Liraglutide attenuates obese-associated breast cancer cell proliferation via inhibiting PI3K/Akt/mTOR signaling pathway.

This study aims to explore the anti-proliferative, pro-apoptotic, and anti-migration activities of liraglutide (LGT) in MCF-7 breast cancer (BC) cells in subjects with obesity, particularly its effects on the PI3K/Akt/mTOR/AMPK pathway. The role of AMPK/SIRT-1, an essential regulator of adipokine production, in the effect of LGT on the production of adipose-derived adipokine was also assessed. MCF-7 cells were incubated in conditioned medium (CM) generated from adipose-derived stem cells (ADSCs) of obese subjects. MCF-7 cells were then treated with LGT for 72 h. Anti-proliferative, pro-apoptotic, and anti-migration activities were investigated using alamarBlue, annexin V stain, and scratch assay, respectively. Protein levels of phosphorylated PI3K, p-Akt, p-mTOR, and p-AMPK were investigated using immunoblotting. Levels of adipokines in ADSCs were determined using RT-PCR before and after transfection of ADSCs using the specific small interference RNA sequences for AMPK and SIRT-1. LGT evoked anti-proliferative, apoptotic, and potential anti-migratory properties on MCF-7 cells incubated in CM from obese ADSCs and significantly mitigated the activity of the PI3K/Akt/mTOR survival pathway-but not AMPK-in MCF-7 cells. Furthermore, the anti-proliferative effects afforded by LGT were similar to those mediated by LY294002 (PI3K inhibitor) and rapamycin (mTOR inhibitor). Our results reveal that transfection of AMPK/SIRT-1 genes did not affect the beneficial role of LGT in the expression of adipokines in ADSCs. In conclusion, LGT elicits anti-proliferative, apoptotic, and anti-migratory effects on BC cells in obese conditions by suppressing the activity of survival pathways; however, this effect is independent of the AMPK/SIRT1 pathway in ADSCs or AMPK in BC cells.

The platinum coordination complex inhibits cell invasion-migration and epithelial-to-mesenchymal transition by altering the TGF-ß-SMAD pathway in colorectal cancer.

Introduction: There is a steady increase in colorectal cancer (CRC) incidences worldwide; at diagnosis, about 20 percent of cases show metastases. The transforming growth factor-beta (TGF-ß) signaling pathway is one of the critical pathways that influence the expression of cadherins allowing the epithelial-to-mesenchymal transition (EMT), which is involved in the progression of the normal colorectal epithelium to adenoma and metastatic carcinoma. The current study aimed to investigate the impact of a novel coordination complex of platinum (salicylaldiminato) PT(II) complex with dimethyl propylene linkage (PT-complex) on TGF-ß and EMT markers involved in the invasion and migration of the human HT-29 and SW620 CRC cell lines. Methods: Functional study and wound healing assay showed PT-complex significantly reduced cell motility and the migration and invasion of CRC cell lines compared to the untreated control. Western blot performed in the presence and absence of TGF-ß demonstrated that PT-complex significantly regulated the TGF-ß-mediated altered expressions of EMT markers. Results and Discussion: PT-complex attenuated the migration and invasion by upregulating the protein expression of EMT-suppressing factor E-cadherin and suppressing EMT-inducing factors such as N-Cadherin and Vimentin. Moreover, PT-complex significantly suppressed the activation of SMAD3 in both CRC cell lines. Further, the microarray data analysis revealed differential expression of genes related to invasion and migration. In conclusion, besides displaying antiproliferative activity, the PT complex can decrease the metastasis of CRC cell lines by modulating TGF-ß-regulated EMT markers. These findings provide new insight into TGF-ß/SMAD signaling as the molecular mechanism involved in the antitumoral properties of novel PT-complex.

In Vivo Analysis of Porous Bioactive Silicon Carbide Scaffold for Craniofacial Bone Augmentation.

