Phyto-Extract-Mediated Synthesis of Silver Nanoparticles (AgNPs) and Their Biological Activities.

Background: Nanotechnology finds broad applications in the field of nanomedicine, an emerging new field used for diagnosis, treatment, prevention of diseases, and improvement of health. Objectives: To synthesize silver nanoparticles (AgNPs) from Withania somnifera and Fagonia indica and to carry out their antimicrobial, insecticidal, and phytotoxic activities, a step toward the new range of nanomedicines. Methods: Silver nanoparticles were synthesized from Withania somnifera and Fagonia indica by chemical reduction method, and further biological activities of these nanoparticles were compared with crude methanolic extract, prepared through cold maceration process, at the concentration of 50 mg/ml. Results: Among all tested bacterial pathogens, crude extract of W. somnifera showed a statistically high significant inhibition zone in millimeter against Pseudomonas aeruginosa (21; p < 0.01). AgNPs showed highly significant result against Streptococcus pneumonia (14; p < 0.01). In comparison with crude extracts, AgNPs showed statistically significant (p < 0.01) results against S. pneumonia (AgNPs, 14; crude, 8.33 mm). Crude extract showed significant inhibition zone against two bacterial strains, P. aeruginosa (crude, 21; AgNPs, 11.67 mm) and Klebsiella pneumoniae (crude, 11.33; AgNPs, 8 mm). Crude extracts of F. indica showed the significant activity against Vibrio cholera (p < 0.01; 11.33 mm). Silver nanoparticles of F. indica exhibited the highest significant activity against Aspergillus flavus and Fusarium oxysporum while AgNPs of W. somnifera were active only against A. flavus. Extracts of W. somnifera and F. indica showed increasing phytotoxic activity with increasing concentrations. The highest significant inhibition was obtained for crude extract (46.7) and AgNPs (45.7) of F. indica at 1000 µg/ml. Insecticidal activity of crude and AgNPs of both plants showed significant inhibition against all tested insects with increasing time intervals, and the highest significant result was obtained at 72 hours with a value of p < 0.01 except T. castaneum. Conclusions: Both crude and AgNPs showed potent activity; however, in comparison, silver nanoparticles showed slightly enhanced activity. Crude and AgNPs of both plants showed good phytotoxic and insecticidal inhibition. Antimicrobial studies of AgNPs on diseases causing pathogens open a door for new antimicrobial agents and could be the answer to antibiotic resistance after further analysis.

Methodologies to Isolate and Purify Clinical Grade Extracellular Vesicles for Medical Applications.

The use of extracellular vesicles (EV) in nano drug delivery has been demonstrated in many previous studies. In this study, we discuss the sources of extracellular vesicles, including plant, salivary and urinary sources which are easily available but less sought after compared with blood and tissue. Extensive research in the past decade has established that the breadth of EV applications is wide. However, the efforts on standardizing the isolation and purification methods have not brought us to a point that can match the potential of extracellular vesicles for clinical use. The standardization can open doors for many researchers and clinicians alike to experiment with the proposed clinical uses with lesser concerns regarding untraceable side effects. It can make it easier to identify the mechanism of therapeutic benefits and to track the mechanism of any unforeseen effects observed.

Heat shock protein 20 promotes sirtuin 1-dependent cell proliferation in induced pluripotent stem cells.

BACKGROUND: Heat shock proteins (HSPs) are molecular chaperones that protect cells against cellular stresses or injury. However, it has been increasingly recognized that they also play crucial roles in regulating fundamental cellular processes. HSP20 has been implicated in cell proliferation, but conflicting studies have shown that it can either promote or suppress proliferation. The underlying mechanisms by which HSP20 regulates cell proliferation and pluripotency remain unexplored. While the effect of HSP20 on cell proliferation has been recognized, its role in inducing pluripotency in human-induced pluripotent stem cells (iPSCs) has not been addressed. AIM: To evaluate the efficacy of HSP20 overexpression in human iPSCs and evaluate the ability to promote cell proliferation. The purpose of this study was to investigate whether overexpression of HSP20 in iPSCs can increase pluripotency and regeneration. METHODS: We used iPSCs, which retain their potential for cell proliferation. HSP20 overexpression effectively enhanced cell proliferation and pluripotency. Overexpression of HSP20 in iPSCs was characterized by immunocytochemistry staining and real-time polymerase chain reaction. We also used cell culture, cell counting, western blotting, and flow cytometry analyses to validate HSP20 overexpression and its mechanism. RESULTS: This study demonstrated that overexpression of HSP20 can increase the pluripotency in iPSCs. Furthermore, by overexpressing HSP20 in iPSCs, we showed that HSP20 upregulated proliferation markers, induced pluripotent genes, and drove cell proliferation in a sirtuin 1 (SIRT1)-dependent manner. These data have practical applications in the field of stem cell-based therapies where the mass expansion of cells is needed to generate large quantities of stem cell-derived cells for transplantation purposes. CONCLUSION: We found that the overexpression of HSP20 enhanced the proliferation of iPSCs in a SIRT1-dependent manner. Herein, we established the distinct crosstalk between HSP20 and SIRT1 in regulating cell proliferation and pluripotency. Our study provides novel insights into the mechanisms controlling cell proliferation that can potentially be exploited to improve the expansion and pluripotency of human iPSCs for cell transplantation therapies. These results suggest that iPSCs overexpressing HSP20 exert regenerative and proliferative effects and may have the potential to improve clinical outcomes.

