Band 12, Heft 1

The advent of genome editing technology promises to transform human health, livestock and agriculture, and to eradicate pest species. This transformative power demands urgent scrutiny and resolution of the ethical conflicts attached to the creation and release of engineered genomes. Here, I discuss the ethics surrounding the transformative CRISPR/Cas9-mediated genome editing technology in the contexts of human genome editing to eradicate genetic disease and of gene drive technology to eradicate animal vectors of human disease.

Doxorubicin (DOX) is an effective chemotherapeutic agent used for the treatment of various types of cancer. However, its poor solubility, undesirable side effects, and short half-life have remained a challenge. We used a formulation based on graphene oxide as an anticancer drug delivery system for DOX in MCF-7 breast cancer cells, to address these issues. In vitro release studies confirmed that the synthesized formulation has an improved release profile in acidic conditions (similar to the tumor microenvironment). Further in vitro studies, including MTT, uptake, and apoptosis assays were performed. The toxic effects of the nanocarrier on the kidney, heart and liver of healthy rats were also evaluated. We observed that the DOX-loaded carrier improved the cytotoxic effect of DOX on the breast cell line compared to free DOX. In summary, our results introduce the DOX-loaded carrier as a potential platform for in vitro targeting of cancer cells and suggest further studies are necessary to investigate its in vivo anti-cancer potential.

Tuberculosis (TB) caused by Mycobacterium tuberculosis ( M. tb ) still remains a devastating infectious disease in the world. There has been a daunting increase in the incidence of Type 2 Diabetes Mellitus (T2DM) worldwide. T2DM patients are three times more vulnerable to M. tb infection compared to healthy individuals. TB-T2DM coincidence is a challenge for global health control. Despite some progress in the research, M. tb still has unexplored characteristics in successfully evading host defenses. The lengthy duration of treatment, the emergence of multi-drug-resistant strains and extensive-drug-resistant strains of M. tb have made TB treatment very challenging. Previously, we have tested the antimycobacterial effects of everolimus within in vitro granulomas generated from immune cells derived from peripheral blood of healthy subjects. However, the effectiveness of everolimus treatment against mycobacterial infection in individuals with T2DM is unknown. Furthermore, the effectiveness of the combination of in vivo glutathione (GSH) supplementation in individuals with T2DM along with in vitro treatment of isolated immune cells with everolimus against mycobacterial infection has never been tested. Therefore, we postulated that liposomal glutathione (L-GSH) and everolimus would offer great hope for developing adjunctive therapy for mycobacterial infection. L-GSH or placebo was administered to T2DM individuals orally for three months. Study subjects’ blood was drawn pre- and post-L-GSH/or placebo supplementation, where Peripheral Blood Mononuclear Cells (PBMCs) were isolated from whole blood to conduct in vitro studies with everolimus. We found that in vitro treatment with everolimus, an mTOR (membrane target of rapamycin) inhibitor, significantly reduced intracellular M. bovis BCG infection alone and in conjunction with L-GSH supplementation. Furthermore, we found L-GSH supplementation coupled with in vitro everolimus treatment produced a greater effect in inhibiting the growth of intracellular Mycobacterium bovis BCG, than with the everolimus treatment alone. We also demonstrated the functions of L-GSH along with in vitro everolimus treatment in modulating the levels of cytokines such as IFN-γ, TNF-α, and IL-2 and IL-6, in favor of improving control of the mycobacterial infection. In summary, in vitro everolimus-treatment alone and in combination with oral L-GSH supplementation for three months in individuals with T2DM, was able to increase the levels of T-helper type 1 (Th1) cytokines IFN-γ, TNF-α, and IL-2 as well as enhance the abilities of granulomas from individuals with T2DM to improve control of a mycobacterial infection.

Previously, we have shown that hepatic lipid accumulation induces the secretion of cathepsin D (CTSD), and that plasma CTSD levels are associated with increased inflammation and disease severity in nonalcoholic fatty liver disease (NAFLD). Although it is clear that the liver is a major source of plasma CTSD, it is unknown whether other metabolically active organs such as the muscle, also associate with plasma CTSD levels in NAFLD patients. Therefore, the aim of this study was to explore the relation between lipid accumulation in the muscle (myosteatosis) and plasma CTSD levels in forty-five NAFLD patients. We observed that hepatic steatosis positively associated with plasma CTSD levels, confirming the previously established link between plasma CTSD and the liver. Furthermore, a positive association between myosteatosis and plasma CTSD levels was observed, which was independent of sex, age, BMI, waist circumference and hepatic steatosis. By establishing a positive association between myosteatosis and plasma CTSD levels, our findings suggest that, in addition to the liver, the muscle is also linked to plasma CTSD levels in NAFLD patients. The observed link between myosteatosis and plasma CTSD levels supports the concept of a significant role of the skeletal muscle in metabolic disturbances in metabolic syndrome-related disorders.

