Towards clinical translation of 'second-generation' regenerative stroke therapies: hydrogels as game changers?

Stroke is an unmet clinical need with a paucity of treatments, at least in part because chronic stroke pathologies are prohibitive to 'first-generation' stem cell-based therapies. Hydrogels can remodel the hostile stroke microenvironment to aid endogenous and exogenous regenerative repair processes. However, no clinical trials have yet been successfully commissioned for these 'second-generation' hydrogel-based therapies for chronic ischaemic stroke regeneration. This review recommends a path forward to improve hydrogel technology for future clinical translation for stroke. Specifically, we suggest that a better understanding of human host stroke tissue-hydrogel interactions in addition to the effects of scaling up hydrogel volume to human-sized cavities would help guide translation of these second-generation regenerative stroke therapies.

The innate immune response of self-assembling silk fibroin hydrogels.

Silk has a long track record of use in humans, and recent advances in silk fibroin processing have opened up new material formats. However, these new formats and their applications have subsequently created a need to ascertain their biocompatibility. Therefore, the present aim was to quantify the haemocompatibility and inflammatory response of silk fibroin hydrogels. This work demonstrated that self-assembled silk fibroin hydrogels, as one of the most clinically relevant new formats, induced very low blood coagulation and platelet activation but elevated the inflammatory response of human whole blood in vitro. In vivo bioluminescence imaging of neutrophils and macrophages showed an acute, but mild, local inflammatory response which was lower than or similar to that induced by polyethylene glycol, a benchmark material. The time-dependent local immune response in vivo was corroborated by histology, immunofluorescence and murine whole blood analyses. Overall, this study confirms that silk fibroin hydrogels induce a similar immune response to that of PEG hydrogels, while also demonstrating the power of non-invasive bioluminescence imaging for monitoring tissue responses.

Structural insights into the camel milk lactoperoxidase: Homology modeling and molecular dynamics simulation studies.

Lactoperoxodase (LPO) is a heme peroxidase enzyme present in mammalian milk. It is an antimicrobial protein with wide range of industrial applications. Although the three dimensional structure of LPO from various mammalian species has been reported, but its structure from camel source is still unknown. So far, the crystallization attempts have not been successful in determining camel LPO (cLPO) structure. Herein, we developed the three dimensional structure of cLPO by homology modeling approach using prime module available in Schrodinger suite. The developed model in complex with ligand hypothiocyanate (OSCN-) was further validated by Ramachandran plot followed by molecular dynamics (MD) simulation studies using Desmond module of Schrodinger. cLPO model exhibited overall structural similarity with template crystal structure, however, it displayed different interaction pattern of amino acid residues with ligand OSCN- in comparison to template crystal structure. Moreover, the ligand binding site environment in cLPO is more polar, less hydrophobic, and harbours more number of charged residues than template crystal structure. The substrate binding pocket environment of cLPO shows a considerable difference from template crystal structure. This subsequently resulted in dissimilar behaviour of ligand during the course of MD simulation studies.

Structural studies on inhibitory mechanisms of antibiotic, corticosteroid and catecholamine molecules on lactoperoxidase.

