Crisaborole-Enthused Glycerosomal Gel for an Augmented Skin Permeation.

BACKGROUND: Crisaborole (CB), a boron-based compound, is the first topical PDE4 inhibitor to be approved by the US Food and Drug Administration (2016) for the treatment of Atopic Dermatitis. It is marketed as a 2% ointment (Eucrisa, Pfizer). However, CB is insoluble in water; therfore, CB glycersomes were formulated to enhance its permeation flux across the skin. OBJECTIVE: We developed a glycerosomal gel of CB and compared its in vitro release and permeation flux with the 2% conventional ointment. METHODS: Glycerosomes were prepared using a thin film hydration method employing CB, soya phosphatidylcholine, and cholesterol. The formed film was further hydrated employing a mixture of phosphate buffer pH 7.4 /glycerin solution containing varying percentages (20,30, 40, and 50 %) of glycerol. The glycerosomes obtained were characterized by their size, polydispersity index (PDI), and Zeta potential. The entrapment efficiency of the optimized formulation (F 1) was determined. The in vitro release of F1 was compared with its 2% conventional ointment. F1 was further incorporated into carbopol 934 P gel. The gel was characterized by pH, viscosity, spreadability, and drug content. The permeability flux of the glycerosomal gel was compared with its 2% conventional ointment. RESULTS: The optimized CB glycerosomes had a vesicle size of 137.5 ± 50.58 nm, PDI 0.342, and zeta potential -65.4 ± 6.75 mV. CB glycerosomal gel demonstrated a 2.13-fold enhancement in the permeation flux. CONCLUSION: It can thereby be concluded that glycerosomes can be an effective delivery system to enhance the penetration of CB across the skin.

MSMEG_0311 is a conserved essential polar protein involved in mycobacterium cell wall metabolism.

Cell wall synthesis and cell division are two closely linked pathways in a bacterial cell which distinctly influence the growth and survival of a bacterium. This requires an appreciable coordination between the two processes, more so, in case of mycobacteria with an intricate multi-layered cell wall structure. In this study, we investigated a conserved gene cluster using CRISPR-Cas12 based gene silencing technology to show that knockdown of most of the genes in this cluster leads to growth defects. Investigating conserved genes is important as they likely perform vital cellular functions and the functional insights on such genes can be extended to other mycobacterial species. We characterised one of the genes in the locus, MSMEG_0311. The repression of this gene not only imparts severe growth defect but also changes colony morphology. We demonstrate that the protein preferentially localises to the polar region and investigate its influence on the polar growth of the bacillus. A combination of permeability and drug susceptibility assay strongly suggests a cell wall associated function of this gene which is also corroborated by transcriptomic analysis of the knockdown where a number of cell wall associated genes, particularly iniA and sigF regulon get altered. Considering the gene is highly conserved across mycobacterial species and appears to be essential for growth, it may serve as a potential drug target.

A Comprehensive Review on Polyphenols Based Nanovesicular System for Topical Delivery.

BACKGROUND: Polyphenols are naturally occurring compounds having more than one hydroxy functional group. They are ubiquitous secondary plant metabolites possessing a wide range of pharmacological activity. Brightly colored fruits and vegetables are the natural source of polyphenols. Majorly, they possess antioxidant, anti-inflammatory and antimicrobial properties which make them suitable candidates to target skin related disorders. OBJECTIVE: This study is focused to explore the potential of polyphenols loaded nanovesicles for skin related disorders. The aim of the study is to review the applicability and efficacy of different vesicular systems encapsulated with various classes of polyphenols for skin related disorders, thus opening the opportunity for future studies based on these drug delivery systems. METHOD: Web of Science, PubMed, Scopus database, and the search engine Google Scholar were accessed for the literature search. The results were then filtered based on the titles, abstracts, and accessibility of the complete texts. RESULTS: The expository evaluation of the literature revealed that various nanovesicles like liposomes, niosomes, ethosomes and transferosomes incorporating polyphenol have been formulated to address issues pertaining to delivery across the skin. These developed nano vesicular systems have shown improvement in the physicochemical properties and pharmacological action. CONCLUSION: Polyphenol based nano-vesicular formulations have proved to be an effective system for topical delivery and henceforth, they might curtail the use of other skin therapies having limited applicability.

