Diabetes mellitus represents a major independent risk factor for developing fatal cardiovascular diseases (CVDs) presumably through accelerating atherosclerosis; the underlying cause of most CVDs. Notably, this relative risk is reported to be higher in women than men. Endeavors directed towards identifying novel reliable predictive biomarkers are immensely thereby urged to improve the long-term outcome in these diabetic female patients. Sclerostin (SOST) is a Wnt signaling antagonist whereas irisin is a muscle-derived factor released after exercising which enhances browning of white adipose tissue. Emerging lines of evidence hint at potential crosstalk between them and CVDs. The present study aimed to assess the serum levels of SOST and irisin in Egyptian type 2 diabetic (T2DM) female patients with and without atherosclerosis and explore the possible relationship between both markers and other studied parameters among the studied cohorts. In this case-control study, 69 female subjects were enrolled; 39 type 2 diabetes patients with atherosclerosis (T2DM+ATHR), 22 type 2 diabetes patients without atherosclerosis (T2DM-ATHR) and 8 healthy controls. Their serum levels of SOST and irisin were assessed using ELISA. Significant increase in SOST levels were found in T2DM+ATHR compared to T2DM-ATHR and control (259.9 ±17.98 vs. 165.8±13.12 and 142.0±13.31 pg/mL respectively, P<0.001). Conversely, irisin levels were significantly lower in T2DM+ATHR (P<0.001) and T2DM-ATHR (P<0.01) compared to the control group (32.91±2.545 and 58.55±13.19 vs. 473.6±112.7 pg/mL). Interestingly, significant correlations between the levels of SOST and both irisin and fasting blood glucose were noticed in T2DM+ATHR group (r = 0.3754 and 0.3381 respectively, P<0.05). In conclusion, to the best of our knowledge, this study is the first to demonstrate the correlation between SOST and irisin levels in atherosclerotic T2DM female patients implying their potential implication in diabetic cardiovascular pathophysiology and supporting their use as reliable diagnostic/prognostic biomarkers for monitoring and preventing CVDs progression of T2DM female patients.

Combining sonochemistry with phytochemistry is a modern trend in the biosynthesis of metallic nanoparticles (NPs), which contributes to the sustainability of chemical processes and minimizes hazardous effects. Herein, titanium dioxide (TiO₂) NPs were bioengineered using a novel and facile ultrasound-assisted approach utilizing the greenly extracted essential oil of Ocimum basilicum. FTIR and UV-Vis spectrophotometry were used to confirm the formation of TiO₂ NPs. The X-ray diffraction (XRD) analysis showed the crystalline nature of TiO₂ NPs. TEM analysis revealed the spherical morphology of the NPs with sizes ranging from 5.55 to 13.89 nm. Energy-dispersive X-ray (EDX) confirmed the purity of the greenly synthesized NPs. TiO₂ NPs demonstrated outstanding antitumor activity against breast (MCF-7) and lung (A-549) cancer cells with estimated IC₅₀ values of 1.73 and 4.79 μg mL⁻¹. The TiO₂ NPs were cytocompatible to normal cells (MCF-10A) with a selectivity index (SI) of 8.77 for breast and 3.17 for lung cancer. Biological assays revealed a promising potential for TiO₂ NPs to induce apoptosis and arrest cells at the sub-G1 phase of the cell cycle phase in both cancer cell lines. Molecular investigations showed the ability of TiO₂ NPs to increase apoptotic genes' expression (Bak and Bax) and their profound ability to elevate the expression of apoptotic proteins (caspases 3 and 7). Molecular docking demonstrated strong binding interactions for TiO₂ NPs with caspase 3 and EGFR-TK targets. In conclusion, the greenly synthesized TiO₂ NPs exhibited potent antitumor activity and mitochondrion-based cell death against breast and lung cancer cell lines while maintaining cytocompatibility against normal cells.

The highly extreme conditions of the lower convective layer in the Atlantis II (ATII) Deep brine pool of the Red Sea make it an ideal environment for the search for novel enzymes that can function under extreme conditions. In the current study, we isolated a novel sequence of a thioredoxin reductase (TrxR) enzyme from the metagenomic dataset established from the microbial community that resides in the lower convective layer of Atlantis II. The gene was cloned, expressed and characterized for redox activity, halophilicity, and thermal stability. The isolated thioredoxin reductase (ATII-TrxR) was found to belong to the high-molecular-weight class of thioredoxin reductases. A search for conserved domains revealed the presence of an extra domain (Crp) in the enzyme sequence. Characterization studies of ATII-TrxR revealed that the enzyme was halophilic (maintained activity at 4 M NaCl), thermophilic (optimum temperature was 65°C) and thermostable (60% of its activity was retained at 70°C). Additionally, the enzyme utilized NADH in addition to NADPH as an electron donor. In conclusion, a novel thermostable and halophilic thioredoxin reductase has been isolated with a unique sequence that adapts to the harsh conditions of the brine pools making this protein a good candidate for biological research and industrial applications.

