A mechanistic study on the tolerance of PAM distal end mismatch by SpCas9.

The therapeutic application of CRISPR-Cas9 is limited due to its off-target activity. To have a better understanding of this off-target effect, we focused on its mismatch-prone PAM distal end. The off-target activity of SpCas9 depends directly on the nature of mismatches, which in turn results in deviation of the active site of SpCas9 due to structural instability in the RNA-DNA duplex strand. In order to test the hypothesis, we designed an array of mismatched target sites at the PAM distal end and performed in vitro and cell line-based experiments, which showed a strong correlation for Cas9 activity. We found that target sites having multiple mismatches in the 18th to 15th position upstream of the PAM showed no to little activity. For further mechanistic validation, Molecular Dynamics simulations were performed, which revealed that certain mismatches showed elevated root mean square deviation values that can be attributed to conformational instability within the RNA-DNA duplex. Therefore, for successful prediction of the off-target effect of SpCas9, along with complementation-derived energy, the RNA-DNA duplex stability should be taken into account.

Current Strategies for Combating Biofilm-Forming Pathogens in Clinical Healthcare-Associated Infections.

The biofilm formation by various pathogens causes chronic infections and poses severe threats to industry, healthcare, and society. They can form biofilm on surfaces of medical implants, heart valves, pacemakers, contact lenses, vascular grafts, urinary catheters, dialysis catheters, etc. These biofilms play a central role in bacterial persistence and antibiotic tolerance. Biofilm formation occurs in a series of steps, and any interference in these steps can prevent its formation. Therefore, the hunt to explore and develop effective anti-biofilm strategies became necessary to decrease the rate of biofilm-related infections. In this review, we highlighted and discussed the current therapeutic approaches to eradicate biofilm formation and combat drug resistance by anti-biofilm drugs, phytocompounds, antimicrobial peptides (AMPs), antimicrobial lipids (AMLs), matrix-degrading enzymes, nanoparticles, phagebiotics, surface coatings, photodynamic therapy (PDT), riboswitches, vaccines, and antibodies. The clinical validation of these findings will provide novel preventive and therapeutic strategies for biofilm-associated infections to the medical world.

Cronobacter sakazakii Cue for the Attraction and Its Impact on the Immunity of Caenorhabditis elegans.

Cronobacter sakazakii, an opportunistic foodborne pathogen prevalently detected in contaminated powdered infant formula, is associated with different diseases, including meningitis. It can cross the blood-brain barrier and affects the CNS. The impact of C. sakazakii on host neuronal cells and behavior is largely unknown. Hence, detailed molecular data are required to understand its severity. Caenorhabditis elegans is a unique model for studying chemical communication, as it relies on chemosensation for searching nutritional supplements. Although, C. sakazakii is pathogenic to C. elegans, our analysis indicated that C. elegans was highly attracted toward C. sakazakii compared to its food source, E. coli OP50. To study the cue for the attraction, bioactive components (RNA/Protein/Lipopolysaccharides/Metabolites) of C. sakazakii were isolated and used for observing the chemotaxis behavior of C. elegans. The results signified that C. elegans was more attracted toward acid extracted metabolites than those of the other extraction methods. The combined action of acid extracted metabolites of C. sakazakii and a candidate pathogen drastically reduced the survival of C. elegans. In addition, qPCR analysis suggested that the exposure of isolated metabolites through acid extraction to C. elegans for 24 h modified the candidate immune regulatory genes involved in pathogen recognition and kinase activity such as clec-60, clec-87, lys-7, akt-2, pkc-1, and jnk-1.

Glucogallin Attenuates the LPS-Induced Signaling in Macrophages and Protects Mice against Sepsis.

