ZIF-8 Nanoparticle: A Valuable Tool for Improving Gene Delivery in Sperm-Mediated Gene Transfer.

Metal-organic frameworks (MOFs) are porous materials with unique characteristics that make them well-suited for drug delivery and gene therapy applications. Among the MOFs, zeolitic imidazolate framework-8 (ZIF-8) has emerged as a promising candidate for delivering exogenous DNA into cells. However, the potential of ZIF-8 as a vector for sperm-mediated gene transfer (SMGT) has not yet been thoroughly explored.This investigation aimed to explore the potential of ZIF-8 as a vector for enhancing genetic transfer and transgenesis rates by delivering exogenous DNA into sperm cells. To test this hypothesis, we employed ZIF-8 to deliver a plasmid expressing green fluorescent protein (GFP) into mouse sperm cells and evaluated the efficiency of DNA uptake. Our findings demonstrate that ZIF-8 can efficiently load and deliver exogenous DNA into mouse sperm cells, increasing GFP expression in vitro. These results suggest that ZIF-8 is a valuable tool for enhancing genetic transfer in SMGT, with important implications for developing genetically modified animals for research and commercial purposes. Additionally, our study highlights the potential of ZIF-8 as a novel class of vectors for gene delivery in reproductive biology.Overall, our study provides a foundation for further research into using ZIF-8 and other MOFs as gene delivery systems in reproductive biology and underscores the potential of these materials as promising vectors for gene therapy and drug delivery.

Culture strategy as a modulator of target assessments: Functionality of suspension versus hanging drop-derived choriocarcinoma spheroids as in vitro model of embryo implantation.

The choriocarcinoma spheroid model has been amply applied to study the underlying molecular mechanism of implantation. Reproducibility and functionality of spheroid tumor models were addressed precisely. To mimic embryo-endometrium crosstalk, no functional characteristics of spheroids have been provided based on culture strategies. In this study, choriocarcinoma spheroids were provided as suspension culture (SC) or hanging drop culture (HDC). Primary assessments were performed based on morphology, cellular density, and hormonal secretion. Spheroid-endometrial cross talk was assessed as coculture procedures. Further, alkaline phosphatase (ALP) activity and expression of genes involved in attachment, invasion, and inducing migration were quantified. We found HDC spheroids provided a homogenous-shaped aggregate with a high grade of viability, cellular integration, hormonal secretion, and the dominant role of WNTs expression in their microarchitecture. SC spheroids showed a higher level of ALP activity and the expression of integrated genes in modulating attachment, invasion, and migration abilities. Spheroid confrontation assays clearly clarified the superiority of SC spheroids to crosstalk with epithelial and stromal cells of endometrium in addition to motivating an ideal endometrial response. Conclusively, culture strategies by affecting various molecular signaling pathways should be chosen precisely according to specific target assessments. Specifically, SC assumed as an ideal model in spheroid-endometrial cross talk.

Photobiomodulation preconditioned human semen protects sperm cells against detrimental effects of cryopreservation.

The biological consequences of semen samples preconditioning with photobiomodulation (PBM) were studied on human sperm cells post cryopreservation. Donated semen samples were collected from 22 married men with normal sperm parameters according to World Health Organization (WHO) criteria. Included samples were divided into control and PBM-preconditioning (one session, 810 nm, diode laser, and 0.6 J/cm2) groups before cryopreservation procedure. Progressive sperm motility (PSM), morphology, viability, sperm mitochondrial membrane potential(MMP), intracellular reactive oxygen species (ROS) and lipid peroxidation of sperm cells were assessed post thawing. PBM preconditioning of cryopreserved semen samples most prominently increased the PSM percentage 30 min post thawing (p = 0.000).Application of PBM before cryopreservation significantly increased the number of viable spermatozoa (p = 0.000), increased significantly the number of spermatozoa with high MMP (p = 0.004) and decreased significantly the number of spermatozoa with low MMP post-thawing(P = 0. 007)compared to control group. Cryopreserved human sperm cells with PBM preconditioning showed significant decrease in the levels of intracellular ROS (47.66 ± 2.14 versus 60.42 ± 3.16, p = 0.002) and lipid peroxidation (3.06 ± 0.13 versus 3.68 ± 0.27, p = 0.05)compared to control group. Our findings, as the first evidence, indicated that PBM-preconditioning of human semen before cryopreservation provides a real and substantial advantage. This might lead to a novel strategy in improving PBM application in the procedures of assisted reproductive technologies.