BACKGROUND: Bone augmentation is a vital area of research because of its high clinical demand and the reported complications associated with the available biomaterials. Purpose: The study assess the role of decellurized skeletal muscle (DSM) when combined with synthesized porous bioactive silicon carbide (SiC) ceramic and evaluated its ability to augment bone calvaria in a rat model. MATERIAL AND METHODS: Eighteen rats were divided into 2 groups; group 1 (n=9), SiC discs (10 × 0.2 mm) pre-treated with 20% NaOH were placed as an onlay grafts on calvarial bone. Meanwhile, in group 2 (n=9), SiC discs pre-treated with 20% NaOH (10 × 0.2 mm) were covered with DSM. After 12 weeks, the grafted tissues were harvested and examined using cone-beam computed tomography, mechanical testing, and histologic analysis. RESULTS: Cone-beam computed tomography for group 2 showed more radio-opacity for the remnant of SiC compared with native bone. The surface area and volume of radio-opacity were 2.48 mm2 ± 1.6 and 14.9 ± 7.8 mm3, respectively. The estimated quantitative average surface area of the radio-opacity for group 1 and volume were 2.55 mm2 ± (Sd=3.7) and 11.25 ± (Sd=8.9), respectively. Mechanically, comparable values of the flexural strength and statistically significant higher modulus of elasticity of calvaria in group 1 compared with group 2 and control (P<0.001). Histologically, group 2 region of woven bone was seen close to the lamellar bone (native bone), and there was immature bone present near the implanted SiC. CONCLUSION: The tested construct made of SiC/DSM has potential to osteointegrate into native bone, making it a suitable material for bone augmentation.

Comparison of the Protective Effects of Nebivolol and Metoprolol against LPS-Induced Injury in H9c2 Cardiomyoblasts.

Here, we, for the first time, compared the cardioprotective effects of third-generation vasodilating beta-blocker nebivolol (Neb) and conventional beta-blocker metoprolol (Met) on LPS-induced injury in H9c2 cardiomyoblasts. Our findings denoted that Neb and Met pretreatment diminish LPS-mediated cytotoxicity and oxidative stress. Concomitantly, LPS-triggered inflammatory cytokines activation was significantly suppressed by Neb but not by Met. Pretreatment with either Neb or Met alleviated LPS-mediated mitochondrial impairment by enhancing the expression of genes related to its biogenesis such as PGC-1α, NRF1, and TFAM. On the contrary, Neb but not Met-upregulated mitochondrial fusion-related genes such as OPA, and MFN2. In summary, our findings suggest that Neb and Met treatment significantly ameliorated the LPS-induced cytotoxicity and oxidative stress. Additionally, these findings suggest that Neb but not Met significantly down-regulates LPS-induced proinflammatory factors, probably by enhancing mitochondrial biogenesis and fusion.

The effect of ascorbic acid and cranberry on the bond strength, surface roughness, and surface hardness of bleached enamel with hydrogen peroxide and zinc phthalocyanine activated by photodynamic therapy.

AIM: To evaluate the effect of different bleaching methods 40% (hydrogen peroxide) HP and Zinc Phthalocyanine (ZP) activated by photodynamic therapy (PDT) with the utilization of diverse procedures of reversal (10% ascorbic acid and 6% cranberry solution) on bond values, surface microhardness and surface roughness of bleached enamel surface. MATERIAL AND METHODS: An aggregate of 60 extracted human mandibular molars was gathered and the buccal surface of each specimen was exposed to 2 mm of enamel surface for bleaching with chemical and photoactivated agents with the use of reversal solutions. Specimens were divided into six groups (n = 10) at random- Group 1: samples bleached with 40% HP with 10% ascorbic acid (reversal agent), group 2: ZP activated by PDT with 10% ascorbic acid (reversal agent), group 3: 40% HP with 6% cranberry solution as a reversal agent, group 4: ZP activated by PDT with 6% cranberry solution, group 5: 40% HP and group 6: ZP activated by PDT with no reversal agents. Resin cement restoration was performed via etch and rinse technique and SBS was estimated by using the universal testing machine, SMH by using Vickers hardness tester, and Ra by stylus profilometer. Statistical analysis was executed using the ANOVA test and the Tukey multiple tests (p<0.05). RESULTS: Enamel surface bleached with 40% HP reversed with 10% ascorbic acid displayed the highest SBS while 40% HP with no reversal agent use showed the least SBS. For SMH, ZP activated by PDT when applied on the enamel surface and reversed with 10% ascorbic acid showed the highest SMH while when bleached with 40% HP and reversed with 6% cranberry solution showed the least SMH value. For Ra, Group 3: samples bleached with 40% HP with 6% cranberry solution as reversal agent showed the highest Ra value while bleaching of enamel surface with ZP activated by PDT with 6% cranberry displayed the least Ra value. CONCLUSION: Bleached enamel surface with Zinc Phthalocyanine activated by PDT with the application of 10% ascorbic acid as reversal solution has demonstrated the highest SBS and SMH with acceptable surface roughness for bonding adhesive resin to the enamel surface.