The anti-oxidant and anti-inflammatory effect of beta-glucogallin (BGG), a plant-derived natural product, was evaluated in both in vitro and in vivo studies. For the in vitro study, the ability of BGG pre-treatment to quench LPS-induced effects compared to LPS alone in macrophages was investigated. It was found that BGG pre-treatment showed a significant decrease in ROS, NO, superoxide, and pro-inflammatory cytokines (TNF-alpha, IL-4, IL-17, IL-1ß, and IL-6) and increased reduced glutathione coupled with the restoration of mitochondrial membrane potential. Gene profiling and further validation by qPCR showed that BGG pre-treatment downregulated the LPS-induced expression of c-Fos, Fas, MMP-9, iNOS, COX-2, MyD88, TRIF, TRAF6, TRAM, c-JUN, and NF-κB. We observed that BGG pre-treatment reduced nuclear translocation of LPS-activated NF-κB and thus reduced the subsequent expressions of NLRP3 and IL-1ß, indicating the ability of BGG to inhibit inflammasome formation. Molecular docking studies showed that BGG could bind at the active site of TLR4. Finally, in the LPS-driven sepsis mouse model, we showed that pre-treatment with BGG sustained toxic shock, as evident from their 100% survival. Our study clearly showed the therapeutic potential of BGG in toxic shock syndrome.

Glucogallin Attenuates RAW 264.7 Cells from Arsenic Trioxide Induced Toxicity via the NF-Ò¡B/NLRP3 Pathway.

Chronic arsenic (As) poisoning is mostly due to subsoil water contaminated with As and its salts. Exposure to As has been found to cause an elevation in reactive oxygen species (ROS), leading to the damage of DNA and proteins, and it also causes immunotoxicity. Treatment regimens are primarily based on chelation therapy and amino acid and vitamin supplementations. Recent studies have established that natural products display effective and progressive relief from arsenicosis without any side effects. ß-glucogallin (BGG), a gallo-tannin natural product, is reported to possess anti-oxidant and anti-inflammatory properties. In the present study, we aim to observe the protective role of BGG against As-induced cytotoxicity, apoptosis, mitochondrial dysfunction, and the underlying mechanisms in RAW 264.7 macrophage cells. We found that BGG alleviates As-induced ROS, apoptosis, and mitochondrial dysfunction in RAW 264.7 macrophage cells. Thus, BGG can be used therapeutically to prevent As-induced toxicity.

In Vitro Anticancer Activity of Methanolic Extract of Justicia adhatoda Leaves with Special Emphasis on Human Breast Cancer Cell Line.

Natural products are being targeted as alternative anticancer agents due to their non-toxic and safe nature. The present study was conducted to explore the in vitro anticancer potential of Justicia adhatoda (J. adhatoda) leaf extract. The methanolic leaf extract was prepared, and the phytochemicals and antioxidant potential were determined by LCMS analysis and DPPH radical scavenging assay, respectively. A docking study performed with five major alkaloidal phytoconstituents showed that they had a good binding affinity towards the active site of NF-κB. Cell viability assay was carried out in five different cell lines, and the extract exhibited the highest cytotoxicity in MCF-7, a breast cancer cell line. Extract-treated cells showed a significant increase in nitric oxide and reactive oxygen species production. Cell cycle analysis showed an arrest in cell growth at the Sub-G0 phase. The extract successfully inhibited cell migration and colony formation and altered mitochondrial membrane potential. The activities of superoxide dismutase and glutathione were also found to decrease in a dose-dependent manner. The percentage of apoptotic cells was found to increase in a dose-dependent manner in MCF-7 cells. The expressions of caspase-3, Bax, and cleaved-PARP were increased in extract-treated cells. An increase in the expression of NF-κB was found in the cytoplasm in extract-treated cells. J. adhatoda leaf extract showed a potential anticancer effect in MCF-7 cells.

Survival upon Staphylococcus aureus mediated wound infection in Caenorhabditis elegans and the mechanism entailed.

Infection following injury is one of the major threats which causes huge economic burden in wound care management all over the world. Injury often results with poor healing when coupled by following infection. In contrast to this, we observed enhanced survival of wound infected worms compared to wounded worms in Caenorhabditis elegans wound model while infecting with Staphylococcus aureus. Hence, the study was intended to identify the mechanism for the enhanced survival of wound infected worms through LCMS/MS based high throughput proteomic analysis. Bioinformatics analyses of the identified protein players indicated differential enrichment of several pathways including MAPK signaling, oxidative phosphorylation and phosphatidylinositol signaling. Inhibition of oxidative phosphorylation and phosphatidylinositol signaling through chemical treatment showed complete reversal of the enhanced survival during wound infection nevertheless mutant of MAPK pathway did not reverse the same. Consequently, it was delineated that oxidative phosphorylation and phosphatidylinositol signaling are crucial for the survival. In this regard, elevated calcium signals and ROS including O- and H2O2 were observed in wounded and wound infected worms. Consequently, it was insinuated that presence of pathogen stress could have incited survival in wound infected worms with the aid of elevated ROS and calcium signals.