Pharmaceutical characterization and exploration of Arkeshwara rasa in MDA-MB-231 cells.

BACKGROUND: The diverse specificity mode of cancer treatment targets and chemo resistance demands the necessity of drug entities which can address the devastating dynamicity of the disease. OBJECTIVES: To check the anti-tumour potential of traditional medicine rich in polyherbal components and metal nanoparticle namely Arkeshwara rasa (AR). MATERIAL METHODS: The AR was prepared in a modified version with reference from Rasaratna Samuchaya and characterized using sophisticated instrumental analysis including XRD, SEM-EDAX, TEM, TGA-DSC, and LC-MS and tested against the MDA-MB-231 cell line to screen cell viability and the cytotoxicity with MTT, SRB and the AO assay. RESULTS: XRD pattern shows cubic tetrahedrite structure with Sb, Cu, S peaks and trace elements like Fe, Mg, etc. The particle size of AR ranges between 20 and 30 nm. The TGA points thermal decomposition at 210 °C and the metal sulphide peaks in DSC. LC-MS analysis reveals the components of the formulation more on the flavonoid portion. The IC50 value of MTT and SRB are 25.28 µg/mL and 31.7 µg/mL respectively. The AO colorimeter substantiated the cell viability and the apoptosis figures of the same cell line. The AR exhibits cytotoxicity and reaffirms the apoptosis fraction with SRB assay. CONCLUSIONS: The Hesperidine, Neohesperidin, Rutin components in the phytochemical pool can synergize the anti-tumour potential with either influencing cellular pathways or decreasing chemo resistance to conventional treatment. AR need to be further experimented with reverse transcription, flow cytometry, western blotting, etc.

Ocimum sanctum Alters the Lipid Landscape of the Brain Cortex and Plasma to Ameliorate the Effect of Photothrombotic Stroke in a Mouse Model.

Stroke-like injuries in the brain result in not only cell death at the site of the injury but also other detrimental structural and molecular changes in regions around the stroke. A stroke-induced alteration in the lipid profile interferes with neuronal functions such as neurotransmission. Preventing these unfavorable changes is important for recovery. Ocimum sanctum (Tulsi extract) is known to have anti-inflammatory and neuroprotective properties. It is possible that Tulsi imparts a neuroprotective effect through the lipophilic transfer of active ingredients into the brain. Hence, we examined alterations in the lipid profile in the cerebral cortex as well as the plasma of mice with a photothrombotic-ischemic-stroke-like injury following the administration of a Tulsi extract. It is also possible that the lipids present in the Tulsi extract could contribute to the lipophilic transfer of active ingredients into the brain. Therefore, to identify the major lipid species in the Tulsi extract, we performed metabolomic and untargeted lipidomic analyses on the Tulsi extract. The presence of 39 molecular lipid species was detected in the Tulsi extract. We then examined the effect of a treatment using the Tulsi extract on the untargeted lipidomic profile of the brain and plasma following photothrombotic ischemic stroke in a mouse model. Mice of the C57Bl/6j strain, aged 2-3 months, were randomly divided into four groups: (i) Sham, (ii) Lesion, (iii) Lesion plus Tulsi, and (iv) Lesion plus Ibuprofen. The cerebral cortex of the lesioned hemisphere of the brain and plasma samples were collected for untargeted lipidomic profiling using a Q-Exactive Mass Spectrometer. Our results documented significant alterations in major lipid groups, including PE, PC, neutral glycerolipids, PS, and P-glycerol, in the brain and plasma samples from the photothrombotic stroke mice following their treatment with Tulsi. Upon further comparison between the different study groups of mice, levels of MGDG (36:4), which may assist in recovery, were found to be increased in the brain cortexes of the mice treated with Tulsi when compared to the other groups (p < 0.05). Lipid species such as PS, PE, LPG, and PI were commonly altered in the Sham and Lesion plus Tulsi groups. The brain samples from the Sham group were specifically enriched in many species of glycerol lipids and had reduced PE species, while their plasma samples showed altered PE and PS species when compared to the Lesion group. LPC (16:1) was found in the Tulsi extract and was significantly increased in the brains of the PTL-plus-Tulsi-treated group. Our results suggest that the neuroprotective effect of Tulsi on cerebral ischemia may be partially associated with its ability to regulate brain and plasma lipids, and these results may help provide critical insights into therapeutic options for cerebral ischemia or brain lesions.