Weight-related disorders affect more than half of the adult population worldwide; they are also concomitant with a state of chronic low-grade inflammation manifesting in abnormal cytokine production. The present study evaluated the effect of polyphenol and flavonoid extract from Passiflora ligularis (granadilla) on low-grade inflammation and body weight in overweight Wistar rats. To induce weight-gain, rats were fed a chow diet with 30% sucrose water and supplemented with 2.0, 2.5, and 3.0 g/L polyphenol extracts ( n = 16). The design was a 3 +1 factorial model performed for 42 days (granadilla polyphenols, 3 levels of supplementation, and 1 control group). In addition to total polyphenol and total flavonoid content, the major identified and quantified polyphenol, via UHPLC, was ferulic acid. Interleukin 6 (IL-6), and cytokine tumor necrosis factor-alpha (TNF-α) were evaluated in serum. A decline in the concentration of TNF-α and in weight-gain was found in P. ligularis (granadilla) groups treated with the 2.5 g/L dose. Consumption of polyphenol extracts from granadilla inhibits interleukin-activity as an indicator of inflammation and aids in body-weight control, considering similar food intake, in overweight Wistar rats.

With a disease as widespread and destructive as tuberculosis, more effective drugs and healthcare strategies, in addition to the current antibiotics regimen, are crucial for the enhanced well-being of millions of people suffering from the disease. Host-directed therapy is a new and emerging concept in treating chronic infectious diseases, such as tuberculosis. Repurposing of anti-cancer drugs, such as everolimus, may be an effective way to supplement the standard antibiotic treatment. Individuals with type 2 diabetes are increasingly susceptible to co-morbidities and co-infections including Mycobacterium tuberculosis, the causative agent of tuberculosis. We demonstrated in this study that in vitro everolimus treatment of granulomas from individuals with type 2 diabetes caused significant reduction in the viability of Mycobacterium tuberculosis. Further investigations revealed the effects of everolimus in targeting foamy macrophages, a macrophage phenotype that forms around granulomas, and is characterized by a higher lipid accumulation inside the cells. These foamy macrophages are thought to harbor dormant bacilli, which are potential sources of disease reactivation. Therefore, blocking foamy macrophage formation would help better killing of intracellular bacteria. Here, we report the potential of everolimus treatment to downregulate lipid content within the foamy macrophages of in vitro granulomas, thus leading to a potential decrease in the number of foamy macrophages and a more robust response to Mycobacterium tuberculosis .

Abdominal aortic aneurysm (AAA) is a complex degenerative vascular disease, with considerable morbidity and mortality rates among the elderly population. The mortality of AAA is related to aneurysm expansion (the enlargement of the aortic diameter up to 30 mm and above) and the subsequent rupture. The pathogenesis of AAA involves several biological processes, including aortic mural inflammation, oxidative stress, vascular smooth muscle cell apoptosis, elastin depletion, and degradation of the extracellular matrix. Mitochondrial dysfunction was also found to be associated with AAA formation. The evidence accumulated to date supports a close relationship between environmental and genetic factors in AAA initiation and progression. However, a comprehensive pathophysiological understanding of AAA formation remains incomplete. The open surgical repair of AAA is the only therapeutic option currently available, while a specific pharmacotherapy is still awaited. Therefore, there is a great need to clarify pathophysiological cellular and molecular mechanisms underlying AAA formation that would help to develop effective pharmacological therapies. In this review, pathophysiological aspects of AAA development with a special focus on mitochondrial dysfunction and genetic associations were discussed.

Protein therapeutics are in great demand due to their effectiveness towards hard-to-treat diseases. Despite their high demand, these bio-therapeutics are very susceptible to degradation via aggregation, fragmentation, oxidation, and reduction, all of which are very likely to affect the quality and efficacy of the product. Mechanisms and modelling of these degradation (aggregation and fragmentation) pathways is critical for gaining a deeper understanding of stability of these products. This review aims to provide a summary of major developments that have occurred towards unravelling the mechanisms of size-based protein degradation (particularly aggregation and fragmentation), modelling of these size-based degradation pathways, and their control. Major caveats that remain in our understanding and control of size-based protein degradation have also been presented and discussed.