Sortase A: A chemoenzymatic approach for the labeling of cell surfaces.

Sortase A, a transpeptidase enzyme is present in many Gram-positive bacteria and helps in the recruitment of the cell surface proteins. Over the last two decades, Sortase A has become an attractive tool for performing in vivo and in vitro ligations. Sortase A-mediated ligation has continuously been used for its specificity, robustness, and highly efficient nature. These properties make it a popular choice among protein engineers as well as researchers from different fields. In this review, we give an overview of Sortase A-mediated ligation of various molecules on the cell surfaces, which can have diverse applications in interdisciplinary fields.

Protease Inhibitory Effect of Natural Polyphenolic Compounds on SARS-CoV-2: An In Silico Study.

The current pandemic, caused by SARS-CoV-2 virus, is a severe challenge for human health and the world economy. There is an urgent need for development of drugs that can manage this pandemic, as it has already infected 19 million people and led to the death of around 711,277 people worldwide. At this time, in-silico studies are providing lots of preliminary data about potential drugs, which can be a great help in further in-vitro and in-vivo studies. Here, we have selected three polyphenolic compounds, mangiferin, glucogallin, and phlorizin. These compounds are isolated from different natural sources but share structural similarities and have been reported for their antiviral activity. The objective of this study is to analyze and predict the anti-protease activity of these compounds on SARS-CoV-2main protease (Mpro) and TMPRSS2 protein. Both the viral protein and the host protein play an important role in the viral life cycle, such as post-translational modification and viral spike protein priming. This study has been performed by molecular docking of the compounds using PyRx with AutoDock Vina on the two aforementioned targets chosen for this study, i.e., SARS-CoV-2 Mpro and TMPRSS2. The compounds showed good binding affinity and are further analyzed by (Molecular dynamic) MD and Molecular Mechanics Poisson-Boltzmann Surface Area MM-PBSA study. The MD-simulation study has predicted that these natural compounds will have a great impact on the stabilization of the binding cavity of the Mpro of SARS-CoV-2. The predicted pharmacokinetic parameters also show that these compounds are expected to have good solubility and absorption properties. Further predictions for these compounds also showed no involvement in drug-drug interaction and no toxicity.

A Competitive Pull-Down Assay Using G-quadruplex DNA Linked Magnetic Nanoparticles To Determine Specificity of G-quadruplex Ligands.

Herein, we develop a competitive screening method in which G-quadruplex DNA linked magnetic nanoparticles pull down selective ligands for a particular quadruplex topology from a series of small molecules. The screening strategy is first optimized with known G-quadruplex ligands and then used with a new series of G-quadruplex interactive bis-triazolyl ligands that are synthesized by Cu(I)-catalyzed azide-alkyne cycloaddition. The assay enables the identification of c-MYC and BCL2 G-quadruplex selective bis-triazole ligands that specifically target promoter G-quadruplexes in cancer cells.

A 3D in-vitro biomimicking Caco-2 intestinal permeability model-based assessment of physically modified telmisartan towards an alkalizer-free formulation development.

Efficient telmisartan delivery for hypertension management requires the incorporation of meglumine and/or sodium hydroxide as an alkalizer in the formulation. Long-term use of powerful alkalis with formulation as part of chronic therapy can cause metabolic alkalosis, ulcers, diarrhea, and body pain. Here, we aimed to design a telmisartan formulation without alkalizers. Telmisartan properties were tailor-made by microfluidizer-based physical modification. After microfluidization, telmisartan nanosuspension was lyophilized to obtain telmisartan premix powder. The optimized telmisartan nanosuspension had an average particle size of 579.85 ± 32.14 nm. The lyophilized premix was characterized by FT-IR, DSC, and PXRD analysis to ensure its physicochemical characteristics. The solubility analysis of premix showed 2.2 times, 2.3 times, and 6 times solubility improvement in 0.1 N HCl, phosphate buffer pH 7.5, and pH 6.8 compared to pure telmisartan. A 3D in-vitro Caco-2 model was developed to compare apparent permeability of API and powder premix. It showed that the powder premix was more permeable than pure API. The tablet formulation prepared from the telmisartan premix showed a dissolution profile comparable to that of the marketed formulation. The technique present herein can be used as a platform technology for solubility and permeability improvement of similar classes of molecules.

