Evaluation of phenolic and antioxidant profiles of pink Guava peel (Psidium guajava L. cv Arka kiran) during fruit ripening and its in silico Anti SARS-CoV-2 property

Guava (Psidium guajava L.) is a highly nutritious and economically important fruit. Although fruit peel is generally regarded as a waste, researchers believe that the peel of the guava is rich in bioactive constituents, even higher than the fruit's flesh. The present study aimed to estimate phenolic content (total phenolic and total flavonoid) and assess antioxidant properties of guava fruit peel (pink variety, cv Arka kiran) by 2,2-di (4-tert-octylphenyl)-1-picryl-hydrazyl (DPPH), 2,2′-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid (ABTS) and Ferric Reducing Antioxidant Potential (FRAP) assays at five different ripening stages (stage 1 to 5). The TPC and TFC assays were performed by Folin-Ciocalteu and aluminium chloride (AlCl3) methods, respectively. The molecular docking experiment between the major phenolic of guava peel, Catechin and the spike protein of SARS-CoV-2 was performed by the Dockthor online server. Results showed that the peel had high phenolic (highest TPC and TFC, 7307.3 mg gallic acid equivalent/g dry weight [DW] and 433.9 mg quercetin equivalent/g DW, respectively) and antioxidant values (highest DPPH, ABTS and FRAP values 4784.8, 206.6 and 2451 mg ascorbic acid equivalent/g DW, respectively) throughout all stages, although there was a gradual decline in the activity at the later stages. Furthermore, it was found that catechin had a strong binding affinity (-7.591 kcal mol-1) with the spike protein, in silico when compared with the control drug ceftazidime (-7.250 kcal mol-1). The overall outcome of our experiemnts revealed that guava peel could be explored for future pharmacological applications through in vivo studies, and the ‘green mixed with the yellow’ stage of ripening is optimum for such studies.

Coenzyme Q10 deficiencies: pathways in yeast and humans.

Coenzyme Q (ubiquinone or CoQ) is an essential lipid that plays a role in mitochondrial respiratory electron transport and serves as an important antioxidant. In human and yeast cells, CoQ synthesis derives from aromatic ring precursors and the isoprene biosynthetic pathway. Saccharomyces cerevisiae coq mutants provide a powerful model for our understanding of CoQ biosynthesis. This review focusses on the biosynthesis of CoQ in yeast and the relevance of this model to CoQ biosynthesis in human cells. The COQ1-COQ11 yeast genes are required for efficient biosynthesis of yeast CoQ. Expression of human homologs of yeast COQ1-COQ10 genes restore CoQ biosynthesis in the corresponding yeast coq mutants, indicating profound functional conservation. Thus, yeast provides a simple yet effective model to investigate and define the function and possible pathology of human COQ (yeast or human gene involved in CoQ biosynthesis) gene polymorphisms and mutations. Biosynthesis of CoQ in yeast and human cells depends on high molecular mass multisubunit complexes consisting of several of the COQ gene products, as well as CoQ itself and CoQ intermediates. The CoQ synthome in yeast or Complex Q in human cells, is essential for de novo biosynthesis of CoQ. Although some human CoQ deficiencies respond to dietary supplementation with CoQ, in general the uptake and assimilation of this very hydrophobic lipid is inefficient. Simple natural products may serve as alternate ring precursors in CoQ biosynthesis in both yeast and human cells, and these compounds may act to enhance biosynthesis of CoQ or may bypass certain deficient steps in the CoQ biosynthetic pathway

Diversity and Distribution of Archaea in the Mangrove Sediment of Sundarbans

Mangroves are among the most diverse and productive coastal ecosystems in the tropical and subtropical regions. Environmental conditions particular to this biome make mangroves hotspots for microbial diversity, and the resident microbial communities play essential roles in maintenance of the ecosystem. Recently, there has been increasing interest to understand the composition and contribution of microorganisms in mangroves. In the present study, we have analyzed the diversity and distribution of archaea in the tropical mangrove sediments of Sundarbans using 16S rRNA gene amplicon sequencing. The extraction of DNA from sediment samples and the direct application of 16S rRNA gene amplicon sequencing resulted in approximately 142 Mb of data from three distinct mangrove areas (Godkhali, Bonnie camp, and Dhulibhashani). The taxonomic analysis revealed the dominance of phyla Euryarchaeota and Thaumarchaeota (Marine Group I) within our dataset. The distribution of different archaeal taxa and respective statistical analysis (SIMPER, NMDS) revealed a clear community shift along the sampling stations. The sampling stations (Godkhali and Bonnie camp) with history of higher hydrocarbon/oil pollution showed different archaeal community pattern (dominated by haloarchaea) compared to station (Dhulibhashani) with nearly pristine environment (dominated by methanogens). It is indicated that sediment archaeal community patterns were influenced by environmental conditions