Artificial intelligence and guidance of medicine in the bubble.

Microbubbles are nanosized gas-filled bubbles. They are used in clinical diagnostics, in medical imaging, as contrast agents in ultrasound imaging, and as transporters for targeted drug delivery. They can also be used to treat thrombosis, neoplastic diseases, open arteries and vascular plaques and for localized transport of chemotherapies in cancer patients. Microbubbles can be filled with any type of therapeutics, cure agents, growth factors, extracellular vesicles, exosomes, miRNAs, and drugs. Microbubbles protect their cargo from immune attack because of their specialized encapsulated shell composed of lipid and protein. Filled with curative medicine, they could effectively circulate through the whole body safely and efficiently to reach the target area. The advanced bubble-based drug-delivery system, integrated with artificial intelligence for guidance, holds great promise for the targeted delivery of drugs and medicines.

Microbubbles versus Extracellular Vesicles as Therapeutic Cargo for Targeting Drug Delivery.

Extracellular vesicles (EVs) and microbubbles are nanoparticles in drug-delivery systems that are both considered important for clinical translation. Current research has found that both microbubbles and EVs have the potential to be utilized as drug-delivery agents for therapeutic targets in various diseases. In combination with EVs, microbubbles are capable of delivering chemotherapeutic drugs to tumor sites and neighboring sites of damaged tissues. However, there are no standards to evaluate or to compare the benefits of EVs (natural carrier) versus microbubbles (synthetic carrier) as drug carriers. Both drug carriers are being investigated for release patterns and for pharmacokinetics; however, few researchers have focused on their targeted delivery or efficacy. In this Perspective, we compare EVs and microbubbles for a better understanding of their utility in terms of delivering drugs to their site of action and future clinical translation.

Clinical Relevance of RNA Editing to Early Detection of Cancer in Human.

DNA encodes RNA and is responsible for protein production in cells. RNA editing is the process by which genetic information is altered in the RNA molecule. RNA editing in cancer initiation, progression and development has been well documented and play an important role in tumorigenesis. Studying RNA editing and its application to change genetic information after transcription, RNA-editing technology could be an important innovation in cancer and has the potential for more effective precision treatment. Bioengineering integration approach and artificial intelligence could revolutionize the entire field of RNA editing for early detection of cancer.

Clinical Applications of RNA Editing Technology for the Early Detection of Cancer and Future Directions.

Early detection of cancer has great clinical importance and potentially improves cure, survival rate and treatment outcome. RNA editing technology can be used as targeted and precise molecular scissors to cut and replace disease-causing genes with healthy ones. This is a post transcriptional modification that can lead to the recoding of proteins. RNA editing technology is in its infancy, but it can be used for early diagnoses and effective treatment of cancer. The full potential of precision medicine will be achieved by using the knowledge of RNA reversible-recoding to edit the protein. RNA editing technology could be used to expose chemo resistant cancer cells, dormant cancer stem cells and other malignant tumors. RNA editing generates RNA and protein diversity to accelerate and enhance the screening window for early detection of cancer. We propose that the RNA editing sites could be used as a novel tool for early detection of cancer.

Mesenchymal stem cells confer chemoresistance in breast cancer via a CD9 dependent mechanism.

The development of chemotherapy drug resistance remains a significant barrier for effective therapy in several cancers including breast cancer. Bone marrow-derived mesenchymal stem cells (BMMSCs) have previously been shown to influence tumor progression and the development of chemoresistance. In the present study, we showed that when GFP labelled BMMSCs and RFP labelled HCC1806 cells are injected together in vivo, they create tumors which contain a new hybrid cell that has characteristics of both BMMSCs and HCC1806 cells. By labelling these cells prior to their injection, we were then able to isolate new hybrid cell from harvested tumors using FACS (DP-HCC1806:BMMSCs). Interestingly, when DP-HCC1806:BMMSCs were then injected into the mammary fat pad of NOD/SCID mice, they produced xenograft tumors which were smaller in size, and exhibited resistance to chemotherapy drugs (i.e. doxorubicin and 5-fluorouracil), when compared tumors from HCC1806 cells alone. This chemoresistance was shown to associated with an increased expression of tetraspanins (CD9, CD81) and drug resistance proteins (BCRP, MDR1). Subsequent siRNA-mediated knockdown of BMMSC-CD9 in DP-HCC1806:BMMSCs resulted in an attenuation of doxorubicin and 5-fluorouracil chemoresistance associated with decreased BCRP and serum cytokine expression (CCL5, CCR5, CXCR12). Our findings suggest that within the tumor microenvironment, CD9 is responsible for the crosstalk between BMMSCs and HCC1806 breast cancer cells (via CCL5, CCR5, and CXCR12) which contributes to chemoresistance. Hence, BMMSC-CD9 may serve as an important therapeutic target for the treatment of breast cancer.