AIM: Lactoperoxidase (LPO) is an essential protein with broad spectrum antimicrobial activity present in mammalian milk. It imparts immunity to infants against wide range of pathogenic infections. Several in vitro studies have shown inhibition of LPO activity by pharmaceutical compounds including commonly used antibiotics such as ampicillin and gentamicin, and molecules like prednisolone, norepinephrine, etc. Prescription of such drugs to lactating mothers might have adverse health effects on infants. The aim of our study was the elucidation of the structural aspects of the inhibitory mechanism of ampicillin, gentamicin, amoxicillin, prednisolone and norepinephrine on LPO. MATERIAL AND METHODS: Three dimensional structure of camel LPO (cLPO) was developed using homology modeling and used for in silico experimental studies. The Schrödinger induced fit docking along with binding affinity estimation experiments were performed. The cLPO and Ligands were prepared using Protein Preparation Wizard and Ligprep modules available in Schrodinger suite. For estimating Binding affinity Prime Molecular Mechanics with Generalized Born and Surface Area (MMGB-SA) module was used. KEY RESULTS: The five drug ligands formed three to five hydrogen bonding interactions with cLPO. Amino acids Arg-231, Asp-232, Ser-370, Arg-371 and Glu-374 of cLPO were crucial for these interactions. The binding affinity values for gentamicin were highest and for norepinephrine were the lowest. SIGNIFICANCE: This study concludes that the five drug molecules show potential ability to inhibit the LPO activity. Further, a very high sequence similarity of cLPO with human LPO imparts high significance to these conclusions in relation to human health especially in new born infants.

Molecular characterization of exonic rearrangements and frame shifts in the dystrophin gene in Duchenne muscular dystrophy patients in a Saudi community.

BACKGROUND: In individuals with Duchenne muscular dystrophy (DMD), exon skipping treatment to restore a wild-type phenotype or correct the frame shift of the mRNA transcript of the dystrophin (DMD) gene are mutation-specific. To explore the molecular characterization of DMD rearrangements and predict the reading frame, we simultaneously screened all 79 DMD gene exons of 45 unrelated male DMD patients using a multiplex ligation-dependent probe amplification (MLPA) assay for deletion/duplication patterns. Multiplex PCR was used to confirm single deletions detected by the MLPA. RESULTS: There was an obvious diagnostic delay, with an extremely statistically significant difference between the age at initial symptoms and the age of clinical evaluation of DMD cases (t value, 10.3; 95% confidence interval 5.95-8.80, P < 0.0001); the mean difference between the two groups was 7.4 years. Overall, we identified 147 intragenic rearrangements: 46.3% deletions and 53.7% duplications. Most of the deletions (92.5%) were between exons 44 and 56, with exon 50 being the most frequently involved (19.1%). Eight new rearrangements, including a mixed deletion/duplication and double duplications, were linked to seven cases with DMD. Of all the cases, 17.8% had duplications with no hot spots. In addition, confirmation of the reading frame hypothesis helped account for new DMD rearrangements in this study. We found that 81% of our Saudi patients would potentially benefit from exon skipping, of which 42.9% had a mutation amenable to skipping of exon 51. CONCLUSIONS: Our study could generate considerable data on mutational rearrangements that may promote future experimental therapies in Saudi Arabia.

Intrinsic disorder in biomarkers of insulin resistance, hypoadiponectinemia, and endothelial dysfunction among the type 2 diabetic patients.

Type 2 diabetes mellitus (T2DM) is a chronic and progressive disease that is strongly associated with various complications including cardiovascular diseases and related mortality. The present study aimed to analyze the abundance and functionality of intrinsically disordered regions in several biomarkers of insulin resistance, adiponectin, and endothelial dysfunction found in the T2DM patients. In fact, in comparison to controls, obese T2DM patients are known to have significantly higher levels of inter-cellular adhesion molecule (iCAM-1), vascular cell adhesion molecule (vCAM-1), and E-selectin, whereas their adiponectin levels are relatively low. Bioinformatics analysis revealed that these selected biomarkers (iCAM-1, vCAM-1, E-selectin, and adiponectin) are characterized by the noticeable levels of intrinsic disorder propensity and high binding promiscuity, which are important features expected for proteins serving as biomarkers. Within the limit of studied groups, there is an association between insulin resistance and both hypoadiponectinemia and endothelial dysfunction.

The impact of common tumor necrosis factor haplotypes on the development of asthma in children: an Egyptian model.