Analysis and characterization of the biosynthetic pathway of Coenzyme Q in eukaryotes, and the role of ring precursors and key intermediates

Coenzyme Q (known by various names that include ubiquinone, CoQ or simply Q) is a crucial redox-active lipid that consists of a fully substituted benzenoid head group and a polyisoprenoid tail. The benzenoid head group moiety that resembles a quinone, can undergo reversible redox reactions interconverting from the fully oxidized quinone through a radical semi-quinone intermediate to the fully reduced quinol. This structural feature aids in the essential role that Q plays in cellular respiration, wherein it transports electrons from NADH and succinate to cytochrome c (Respiratory complex I and II to III respectively in eukaryotes). Q also acts as a lipid soluble chain terminating anti-oxidant. Thus, complete lack of Q is embryonically fatal and sufficient de novo Q biosynthesis is crucial for proper health maintenance in humans. Q deficiency has been directly or indirectly linked to a wide spectrum of health disorders in humans, including kidney disease, neurodegenerative diseases, cerebellar ataxia, and cardiovascular complications. Additionally, decreased Q levels have been linked to aging. Current therapeutic strategies to treat Q deficiency related complications involve direct oral supplementation of Q, which has its challenges due to the hydrophobicity and low bio-availability of Q. Therefore, our research is aimed at characterizing the biosynthesis and metabolism of Q in living cells, thereby potentially leading the way to novel therapeutic techniques. Saccharomyces cerevisiae (yeast) serves as a highly useful model for research on Q, due to its widely studied molecular genetics and its close homology to human Q biosynthesis, metabolism and function. Q biosynthesis in S. cerevisiae (which makes Q6 with six isoprene units, versus humans whose Q10 has ten isoprene units) takes place in the mitochondria. Chapter 1 highlights the currently known Q biosynthetic steps along with phenotypes observed from deletion and malfunction of Q biosynthesis in yeast and humans. The primary precursor molecule that is utilized by eukaryotes to biosynthesize Q is 4-hydroxybenzoic acid (4HB). The latter is in turn immediately preceded by 4-hydroxybenzaldehyde (4HBz) in the Q biosynthetic pathway. Fourteen known proteins that localize in the mitochondria are responsible for catalyzing different steps in this process—Coq1-Coq11, Yah1 (ferredoxin), Arh1 (ferredoxin reductase), and Hfd1 (aldehyde dehydrogenase). In yeast 4HBz is biosynthesized from the precursor amino acid Tyrosine (Tyr). However, this pathway lacks proper characterization and only the deaminases Aro8 and Aro9 and the aldehyde dehydrogenase Hfd1 have been identified. The human homolog of Hfd1 is ALDH3A1 which can serve as a potential target when attempting to screen for Q deficiency in humans.Chapter 2 investigates the role of key proteins and intermediates in the biosynthesis of Q in yeast. In addition to 4HB yeast is also capable of utilizing p-aminobenzoic acid (pABA) as a Q ring precursor. However, the exact steps by which the two pathways converge was not fully characterized. It was postulated that the deamination followed by hydroxylation of the pABA phenyl ring occurs via Schiff base chemistry. Additionally, high performance liquid chromatography tandem mass spectrometry (HPLC-MS/MS) analysis of yeast mutants with deletions in selected Coq genes, showed accumulation of Q intermediates. These intermediates included 3-hexaprenyl-4-hydroxyphenol (4-HP), 3-hexaprenyl-4-aminophenol (4-AP), demethyl demethoxy Q6 (DDMQ6), imino demethyl demethoxy Q6 (IDDMQ6), demethoxy Q6 (DMQ6), and imino demethoxy Q6 (IDMQ6). In order to test the hypothesis of the Schiff base chemistry responsible for Q biosynthesis from pABA, and to investigate whether the above mentioned intermediates are actual productive Q intermediates or just dead-end intermediates, farnesylated analogs (wherein the hexaprenyl tail of Q intermediates is changed to a farnesyl tail consisting of three isoprene units) of 4-HP, DDMQ6 and DMQ6 along with the reduced intermediate IDDMQ6H2, were chemically synthesized. Thus 2-farnesyl-4-dyroxyphenol (4-HFP), demethyl demethoxy Q3 (DDMQ3), demethoxy Q3 (DMQ3) and reduced imino demethyl demethoxy Q3 (IDDMQ3H2) were correspondingly obtained. These intermediates were fed to yeast in biochemical feeding assays and their corresponding potential transformation to Q3 was analyzed via HPLC-MS/MS studies. DMQ3 showed ready conversion to Q3. However, DDMQ3 showed very limited Q3 generation, whereas, 4-HFP and IDDMQ3H2 failed to show detectable levels of Q3. Thus the role of DMQ6 as a Q biosynthetic intermediate was further elucidated. It was demonstrated by Dr. Fabien Pierrel’s research group that the Schiff base chemistry hypothesis for the convergence of the pABA and 4HB pathways