Improvement in viability and mineralization of osteoporotic bone marrow mesenchymal stem cell through combined application of photobiomodulation therapy and oxytocin.

The probable positive effects of photobiomodulation therapy (PBMT) and oxytocin (OT) treatments together or alone were evaluated on cell viability along with the changes in the gene expression of Osteocalcin (OC), Osteoprotegerin (OPG), and Runt-related transcription factor 2 (Runx2) levels of sham (healthy)-Bone marrow mesenchymal stem cell(BMMSC) and ovariectomy-induced osteoporosis (OVX)-BMMSC. BMMSC was harvested from healthy and OVX rats and was cultured in osteogenic induction medium (OIM). There were five groups of BMMSCs: (1) sham -BMMSCs; (2) control -OVX-BMMSCs; (3) OT-treated-OVX-BMMSCs; (4) PBMT-treated-OVX-BMMSCs, and (5) OT + PBMT-OVX-BMMSCs. In all 5 groups, BMMSC viability and proliferation as well as gene expression of OC, OPG, and RUNX2 were evaluated. PBMT and PBMT + OT treatments showed a promising effect on the increased viability of OVX-BMMSC (ANOVA test; LSD test, p = 0.01, p = 0.002). The results of gene expression analysis revealed that the sham- BMMSCs responded optimally to OT treatment. It was also found that OVX-BMMSCs responded optimally to PBMT + OT and PBMT treatments at early and middle stages of osteogenic induction process. Nevertheless, they responded optimally to PBMT + OT and OT especially at the late stage of osteogenic induction process. PBMT and PBMT + OT treatments significantly increased viability of OVX-BMMSC in OIM in vitro. Both PBMT and PBMT + OT treatments could promote mineralization of OVX-BMMSC in the culture medium at early and middle stages of osteogenic induction process. Both OT and PBMT + OT treatments could promote mineralization of OVX-BMMSC in vitro at late stages of osteogenic induction process.

Intraovarian injection of miR-224 as a marker of polycystic ovarian syndrome declines oocyte competency and embryo development.

miR-224 is associated with polycystic ovary syndrome (PCOS) that is an epidemic in reproductive age women. Most studies of miR-224 have focused on in vitro analyses, whereas the in vivo effects are not widely understood. In this study, we have conducted in silico analysis and found two potential miR-224 target genes, Ptx3 and Smad4 that have roles in folliculogenesis. Because patients with PCOS have decreased numbers of follicular cells related to cell apoptosis, we also investigated two apoptotic genes, Bax and Bcl2. We used the intraovarian injection method to deliver miR-224 into a mouse model. Histological examination of the ovaries was done by fluorescent microscope. Fertilization, cleavage, and developmental competence rates were counted under a stereomicroscope and compared between the studied groups. Quantitative real-time polymerase chain reaction (qRT-PCR) analysis of miR-224 was conducted to determine the levels of the studied genes in the oocytes, cumulus cells, and blastocysts. The numbers of oocytes and fertilization rate indicated a higher apoptosis index ( p < 0.05) and increased numbers of degenerated embryos with irregular blastomeres and fragmented cytoplasm in the experimental group. RT-PCR results indicated a significant increase in miR-224 levels in the manipulated group. Of the four analyzed genes, Ptx3, Smad4, and Bcl2 had decreased levels in the transfected group, with increased Bax expression ( p < 0.05). This data showed that miR-224 negatively affected ovulation in the mouse model by decreasing Ptx3 and Smad4 expressions. The changes in Bcl2 and Bax expression levels, as apoptosis biomarkers, showed that apoptosis was a secondary outcome of the effect of miR-224.