Bacillus cereus is considered to be an important food poisoning agent causing diarrhea and vomiting. In this study, the occurrence of B. cereus bacteriophages in Thai fermented soybean products (Thua Nao) was studied using five B. cereus sensu lato indicator strains (four B. cereus strains and one B. thuringiensis strain). In a total of 26 Thua Nao samples, there were only two bacteriophages namely BaceFT01 and BaceCM02 exhibiting lytic activity against B. cereus . Morphological analysis revealed that these two bacteriophages belonged to the Myoviridae . Both phages were specific to B. cereus and not able to lyse other tested bacteria including B. licheniformis and B. subtilis . The two phages were able to survive in a pH range between 5 and 12. However, both phages were inactive either by treatment of 50°C for 2 h or exposure of UV for 2 h. It should be noted that both phages were chloroform-insensitive, however. This is the first report describing the presence of bacteriophages in Thua Nao products. The characterization of these two phages is expected to be useful in the food industry for an alternative strategy including the potential use of the phages as a biocontrol candidate against foodborne pathogenic bacteria.

We previously reported that M. tb on its own as well as together with HIV inhibits macrophage apoptosis by upregulating the expression of Bcl2 and Inhibitor of Apoptosis (IAP). In addition, recent reports from our lab showed that stimulation of either macrophages or BMDCs results in the significant upregulation of Bcl2. In this report, we delineate the role of Bcl2 in mediating defense responses from dendritic cells (BMDCs) during mycobacterial infection. Inhibiting Bcl2 led to a significant decrease in intracellular bacterial burden in BMDCs. To further characterize the role of Bcl2 in modulating defense responses, we inhibited Bcl2 in BMDCs as well as human PBMCs to monitor their activation and functional status in response to mycobacterial infection and stimulation with M. tb antigen Rv3416. Inhibiting Bcl2 generated protective responses including increased expression of co-stimulatory molecules, oxidative burst, pro-inflammatory cytokine expression and autophagy. Finally, co-culturing human PBMCs and BMDCs with antigen-primed T cells increased their proliferation, activation and effector function. These results point towards a critical role for Bcl2 in regulating BMDCs defense responses to mycobacterial infection.

Previous studies associated plasma cathepsin D (CTSD) activity with hepatic insulin resistance in overweight and obese humans. Insulin resistance is a major feature of non-alcoholic fatty liver disease (NAFLD) and is one of the multiple hits determining the progression towards non-alcoholic steatohepatitis (NASH). In line, we have previously demonstrated that plasma CTSD levels are increased in NASH patients. However, it is not known whether insulin resistance associates with plasma CTSD activity in NAFLD. To increase our understanding regarding the mechanisms by which insulin resistance mediates NAFLD, fifty-five liver biopsy or MRI-proven NAFLD patients (BMI>25kg/m 2 ) were included to investigate the link between plasma CTSD activity to insulin resistance in NAFLD. We concluded that HOMA-IR and plasma insulin levels are independently associated with plasma CTSD activity in NAFLD patients (standardized coefficient β: 0.412, 95% Cl: 0.142~0.679, p=0.004 and standardized coefficient β: 0.495, 95% Cl: 0.236~0.758, p=0.000, respectively). Together with previous studies, these data suggest that insulin resistance may link to NAFLD via elevation of CTSD activity in plasma. As such, these data pave the way for testing CTSD inhibitors as a pharmacological treatment of NAFLD.

Tuberculosis (TB) is a global health threat that affects 10 million people worldwide. Human Immunodeficiency Virus (HIV) remains one of the major contributors to the reactivation of asymptomatic latent tuberculosis (LTBI). Over the recent years, there has been a significant focus in developing in-vitro 3D models mimicking early events of Mycobacterium tuberculosis (Mtb) pathogenesis, especially formation of the granuloma. However, these models are low throughput and require extracellular matrix. In this article, we report the generation of a matrix-free 3D model, using THP-1 human monocyte/macrophage cells and mCherry-expressing Mycobacterium bovis BCG (Bacilli Camille Guérin), henceforth referred as 3D spheroids, to study the host cell-bacterial interactions. Using mCherry-intensity-based tracking, we monitored the kinetics of BCG growth in the 3D spheroids. We also demonstrate the application of the 3D spheroids for testing anti-TB compounds such as isoniazid (INH), rifampicin (RIF), as well as a host-directed drug, everolimus (EVR) as single and combinational treatments. We further established a dual infection 3D spheroid model by coinfecting THP-1 macrophages with BCG mCherry and pseudotype HIV. In this HIV-TB co-infection model, we found an increase in BCG mCherry growth within the 3D spheroids infected with HIV pseudotype. The degree of disruption of the granuloma was proportional to the virus titers used for co-infection. In summary, this 3D spheroid assay is an useful tool to screen anti-TB response of potential candidate drugs and can be adopted to model HIV-TB interactions.