leading to Q biosynthesis, was incorrect, and the deamination of the ring of pABA occurs further upstream than formerly postulated. Therefore efforts to further synthesize and investigate farnesylated analogs of Q intermediates were postponed and instead investigations were carried out to test the role of ring precursors in addition to 4HB and pABA in Q biosynthesis. Chapter 3 gives details of the studies conducted on selected alternate Q ring precursors, and the discovery of kaempferol (a plant derived flavonol), as a novel Q ring precursor in mammalian cells. Mouse kidney proximal tubule epithelial (Tkpts) cells and human embryonic kidney cells 293 (HEK 293) were treated with several types of polyphenols, and kaempferol produced the largest increase in Q levels. Experiments with stable isotope 13C-labeled kaempferol demonstrated a previously unrecognized role of kaempferol as an aromatic ring precursor in Q biosynthesis. Investigations of the structure-function relationship of related flavonols showed the importance of two hydroxyl groups, located at C3 of the C ring and C4′ of the B ring, both present in kaempferol, as important determinants of kaempferol as a Q biosynthetic precursor. Concurrently, through a mechanism not related to the enhancement of Q biosynthesis, kaempferol also augmented mitochondrial localization of Sirt3. The role of kaempferol as a precursor that increases Q levels, combined with its ability to upregulate Sirt3, identify kaempferol as a potential candidate in the design of interventions aimed on increasing endogenous Q biosynthesis, particularly in kidney.In addition to kaempferol, other phenolic molecules were previously shown to act as Q ring precursors in yeast and mammalian cells. This included p-coumaric acid. Chapter 4 reports a detailed investigation into the role of p-coumaric acid as a Q ring precursor in yeast. Stable isotope labeled [13C6-ring]-p-coumaric acid was chemically synthesized. This was tested on BY4741 and W303 genetic backgrounds of wild type (WT) yeast to analyze corresponding [13C6-ring]-Q levels via HPLC-MS/MS. Different growth media conditions and times of incubation were utilized in order to fully assess the role of p-coumaric acid as an alternate Q ring precursor. It was discovered that the W303 genetic background of yeast has a much higher efficiency of p-coumaric acid uptake and subsequent conversion to Q. Furthmore, attempts were made to test the pathway by which p-coumaric acid is biosynthesized to Q. It was postulated that this occurs via intermediary biosynthesis of the former to 4HB. To investigate this possibility the Hfd1 gene was knocked out in the W303 genetic background, and the corresponding hfd1 null strain was assayed with [13C6-ring]-p-coumaric acid. Chapter 5 provides insight and perspectives into projects being currently pursued and potential experiments to be conducted in the future. In particular, we are probing further into the role of kaempferol as a Q ring precursor in mammalian cells. It was hypothesized that the B ring of kaempferol underwent cleavage from the rest of the molecule and was utilized to generate the ring of Q in mammalian cells. This hypothesis was further strengthened when Dr. Gilles Basset was able to confirm the utilization of the B ring of kaempferol to generate Q in Arabidopsis thaliana. Moreover, Dr. Bassett showed that this occurs through a peroxidative cleavage mechanism, whereby the B ring of kaempferol is converted to 4HB, which in turn is used to generate Q. Attempts are being made to explore a similar potential peroxidative mechanism occurring in mammalian cells. An in vitro peroxidation assay similar to the one used by Dr. Bassett is in the process of being set up on mammalian cell extracts incubated with kaempferol. Methods have been generated to detect 4HB (synthesized by kaempferol peroxidation in the cell extracts) by HPLC-MS/MS via a derivatization strategy. In addition, mouse kidney cells grown in presence of kaempferol with the B ring selectively labeled with stable 13C isotope (generated by Dr. Bassett), have shown production of 13C stable isotope ring labeled Q. Finally, the Appendix contains two additional publications. The first explores alternative splicing in yeast and the role it plays in Q biosynthesis. PTC7 encodes the phosphatase responsible for the dephosphorylation of Coq7 undergoes alternative splicing, which is rare in yeast. The study also implicated SNF2 as the gene that is responsible for this alternative splicing event and showed that deletion of SNF2 leads to increased Q levels in yeast. The second publication explores the rescue of the clinical phenotypes associated with Coq6 deletion in mice by supplementation with 2,4-dihydroxybenzoic acid. In particular it was shown that steroid resistance nephrotic syndrome which develops in mice with Coq6 deletion, can be ameliorated by treatment with 2,4-dihydroxybenzoic acid