Sirt1 antisense transcript is down-regulated in human tumors.

Natural antisense transcripts (NATs) have recently been associated with the development of human cancers. Recent studies have shown that a natural antisense transcript (NAT) is present in Sirt1 gene which encodes a NAD-dependent deacetylase. Interestingly, expression of Sirt1 mRNA changes during development and progression of human cancers. However, it remains unclear to what extent Sirt1 antisense transcript (AS) may contribute to changes in the expression of Sirt1 mRNA. To determine this, we used quantitative measurement of RNA to reveal relationship between Sirt1 mRNA and Sirt1-AS across human cancer tissues, cell lines and stem cells. While Sirt1 mRNA level was increased in cancer cell lines and cancer tissues, the expression level of Sirt1-AS was lower in cancers compared to controls. This inverse correlation was observed in the expression of Sirt1 sense and antisense transcripts in normal and cancer tissues suggesting a functional role for Sirt1-AS in regulation of Sirt1 mRNA.

Dimethyl sulphoxide and electrolyte-free medium improve exogenous DNA uptake in mouse sperm and subsequently gene expression in the embryo.

SummaryOne of the methods to generate transgenic animals is called sperm-mediated gene transfer (SMGT). Mature sperm cells can take up exogenous DNA molecules intrinsically and transfer them into the oocyte during fertilization. This study assessed the effect of dimethyl sulfoxide (DMSO) and electrolyte-free medium (EFM) on DNA uptake (EGFP-N1plasmid) in mouse sperm. Sperms cells cultured in human tubular fluid (HTF) without any treatment were considered as the control group. Sperms cells that were incubated in EFM and HTF with DNA/DMSO at 4°C were classified into EFM and HTF groups. Sperm motility and viability were assessed following treatment. In vitro fertilization (IVF) with sperm in all groups was performed. Fertilization, embryo development and GFP-positive blastocyst rates were analyzed and compared. The result showed that sperm motility and viability in EFM were better than those in the HTF group. The rate of development to reach the blastocyst stage and GFP-positive blastocysts was significantly higher in the EFM group compared with the HTF group (P<0.05). Our data demonstrate that sperm stored in the EFM group can improve the efficiency of SMGT for the generation of GFP-positive blastocysts.

Formulation and evaluation of atovaquone-loaded macrophage-derived exosomes against Toxoplasma gondii: in vitro and in vivo assessment.

IMPORTANCE: This study is the first of its kind that suggests exosomes as a nano-carrier loaded with atovaquone (ATQ), which could be considered as a new strategy for improving the effectiveness of ATQ against acute and chronic phases of Toxoplasma gondii.

Recent Advances in CRISPR/Cas9-Mediated Genome Editing in Leishmania Strains.

BACKGROUND: Genome manipulation of Leishmania species and the creation of modified strains are widely employed strategies for various purposes, including gene function studies, the development of live attenuated vaccines, and the engineering of host cells for protein production. OBJECTIVE: Despite the introduction of novel manipulation approaches like CRISPR/Cas9 technology with significant advancements in recent years, the development of a reliable protocol for efficiently and precisely altering the genes of Leishmania strains remains a challenging endeavor. Following the successful adaptation of the CRISPR/Cas9 system for higher eukaryotic cells, several research groups have endeavored to apply this system to manipulate the genome of Leishmania. RESULTS: Despite the substantial differences between Leishmania and higher eukaryotes, the CRISPR/Cas9 system has been effectively tested and applied in Leishmania.  CONCLUSION: This comprehensive review summarizes all the CRISPR/Cas9 systems that have been employed in Leishmania, providing details on their methods and the expression systems for Cas9 and gRNA. The review also explores the various applications of the CRISPR system in Leishmania, including the deletion of multicopy gene families, the development of the Leishmania vaccine, complete gene deletions, investigations into chromosomal translocations, protein tagging, gene replacement, large-scale gene knockout, genome editing through cytosine base replacement, and its innovative use in the detection of Leishmania. In addition, the review offers an up-to-date overview of all double-strand break repair mechanisms in Leishmania.