A unique feature of eukaryote initiation of protein translation is a so-called scanning of 5′-untranslated region (5′-UTR) by a ribosome initiation complex to enable bound Met-tRNA i access to the initiation codon located further downstream. Here, we propose a universal scanning-free translation initiation model that is independent of 5′-UTR length and applicable to both 5′-m 7 G (capped) and uncapped mRNAs.

Stromal cell-derived factor 1 (SDF-1) is known to influence bone marrow stromal cell (BMSC) migration, osteogenic differentiation, and fracture healing. We hypothesize that SDF-1 mediates some of its effects on BMSCs through epigenetic regulation, specifically via microRNAs (miRNAs). MiRNAs are small non-coding RNAs that target specific mRNA and prevent their translation. We performed global miRNA analysis and determined several miRNAs were differentially expressed in response to SDF-1 treatment. Gene Expression Omnibus (GEO) dataset analysis showed that these miRNAs play an important role in osteogenic differentiation and fracture healing. KEGG and GO analysis indicated that SDF-1 dependent miRNAs changes affect multiple cellular pathways, including fatty acid biosynthesis, thyroid hormone signaling, and mucin-type O-glycan biosynthesis pathways. Furthermore, bioinformatics analysis showed several miRNAs target genes related to stem cell migration and differentiation. This study's findings indicated that SDF-1 induces some of its effects on BMSCs function through miRNA regulation.

The BCR-ABL oncogene is a tyrosine kinase gene that is over-expressed in CML. It inhibits the TGF-β1 signaling pathway. Due to resistance of cells to the tyrosine kinase inhibitor, STI-571, the combined effect of STI-571 and TGF-β1 on K562 cells was studied in the present research. Results revealed that the TGF-β1 cell signaling pathway, which is activated in K562 cells treated with TGF-β1, activates collective cell signaling pathways involved in survival and apoptosis. It is noteworthy that treating K562 cells with STI-571 triggered apoptotic pathways, accompanied by a reduction in proteins such as Bcl-xL, Bcl-2, p-AKT, p-Stat5, p-FOXO3, and Mcl-1 and an increase in the pro-apoptotic proteins PARP cleavage, and p27, leading to an increase in sub-G1 phase-arrested and Annexin-positive cells. Interestingly, the proliferation behavior of TGF-β1-induced cells was changed with the combination therapy, and STI-571-induced apoptosis was also prompted by this combination. Thus, combination treatment appears to promote sub-G1 cell cycle arrest compared to individually treated cells. Furthermore, it strongly triggered apoptotic signaling. In conclusion, TGF-β1 did not negatively impact the effect of STI-571, based on positive annexin cells, and AKT protein phosphorylation remains effective in apoptosis.

Studies published earlier this year demonstrated the association of the solute carrier SLC6A20 gene with the risk and severity of COVID-19. The SLC6A20 protein product (Sodium-dependent Imino Transporter 1 (SIT1)) is involved in the transport of amino acids, including glycine. Here we summarized the results of recent studies demonstrating the interaction of SIT1 with the ACE2 receptor for SARS-CoV-2 as well as an observed association of SLC6A20 with the risk and traits of Type 2 diabetes (T2D). Recently, it was also proposed that SLC6A20 represents the novel regulator of glycine levels and that glycine has beneficial effects against the proinflammatory cytokine secretion induced by SARS-CoV-2 infection. Ivermectin, as a partial agonist of glycine-gated chloride channels, was also recently suggested to interfere with the COVID-19 cytokine storm by inducing the activation of glycine receptors. Furthermore, plasma glycine levels are found to be decreased in diabetic patients. Thus, further clinical trials are warranted to confirm the potential favorable effects of targeting the SIT1 transporter and glycine levels in the treatment of COVID-19, particularly for the severe case of disease associated with hyperglycemia, inflammation, and T2D. These findings suggest that SIT1 may potentially represent one of the missing pieces in the complex puzzle observed between these two pandemic diseases and the potential novel target for their efficient treatment.