Transient downregulation of NR4A1 leads to impaired osteoblast differentiation through the TGF-ß pathway, and Elesclomol (STA-4783) rescues this phenotype.

Bone formation is regulated by numerous factors, such as transcription factors, cytokines, and extracellular matrix molecules. Human hormone nuclear receptors (hHNR) are a family of ligand-regulated transcription factors that are activated by steroid hormones, such as estrogen and progesterone, and various lipid-soluble signals, including retinoic acid, oxysterols, and thyroid hormone. We found that an hHNR called NR4A1 was the most highly expressed after human MSC differentiation into osteoblasts by whole-genome microarray. NR4A1 knockout decreased the osteoblastic differentiation of hMSCs in terms of ALPL expression and key marker gene expression. Whole-genome microarray analysis further confirmed the decrease in key pathways when we knocked down NR4A1. Further studies with small molecule activators identified a novel molecule called Elesclomol (STA-4783), which could activate and enhance osteoblast differentiation. Elesclomol activation of hMSCs also induced the gene expression of NR4A1 and rescued the phenotype of NR4A1 KD. In addition, Elesclomol activated the TGF-ß pathway by regulating key marker genes. In conclusion, we first identified the role of NR4A1 in osteoblast differentiation and that Elesclomol is a positive regulator of NR4A1 through activation of the TGF-ß signalling pathway.

Profiling of G-Protein Coupled Receptors in Adipose Tissue and Differentiating Adipocytes Offers a Translational Resource for Obesity/Metabolic Research.

G protein-coupled receptors (GPCRs) are expressed essentially on all cells, facilitating cellular responses to external stimuli, and are involved in nearly every biological process. Several members of this family play significant roles in the regulation of adipogenesis and adipose metabolism. However, the expression and functional significance of a vast number of GPCRs in adipose tissue are unknown. We used a high-throughput RT-PCR panel to determine the expression of the entire repertoire of non-sensory GPCRs in mouse white, and brown adipose tissue and assess changes in their expression during adipogenic differentiation of murine adipocyte cell line, 3T3-L1. In addition, the expression of GPCRs in subcutaneous adipose tissues from lean, obese, and diabetic human subjects and in adipocytes isolated from regular chow and high-fat fed mice were evaluated by re-analyzing RNA-sequencing data. We detected a total of 292 and 271 GPCRs in mouse white and brown adipose tissue, respectively. There is a significant overlap in the expression of GPCRs between the two adipose tissue depots, but several GPCRs are specifically expressed in one of the two tissue types. Adipogenic differentiation of 3T3-L1 cells had a profound impact on the expression of several GPCRs. RNA sequencing of subcutaneous adipose from healthy human subjects detected 255 GPCRs and obesity significantly changed the expression of several GPCRs in adipose tissue. High-fat diet had a significant impact on adipocyte GPCR expression that was similar to human obesity. Finally, we report several highly expressed GPCRs with no known role in adipose biology whose expression was significantly altered during adipogenic differentiation, and/or in the diseased human subjects. These GPCRs could play an important role in adipose metabolism and serve as a valuable translational resource for obesity and metabolic research.

Inventing Engineered Organoids for end-stage liver failure patients.