The hypersaline Kebrit Deep brine pool in the Red Sea is characterized by high levels of toxic heavy metals. Here we describe two structurally related mercuric reductases (MerAs) from this site, which were expressed in Escherichia coli. Sequence similarities suggest that both genes are derived from Proteobacteria, most likely β- or γ-Proteobacteria. We show that one of the enzymes (K35NH) is strongly inhibited by NaCl, while the other (K09H) is activated in a NaCl-dependent manner. We infer from this difference that the two forms might support detoxification of mercury in bacterial microorganisms that employ the compatible solutes and salt-in strategies, respectively. Three-dimensional structure modeling shows that all amino-acid substitutions unique to each type are located outside the domain responsible for formation of the active MerA homodimer, and the vast majority of these are found on the surface of the molecule. Moreover, K09H exhibits the predominance of acidic over hydrophobic side-chains that is typical of halophilic, salt-dependent proteins. These findings enhance our understanding of how selection pressures imposed by two environmental stressors have endowed MerA enzymes with catalytic properties that can potentially function in microorganisms that utilize distinct mechanisms for osmotic balance in hypersaline environments. IMPORTANCE Analysis of two structurally homologous but catalytically distinct mercuric reductases from the Kebrit Deep brine in the Red See sheds light on the adaptations that enable microorganisms to cope simultaneously with extreme salinity and toxic mercury compounds. One is strongly inhibited by high NaCl concentrations, while the other exhibits NaCl-dependent activation. Their different activity profiles imply that they may derive from bacterial microorganisms that utilize compatible solutes and salt-in strategies, respectively, to maintain osmotic balance. Three-dimensional modeling reveals that regions not involved in formation of the active homodimer are conserved between the two. However, in the NaCl-dependent form, distinct amino-acid substitutions are found in areas that are critical for stability in high salt. The work provides insights into how two environmental stressors have shaped the structure of orthologous enzymes through selection and adaptation, enabling them to retain their catalytic function in what may be very different cellular contexts.

The lower convective layer (LCL) of the Atlantis II brine pool of the Red sea is a unique environment in terms of high salinity, temperature, and high concentrations of heavy metals. Mercuric reductase enzymes functional in such extreme conditions could be considered as a potential tool in the environmental detoxification of mercurial poisoning and might alleviate ecological hazards in the mining industry. Here, we constructed a mercuric reductase library from Atlantis II, from which we identified genes encoding two thermostable mercuric reductase (MerA) isoforms: one is halophilic (designated ATII-LCL) while the other is not (designated ATII-LCL-NH). The ATII-LCL MerA has a short motif composed of four aspartic acids (4D_414-417) and two characteristic signature boxes that played a crucial role in its thermal stability. To further understand the mechanism behind the thermostability of the two studied enzymes, we mutated the isoform ATII-LCL-NH and found that the substitution of 2D at positions 415/416 enhanced the thermal stability, while other mutations had the opposite effect. The 2D mutant showed superior thermal tolerance as it retained 81% of its activity after 10 minutes incubation at 70°C. Three-dimensional structure prediction revealed newly formed salt-bridges and H-bonds in the 2D mutant, compared to the parent molecule. To the best of our knowledge, this study is the first to rationally design a mercuric reductase with enhanced thermal stability, which we propose to have a strong potential in bioremediation of mercurial poisoning. Importance The Red Sea is an attractive environment for bioprospecting. There are 25 brine-filled deeps in the Red Sea. The Atlantis II brine pool is the biggest and hottest of such hydrothermal ecosystems. We generated an environmental mercuric reductase library from the lowermost layer of the Atlantis II brine pool, in which we identified two variants of the mercuric reductase enzyme (MerA). One is the previously described halophilic and thermostable ATII-LCL MerA and the other is a non-halophilic, relatively less thermostable, designated ATII-LCL-NH MerA enzyme. We used the ATII-LCL-NH enzyme as a parent molecule to locate the amino acid residues involved in the noticeable higher thermo-tolerance of the homolog ATII-LCL MerA. Moreover, we designed a novel enzyme with superior thermal stability. This enzyme could have strong potential in bioremediation of mercuric toxicity.