Identification of co-regulated genes associated with doxorubicin resistance in the MCF-7/ADR cancer cell line.

Introduction: The molecular mechanism of chemotherapy resistance in breast cancer is not well understood. The identification of genes associated with chemoresistance is critical for a better understanding of the molecular processes driving resistance. Methods: This study used a co-expression network analysis of Adriamycin (or doxorubicin)-resistant MCF-7 (MCF-7/ADR) and its parent MCF-7 cell lines to explore the mechanisms of drug resistance in breast cancer. Genes associated with doxorubicin resistance were extracted from two microarray datasets (GSE24460 and GSE76540) obtained from the Gene Expression Omnibus (GEO) database using the GEO2R web tool. The candidate differentially expressed genes (DEGs) with the highest degree and/or betweenness in the co-expression network were selected for further analysis. The expression of major DEGs was validated experimentally using qRT-PCR. Results: We identified twelve DEGs in MCF-7/ADR compared with its parent MCF-7 cell line, including 10 upregulated and 2 downregulated DEGs. Functional enrichment suggests a key role for RNA binding by IGF2BPs and epithelial-to-mesenchymal transition pathways in drug resistance in breast cancer. Discussion: Our findings suggested that MMP1, VIM, CNN3, LDHB, NEFH, PLS3, AKAP12, TCEAL2, and ABCB1 genes play an important role in doxorubicin resistance and could be targeted for developing novel therapies by chemical synthesis approaches.

Identification and differentiation of Fasciola hepatica and F. gigantica using multiplex PCR technique.

We aimed to present an alternate method instead of PCR-RFLP and also develop an optimized method for rapid, time-saving and affordable molecular-based approach to discriminate species of liver fluke, Fasciola hepatica and F. gigantica. Seventy-six samples of F. hepatica and 28 F. gigantica were collected from the slaughterhouses of endemic regions in Iran. Following a comprehensive analysis of the mitochondrial complete sequences of both F. hepatica and F. gigantica, the extracted DNAs from all samples were used as templates in multiplex PCR reactions containing two sets of primers specific for cytochrome c oxidase I (cox I) gene of both species. In a parallel experiment, PCR-RFLP was performed for each sample using internal transcribed spacer (ITS1) sequence. Furthermore, following a PCR amplification for cox I gene, the amplicons were purified for sequencing. To assess the validity of the multiplex PCR approach, the obtained data from the multiplex PCR and PCR-RFLP experiments were compared with each other. By sequence analysis of 104 samples, 76 and 28 samples were identified as F. hepatica and F. gigantica, respectively. Results revealed 100% and 92% of accuracy as for multiplex PCR and PCR-RFLP. The designed multiplex PCR strategy offers a valid alternative approach to the conventional methods with distinctive features including convenience, cost-effectiveness, time-saving (3 hours from sampling to obtain final results) and high efficacy.

Polycistronic Expression of Multi-Subunit Complexes in the Eukaryotic Environment: A Narrative Review.