End-stage liver disease (ESLD) is a term used clinically in reference to a group of liver diseases with liver transplantation as the choice of treatment. Due to the limitations of liver transplantation, alternative treatments are needed. The use of primary human hepatocytes represents a valid alternative treatment, but the limitations related to hepatocyte quality, viability, function, conservation, and storage need to be overcome. Transplanted hepatocytes have only been followed for 6-9 months. Therefore, long-term causes of failures are not yet established, including rejection, apoptosis, or other causes. Other alternative therapies to replace liver transplantation include plasmapheresis, hemodiafiltration, and artificial livers. Unfortunately, these methods are highly limited due to availability, high cost, anaphylaxis reaction, development-deposition of immune-complexes, and restricted functionality. Liver organoids, which utilize stem cells instead of 'impractical' adult hepatocytes, may be a solution for the development of a complex bioartificial liver. Recent studies have explored the benefits of differentiating mature hepatocytes from stem cells inside a bioreactor. When the use of human-induced Hepatocytes (hiHeps) was investigated in mouse and pig models of liver failure, liver failure markers were decreased, hepatocyte function indicated by albumin synthesis improved, and survival time increased. Bioartificial liver treatment may decrease the infiltration of inflammatory cells into liver tissue by down-regulating pro-inflammatory cytokines.

Vitamin C stimulates RNA expression of human gingival fibroblasts proliferation and adhesion in cigarette smokers: An in vitro study.

Background: Smoking and the severity of periodontal disease have long been associated. In Saudi Arabia, tobacco smoking is rising, contributing to the increased demand for products that counter its detrimental effects. The antioxidant properties of vitamin C (vit C) make it a powerful countermeasure to tobacco toxicity. Observation of these effects on human gingival fibroblasts (hGFs) would suggest use of vitamin C in future dental applications. Aim: To examine the proliferation, adhesion, and expression of extracellular RNA in human gingival fibroblasts extracted from cigarette smokers when compared to never-smokers, in association with vitamin C. Materials and Methods: Human gingival fibroblasts were extracted from Periodontal free sites of healthy adult male participants. Group 1; consisted of Heavy cigarette smokers (n = 1) while group 2 was never-smokers (n = 1). Collected cells were cultured and subcultured in supplemented growth medium. Vitamin C was then induced in the medium at the experimental sixth passage. RNA expression analysis using quantitative reverse transcriptase-polymerase chain reaction was performed to analyze the adhesion, proliferation, and extracellular matrix expression. Results: Expression of the adhesion gene (CD44) in the smoker group was significantly downregulated than never-smoker group (p-value = 0.024). After the induction of vitamin C, the smoker samples showed a significant improvement in their gene expression levels. The extracellular genes involved in this study (COL1A1, LAMA3, and TGFB3) were significantly affected by the smoking status. In addition, the proliferation of MK167 and CCNB1 genes in smokers and never-smokers was increased. Conclusion: Cigarette smoking affects the overall properties of human gingival fibroblasts' adhesion, proliferation, and extra-cellular matrix formation. Furthermore, the addition of vitamin C affects these cellular properties in a positive manner.

The additive effect of iloprost on the biological properties of Mineral trioxide aggregate on mesenchymal stem cells.

BACKGROUND/PURPOSE: Iloprost has been proposed as a potential biomaterial owing to angiogenic and odontogenic properties. However, the liquid form can limit its use during clinical applications. Mineral trioxide aggregate (MTA) has been used for various dental applications in which cell-material interaction is essential. This study aimed to investigate additive effects of iloprost on the biological properties of MTA on the viability, attachment, migration and differentiation of human mesenchymal stem cells (hMSCs). MATERIALS AND METHODS: Standardized human dentin disks were prepared. MTA was prepared by mixing distilled water or iloprost solution, and the lumen of the disks was filled with MTA or MTA-iloprost. hMSCs on disk alone and hMSCs on culture plates were used as controls. Cell viability and attachment were measured after 1, 7 and 14 days using AlamarBlue assay and scanning electron microscopy (SEM). Cell migration in MTA or MTA-iloprost extracts was determined using a wound-healing model.Osteogenic differentiation was evaluated by real-time reverse transcriptase polymerase chain reaction for alkaline phosphatase (ALP), bone sialoprotein (BSP), osteocalcin (OCN), and osteopontin (OSP) gene expressions after 7 and 14 days of osteogenic induction. RESULTS: Cells on MTA-iloprost surface showed similar viability with MTA at 1 and 14 days but enhanced cellular viability and cell spreading compared to MTA at 7 days (p < 0.05). Cell migration was similar by MTA-iloprost and MTA extracts (p > 0.05). MTAiloprost significantly upregulated BSP, OCN and OSP expressions compared to MTA (p < 0.05). CONCLUSION: The addition of iloprost to MTA improved the initial cell viability and osteogenic potential of hMSCs.