Decellularized extracellular matrix-based bioengineered 3D breast cancer scaffolds for personalized therapy and drug screening.

Breast cancer (BC) is the second deadliest cancer after lung cancer. Similar to all cancers, it is also driven by a 3D microenvironment. The extracellular matrix (ECM) is an essential component of the 3D tumor micro-environment, wherein it functions as a scaffold for cells and provides metabolic support. BC is characterized by alterations in the ECM. Various studies have attempted to mimic BC-specific ECMs using artificial materials, such as Matrigel. Nevertheless, research has proven that naturally derived decellularized extracellular matrices (dECMs) are superior in providing the essential in vivo-like cues needed to mimic a cancer-like environment. Developing in vitro 3-D BC models is not straightforward and requires extensive analysis of the data established by researchers. For the benefit of researchers, in this review, we have tried to highlight all developmental studies that have been conducted by various scientists so far. The analysis of the conclusions drawn from these studies is also discussed. The advantages and drawbacks of the decellularization methods employed for generating BC scaffolds will be covered, and the review will shed light on how dECM scaffolds help develop a BC environment. The later stages of the article will also focus on immunogenicity issues arising from decellularization and the origin of the tissue. Finally, this review will also discuss the biofabrication of matrices, which is the core part of the bioengineering process.

Dehydrozingerone ameliorates arsenic-induced reproductive toxicity in male Wistar rats.

Arsenic (As3+), a significant environmental pollutant that has garnered global attention, is widely recognized for its adverse effects on reproductive health. This study assesses the aphrodisiac activity of Dehydrozingerone (DHZ) against As3+ induced sexual dysfunction in male Wistar rats. Male Wistar rats were divided into control, As3+, and As3++DHZ groups. The As3+ group received 5 mg/kg sodium arsenite (NaAsO2) orally while As3++DHZ group received 50 mg/kg synthesized DHZ along with As3+ for 42 days. Following administration, mount and intromission latency, frequency, and average time were measured to assess aphrodisiac and reproductive toxicity in male Wistar rats which had 1:1 coitus with female rats. On days 14th, 28th, and 42nd, sexual behaviour was measured. Further on 43rd day, animals were sacrificed, blood was collected to measure oxidative parameters and LH hormone, and then testes were collected to profile reproductive damage. As3+ treated rats had lower sperm counts, motility, and abnormalities. These alterations reduced sexual hormones. In addition, As3+ toxicity depleted antioxidant indicators including SOD, GSH and elevated ROS. Compared to the As3+ group, As3++DHZ showed a substantial (p < 0.05) increase in sperm count, motility, and reduced abnormalities. DHZ also reversed the rise in luteinizing hormone caused by As3+ therapy, restored oxidative indicators, and improved seminiferous tubule structural damage. 42 days As3+ exposure slightly increased rats' sexual desire but not sperm quality. However, As3++DHZ lower libido and sperm quality. Thus, DHZ therapy enhanced rat sexual desire and sperm quality compared to As3+.

Surface engineered nanodiamonds: mechanistic intervention in biomedical applications for diagnosis and treatment of cancer.

In terms of biomedical tools, nanodiamonds (ND) are a more recent innovation. Their size typically ranges between 4 to 100 nm. ND are produced via a variety of methods and are known for their physical toughness, durability, and chemical stability. Studies have revealed that surface modifications and functionalization have a significant influence on the optical and electrical properties of the nanomaterial. Consequently, surface functional groups of NDs have applications in a variety of domains, including drug administration, gene delivery, immunotherapy for cancer treatment, and bio-imaging to diagnose cancer. Additionally, their biocompatibility is a critical requisite for theirin vivoandin vitrointerventions. This review delves into these aspects and focuses on the recent advances in surface modification strategies of NDs for various biomedical applications surrounding cancer diagnosis and treatment. Furthermore, the prognosis of its clinical translation has also been discussed.

Recent Developments in the Design of New Water-Soluble Boron Dipyrromethenes and Their Applications: An Updated Review.