Crisaborole loaded nanoemulgel for the mitigation of atopic dermatitis in mice model.

OBJECTIVE: The present work aims to formulate nanoemulgel of crisaborole (CB) and evaluate its effectiveness against 2,4-Di-nitrochlorobenzene induced (DNCB) atopic dermatitis (AD) in mice. SIGNIFICANCE: AD is a chronic inflammation of the skin affecting the quality of life. CB is a topical PDE4 inhibitor marketed as a 2% ointment. It, however, possesses poor aqueous solubility. An o/w nanoemulsion shall exhibit an enhanced therapeutic effect owing to the increased solubility of CB and an augmented skin penetration. The addition of a gelling agent to form a nanoemulgel further provides ease of application to the patients. METHODS: Nanoemulsion was prepared by aqueous titration method using caproyl PGMC, cremophore EL and propylene glycol as the oil, surfactant, and cosurfactant respectively. The formulations were characterized by their size, zeta potential and polydispersity index (PDI). 1% Carbopol 934 was used as the gelling agent to formulate nanoemulgel comprising of optimized nanoemulsion (NE 9). Ex vivo skin permeation of the CB nanoemulgel was compared with the CB ointment. Its therapeutic effect was evaluated in Balb/c mice. RESULTS: NE 9 comprised of 7.49% oil, 37.45% Smix (1:3) and water 55.06%. Its particle size, PDI and zeta potential were 15.45 ± 5.265 nm, 0.098 and -17.9 ± 8.00 mV respectively. The nanoemulgel exhibited a 3-fold higher permeation flux as compared to the ointment. In vivo studies demonstrated that the nanoemulgel provided better therapeutic effect than the ointment. CONCLUSION: We can thereby conclude that nanoemulgel formulation can be a successful drug delivery strategy for enhancing the therapeutic effect of CB.

Efficacy of LAMP assay for Mycobacterial spp. detection to prevent treatment delays and onset of drug resistance: a systematic review and meta-analysis.

Background: Tuberculosis (TB) remains a deadly disease affecting one-third population globally. Long turnaround time and poor sensitivity of the conventional diagnostics are the major impediments for faster diagnosis of Mycobacterial spp to prevent drug resistance. To overcome these issues, molecular diagnostics have been developed. They offer enhanced sensitivity but require sophisticated infrastructure, skilled manpower and remain expensive. Methods: In that context, loop-mediated isothermal amplification (LAMP) assay, recommended by the WHO in 2016 for TB diagnosis, sounds as a promising alternative that facilitates visual read outs. Therefore, the aim of the present study is to conduct a meta-analysis to assess the diagnostic efficiency of LAMP for the detection of a panel of Mycobacterium spp. following PRISMA guidelines using scientific databases. From 1600 studies reported on the diagnosis of Mycobacterium spp., a selection of 30 articles were identified as eligible to meet the criteria of LAMP based diagnosis. Results: It was found that most of the studies were conducted in high disease burden nations such as India, Thailand, and Japan with sputum as the most common specimen to be used for LAMP assay. Furthermore, IS6110 gene and fluorescence-based detections ranked as the most used target and method respectively. The accuracy and precision rates mostly varied between 79.2% to 99.3% and 73.9% to 100%, respectively. Lastly, a quality assessment based on QUADAS-2 of bias and applicability was conducted. Conclusion: LAMP technology could be considered as a feasible alternative to current diagnostics considering high burden for rapid testing in low resource regions.

Diagnostic Efficacy of LAMP Assay for Human Fungal Pathogens: a Systematic Review and Meta-analysis.