Protein complexes are involved in many vital biological processes. Therefore, researchers need these protein complexes for biochemical and biophysical studies. Several methods exist for expressing multi-subunit proteins in eukaryotic cells, such as 2A sequences, IRES, or intein. Nevertheless, each of these elements has several disadvantages that limit their usage. In this article, we suggest a new system for expressing multi-subunit proteins, which have several advantages over existing methods meanwhile it, lacks most of their disadvantages. Leishmania is a unicellular eukaryote and member of the Trypanosomatidae family. In the expression system of Leishmania, pre-long RNAs that contain several protein sequences transcribe. Then these long RNAs separate into mature mRNAs in the process named trans splicing. For producing multi-subunit protein, Leishmania transformed with a vector containing the sequences of all subunits. Therefore, those subunits translate and form the complex under eukaryotic cell conditions. The sequence of each protein must separate by the spatial sequence needed for trans splicing. Based on a Leishmania expression pattern, not only is it possible to produce the complexes with the correct structures and post-translational modifications, but also it is possible to overcome previous method problems.

The Influence of Different Culture Media on the Growth and Recombinant Protein Production of Iranian Lizard Leishmania Promastigote.

Background: Leishmania is a eukaryotic protozoan parasite belonging to the Trypanosomatidae family. The Iranian Lizard Leishmania (I.L.L.), which is nonpathogenic to mammals, shows great promise to be used as an expression system for recombinant protein production. Unlike other Leishmania strains, the ideal culture medium for I.L.L. has not been established, although it is commonly cultured in the RPMI1640 medium. Methods: We investigated the growth rate of the wild and recombinant I.L.L. in BHI, RPMI1640, LB, and M199 media with and without FBS, hemin, or lyophilized rabbit serum. Subsequently, the expression rate of the recombinant protein in these media was compared. Results: The growth rate of I.L.L. in RPMI1640 medium and LB broth was similar and supplementation with 10% FBS did not affect the growth rate. The amount of protein expression in the LB medium was higher than in the other three media. Conclusion: The LB broth is an appropriate medium for I.L.L. culture and recombinant protein production.

Identifying Key Lysosome-Related Genes Associated with Drug-Resistant Breast Cancer Using Computational and Systems Biology Approach.

Background: Drug resistance in breast cancer is an unsolved problem in treating patients. It has been recently discussed that lysosomes contribute to the invasion and angiogenesis of cancer cells. There is evidence that lysosomes can also cause multi-drug resistance. We analyzed this emerging concept in breast cancer through computational and systems biology approaches. Objectives: We aimed to identify the key lysosome-related genes associated with drug-resistant breast cancer. Methods: All genes contributing to the structure and function of lysosomes were inquired through the Human Lysosome Gene Database. The prioritized top 51 genes from the provided lists of Endeavour, ToppGene, and GPSy as prioritization tools were selected. All lysosomal genes and 12 breast cancer-related genes aligned to identify the most similar genes to breast cancer-related genes. Different centralities were applied to score each human protein to calculate the most central lysosomal genes in the human protein-protein interaction (PPI) network. Common genes were extracted from the results of the mentioned methods as a selected gene set. For Gene Ontology enrichment, the selected gene set was analyzed by WebGestalt, DAVID, and KOBAS. The PPI network was constructed via the STRING database. The PPI network was analyzed utilizing Cytoscape for topology network interaction and CytoHubba to extract hub genes. Results: Based on biological studies, literature reviews, and comparing all mentioned analyzing methods, six genes were introduced as essential in breast cancer. This computational approach to all lysosome-related genes suggested that candidate genes include PRF1, TLR9, CLTC, GJA1, AP3B1, and RPTOR. The analyses of these six genes suggest that they may have a crucial role in breast cancer development, which has rarely been evaluated. These genes have a potential therapeutic implication for new drug discovery for chemo-resistant breast cancer. Conclusions: The present work focused on all the functional and structural lysosome-related genes associated with breast cancer. It revealed the top six lysosome hub genes that might serve as therapeutic targets in drug-resistant breast cancer. Since these genes play a pivotal role in the structure and function of lysosomes, targeting them can effectively overcome drug resistance.

Mission impossible for cellular internalization: When porphyrin alliance with UiO-66-NH2 MOF gives the cell lines a ride.

