Dynamics and recognition of homeodomain containing protein-DNA complex of IRX4.

Iroquois Homeobox 4 (IRX4) belongs to a family of homeobox TFs having roles in embryogenesis, cell specification, and organ development. Recently, large scale genome-wide association studies and epigenetic studies have highlighted the role of IRX4 and its associated variants in prostate cancer. No studies have investigated and characterized the structural aspect of the IRX4 homeodomain and its potential to bind to DNA. The current study uses sequence analysis, homology modeling, and molecular dynamics simulations to explore IRX4 homeodomain-DNA recognition mechanisms and the role of somatic mutations affecting these interactions. Using publicly available databases, gene expression of IRX4 was found in different tissues, including prostate, heart, skin, vagina, and the protein expression was found in cancer cell lines (HCT166, HEK293), B cells, ascitic fluid, and brain. Sequence conservation of the homeodomain shed light on the importance of N- and C-terminal residues involved in DNA binding. The specificity of IRX4 homodimer bound to consensus human DNA sequence was confirmed by molecular dynamics simulations, representing the role of conserved amino acids including R145, A194, N195, S190, R198, and R199 in binding to DNA. Additional N-terminal residues like T144 and G143 were also found to have specific interactions highlighting the importance of N-terminus of the homeodomain in DNA recognition. Additionally, the effects of somatic mutations, including the conserved Arginine (R145, R198, and R199) residues on DNA binding elucidated the importance of these residues in stabilizing the protein-DNA complex. Secondary structure and hydrogen bonding analysis showed the roles of specific residues (R145, T191, A194, N195, R198, and R199) in maintaining the homogeneity of the structure and its interaction with DNA. The differences in relative binding free energies of all the mutants shed light on the structural modularity of this protein and the dynamics behind protein-DNA interaction. We also have predicted that the C-terminal sequence of the IRX4 homeodomain could act as a potential cell-penetrating peptide, emphasizing the role these small peptides could play in targeting homeobox TFs.

Unravelling the Role of Iroquois Homeobox 4 and its Interplay with Androgen Receptor in Prostate Cancer.

Genome-wide association studies have linked Iroquois-Homeobox 4 (IRX4) as a robust expression quantitative-trait locus associated with prostate cancer (PCa) risk. However, the intricate mechanism and regulatory factors governing IRX4 expression in PCa remain poorly understood. Here, we unveil enrichment of androgen-responsive gene signatures in metastatic prostate tumors exhibiting heightened IRX4 expression. Furthermore, we uncover a novel interaction between IRX4 and the androgen receptor (AR) co-factor, FOXA1, suggesting that IRX4 modulates PCa cell behavior through AR cistrome alteration. Remarkably, we identified a distinctive short insertion-deletion polymorphism (INDEL), upstream of the IRX4 gene that differentially regulates IRX4 expression through the disruption of AR binding. This INDEL emerges as the most significant PCa risk-associated variant within the 5p15 locus, in a genetic analysis involving 82,591 PCa cases and 61,213 controls and was associated with PCa survival in patients undergoing androgen-deprivation therapy. These studies suggest the potential of this INDEL as a prognostic biomarker for androgen therapy in PCa and IRX4 as a potential therapeutic target in combination with current clinical management.

Towards Real-time Multiplexed Bioimpedance Tumour-Tissue Margin Analysis.

Bioimpedance varies with physical tissue characteristics. As such it can be used for real-time tissue discrimination. This has led to its application as a surgical mapping tool to differentiate between healthy and abnormal tissue intraoperatively during tumour resection. Here, we build on previous work implementing a probe-based tetrapolar bioimpedance systems demonstrator, now extracting additional information for margin analysis with imperfect bioimpedance visibility. Through finite element analysis, we show preliminary findings using a single measurement with a multiplexed tetrapolar bioimpedance probe for identifying tissue boundaries, applied to porcine tissue as a surrogate for a tumour-tissue interface.

In vitro screening of EPS-producing Streptococcus thermophilus strains for their probiotic potential from Dahi.