Purpose: Human fungal infections particularly caused by Candida and Aspergillus have emerged as major public health burden. Long turnaround time and poor sensitivity of the conventional diagnostics are the major impediments for faster diagnosis of human fungal pathogens. Recent Findings: To overcome these issues, molecular-based diagnostics have been developed. They offer enhanced sensitivity but require sophisticated infrastructure, skilled manpower, and remained expensive. In that context, loop-mediated isothermal amplification (LAMP) assay represents a promising alternative that facilitates visual read outs. However, to eradicate fungal infections, all forms of fungi must be accurately detected. Thus, a need for alternative testing methodologies is imperative that should be rapid, accurate and facilitate widespread adoption. Therefore, the aim of the present study is to conduct a meta-analysis to assess the diagnostic efficiency of LAMP in the detection of a panel of human fungal pathogens following PRISMA guidelines using scientific databases viz. PubMed, Google Scholar, Science Direct, Scopus, BioRxiv, and MedRxiv. Summary: From various studies reported on the diagnosis of fungi, only 9 articles were identified as eligible to meet the criteria of LAMP based diagnosis. Through this meta-analysis, it was found that most of the studies were conducted in China and Japan with sputum and blood as the most common specimens to be used for LAMP assay. The collected data underlined that ITS gene and fluorescence-based detections ranked as the most used target and method. The pooled sensitivity values of meta-analysis ranged between 0.71 and 1.0 and forest plot and SROC (summary receiver operating characteristic) curve revealed a pooled specificity values between 0.13 and 1.0 with the confidence interval of 95%, respectively. The accuracy and precision rates of eligible studies mostly varied between 70 to 100% and 68 to 100%, respectively. A quality assessment based on QUADAS-2 (Quality Assessment of Diagnostic Accuracy Studies) of bias and applicability was conducted which depicted low risk of bias and applicability concerns. Together, LAMP technology could be considered as a feasible alternative to current diagnostics considering high fungal burden for rapid testing in low resource regions.

Quantitative analysis of the bioenergetics of Mycobacterium tuberculosis along with Glyoxylate cycle as a drug target under inhibition of enzymes using Petri net.

The bacteria Mycobacterium tuberculosis is responsible for the infectious disease Tuberculosis. Targeting the tubercule bacteria is an important challenge in developing the antimycobacterials. The glyoxylate cycle is considered as a potential target for the development of anti-tuberculosis agents, due to its absence in the humans. Humans only possess tricarboxylic acid cycle, while this cycle gets connected to glyoxylate cycle in microbes. Glyoxylate cycle is essential to the Mycobacterium for its growth and survival. Due to this reason, it is considered as a potential therapeutic target for the development of anti-tuberculosis agents. Here, we explore the effect on the behavior of the tricarboxylic acid cycle, glyoxylate cycle and their integrated pathway with the bioenergetics of the Mycobacterium, under the inhibition of key glyoxylate cycle enzymes using Continuous Petri net. Continuous Petri net is a special Petri net used to perform the quantitative analysis of the networks. We first study the tricarboxylic acid cycle and glyoxylate cycle of the tubercule bacteria by simulating its Continuous Petri net model under different scenarios. Both the cycles are then integrated with the bioenergetics of the bacteria and the integrated pathway is again simulated under different conditions. The simulation graphs show the metabolic consequences of inhibiting the key glyoxylate cycle enzymes and adding the uncouplers on the individual as well as integrated pathway. The uncouplers that inhibit the synthesis of adenosine triphosphate, play an important role as anti-mycobacterials. The simulation study done here validates the proposed Continuous Petri net model as compared with the experimental outcomes and also explains the consequences of the enzyme inhibition on the biochemical reactions involved in the metabolic pathways of the mycobacterium.

Global Lipidome Profiling Revealed Multifaceted Role of Lipid Species in Hepatitis C Virus Replication, Assembly, and Host Antiviral Response.