Is it possible to accelerate cell internalization by hybridization of nanomaterials? Herein we support the realization of using metal-organic frameworks (MOFs) with the assistance of rigid porphyrin structure (H2TMP) aimed at drug loading, drug release, relative cell viability, and targeted in vitro drug delivery. There are several MOFs, i.e., UiO-66-NH2 (125 ± 12.5 nm), UiO-66-NH2 @H2TMP (160 ± 14 nm), UiO-66-NH2 @H2TMP@DOX, and UiO-66-NH2 @H2TMP@DOX@RO were synthesized and characterized applying HEK-293, HT-29, MCF-7, and MCF-10A cell lines. MTT investigations proved a significantly higher relative cell viability for H2TMP-aided leaf-extract-coated nanocarriers (above 62 % relative cell viability). Furthermore, the rigid H2TMP structure improved drug loading capacity by 24 % through an enhanced hydrogen bond, van der Waals, and π-π interactions. The in vitro targeted drug delivery experiments were conducted on HT-29 and MCF-7 cell lines. First, nanocarriers were treated with HT-29 cells, where UiO-66-NH2 @H2TMP@DOX@RO appeared as the best nanocarrier. Then, the selected nanocarrier was extracted from the HT-29 cell line and treated with the MCF-7 cell line. For the first time, the DOX remained inside the UiO-66-NH2 @H2TMP@DOX@RO after successful delivery to the HT-29 cell lines was observed on the MCF-7 cell line, and the second targeted drug delivery was performed. The results of this survey can enlighten the future ahead of cell internalization in MOF-based hybrid nanostructures.

Comparative Effect of Photobiomodulation on Human Semen Samples Pre- and Post-Cryopreservation.

The primary objective of this study is to evaluate and to compare the effects of photobiomodulation (PBM) on sperm parameters both before and after cryopreservation. In this regard, 24 freshly ejaculated semen samples from normozoospermic men were included in this study. Each semen sample was randomly divided into three groups (1 ml aliquot for each group): the control group (group one) underwent conventional sperm cryopreservation (n = 24), group two underwent pre-freezing PBM exposure (810 nm, diode laser, and 0.6 J/cm2) (n = 24), and group three underwent post freezing and thawing PBM exposure (n = 24). Indicators of sperm quality, including total sperm motility (TSM), progressive sperm motility (PSM), DNA fragmentation, lipid peroxidation levels, apoptosis-like changes, and gene expression levels of protamine (PRM) 1, PRM2, and adducin 1 alpha (ADD1), were investigated in a blinded style. Due to the beneficial effect of pre-freezing PBM therapy, group 2 exhibited the highest TSM and PSM levels compared to groups 1 and 3. At the same time, DNA fragmentation and lipid peroxidation were significantly reduced in the group 2 compared to the group 1 (p = 0.024 p = 0.016, respectively). Evaluation of apoptotic/necrotic changes revealed that parameters including early apoptosis, dead, and necrotic cells decreased in the group 2 compared to the either groups 1 (p = 0. 008, p = 0. 032, p = 0. 02, respectively) or group 3 (p = 0.037, p = 0.108, p = 0.083). There were no significant differences in the expression levels of PRM1, PRM2, and ADD1 among the study groups. Based on our results, PBM therapy prior to cryopreservation, even in the normal semen samples, plays a significant protective role against cryo-damage by preserving the functional parameters of spermatozoa.

Therapeutic potentials of CRISPR-Cas genome editing technology in human viral infections.

Viral infections are a common cause of morbidity worldwide. The emergence of Coronavirus Disease 2019 (COVID-19) has led to more attention to viral infections and finding novel therapeutics. The CRISPR-Cas9 system has been recently proposed as a potential therapeutic tool for the treatment of viral diseases. Here, we review the research progress in the use of CRISPR-Cas technology for treating viral infections, as well as the strategies for improving the delivery of this gene-editing tool in vivo. Key challenges that hinder the widespread clinical application of CRISPR-Cas9 technology are also discussed, and several possible directions for future research are proposed.