Dahi is a very common and traditional fermented dairy product in Pakistan and its neighboring countries, it represents a rich source for the isolation of many new strains of lactic acid bacteria (LAB). The major objective of this study was to evaluate the probiotic potential of novel exopolysaccharide (EPS)-producing strains of S. thermophilus isolated from Dahi, sold in the local markets of Rawalpindi and Islamabad, Pakistan. In this study, 32 isolates of S. thermophilus were initially isolated from Dahi and out of these, 10 identified strains were further screened for their EPS-producing ability. Maximum EPS production was estimated for RIY strain (133.0 ± 0.06), followed by RIH4 strain (103.83 ± 0.76) and RIRT2 strain (95.77 ± 0.22), respectively. Thereafter, in vitro studies revealed that these newly identified EPS-producing strains of S. thermophilus fulfilled the basic requirements for probiotic functions; including resistance to harsh conditions of GIT, good cell surface hydrophobicity, auto-aggregation, and co-aggregation, especially against L. monocytogenes. Finally, the safety assessment displayed that these strains were also sensitive to clinical antibiotics, including vancomycin. Thus, these selected EPS strains of S. thermophilus act as potential candidates for biostabilizers in the preparation of consumer-friendly fermented probiotic milk products.

In Vitro Digestion and Colonic Fermentation of UHT Treated Faba Protein Emulsions: Effects of Enzymatic Hydrolysis and Thermal Processing on Proteins and Phenolics.

Faba bean (Vicia faba L.) protein is a new plant protein alternative source with high nutrient content especially protein and phenolic compounds. The present study investigated physicochemical properties, phenolic content, antioxidant potential, and short chain fatty acids (SCFAs) production during in vitro digestion and colonic fermentation of faba bean hydrolysates and oil-in-water (O/W) emulsions. Results indicate that the enzymic hydrolysates of faba proteins exhibited higher protein solubility, increased electronegativity, and decreased surface hydrophobicity than native faba protein. O/W emulsions showed improved colloidal stability for the faba protein hydrolysates after ultra-high temperature processing (UHT). Furthermore, UHT processing preserved total phenolic content, DPPH and ABTS radical scavenging abilities while decreasing total flavonoid content and ferric reducing power. Besides, the release of phenolic compounds in faba bean hydrolysates (FBH) and emulsions (FBE) improved after intestinal digestion by 0.44 mg GAE/g and 0.55 mg GAE/g, respectively. For colonic fermentation, FBH demonstrated an approximately 10 mg TE/g higher ABTS value than FBE (106.45 mg TE/g). Total SCFAs production of both FBH and FBE was only 0.03 mM. The treatment of FBH with 30 min enzymatic hydrolysis displayed relatively higher antioxidant capacities and SCFAs production, indicating its potential to bring more benefits to gut health. Overall, this study showed that enzymic hydrolysis of faba proteins not only improved the colloidal emulsion stability, but also released antioxidant capacity during in vitro digestibility and colonic fermentation. Colonic fermentation metabolites (SCFAs) were related to the degree of hydrolysis for both FBH and FBE. Additional studies are required to further elucidate and differentiate the role of phenolics during faba protein processing and digestion stages in comparison to contributions of peptides, amino acids and microelements to digestion rates, antioxidant capacities and colonial SCFA production.

Design and Characterization of a Cell-Penetrating Peptide Derived from the SOX2 Transcription Factor.