CLINICAL VALIDATION OF ANKRD36 MUTATIONS AS A NOVEL BIOMARKER FOR MONITORING EARLY PROGRESSION AND TIMELY CLINICAL INTERVENTIONS IN BLAST CRISIS CML.

Background: Chronic Myeloid Leukemia (CML) is initiated in the bone marrow due to the chromosomal translocation t(9;22), resulting in the fusion oncogene BCR-ABL. Tyrosine kinase inhibitors (TKIs) targeting BCR-ABL have transformed fatal CML into an almost curable disease. However, TKIs lose efficacy during disease progression, and the mechanism of CML progression remains to be fully understood. Additionally, common molecular biomarkers for CML progression are lacking. Our studies previously detected ANKRD36 (c.1183_1184 delGC and c.1187_1188 dupTT) associated exclusively with advanced phase CML. However, clinical validation of this finding was pending. Therefore, this study aimed to clinically validate mutated ANKRD36 as a novel biomarker of CML progression. Materials and Methods: The study enrolled 124 patients in all phases of CML, recruited from Mayo Hospital and Hameed Latif Hospital in Lahore, Punjab, between January 2019 and August 2021. All response criteria were adopted from the European LeukemiaNet guideline 2020. Informed consent was obtained from all study subjects. The study was approved by scientific and ethical review committees of all participating centers.Sanger sequencing was employed to detect ANKRD36 mutations in CML patients in accelerated phase (AP) (n=11) and blast crisis (BC) (n=10), with chronic-phase CML (CP-CML) patients as controls (n=103). Samples were processed using Big Dye Terminator Cycle Sequencing Ready Reaction kits and sequenced using ABI Prism 3730 Genetic Analyzer, and sequencing using forward and reverse primers for ANKRD36. Results: During our study, 17% of CML patients progressed to advanced phases AP-CML n=11 (8.9%) and BC-CML n=10 (8.1%). The chronic- and advanced-phase patients showed significant difference with respect to male-to-female ratio, hemoglobin level, WBC count, and platelet count. Sanger sequencing detected ANKRD36 mutations c. 1183 1184 delGC and c. 1187 1185 dupTT exclusively in all AP- and BC-CML patients but in none of the CP-CML patients. Nevertheless, mutations status was not associated with male-to-female ratio, hemoglobin level, WBC count, and platelet count, which makes ANKRD32 as an independent predictor of early and terminal disease progression in CML. Conclusions: The study confirms ANKRD36 as a novel genomic biomarker for early and late CML progression. Further prospective studies should be carried out in this regard. ANKRD36, although fully uncharacterized in humans, shows the highest expression in bone marrow, particularly myeloid cells. Functional integrated genomic studies are recommended to further explore the role of ANKRD36 in the biology and pathogenesis of CML.

Integrated Genomic Analysis Identifies ANKRD36 Gene as a Novel and Common Biomarker of Disease Progression in Chronic Myeloid Leukemia.

Background: Chronic myeloid leukemia (CML) is initiated in bone marrow due to chromosomal translocation t(9;22) leading to fusion oncogene BCR-ABL. Targeting BCR-ABL by tyrosine kinase inhibitors (TKIs) has changed fatal CML into an almost curable disease. Despite that, TKIs lose their effectiveness due to disease progression. Unfortunately, the mechanism of CML progression is poorly understood and common biomarkers for CML progression are unavailable. This study was conducted to find novel biomarkers of CML progression by employing whole-exome sequencing (WES). Materials and Methods: WES of accelerated phase (AP) and blast crisis (BC) CML patients was carried out, with chronic-phase CML (CP-CML) patients as control. After DNA library preparation and exome enrichment, clustering and sequencing were carried out using Illumina platforms. Statistical analysis was carried out using SAS/STAT software version 9.4, and R package was employed to find mutations shared exclusively by all AP-/BC-CML patients. Confirmation of mutations was carried out using Sanger sequencing and protein structure modeling using I-TASSER followed by mutant generation and visualization using PyMOL. Results: Three novel genes (ANKRD36, ANKRD36B and PRSS3) were mutated exclusively in all AP-/BC-CML patients. Only ANKRD36 gene mutations (c.1183_1184 delGC and c.1187_1185 dupTT) were confirmed by Sanger sequencing. Protein modeling studies showed that mutations induce structural changes in ANKRD36 protein. Conclusions: Our studies show that ANKRD36 is a potential common biomarker and drug target of early CML progression. ANKRD36 is yet uncharacterized in humans. It has the highest expression in bone marrow, specifically myeloid cells. We recommend carrying out further studies to explore the role of ANKRD36 in the biology and progression of CML.