Conflicting results have arisen among different ethnic populations with regard to the ability of tumor necrosis factor (TNF) to control the development of bronchial asthma. We examined common TNF polymorphisms (TNFA -1031C>T, TNFA -308G>A, and TNFB +252A>G) to develop a model of the associations between these genetic markers and the development of the disease in Egypt. Amplified DNA from buccal mucosa was genotyped for 240 children using polymerase chain reaction-restriction fragment length polymorphism. Skin prick test, total serum immunoglobulin E levels, and assessment of pulmonary functions were investigated. The onset age for one-third of the asthma patients in our study was between 7 and 10 years. The TNFA -1031C>T and TNFA -308G>A polymorphisms were strongly associated with the risk of asthma (p = 0.007, and p = 0.000, respectively), but the TNFB +252A>G polymorphism was not (p = 0.6). We detected a significant linkage between the +252A>G and -1031C>T, and another between the +252A>G and the -308G>A (p < 0.0001 for both). The -1031C>T and -308G>A polymorphisms were not linked (p = 0.14). The -308A/A genotype was absent, and the -308A allele was expressed only in patients with -308G/A heterozygosity (13%). All but the +252G/A genotype were also strongly associated with the severity of disease. Environmental factors, as genetic variations, clearly influence susceptibility, the onset, progression, and severity of bronchial asthma. More information is needed to develop genetic models of susceptibility for different ethnic populations.

Association between ß+252 tumour necrosis factor polymorphism and asthma in western Saudi children.

There is strong evidence that supports the role of tumour necrosis factors (TNF-alpha/beta) as common genetic factors, located on 6p21.1-6p21.3 loci, in the pathogenesis of asthma disease. In this study, we extended our research work on TNFA to include the genotyping of Saudi asthmatic children as regards to TNFB gene (namely as lymphotoxin-α, LTA). We examined 60 asthmatic Saudi children compared to 56 healthy non-asthmatics using the PCR-RFLP analyses to identify the polymorphism +252A>G in intron 1 in lymphotoxin-α gene. We identified 55% of the allele A of the LTA∗NcoI polymorphism in subjects with asthma disease, and 45% of the allele G. In this study, the frequency of the LTA∗NcoI-A/A genotype was 40% preferably to the LTA∗NcoI-G/A and LTA∗NcoI-G/G genotypes. In addition, the severe persistent asthmatic cases were associated with the LTA∗NcoI-AA genotype at a frequency of 80%, while the genotype LTA∗NcoI-GG are associated with the mildest form of the disease. Consequently, one could predict the severity of asthma and hence the polymorphism of the LTA∗NcoI. Herein, we stated that more than 93% of Saudi children under investigation lived in the randomized areas of western regions of Saudi Arabia. In conclusion, genotype frequencies for the LTA+252 polymorphisms were significantly different from the controls. These findings may have implications for future early intervention studies by helping to identify infants at increased risk for wheezing and childhood asthma.

Molecular updating of ß-thalassemia mutations in the Upper Egyptian population.

We have updated the dataset of the molecular spectrum of the ß-thalassemia (ß-thal) in Upper Egypt. Buccal swabs were analyzed from 94 unrelated patients with ß-thal major (ß-TM) using reverse dot-blot and multiplex amplification refractory mutation system-polymerase chain reaction (ARMS-PCR). The most frequent mutation was IVS-I-110 (G>A) (57%). The IVS-I-110, IVS-I-6 (T>C) and IVS-I-1 (G>A) mutations accounted for 87% of the ß-thal anomalies. The codon 39 (C>T) and frameshift codon (FSC) 6 (-A) (GAG>-GG) mutations were only detected in Al-Minya and Qina, respectively. We did not observe the IVS-II-745 (C>G) or -101 (C>T) mutations. Forty-three percent of Upper Egyptians were homozygotes. Our efforts were an important step to complete the mutation map of ß-thal in Egypt restricted to Cairo and the Nile Delta regions. This study will help to develop preventative programs for Upper Egyptians. It addressed the genetic drift of the ß-thal gene mutations in Africa, Asia, and Europe.