SOX2 is an oncogenic transcription factor overexpressed in nearly half of the basal-like triple-negative breast cancers associated with very poor outcomes. Targeting and inhibiting SOX2 is clinically relevant as high SOX2 mRNA levels are positively correlated with decreased overall survival and progression-free survival in patients affected with breast cancer. Given its key role as a master regulator of cell proliferation, SOX2 represents an important scaffold for the engineering of dominant-negative synthetic DNA-binding domains (DBDs) that act by blocking or interfering with the oncogenic activity of the endogenous transcription factor in cancer cells. We have synthesized an interference peptide (iPep) encompassing a truncated 24 amino acid long C-terminus of SOX2 containing a potential SOX-specific nuclear localization sequence, and the determinants of the binding of SOX2 to the DNA and to its transcription factor binding partners. We found that the resulting peptide (SOX2-iPep) possessed intrinsic cell penetration and promising nuclear localization into breast cancer cells, and decreased cellular proliferation of SOX2 overexpressing cell lines. The novel SOX2-iPep was found to exhibit a random coil conformation predominantly in solution. Molecular dynamics simulations were used to characterize the interactions of both the SOX2 transcription factor and the SOX2-iPep with FGF4-enhancer DNA in the presence of the POU domain of the partner transcription factor OCT4. Predictions of the free energy of binding revealed that the iPep largely retained the binding affinity for DNA of parental SOX2. This work will enable the future engineering of novel dominant interference peptides to transport different therapeutic cargo molecules such as anti-cancer drugs into cells.

Integrative Transcriptome-Wide Analyses Uncover Novel Risk-Associated MicroRNAs in Hormone-Dependent Cancers.

BACKGROUND: Hormone-dependent cancers (HDC) are among the leading causes of death worldwide among both men and women. Some of the established risk factors of HDC include unhealthy lifestyles, environmental factors, and genetic influences. Numerous studies have been conducted to understand gene-cancer associations. Transcriptome-wide association studies (TWAS) integrate data from genome-wide association studies (GWAS) and gene expression (expression quantitative trait loci - eQTL) to yield meaningful information on biological pathways associated with complex traits/diseases. Recently, TWAS have enabled the identification of novel associations between HDC risk and protein-coding genes. METHODS: In the present study, we performed a TWAS analysis using the summary data-based Mendelian randomization (SMR)-heterogeneity in dependent instruments (HEIDI) method to identify microRNAs (miRNAs), a group of non-coding RNAs (ncRNAs) associated with HDC risk. We obtained eQTL and GWAS summary statistics from the ncRNA-eQTL database and the National Human Genome Research Institute-European Bioinformatics Institute (NHGRI-EBI) GWAS Catalog. RESULTS: We identified 13 TWAS-significant miRNAs at cis regions (±1 Mb) associated with HDC risk (two, five, one, two, and three miRNAs for prostate, breast, ovarian, colorectal, and endometrial cancers, respectively). Among them, eight novel miRNAs were recognized in HDC risk. Eight protein-coding genes targeted by TWAS-identified miRNAs (SIRT1, SOX4, RUNX2, FOXA1, ABL2, SUB1, HNRNPH1, and WAC) are associated with HDC functions and signaling pathways. CONCLUSION: Overall, identifying risk-associated miRNAs across a group of related cancers may help to understand cancer biology and provide novel insights into cancer genetic mechanisms. This customized approach can be applied to identify significant miRNAs in any trait/disease of interest.

Quality Attributes of Ultra-High Temperature-Treated Model Beverages Prepared with Faba Bean Protein Concentrates.

The objective of this research was to develop a model faba bean drink with a high concentration of protein (>4% w/w). The protein molecular weights and frequency for both faba and soy were assessed using SDS-PAGE. Results showed similarities in the protein molecular weight of both faba and soy (mainly 11S globulin ~Glycinin and 7S globulin ~ß-conglycinin). Thus, faba can be considered as a potential soy replica in plant-based milk beverages. Oil-in-water emulsions (5-8% w/w available protein) were prepared using faba bean protein concentrate (FPC), 1% sunflower oil, and 0.2% sunflower lecithin. These emulsions were used as model beverages and were further investigated for UHT processibility, stability, and physicochemical properties. The physicochemical properties of emulsions at various processing stages viz., coarse emulsification, homogenisation, and UHT, were measured. An increase in the protein concentration and thermal treatment resulted in an increased oil droplet size, coalescence and flocculation, and protein aggregation. Lower protein concentrations viz., 5-6%, showed greater negative ζ-potential, and thereby, high dispersibility through enhanced electrostatic repulsions than those of higher concentrations (7-8%). Furthermore, an increase in protein concentration and UHT treatment resulted in an increased creaming index. In total, 21 different volatile compounds were detected and quantified, representing different chemical classes, namely alcohols, aldehydes, ketones, esters, furan, and acids. These volatiles have major consequences for the overall flavour chemistry of the model beverage product. Overall, this study showed the potential for application of faba bean as a protein source in UHT-treated legume-based beverages and identified areas for further development.