Assessment of novel surgical procedures using decellularised muscle and bioactive ceramic: a histological analysis.

Tissue regeneration and neovascularisation in cases of major bone loss is a challenge in maxillofacial surgery. The hypothesis of the present study is that the addition of resorbable bioactive ceramic Silica Calcium Phosphate Cement (SCPC) to Declluraized Muscle Scaffold (DSM) can expedite bone formation and maturation. Two surgical defect models were created in 18 nude transgenic mice. Group 1(n = 6), with a 2-mm decortication calvarial defect, was treated with a DSM/SCPC sheet over the corticated bone as an onlay then seeded with human Mesenchymal Stromal Cells hMSC in situ. In Group 2 (n = 6), a critical size (4 mm) calvarial defect was made and grafted with DSM/SCPC/in situ human bone marrow stromal cells (hMSCs). The control groups included Group 3 (n = 3) animals, with a 2-mm decortication defect treated with an onlay DSM sheet, and Group 4 (n = 3) animals, treated with critical size defect grafted with plain DSM. After 8 weeks, bone regeneration in various groups was evaluated using histology, immunohistochemistry and histomorphometry. New bone formation and maturation was superior in groups treated with DSM/SCPC/hMSC. The DMS/SCPC scaffold has the ability to augment and induce bone regeneration and neovascularisation in cases of major bone resorption and critical size defects.

An empirical demonstration of the effect of study design on density estimations.

The simultaneous development of technology (e.g. camera traps) and statistical methods, particularly spatially capture-recapture (SCR), has improved monitoring of large mammals in recent years. SCR estimates are known to be sensitive to sampling design, yet existing recommendations about trap spacing and coverage are often not achieved, particularly for sampling wide-ranging and rare species in landscapes that allow for limited accessibility. Consequently, most camera trap studies on large wide-ranging carnivores relies on convenience or judgmental sampling, and often yields compromised results. This study attempts to highlight the importance of carefully considered sampling design for large carnivores that, because of low densities and elusive behavior, are challenging to monitor. As a motivating example, we use two years of snow leopard camera trapping data from the same areas in the high mountains of Pakistan but with vastly different camera configurations, to demonstrate that estimates of density and space use are indeed sensitive to the trapping array. A compact design, one in which cameras were placed much closer together than generally recommended and therefore have lower spatial coverage, resulted in fewer individuals observed, but more recaptures, and estimates of density and space use were inconsistent with expectations for the region. In contrast, a diffuse design, one with larger spacing and spatial coverage and more consistent with general recommendations, detected more individuals, had fewer recaptures, but generated estimates of density and space use that were in line with expectations. Researchers often opt for compact camera configurations while monitoring wide-ranging and rare species, in an attempt to maximize the encounter probabilities. We empirically demonstrate the potential for biases when sampling a small area approximately the size of a single home range-this arises from exposing fewer individuals than deemed sufficient for estimation. The smaller trapping array may also underestimate density by significantly inflating [Formula: see text]. On the other hand, larger trapping array with fewer detectors and poor design induces uncertainties in the estimates. We conclude that existing design recommendations have limited utility on practical grounds for devising feasible sampling designs for large ranging species, and more research on SCR designs is required that allows for integrating biological and habitat traits of large carnivores in sampling framework. We also suggest that caution should be exercised when there is a reliance on convenience sampling.

Selenium Nanoparticles by Moderating Oxidative Stress Promote Differentiation of Mesenchymal Stem Cells to Osteoblasts.