Boron-dipyrromethene (BODIPY) and its derivatives play an important role in the area of organic fluorophore chemistry. Recently, the water-soluble boron-dipyrromethene dyes have increasingly received interest. The structural modification of the BODIPY core by incorporating different neutral and ionic hydrophilic groups makes it water-soluble. The important hydrophilic groups, such as quaternary ammonium, sulfonate, oligoethylene glycol, dicarboxylic acid, and sugar moieties significantly increase the solubility of these dyes in water while preserving their photophysical properties. As a result, these fluorescent dyes are utilized in aqueous systems for applications such as chemosensors, cell imaging, anticancer, biolabeling, biomedicine, metal ion detection, and photodynamic treatment. This review covers the most current developments in the design and synthesis of water-soluble BODIPY derivatives and their wide applications since 2014.

Role of SIRT-1 as a Target for Treatment and Prevention of Diabetic Nephropathy: A Review.

Type-2 diabetes mellitus is a prime factor for the development of Diabetic Nephropathy (DN) that affects the vital organ namely the kidneys, and further alters the functions of the nephron system. DN is nowadays becoming a challenge for scientists towards the world because of its high pervasiveness and complexity of medication. Various risk factors are involved in the initiation of pathogenic DN, which are associated with different pathways against drug activity. Due to this DN becomes an unpredictable query to the researchers. SIRT1 is a silent information regulator factor 2 related enzyme 1 (SIRT1) is nicotinamide adenine dinucleotide (NAD+) dependent deacetylase that functions as an intracellular regulator of transcriptional activity. An activated version of SIRT-1 improves the metabolic diseased conditions associated with other molecular pathways. SIRT1 attenuates diabetic nephropathy in in vitro and in vivo experimental models of diabetes containing Podocytes, Mesangial cells, and Renal proximal tubular cells. SIRT1 shows nephroprotective effects in DN in part through deacetylation of transcription factors i.e., imply in the disease like p53, PTP1B, FOXO, RelA, NF- kß, STAT-3, and PGC-1α/ PPARγ. It has been shown that some natural products like resveratrol and synthetic compounds are activating the SIRT1, this further involved the cascade pathways to prevent the DN. This review will help regarding the effectiveness of SIRT1as target in the prevention and treatment of DN.

Distinct global metabolomic profiles of the model organism Caenorhabditis elegans during interactions with Staphylococcus aureus and Salmonella enterica Serovar Typhi.

The interactive network of hosts with pathogenic microbes is still questionable. It has been hypothesized and reported that the host shows altered regulatory mechanisms for different pathogens. Several studies using transcriptomics and proteomics revealed the altered pathways and sequential regulations displayed by the host during bacterial interactions. Still, there is a gap in understanding the triggering molecule at transcriptomic and proteomic levels due to the lack of the knowledge of the interactive metabolites produced during their interactions. In this study, the global metabolomic approach was performed in the nematode model organism Caenorhabditis elegans upon exposure to a Gram-negative bacteria, Salmonella enterica Serovar Typhi, and a Gram-positive bacteria, Staphylococcus aureus, and the whole metabolome was categorized as endo-metabolome (internally produced) and exo-metabolome (externally releasing). The extracted metabolites were subjected to liquid chromatography mass spectrometry (ESI-LC/qToF-MS/MS). In total 5578, 4554 and 4046 endo-metabolites and 4451, 3625 and 1281 exo-metabolites were identified in C. elegans when exposed to E. coli OP50, S. Typhi and S. aureus, respectively. Both the multivariate and univariate analyses were performed. The variation in endo- and exo-metabolome during candidate bacterial interactions was observed. The results indicated that, during S. aureus interaction, the exclusively enriched metabolites were significantly involved in alpha-linoleic acid metabolism. Similarly, the exclusively enriched metabolites during the interaction of S. Typhi were significantly involved in the phosphatidylinositol signalling system. The whole metabolomic profile presented here will build the scope to understand the role of metabolites and the respective pathways in host response during the early period of bacterial infections.

Exploring novel and fast stability or sameness evaluation tool for different categories of injectable formulations.