SWATH-MS Based Proteomic Profiling of Prostate Cancer Cells Reveals Adaptive Molecular Mechanisms in Response to Anti-Androgen Therapy.

Prostate cancer (PCa) is the second most common cancer affecting men worldwide. PCa shows a broad-spectrum heterogeneity in its biological and clinical behavior. Although androgen targeted therapy (ATT) has been the mainstay therapy for advanced PCa, it inevitably leads to treatment resistance and progression to castration resistant PCa (CRPC). Thus, greater understanding of the molecular basis of treatment resistance and CRPC progression is needed to improve treatments for this lethal phenotype. The current study interrogated both proteomics and transcriptomic alterations stimulated in AR antagonist/anti-androgen (Bicalutamide and Enzalutamide) treated androgen-dependent cell model (LNCaP) in comparison with androgen-independent/castration-resistant cell model (C4-2B). The analysis highlighted the activation of MYC and PSF/SFPQ oncogenic upstream regulators in response to the anti-androgen treatment. Moreover, the study revealed anti-androgen induced genes/proteins related to transcription/translation regulation, energy metabolism, cell communication and signaling cascades promoting tumor growth and proliferation. In addition, these molecules were found dysregulated in PCa clinical proteomic and transcriptomic datasets, suggesting their potential involvement in PCa progression. In conclusion, our study provides key molecular signatures and associated pathways that might contribute to CRPC progression despite treatment with anti-androgens. Such molecular signatures could be potential therapeutic targets to improve the efficacy of existing therapies and/or predictive/prognostic value in CRPC for treatment response.

A New Era of Prostate Cancer Precision Medicine.

Prostate cancer is the second most common male cancer affecting Western society. Despite substantial advances in the exploration of prostate cancer biomarkers and treatment strategies, men are over diagnosed with inert prostate cancer, while there is also a substantial mortality from the invasive disease. Precision medicine is the management of treatment profiles across different cancers predicting therapies for individual cancer patients. With strategies including individual genomic profiling and targeting specific cancer pathways, precision medicine for prostate cancer has the potential to impose changes in clinical practices. Some of the recent advances in prostate cancer precision medicine comprise targeting gene fusions, genome editing tools, non-coding RNA biomarkers, and the promise of liquid tumor profiling. In this review, we will discuss these recent scientific advances to scale up these approaches and endeavors to overcome clinical barriers for prostate cancer precision medicine.

Solidification of liquid Modified Apple Polysaccharide by its adsorption on solid porous carriers through spray drying and evaluation of its potential as binding agent for tablets.

In the present study application of Modified Apple Polysaccharide (MAP) as tablet binder was evaluated. Liquid MAP was extracted from apple and solidified by adsorbing it on porous surface of Aerosil-200 and trehalose and this dispersion was dried using spray dryer. The concentration of excipients as well as spray drying conditions was optimised by using Box Behnken Design to achieve desirable powder characteristics. The optimised batch of solid MAP was characterized by DSC, PXRD, SEM, and FT-IR studies that confirmed complete adsorption of liquid MAP on the surface of Aerosil-200 and trehalose. This solid MAP was investigated for its binding efficacy for tablet formulation and its binding potential was compared with acacia and polyvinyl pyrrolidone K-30. Mesalamine (model drug) granules containing different concentration of binders were prepared by wet granulation. The granules were evaluated for micromeritic properties and results were found within the pharmacopoeial limits. The prepared tablets were subjected for post compression studies such as hardness, friability, disintegration, dissolution, physical stability, content uniformity and percentage elastic recovery and their results were found good. At 2.5% w/w concentration in tablet, the solid MAP has shown shorter disintegration time and faster dissolution profile as compared to other concentrations used including good physico-mechanical properties.

In-vitro and In-vivo Pharmacokinetic Evaluation of Guar Gum-Eudragit® S100 Based Colon-targeted Spheroids of Sulfasalazine Co-administered with Probiotics.