PURPOSE: Redox homeostasis plays an important role in the osteogenic differentiation of human mesenchymal stem cells (hMSCs) for bone engineering. Oxidative stress (OS) is believed to induce osteoporosis by changing bone homeostasis. Selenium nanoparticles (SeNPs), an antioxidant with pleiotropic pharmacological activity, prevent bone loss. However, the molecular mechanism underlying the osteogenic activity during hMSC-SeNP interaction is unclear. METHODS: This study assessed the effects of different concentrations (25, 50, 100, and 300 ng/mL) of SeNPs on the cell viability and differentiation ability of human embryonic stem cell-derived hMSCs. In addition, we analyzed OS markers and their effect on mitogen-activated protein kinase (MAPK) and Forkhead box O3 (FOXO3) during osteogenesis. RESULTS: SeNPs increased the cell viability of hMSCs and induced their differentiation toward an osteogenic over an adipogenic lineage by enhancing osteogenic transcription and mineralization, while inhibiting Nile red staining and adipogenic gene expression. By preventing excessive reactive oxygen species accumulation, SeNPs increased antioxidant levels in hMSCs undergoing osteogenesis compared to untreated cells. In addition, SeNPs significantly upregulated the gene and protein expression of phosphorylated c-Jun N-terminal kinase (JNK) and FOXO3a, with no significant change in the expression levels of extracellular signal-related kinase (ERK) and p38 MAPK. CONCLUSION: The results approved that low concentrations of SeNPs might enhance the cell viability and osteogenic potential of hMSCs by moderating OS. Increased JNK and FOXO3a expression shows that SeNPs might enhance osteogenesis via activation of the JNK/FOXO3 pathway. In addition, SeNP co-supplementation might prevent bone loss by enhancing osteogenesis and, thus, can be an effective candidate for treating osteoporosis through cell-based therapy.

Discovery and Protein Modeling Studies of Novel Compound Mutations Causing Resistance to Multiple Tyrosine Kinase Inhibitors in Chronic Myeloid Leukemia.

OBJECTIVE: BCR-ABL fusion oncogene is the hallmark of chronic myeloid leukemia (CML), causing genomic instability which leads to accumulation of mutations in BCR-ABL as well as other genes. BCR-ABL mutations are the cause of tyrosine kinase inhibitors (TKIs) resistance in CML. Recently, compound BCR-ABL mutations have been reported to resist all FDA approved TKIs. Therefore, finding novel compound BCR-ABL mutations can help and clinically manage CML. Therefore, our objective was to find out novel drug-resistant compound BCR-ABL mutations in CML and carry out their protein modelling studies. METHODOLOGY: Peripheral blood samples were collected from ten imatinib resistant CML patients receiving nilotinib treatment. BCR-ABL transcript mutations were investigated by employing capillary sequencing. Patient follow-up was carried out using European LeukemiaNet guidelines. Protein modeling  studies were carried out for new compound mutations using PyMol to see the effects of mutations at structural level. RESULTS: A novel compound mutation (K245N mutation along with G250W mutation) and previously known T351I utation was detected in two of the nilotinib resistance CML patients respectively while in the rest of 8 nilotinib responders, no resistant mutations were detected. Protein modelling studies indicated changes in BCR-ABL mutant protein which may have negatively impacted its binding with nilotinib leading to drug resistance. CONCLUSION: We report a novel nilotinib resistant BCR-ABL compound mutation (K245N along with G250W mutation) which impacts structural modification in BCR-ABL mutant protein leading to drug resistance. As compound mutations pose a new threat by causing resistance to all FDA approved tyrosine kinase inhibitors in BCR-ABL+ leukemias, our study opens a new direction for in vitro characterization of novel BCR-ABL compound mutations and their resistant to second  generation and third generation TKIs.

Whole exome sequencing identifies a novel FANCD2 gene splice site mutation associated with disease progression in chronic myeloid leukemia: Implication in targeted therapy of advanced phase CML.

Tyrosine Kinase Inhibitors (TKIs) have significantly improved the clinical outcome of BCR-ABL+ Chronic Phase-Chronic Myeloid Leukemia (CP-CML). Nonetheless, approximately one-third of the CP-CML patient's progress to advanced phases of CML (accelerated and blast phase). Impaired DNA repair including mutations in Fanconi anemia (FA) pathway genes are responsible for progression of many cancers. Nevertheless, FA-pathways genes have never been reported in myeloid cancers. Hence, this study was aimed to discover DNA repair genes associated with CML progression. AP-CML patients were subjected to whole exome sequencing along with appropriate controls. A novel splice site FANCD2 mutation was detected. FANCD2 is a well-known FA-pathway gene with established role in DNA repair. This is first report of FA-pathway DNA repair genes in myeloid cancers that can serve as a novel marker of CML progression to clinically intervene CML progression. Further studies are needed to establish the functional role of FANCD2 in CML progression that can provide novel insights into CML pathogenesis. This study also indicates that a combination TKIs and Poly (ADP-ribose) polymerase (PARP) inhibitors like Olaparib (FDA approved anti-cancer drug for FA-pathway gene mutations) could improve the clinical outcome CML patients in accelerated and blast-crisis phases of the disease.