Hepatitis C virus (HCV) is a major human pathogen that requires a better understanding of its interaction with host cells. There is a close association of HCV life cycle with host lipid metabolism. Lipid droplets (LDs) have been found to be crucial organelles that support HCV replication and virion assembly. In addition to their role in replication, LDs also have protein-mediated antiviral properties that are activated during HCV infection. Studies have shown that HCV replicates well in cholesterol and sphingolipid-rich membranes, but the ways in which HCV alters host cell lipid dynamics are not yet known. In this study, we performed a kinetic study to check the enrichment of LDs at different time points of HCV infection. Based on the LD enrichment results, we selected early and later time points of HCV infection for global lipidomic study. Early infection represents the window period for HCV sensing and host immune response while later infection represents the establishment of viral RNA replication, virion assembly, and egress. We identified the dynamic profile of lipid species at early and later time points of HCV infection by global lipidomic study using mass spectrometry. At early HCV infection, phosphatidylinositol phospholipids (PIPs), lysophosphatidic acid (LPA), triacyl glycerols (TAG), phosphatidylcholine (PC), and trihexosylceramides (Hex3Cer) were observed to be enriched. Similarly, free fatty acids (FFA), phosphatidylethanolamine (PE), N-acylphosphatidylethanolamines (NAPE), and tri acylglycerols were enriched at later time points of HCV infection. Lipids enriched at early time of infection may have role in HCV sensing, viral attachment, and immune response as LPA and PIPs are important for immune response and viral attachment, respectively. Moreover, lipid species observed at later infection may contribute to HCV replication and virion assembly as PE, FFA, and triacylglycerols are known for the similar function. In conclusion, we identified lipid species that exhibited dynamic profile across early and later time points of HCV infection compared to mock cells, which could be therapeutically relevant in the design of more specific and effective anti-viral therapies.

Abrogation of efflux pump activity, biofilm formation, and immune escape by candidacidal geraniol in emerging superbug, Candida auris.

During the last decade, Candida auris emerged as a threatening human fungal pathogen that notably caused outbreaks around the globe with high mortality. Considering C. auris species as newly discovered fungi, the evolutionary features remain elusive. The antifungal resistance which is a norm in C. auris underlines the need for innovative therapeutic options. ATP Binding Cassette (ABC) superfamily efflux pumps overexpression and biofilms are known to be major contributors to multidrug resistance (MDR) in C. auris. Therefore, herein, we investigated the antifungal potential of geraniol (Ger) as a promising natural compound in the fight against MDR C. auris. Our experiments proved that Ger was fungicidal in nature and impaired rhodamine 6G (R6G) efflux, confirming the specific effect on ABC transporters. Kinetic studies unravelled the competitive mode of inhibition by Ger for R6G efflux since the apparent Km increased with no change in Vmax value. Mechanistic insights also revealed that Ger depleted ergosterol content in C. auris. Furthermore, Ger led to inhibition in biofilm formation as evident from crystal violet staining, biofilm metabolic and biomass measurements. Additionally, enhanced survival of Caenorhabditis elegans model after C. auris infection demonstrated the in vivo efficacy of Ger. Lastly, the in vivo efficacy was confirmed from a THP-1 cell line model which depicted enhanced macrophage-mediated killing in the presence of Ger. Modulation of C. auris efflux pump activity and biofilm formation by Ger represents a promising approach to combat MDR. Together, this study demonstrated the potential therapeutic insights of Ger as a promising addition to the antifungal armamentarium required to treat emerging and resistant C. auris.

Ocimum sanctum [Tulsi] as a Potential Immunomodulator for the Treatment of Ischemic Injury in the Brain.

Stroke causes brain damage and is one of the main reasons for death. Most survivors of stroke face long-term physical disabilities and cognitive dysfunctions. In addition, they also have persistent emotional and behavioral changes. The two main treatments that are effective are reperfusion with recombinant tissue plasminogen activator and recanalization of penumbra using mechanical thrombectomy. However, these treatments are suitable only for a few patients due to limitations such as susceptibility to hemorrhage and the requirement for administering tissue plasminogen activators within the short therapeutic window during the early hours following a stroke. The paucity of interventions and treatments could be because of the multiple pathological mechanisms induced in the brain by stroke. The ongoing immune response following stroke has been attributed to the worsening brain injury. Hence, novel compounds with immunomodulatory properties that could improve the outcome of stroke patients are required. Natural compounds and medicinal herbs with anti-inflammatory activities and having minimal or no adverse systemic effect could be beneficial in treating stroke. Ocimum sanctum is a medicinal herb that can be considered an effective therapeutic option for ischemic brain injury. Ocimum sanctum, commonly known as holy basil or "Tulsi," is mentioned as the "Elixir of Life" for its healing powers. Since antiquity, Tulsi has been used in the Ayurvedic and Siddha medical systems to treat several diseases; it possesses immuno-modulatory activity, which can alter cellular and humoral immune responses. Tulsi can be considered a potential option as an immuno-modulator for treating various diseases, including brain stroke. In this review, we will focus on the immunomodulatory properties of Tulsi, specifically its effect on both innate and adaptive immunity, as well as its antioxidant and anti-inflammatory properties, which could potentially be effective in treating ongoing immune reactions following ischemic brain injury.