BACKGROUND: Polysaccharide based delivery systems have been successfully used to target drugs to colon. In some recent reports, the superiority of concomitant administration of probiotics with such systems has been established. However, the pharmacokinetics of such symbiotic therapy remain unexplored hitherto. METHODS: This study deciphers the pharmacokinetic parameters of guar gum based colon targeted spheroids of sulfasalazine with co-administration of probiotics in experimental rats. Thirty rats were divided into five groups using Latin square design. These were subjected to treatment with delayed release formulation, uncoated spheroids, coated spheroid and coated spheroids along with probiotics. RESULTS: In case of delayed release formulation, negligible presence of sulfasalazine in plasma was observed in first 2h, followed by significant increase in sulfasalazine concentration after 3h. Higher plasma concentrations of sulfasalazine were detected for uncoated spheroids with and without probiotics. Negligible release of drug upto 5h and delayed Tmax in case of guar-gum coated sulfasalazine spheroids with or without probiotics clearly indicated successful formulation of colon targeted spheroids. Further, for coated spheroids (both with and without probiotics), the value of Tmax is found to be significantly higher than those with the other treatments. CONCLUSION: Colon targeted spheroids were therefore, found to reduce absorption of drug which, in turn, is expected to reduce the side effects as only local action in colon is required for treatment of colitis. This is the first report on pharmacokinetic study of a colon targeted delivery system co-administered with probiotics.

Novel biorelevant dissolution medium as a prognostic tool for polysaccharide-based colon-targeted drug delivery system.

To overcome the limitations of the conventionally used methods for evaluation of orally administered colon-targeted delivery systems, a novel dissolution method using probiotics has been recently reported. In the present study, universal suitability of this medium composed of five different probiotics is established. Different delivery systems - mini tablets, liquisolid compacts, and microspheres coated with different polysaccharides - were prepared and subjected to sequential dissolution testing in medium with and without microbiota. The results obtained from fluid thioglycollate medium (FTM)-based probiotic medium for all the polysaccharide-based formulations showed statistically similar dissolution profile to that in the rat and goat cecal content media. Hence, it can be concluded that the developed FTM-based probiotic medium, once established, may eliminate the need for further animal sacrifice in the dissolution testing of polysaccharide-based colon-targeted delivery system.

Protective effect of co-administration of curcumin and sildenafil in alcohol induced neuropathy in rats.

Neuropathic pain associated with chronic alcohol consumption is a medico-socioeconomical problem that affects both central and peripheral nervous system and has no satisfactory treatment till date. The present study was designed to investigate the protective effect of co-administration of curcumin and sildenafil on alcohol induced neuropathic pain in rats. In order to carry out this, ethanol (35% v/v, 10g/kg, p.o.) was administered for 10 weeks to induce neuropathic pain. Curcumin (30 and 60mg/kg, i.p.) and sildenafil (5 and 10mg/kg, i.p.) were given alone and in combination at their lower doses (30mg/kg curcumin and 5mg/kg, sildenafil, i.p.) to investigate the changes in thermal and mechanical hyperalgesia, allodynia and histopathological parameters. Biochemical estimations of thiobarbituric acid reactive species, glutathione and protein was also carried out to evaluate oxidative stress. The results revealed that chronic alcohol consumption for 10 weeks caused significant thermal and mechanical hyperalgesia, allodynia and increased oxidative stress. Individual administration of both the drugs at their low as well as high doses were able to improve the symptoms of alcohol induced neuropathic pain. Whereas co-administration of curcumin and sildenafil at their lower doses itself were found to significantly improve nerve functions, biochemical and histopathological parameters as compared to their individual administration. It is therefore proposed that co-administration of curcumin and sildenafil may bring new dimension towards attenuation of alcohol induced neuropathic pain affecting central as well as peripheral nervous system.

Herbicide-resistant cotton (Gossypium hirsutum) plants: an alternative way of manual weed removal.