Nowadays, mutations in the epidermal growth factor receptor (EGFR) kinase domain are studied in targeted therapy of non-small cell lung cancer (NSCLC) with EGFR tyrosine kinase inhibitors including gefitinib and erlotinib. The present study reports a rare case of a patient harboring three simultaneous EGFR mutations (L718A, Q849H, and L858R). The development of erlotinib resistance was detected in the subsequent treatment. Using a computational approach, the current study investigated the conformational changes of wild-type and mutant EGFR's kinase domains in the interaction with erlotinib. Their binding modes with erlotinib were elucidated during molecular dynamics simulation, where higher fluctuations were detected in the mutated forms of the EGFR tyrosine kinase domain. Prediction of stability and functional effect of mutations revealed that amino acidic substitutions have decreased the protein stability as well as the binding affinity to erlotinib. These results may be useful for a recommendation of EGFR mutational analysis for patients with NSCLC carcinoma.

The main objective of the current study was to improve the essential oil contents of Thymus vulgaris L. using bio-inoculation with bacterial endophytes. Therefore, out of fourteen endophytic bacterial isolates obtained from roots of T. vulgaris , five isolates were selected based on the highest nitrogen-fixation and phosphate solubilization activity and identified as: Bacillus haynesii T9r, Citrobacter farmeri T10r, Bacillus licheniformis T11r, Bacillus velezensis T12r, and Bacillus velezensis T13r. These five strains have been recorded as ammonia, hydrogen cyanide (HCN), siderophores, and indole-3-acetic acid (IAA) producers. These strains have the efficacy to fix-nitrogen by reduction of acetylene with values of 82.133±1.4–346.6±1.4 n-mole-C 2 H 4 /ml/24 h. The IAA, gibberellic acid, abscisic acid, benzyl, kinten, and ziaten production were confirmed using HPLC. Two strains of T11r and T13r showed the highest plant growth-promoting properties and were selected for bio-inoculation of T. vulgaris individually or in a consortium with different mineral fertilization doses (0, 50, 75, and 100%) under field conditions. The highest growth performance was attained with the endophytic consortium (T11r+T13r) in the presence of 100% mineral fertilization. The GC-MS analysis of thyme oil contents showed the presence of 23 various compounds with varying percentages and the thymol fraction represented the highest percentages (39.1%) in the presence of the bacterial consortium.

Out of seven Fusarium spp. isolated from infected faba bean roots, two Fusarium oxysporum were selected and showed faba bean-wilt disease severity with percentages of 68% and 47% under greenhouse conditions. The F. oxysporum showed the highest wilt disease was selected to complete the current study. Three rhizobacterial strains were isolated and identified as Bacillus velezensis Vb1, B. paramycoides Vb3, and B. paramycoides Vb6. These strains showed the highest in-vitro antagonistic activity by the dual-culture method against selected F. oxysporum with inhibition percentages of 59±0.2, 46±0.3, and 52±0.3% for Vb1, Vb3, and Vb6, respectively. These rhizobacterial strains exhibit varied activity for nitrogen-fixing and phosphate-solubilizing. Moreover, these strains showed positive results for ammonia, HCN, and siderophores production. The phytohormones production (indole-3-acetic acid, ABA, benzyl, kinten, ziaten, and GA 3 ) and secretion of various lytic enzymes were recorded by these strains with varying degrees. Under greenhouse conditions, the rhizobacterial strains Vb1, Vb3, Vb6, and their consortium can protect faba bean from wilt caused by F. oxysporum with percentages of 70, 60, 65, and 82%, respectively. Under field conditions, the inoculation with the rhizobacterial consortium (Vb1+Vb3+Vb6) significantly increases the growth performance of the F. oxysporum -infected faba bean plant and recorded the highest wilt protection (83.3%).

Antibodies have transformed biomedical research and are now being used for different experimental applications. Generally, the interaction of enzymes with their specific antibodies can lead to a reduction in their enzymatic activity. The effect of the antibody is dependent on its narrow i.e. the regions of the enzyme to which it is directed. The mechanism of this inhibition is rarely a direct combination of the antibodies with the catalytic site, but is rather due to steric hindrance, barring the substrate access to the active site. In several systems, however, the interaction with the antibody induces conformational changes on the enzyme that can either inhibit or enhance its catalytic activity. The extent of enzyme inhibition or enhancement is, therefore, a reflection of the nature and distribution of the various antigenic determinants on the enzyme molecule. Currently, the mode of action of many enzymes has been elucidated at the molecular level. We here review the molecular mechanisms and recent trends by which antibodies inhibit the catalytic activity of enzymes and provide examples of how specific antibodies can be useful for the neutralization of biologically active molecules