The establishment of drug product stability and sameness is the heart of generic formulation development. For regulatory filing, various instrumental methods are used on a case basis to establish the generic and innovator product sameness in multiple aspects. Here in the present study, we explored the applicability of the Time-correlated single photon counting (TCS-PC) technique as a fast, reliable, and nondestructive method for establishing the sameness of three different categories of injectable formulations, namely, Amphotericin B liposome for injection, enoxaparin injection, and iron sucrose injection. All three category formulations were evaluated in their native and artificially induced post degradation state to identify the discrimination power of the used instrumental techniques. The degradation of materials were confirmed by high performance liquid chromatography (HPLC). Based on the product category, pre and post-degradation samples were evaluated by selective instrumental methods like differential scanning calorimetry (DSC), nuclear magnetic resonance (NMR), fluorescence spectroscopy, particle size analysis by dynamic light scattering (DLS), small angle X-ray scattering (SAXS), Raman spectroscopy, inductively coupled plasma optical-emission spectrometry (ICP-OES) and circular dichroism study. All pre and post-degradation samples were further analyzed by TCS-PC. We observed that, TCS-PC can identify the differences between the initial and post degradation samples in very less time with promising discrimination power across the different injectable formulation types. Thus TCS-PC can be used as a fast and promising stability or sameness evaluation tool for different injectable drug products.

Harnessing the combined effect of antivirulence agent trans-chalcone with bactericidal curcumin against sortase A enzyme to tackle Gram-positive bacterial infections.

Gram-positive bacteria are responsible for a wide range of infections in humans. In most Gram-positive bacteria, sortase A plays a significant role in attaching virulence factors to the bacteria's cell wall. These cell surface proteins play a significant role in virulence and pathogenesis. Even though antibiotics are available to treat these infections, there is a continuous search for an alternative strategy due to an increase in antibiotic resistance. Thus, using anti-sortase drugs to combat these bacterial infections may be a promising approach. Here, we describe a method for targeting Gram-positive bacterial infection by combining curcumin and trans-chalcone as sortase A inhibitors. We have used curcumin and trans-chalcone alone and in combination as a sortase A inhibitor. We have seen ~78%, ~43%, and ~94% inhibition when treated with curcumin, trans-chalcone, and a combination of both compounds, respectively. The compounds have also shown a significant effect on biofilm formation, IgG binding, protein A recruitment, and IgG deposition. We discovered that combining curcumin and trans-chalcone is more effective against Gram-positive bacteria than either compound alone. The present work demonstrated that a combination of these natural compounds could be used as an antivirulence therapy against Gram-positive bacterial infection.

Role of Flavonoids in Modulation of Mitochondria Dynamics during Oxidative Stress.

BACKGROUND: Flavonoids are a widespread category of naturally occurring polyphenols distinguished by the flavan nucleus in plant-based foods and beverages, known for their various health benefits. Studies have suggested that consuming 150-500 mg of flavonoids daily is beneficial for health. Recent studies suggest that flavonoids are involved in maintaining mitochondrial activity and preventing impairment of mitochondrial dynamics by oxidative stress. OBJECTIVE: This review emphasized the significance of studying the impact of flavonoids on mitochondrial dynamics, oxidative stress, and inflammatory response. METHODS: This review analysed and summarised the findings related to the impact of flavonoids on mitochondria from publicly available search engines namely Pubmed, Scopus, and Web of Science. DESCRIPTION: Any disruption in mitochondrial dynamics can contribute to cellular dysfunction and diseases, including cancer, cardiac conditions, and neurodegeneration. Flavonoids have been shown to modulate mitochondrial dynamics by regulating protein expression involved in fission and fusion events. Furthermore, flavonoids exhibit potent antioxidant properties by lowering the production of ROS and boosting the performance of antioxidant enzymes. Persistent inflammation is a characteristic of many different disorders. This is because flavonoids also alter the inflammatory response by controlling the expression of numerous cytokines and chemokines involved in the inflammatory process. Flavonoids exhibit an impressive array of significant health effects, making them an effective therapeutic agent for managing various disorders. Further this review summarised available mechanisms underlying flavonoids' actions on mitochondrial dynamics and oxidative stress to recognize the optimal dose and duration of flavonoid intake for therapeutic purposes. CONCLUSION: This review may provide a solid foundation for developing targeted therapeutic interventions utilizing flavonoids, ultimately benefiting individuals afflicted with various disorders.