BACKGROUND: Cotton yield has been badly affected by different insects and weed competition. In Past Application of multiple chemicals is required to manage insects and weed control was achieved by different conventional means, such as hand weeding, crop rotation and polyculture, because no synthetic chemicals were available. The control methods shifted towards high input and target-oriented methods after the discovery of synthetic herbicide in the 1930s. To utilise the transgenic approach, cotton plants expressing the codon-optimised CEMB GTGene were produced in the present study. RESULTS: Local cotton variety CEMB-02 containing Cry1Ac and Cry2A in single cassette was transformed by synthetic codon-optimised 5-enolpyruvylshikimate-3-phosphate synthase gene cloned into pCAMBIA 1301 vector under 35S promoter with Agrobacterium tumifaciens. Putative transgenic plants were screened in MS medium containing 120 µmol/L glyphosate. Integration and expression of the gene were evaluated by PCR from genomic DNA and ELISA from protein. A 1.4-kb PCR product for Glyphosate and 167-bp product for Cry2A were obtained by amplification through gene specific primers. Expression level of Glyphosate and Bt proteins in two transgenic lines were recorded to be 0.362, 0.325 µg/g leaf and 0.390, 0.300 µg/g leaf respectively. FISH analysis of transgenic lines demonstrates the presence of one and two copy no. of Cp4 EPSPS transgene respectively. Efficacy of the transgene Cp4 EPSPS was further evaluated by Glyphosate spray (41 %) assay at 1900 ml/acre and insect bioassay which shows 100 %mortality of insect feeding on transgenic lines as compared to control. CONCLUSION: The present study shows that the transgenic lines produced in this study were resistant not only to insects but also equally good against 1900 ml/acre field spray concentration of glyphosate.

Occurrence and Management Strategies for Catheter Entrapment with Onyx Liquid Embolization.

In June 2012, Food and Drug Administration (FDA) issued a warning about the risk of catheter entrapment associated with Onyx embolization. We used our experience, literature review, and FDA Manufacturer and User Facility Device Experience (MAUDE) data review to identify five strategies to address catheter entrapment: 1/. Surgical resection of vessel at point of entrapment of catheter and retraction from exterior portion at the femoral region; 2/. Advancing and closing the loop of snare over the entrapped catheter followed by retraction; 3/. Advancing the distal access catheter over the entrapped catheter and retraction with forward movement of the distal access catheters; 4/. Inflation of balloon catheter coaxial to the entrapped catheter with subsequent retraction; and 5/. Intravascular retention and internalization of microcatheter. In the MAUDE data, there were 77 reports of catheter entrapment with Onyx embolization; microcatheter was retracted by surgical excision in 15, endovascular snare or other retriever devices in 5, deliberately entrapped inside the vessel using stent in 1, and left without intervention within intravascular compartment in 27 patients.

Prevalence of and Factors Associated with Dural Thickness in Patients with Mild Cognitive Impairment and Alzheimer's Disease.

BACKGROUND AND PURPOSE: We performed this study to evaluate the prevalence of and factors associated with dural thickening in patients with mild cognitive impairment and Alzheimer's disease. METHODS: Alzheimer's disease neuroimaging initiative participants with axial FLAIR sequence magnetic resonance imaging (MRI) images were analyzed. Dural thickness was defined by a linear strip of hyperintense tissue signal along the dura mater observed in at least two different images without evidence of leptomeningeal involvement. RESULTS: Dural thickening was seen in 83 (34%) of 242 persons analyzed (mean age [±SD] 74±7 years: 150 were men) with either mild cognitive impairment or Alzheimer's disease. The mini mental score was not different in persons with (26±0.3) and without (26±0.2) dural thickening (p = 0.6). The proportion of patients with moderate or severe cognitive impairment (defined by mini mental status score) was similar at baseline and at 12-month evaluations. The rates of annual progression according to Alzheimer's disease assessment scale (p = 0.06) and clinical dementia scale (p = 0.001) were higher in persons without dural thickening. The annual rate of volume loss in entorhinal cortex was higher among persons with dural thickening. CONCLUSIONS: We found relatively high prevalence of dural thickening in patients with mild cognitive impairment and Alzheimer's disease.