Molecular Characterization of Animal Fasciola Spp. Isolates from Lorestan Province, Western Iran.

Background: We aimed to detect the genetic diversity of samples identified morphologically as Fasciola spp. from sheep, cattle and goat from Lorestan Province, western Iran using PCR-RFLP method. Besides, we evaluated the genetic diversity indices, sequencing and phylogenetic analysis using mitochondrial gene (ND1 and CO1). Methods: PCR-RFLP analysis of ribosomal ITS1 fragment by RsaI restriction enzyme to investigate the genetic characteristics of Fasciola species obtained from different hosts (18 sheep, 21 cattle, and 17goats) was conducted. The samples were sequenced. Sequences were evaluated using BLAST software and the parasite species were identified with similarity percentage and overlap with the species registered in the gene bank. Then similarity and diversity of intra-species and intra-species diversity of Fasciola species were calculated. Results: In Lorestan, based on RFLP pattern, 93% (52) of the Fasciola spp. isolates had a RFLP pattern related to F. hepatica and 7% (4) were F. gigantica. No hybrid forms were detected. The CO1 gene could clarify 19 haplotypes against ND1 gene that found 22 haplotypes among livestock. Sequencing results of the mtDNA showed intra-species identity 98. 5%-100% and Intra-species-diversity: 0-1.5% compared to the GenBank sequences. Conclusion: Using PCR-RFLP method, two species of F. hepatica and F. gigantica, were present in Lorestan Province, but F. hepatica was more prevalent. Mitochondrial genes could better test variability indices in different hosts than ribosomal genes, consequently among mitochondrial genes, the ND1 gene could better examine differences and similarities than CO1.

Calcium-based nanomaterials and their interrelation with chitosan: optimization for pCRISPR delivery.

There have been numerous advancements in the early diagnosis, detection, and treatment of genetic diseases. In this regard, CRISPR technology is promising to treat some types of genetic issues. In this study, the relationship between calcium (due to its considerable physicochemical properties) and chitosan (as a natural linear polysaccharide) was investigated and optimized for pCRISPR delivery. To achieve this, different forms of calcium, such as calcium nanoparticles (CaNPs), calcium phosphate (CaP), a binary blend of calcium and chitosan including CaNPs/Chitosan and CaP/Chitosan, as well as their tertiary blend including CaNPs-CaP/Chitosan, were prepared via both routine and green procedures using Salvia hispanica to reduce toxicity and increase nanoparticle stability (with a yield of 85%). Such materials were also applied to the human embryonic kidney (HEK-293) cell line for pCRISPR delivery. The results were optimized using different characterization techniques demonstrating acceptable binding with DNA (for both CaNPs/Chitosan and CaNPs-CaP/Chitosan) significantly enhancing green fluorescent protein (EGFP) (about 25% for CaP/Chitosan and more than 14% for CaNPs-CaP/Chitosan). Supplementary Information: The online version contains supplementary material available at 10.1007/s40097-021-00446-1.

Green chemistry and coronavirus.

The novel coronavirus pandemic has rapidly spread around the world since December 2019. Various techniques have been applied in identification of SARS-CoV-2 or COVID-19 infection including computed tomography imaging, whole genome sequencing, and molecular methods such as reverse transcription polymerase chain reaction (RT-PCR). This review article discusses the diagnostic methods currently being deployed for the SARS-CoV-2 identification including optical biosensors and point-of-care diagnostics that are on the horizon. These innovative technologies may provide a more accurate, sensitive and rapid diagnosis of SARS-CoV-2 to manage the present novel coronavirus outbreak, and could be beneficial in preventing any future epidemics. Furthermore, the use of green synthesized nanomaterials in the optical biosensor devices could leads to sustainable and environmentally-friendly approaches for addressing this crisis.