Long term outcome of chronic myeloid leukemia patients treated with imatinib: Report from a developing country.

The outcome of chronic myeloid leukemia has been greatly improved by the use of Imatinib (IM), a selective BCR/ABL kinase inhibitor. The aim of present study was to report long term follow-up & outcome of IM-treated CML patients along with their clinicopathological features, risk group stratification, adverse events and to compare it with CML patients reported from western countries. The mean follow-up of 123 CML patients was 5.5 years in present study, who were treated with frontline IM 400mg daily in a tertiary care hospital in Pakistan. Risk stratification scores, response to treatment (ELN guidelines) and survival outcomes estimated by Kaplan-Meier analysis. Mean age: 35 years (9-67 years) and M: F: 1.5:1, mean follow up time: 5.5 years (1-15 years). Overall survival (OS): at 5.5, 8, 10 and 12 years were 93%, 88%, 81% and 73%, respectively. Progressions free survival (PFS) was 95%, 83%, 83% and 78% at 5.5, 8, 10 and 12 years, respectively. OS estimate by Sokal score was significant (P-value: 0.0019). Additional chromosomal aberrations: 1.6%. Eighteen (14.6%) patients progressed to AP/BC. Adverse events were moderate and tolerable. We present findings from a long term follow up of CML patients treated with IM in a developing country. CML mean age at onset was considerably lower than the western populations. Furthermore, 5.5 years OS are comparable to western CML population. IM in our patients as frontline choice proved to be very effective. IM was found to be well tolerated, safe with manageable moderate side effects.

Transgelin is a poor prognostic factor associated with advanced colorectal cancer (CRC) stage promoting tumor growth and migration in a TGFß-dependent manner.

Colorectal cancer (CRC) is the fourth most common cancer type globally. Investigating the signaling pathways that maintain cancer cell phenotype can identify new biomarkers for targeted therapy. Aberrant transforming growth factor-ß (TGFß) signaling has been implicated in CRC progression, however, the exact mechanism by which TGFß exerts its function is still being unraveled. Herein, we investigated TAGLN expression, prognostic value, and its regulation by TGFß in CRC. While TAGLN was generally found to be downregulated in CRC, elevated expression of TAGLN was associated with advanced CRC stage and predicted poor overall survival (hazard ratio (HR) = 1.8, log-rank test P-value = 0.014) and disease-free survival (HR = 1.6, log-rank test P-value = 0.046), hence implicating TAGLN as poor prognostic factor in CRC. Forced expression of TAGLN was associated with enhanced CRC cell proliferation, clonogenic growth, cell migration and in vivo tumor formation in immunocompromised mice, while targeted depletion of TAGLN exhibited opposing biological effects. Global gene expression profiling of TAGLN-overexpressing or TAGLN-deficient CRC cell lines revealed deregulation of multiple cancer-related genes and signaling pathways. Transmission electron microscopy (TEM) revealed ultrastructural changes due to loss of TAGLN, including disruption of actin cytoskeleton organization and aberrant actin filament distribution. Hierarchical clustering, principle component, and ingenuity pathway analyses revealed distinct molecular profile associated with TAGLNhigh CRC patients with remarkable activation of a number of mechanistic networks, including SMARCA4, TGFß1, and P38 MAPK. The P38 MAPK was the top predicted upstream regulator network promoting cell movement through regulation of several intermediate molecules, including TGFß1. Concordantly, functional categories associated with cellular movement and angiogenesis were also enriched in TAGLNhigh CRC, supporting a model for the molecular mechanisms linking TGFß-induced upregulation of TAGLN and CRC tumor progression and suggesting TAGLN as potential prognostic marker associated with advanced CRC pathological stage.