Glibenclamide-malonic acid cocrystal with an enhanced solubility and bioavailability.

OBJECTIVE: The objective of the work is to enhance the solubility, dissolution, and pharmacokinetic properties of glibenclamide (GLB) via cocrystallization technique. SIGNIFICANCE: Glibenclamide is an oral hypoglycemic agent used for treating non-insulin-dependent (type II) diabetes mellitus. It exhibits poor aqueous solubility and oral bioavailability, thereby compromising its therapeutic effect. Therefore, utilizing cocrystal approach for enhancing the solubility will modulate the physicochemical properties of GLB without altering its molecular structure. METHODS: Cocrystal was prepared by solution crystallization method using coformer malonic acid. The cocrystal was characterized by differential scanning calorimetry (DSC), powder X-ray diffraction (PXRD), and Fourier transform infrared (FT-IR) studies. The prepared cocrystal was subjected to solubility, in vitro dissolution, and pharmacokinetic studies. RESULTS: The DSC endotherms, PXRD patterns, and the FT-IR spectra of the cocrystal established the formation of a cocrystal. The formation of eutectic mixture was refuted upon comparing the DSC endotherm and PXRD pattern of the cocrystal with that of the physical mixture. GLB showed a twofold enhancement in solubility and a significant improvement in the rate of dissolution (p < 0.05, independent t-test) after cocrystallization. The pharmacokinetic parameters on male Sprague Drawly rats showed 1.45 enhancement in AUC0-24 and 1.36-fold enhancement in the Cmax of GLB as compared to the pure drug. CONCLUSION: These findings demonstrate that cocrystallization technique was able to tailor the solubility and dissolution profile of GLB leading to an enhanced pharmacokinetic property.

Dihydroartemesinin and Curcumin Based Self-Microemulsifying Drug Delivery System for Antimalarial Activity.

Malaria is a significant global problem which still persists despite the development of various effective antimalarial drugs. It is challenging to treat this disease due to the parasite's complex life cycle and high recrudensce of antimalarial drugs. A new self-micro emulsifying drug delivery system has been developed to improve the solubility of dihydroartemisinin and curcumin. The prepared formulation contained Dihydroartemesinin, curcumin, Groundnut Oil, Cremephor RH, and Tween 80. Self-micro emulsification time, zeta potential, droplet size, polydispersity index, transmission electron microscopy, drug release, and in-vivo studies were performed for characterization. The globule size was found to be 25.59±0.40 nm and the zeta potential was-5.75±0.18 mV. The globules prepared were spherical in shape. The in-vitro dissolution performance of formulation of dihydroartemisinin and curcumin self emulsifying drug delivery system showed significantly (p<0.05, Origin Pro 8.5) higher release as compared to the pure drugs. The results of the study suggested that the prepared self emulsifying drug delivery system combination of Dihydroartemesinin and curcumin has a better potential to cure parasitemia as compared to the individual drug.

Unique roles of aminophospholipid translocase Drs2p in governing efflux pump activity, ergosterol level, virulence traits, and host-pathogen interaction in Candida albicans.

Infections caused by Candida albicans are rising due to increment in drug resistance and a limited arsenal of conventional antifungal drugs. Thus, elucidating the novel antifungal targets still represent an alternative that could overcome the problem of multidrug resistance (MDR). In this study, we have uncovered the distinctive effect of aminophospholipid translocase (Drs2p) deletion on major MDR mechanisms of C. albicans. We determined that efflux activity was diminished in Δdrs2 mutant as revealed by extracellular rhodamine 6G (R6G) efflux and flow cytometry. Moreover, we further unveiled that Δdrs2 mutant displayed decreased ergosterol content and increased membrane fluidity. Furthermore, Drs2p deletion affects the virulence attributes and led to inhibited hyphal growth and reduced biofilm formation. Additionally, THP-1 cell lines' mediated host-pathogen interaction studies revealed that Δdrs2 mutant displayed enhanced phagocytosis and altered cytokine production leading to increased IL-6 and decreased IL-10 production. Taken together, the present study demonstrates the relevance of Drs2p in C. albicans and consequently disrupting pathways known for mediating drug resistance and immune recognition. Comprehensive studies are further required to authenticate Drs2p as a novel antifungal drug target.

