibiting GATA-3 improves adipocytes differentiation, modulates the secretion of inflammatory cytokines and improves insulin sensitivity in insulin resistant cells. Suppression of GATA-3 could be a promising tool to improve adipogenesis, restore insulin sensitivity and lower obesity-associated inflammation in insulin resistant individuals.

In vitro studies on CNGRC-CPG2 fusion proteins for ligand-directed enzyme prodrug therapy for targeted cancer therapy.

The sequence asparagine-glycine arginine (NGR), flanked by Cysteine (Cys) residues so as to form a disulfide-bridge (CNGRC), has previously been found to target and bind specifically to aminopeptidase N (APN), which is highly expressed on the surface of tumor cells. The goal of this study was to develop and evaluate the potential of fusion proteins carrying the CNGRC sequence linked to the enzyme carboxypeptidase G2 (CPG2) for targeted cancer therapy. We refer to this strategy as ligand-directed enzyme prodrug therapy (LDEPT). We constructed two forms of the CNGRC-CPG2 fusions, containing one or two copies of the cyclic NGR motif and designated CNGRC-CPG2 (X-CPG2) and CNGRC-CPG2-CNGRC (X-CPG2-X), respectively. In vitro binding assays of the purified constructs showed that both X-CPG2 and X-CPG2-X bound with high affinity to cancer cells expressing high levels of APN, compared to their binding to cells expressing low levels of APN. Further in vitro studies of the constructs to assess the therapeutic potential of LDEPT were carried out using cells expressing high and low levels of APN. Using methotrexate, it was demonstrated that cancer cell survival was significantly higher in the presence of the fusion proteins, due to the hydrolysis of this cytotoxic drug by CPG2. Conversely, when the prodrug ZD2767P was used, cancer cell killing was higher in the presence of the fused CPG2 constructs than in their absence, which is consistent with CPG2-mediated release of the cytotoxic drug from the prodrug. Furthermore, the doubly-fused CPG2 construct (X-CPG2-X) was significantly more effective than the singly-fused construct (X-CPG2).

Production of "biobetter" glucarpidase variants to improve drug detoxification and antibody directed enzyme prodrug therapy for cancer treatment.

Recombinant glucarpidase (formerly: Carboxypeptidase G2, CPG2) is used in Antibody Directed Enzyme Prodrug Therapy (ADEPT) for the treatment of cancer. In common with many protein therapeutics, glucarpidase has a relatively short half-life in serum and, due to the need for the repeated cycles of the ADEPT, its bioavailability may be further diminished by neutralizing antibodies produced by patients. PEGylation and fusion with human serum albumin (HSA) are two approaches that are commonly employed to increase the residency time of protein therapeutics in blood, and also to increase the half-lives of the proteins in vivo. To address this stability and the immunogenicity problems, 'biobetter' glucarpidase variants, mono-PEGylated glucarpidase, and HSA fused glucarpidase by genetic fusion with albumin, were produced. Biochemical and bioactivity analyses, including anti-proliferation, bioassays, circular dichroism, and in vitro stability using human blood serum and immunoassays, demonstrated that the functional activities of the designed glucarpidase conjugates were maintained. The immunotoxicity studies indicated that the PEGylated glucarpidase did not significantly induce T-cell proliferation, suggesting that glucarpidase epitopes were masked by the PEG moiety. However, free glucarpidase and HSA-glucarpidase significantly increased T-cell proliferation compared with the negative control. In the latter case, this might be due to the type of expression system used or due to trace impurities associated with the highly purified (99.99%) recombinant HSA-glucarpidase. Both PEGylated glucarpidase and HAS-glucarpidase exhibit more stability in human serum and were more resistant to key human proteases relative to native glucarpidase. To our knowledge, this study is the first to report stable and less immunogenic glucarpidase variants produced by PEGylation and fusion with HSA. The results suggest that they may have better efficacy in drug detoxification and ADEPT, thereby improving this cancer treatment strategy.

Optimized osteogenic differentiation protocol from R1 mouse embryonic stem cells in vitro.

Embryonic stem cells (ESCs) are a unique model that allows the study of molecular pathways underlying commitment and differentiation. One such lineage is osteoblasts, which are responsible for forming bone tissue in the body. There are many osteogenic differentiation protocols in the literature utilizing different soluble factors. The goal of the present study was to increase the efficacy of our osteogenic differentiation protocol from R1 cells. We have studied the effects of the addition of the following factors: dexamethasone, retinoic acid, and peroxisome-proliferator-activated receptor-gamma inhibitor, which have been reported to enhance osteogenesis. We found that among the 6 different protocols that were tested, the addition of retinoic acid with later addition of dexamethasone gives the most enrichment of osteogenic lineage cells. Thus, our findings provide valuable guidelines for culture condition to differentiate mouse R1 ESCs to osteoblastic cells in vitro.