Detection, virulence and genetic diversity of Fusarium species infecting tomato in Northern Pakistan.

In addition to the well-known Fusarium oxysporum f.sp. lycopersici, several other Fusarium species are known to cause extensive worldwide crop losses in tomatoes. Prevalence and identities of Fusarium species infecting tomatoes in Northwest Pakistan is currently not known. In this study, we surveyed and characterized Fusarium species associated with symptomatic tomatoes in Northwest Pakistan using morphological and molecular analyses. Pathogenicity tests revealed varying degrees of virulence with some Fusarium sp. causing severe disease symptoms whereas others displaying mild symptoms. Molecular identification based on Internal Transcribed Spacer (ITS) region and TEF-1α gene sequencing classified all isolates into four major species with a majority (68.9%) belonging to Fusarium incarnatum-equiseti species complex (FIESC), followed by F. graminearum (20.7%), F. acuminatum (6.8%), and F. solani (6.8%). ISSR analyses revealed substantial genetic variability among all the Fusarium population infecting tomatoes. Genetic distance between populations from the central region and the type strain F.o. f.sp. lycopersici from Florida was the highest (0.3662), whereas between the south and central region was the lowest (0.0298), which showed that genetic exchange is negatively effected by distance. High genetic variability suggests that these Fusarium species have the potential to become a major production constraint for tomato growers. Findings in this report would greatly facilitate identification of Fusarium species in developing countries and would provide groundwork for devising and implementing disease management measures for minimizing losses caused by Fusarium species in tomatoes.

A glucanolytic Pseudomonas sp. associated with Smilax bona-nox L. displays strong activity against Phytophthora parasitica.

Biological control is an eco-friendly strategy for mitigating and controlling plant diseases with negligible effects on human health and environment. Biocontrol agents are mostly isolated from field crops, and microbiomes associated with wild native plants is underexplored. The main objective of this study was to characterize the bacterial isolates associated with Smilax bona-nox L, a successful wild plant with invasive growth habits. Forty morphologically distinct bacterial isolates were recovered from S. bona-nox. Based on 16S rRNA gene sequencing, these isolates belonged to 12 different genera namely Burkholderia, Pseudomonas, Xenophilus, Stenotrophomonas, Pantoea, Enterobactriaceae, Kosakonia, Microbacterium, Curtobacterium, Caulobacter, Lysinibacillus and Bacillus. Among them, Pseudomonas sp. EA6 and Pseudomonas sp. EA14 displayed the highest potential for inhibition of Phytophthora. Based on sequence analysis of rpoD gene, these isolates revealed a 97% identity with a Pseudomonas fluorescence strain. Bioactivity-driven assays for finding bioactive compounds revealed that crude proteins of Pseudomonas sp. EA6 inhibited mycelial growth of P. parasitica, whereas crude proteins of Pseudomonas sp. EA14 displayed negligible activity. Fractionation and enzymatic analyses revealed that the bioactivity of Pseudomonas sp. EA6 was mostly due to glucanolytic enzymes. Comparison of chromatographic profile and bioactivity assays indicated that the secreted glucanolytic enzymes consisted of ß-1,3 and ß-1,4 glucanases, which acted together in hydrolyzing Phytophthora cell walls. Since the biological activity of the crude glucanolytic extract was >60-fold higher than the purified ß-1,3 glucanase, the glucanolytic enzyme system of Pseudomonas sp. EA6 likely acts synergistically in cell wall hydrolysis of P. parasitica.

A highly efficient ligation-independent cloning system for CRISPR/Cas9 based genome editing in plants.

BACKGROUND: Most current methods for constructing guide RNAs (gRNA) for the CRISPR/Cas9 genome editing system, depend on traditional cloning using specific type IIS restriction enzymes and DNA ligation. These methods consist of multiple steps of cloning, and are time consuming, resource intensive and not flexible. These issues are particularly exacerbated when multiple guide RNAs need to be assembled in one plasmid such as for multiplexing or for the paired nickases approach. Furthermore, identification of functional gRNA clones usually requires expensive in vitro screening. Addressing these issues will greatly facilitate usage and accessibility of CRISPR/Cas9 genome editing system to resource-limited laboratories. RESULTS: To improve efficiency of cloning multiple guide RNAs for the CRISPR/Cas9 system, we developed a restriction enzyme- and ligation-independent strategy for cloning gRNAs directly in plant expression vectors in one step. Our method relies on a negative selection marker and seamless cloning for combining multiple gRNAs directly in a plant expression vector in one reaction. In addition, using the Agrobacterium-mediated transient assays, this method provides a simple in planta procedure for assaying the effectiveness of multiple gRNAs very rapidly. CONCLUSIONS: For a fraction of resources used in the type IIS restriction enzyme-based cloning method and in vitro screening assays, the system reported here allows efficient construction and testing several ready-to-transfect gRNA constructs in < 3 days. In addition, this system is highly versatile and flexible, and by designing only two additional target-specific primers, multiple gRNAs can be easily assembled in any plasmid in a single reaction.