Magnesium impairs Candida albicans immune evasion by reduced hyphal damage, enhanced ß-glucan exposure and altered vacuole homeostasis.

With a limited arsenal of available antifungal drugs and drug-resistance emergence, strategies that seek to reduce Candida immune evasion and virulence could be a promising alternative option. Harnessing metal homeostasis against C. albicans has gained wide prominence nowadays as a feasible antifungal strategy. Herein, the effect of magnesium (Mg) deprivation on the immune evasion mechanisms of C. albicans is demonstrated. We studied host pathogen interaction by using the THP-1 cell line model and explored the avenue that macrophage-mediated killing was enhanced under Mg deprivation, leading to altered cytokine (TNFα, IL-6 and IL10) production and reduced pyroptosis. Insights into the mechanisms revealed that hyphal damage inside the macrophage was diminished under Mg deprivation. Additionally, Mg deprivation led to cell wall remodelling; leading to enhanced ß-1,3-glucan exposure, crucial for immune recognition, along with concomitant alterations in chitin and mannan levels. Furthermore, vacuole homeostasis was disrupted under Mg deprivation, as revealed by abrogated morphology and defective acidification of the vacuole lumen. Together, we demonstrated that Mg deprivation affected immune evasion mechanisms by: reduced hyphal damage, enhanced ß-1,3-glucan exposure and altered vacuole functioning. The study establishes that Mg availability is indispensable for successful C. albicans immune evasion and specific Mg dependent pathways could be targeted for therapy.

Phytochemical: a treatment option for heavy metal induced neurotoxicity.

Heavy metals are known to be carcinogenic, mutagenic, and teratogenic. Some heavy metals are necessary while present in the growing medium in moderate concentrations known to be essential heavy metals as they required for the body functioning as a nutrient. But there are some unwanted metals and are also toxic to the environment and create a harmful impact on the body, which termed to be non-essential heavy metals. Upon exposure, the heavy metals decrease the major antioxidants of cells and enzymes with the thiol group and affect cell division, proliferation, and apoptosis. It interacts with the DNA repair mechanism and initiates the production of reactive oxygen species (ROS). It subsequently binds to the mitochondria and may inhibit respiratory and oxidative phosphorylation in even low concentrations. This mechanism leads to damage antioxidant repair mechanism of neuronal cells and turns into neurotoxicity. Now, phytochemicals have led to good practices in the health system. Phytochemicals that are present in the fruits and herbs can preserve upon free radical damage. Thus, this review paper summarized various phytochemicals which can be utilized as a treatment option to reverse the effect of the toxicity caused by the ingestion of heavy metals in our body through various environmental or lifestyles ways.

Mechanistic insights into the antimycobacterial action of unani formulation, Qurs Sartan Kafoori.

Background and aim: Tuberculosis (TBC) is a deadly disease and major health issue in the world. Emergence of drug resistant strains further worsens the efficiency of available anti-TBC drugs. Natural compounds and particularly traditional medicines such as Unani drugs are one of the promising alternatives that have been widely used nowadays. This study aims to evaluate the efficacy of unani drug Qurs-e-Sartan Kafoori (QSK) on Mycobacterium tuberculosis (MTB). Experimental procedures: Drug susceptibilities were estimated by broth microdilution assay. Cell surface integrity was assessed by ZN staining, colony morphology and nitrocefin hydrolysis. Biofilms were visualized by crystal violet staining and measurement of metabolic activity and biomass. Lipidomics analysis was performed using mass spectrometry. Host pathogen interaction studies were accomplished using THP-1 cell lines to estimate cytokines by ELISA kit, apoptosis and ROS by flow cytometry. Results: QSK enhanced the susceptibilities of isoniazid and rifampicin and impaired membrane homeostasis as depicted by altered cell surface properties and enhanced membrane permeability. In addition, virulence factor, biofilm formation was considerably reduced in presence of QSK. Lipidomic analysis revealed extensive lipid remodeling. Furthermore, we used a THP-1 cell line model, and investigated the immunomodulatory effect by estimating cytokine profile and found change in expressions of TNF-α, IL-6 and IL-10. Additionally, we uncover reduced THP-1 apoptosis and enhanced ROS production in presence of QSK. Conclusion: Together, this study validates the potential of unani formulation (QSK) with its mechanism of action and attempts to highlight its significance in MDR reversal.

CLEVER assay: A visual and rapid RNA extraction-free detection of SARS-CoV-2 based on CRISPR-Cas integrated RT-LAMP technology.

AIM: The current scenario of COVID-19 pandemic has presented an almost insurmountable challenge even for the most sophisticated hospitals equipped with modern biomedical technology. There is an urgency to develop simple, fast and highly accurate methods for the rapid identification and isolation of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infected patients. To address the ongoing challenge, the present study offers a CLEVER assay (CRISPR-Cas integrated RT-LAMP Easy, Visual and Extraction-free RNA) which will allow RNA extraction-free method to visually diagnose COVID-19. RNA extraction is a major hurdle in preventing rapid and large-scale screening of samples particularly in low-resource regions because of the logistics and costs involved. METHOD AND RESULT: Herein, the visual SARS-CoV-2 detection method consists of RNA extraction-free method directly utilizing the patient's nasopharyngeal and oropharyngeal samples for reverse transcription loop-mediated isothermal amplification (RT-LAMP). Additionally, the assay also utilizes the integration of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-Cas12-based system using different guide RNAs of N, E and an internal control POP7 (human RNase P) genes along with visual detection via lateral flow readout-based dip sticks with unaided eye (~100 min). Overall, the clinical sensitivity and specificity of the CLEVER assay were 89.6% and 100%, respectively. CONCLUSION: Together, our CLEVER assay offers a point-of-care tool with no equipment dependency and minimum technical expertise requirement for COVID-19 diagnosis. SIGNIFICANCE AND IMPACT OF THE STUDY: To address the challenges associated with COVID-19 diagnosis, we need a faster, direct and more versatile detection method for an efficient epidemiological management of the COVID-19 outbreak. The present study involves developing a method for detection of SARS-CoV-2 in human body without RNA isolation step that can visually be detected with unaided eye. Taken together, our assay offers to overcome one major defect of the prior art, that is, RNA extraction step, which could limit the deployment of the previous assays in a testing site having limited lab infrastructure.

Efficacy of Flavonoids in Combating Fluconazole Resistant Oral Candidiasis.

BACKGROUND: Candida is an opportunistic fungus often present in the oral mucosa. In the compromised immune system, it may become pathogenic and cause oral candidiasis. This infection is more common with Candida albicans; though, non-albicans Candida spp also have significant relevance. Current treatment guidelines include polyenes, azoles and echinocandins, where fluconazole is the primary therapeutic option. However, both inherited and acquired resistance to fluconazole is exhaustively reported. The development of resistance has resulted in the worsening of the original and re-emergence of new fungal diseases. Thus, the development of an anti-candidiasis therapy with a satisfactory outcome is the urgent need of the hour. OBJECTIVE: This review article aims to stimulate research in establishing the synergistic efficacy of various flavonoids with fluconazole to combat the resistance and develop an effective pharmacotherapy for the treatment of oral candidiasis. Further, in this article, we discuss in detail the mechanisms of action of fluconazole, along with the molecular basis of the development of resistance in Candida species. METHODS: PubMed and other databases were used for literature search. RESULTS: The designing of natural drugs from the plant-derived phytochemicals are the promising alternatives in modern medicine. The challenge today is the development of alternative anti-oral candidiasis drugs with increased efficacy, bioavailability and better outcome which can combat azole resistance. Identifying the flavonoids with potential antifungal action at low concentrations seems to meet the challenges. CONCLUSION: Phyto-active constituents, either alone or in combination with conventional antibiotics may be an effective approach to deal with global antimicrobial resistance. The efficacy of herbal therapy for decades suggests that bacteria, fungi, and viruses may have a reduced ability to adapt and resistance to these natural antimicrobial regimes.