Antioxidant phenolics of Justicia adhatoda L. and Cordia dichotoma Frost. Promote thrombolytic activity through binding to a serine protease, tissue plasminogen activator protein

Background: The tissue plasminogen activator (tPA) protein dissolutes fibrin clots and prevents the disease like thrombosis. The current study aimed to study the tPA-promoting activity of bioactive molecules of Justicia adhatoda L (JA) and Cordia dichotoma Frost (CD). Methods: The phytochemical characterization of methanolic and aqueous extracts of JA and CD stems was performed through qualitative analysis, Fourier-transform infrared spectroscopy (FTIR), and biochemical tests (total phenolic and total flavonoid content [TPC and TFC]). The bioactivity of the extracts was studied through total antioxidant capacity (TAC) and ferric-reducing antioxidant potential (FRAP) assays. Finally, forty phytocompounds from JA and CD were identified from the literature, and in silico molecular docking study was performed to target tPA protein (PDB id 1A5H, Chain A, X-ray diffraction, resolution 2.90 Å). Results: Various phytochemical classes were identified from extracts, through qualitative and FTIR analysis. TPC and TFC were estimated from the JA and CD extracts within the range of 9.34–28.67 mg gallic acid equivalent/100 g of extract weight (EW) and 2.48–16.17 mg quercetin equivalent/100 g of EW, respectively. The aqueous extract of CD showed the highest TAC of 14.90 ascorbic acid equivalent (AAE)/100 g of EW, and the methanolic extract of JA had the highest FRAP activity of 27.77 mg AAE/100 g EW. The molecular docking study showed that apigenin 6,8-di-glucopyranoside had the highest binding potential toward the tPA (−9.380 kcal/mol). Conclusion: It can be concluded that antioxidant phytochemicals of JA and CD could promote the tPA activity, thereby promoting thrombolytic activity

Intragenic suppressor mutations of the COQ8 protein kinase homolog restore coenzyme Q biosynthesis and function in Saccharomyces cerevisiae.

Saccharomyces cerevisiae Coq8 is a member of the ancient UbiB atypical protein kinase family. Coq8, and its orthologs UbiB, ABC1, ADCK3, and ADCK4, are required for the biosynthesis of coenzyme Q in yeast, E. coli, A. thaliana, and humans. Each Coq8 ortholog retains nine highly conserved protein kinase-like motifs, yet its functional role in coenzyme Q biosynthesis remains mysterious. Coq8 may function as an ATPase whose activity is stimulated by coenzyme Q intermediates and phospholipids. A key yeast point mutant expressing Coq8-A197V was previously shown to result in a coenzyme Q-less, respiratory deficient phenotype. The A197V substitution occurs in the crucial Ala-rich protein kinase-like motif I of yeast Coq8. Here we show that long-term cultures of mutants expressing Coq8-A197V produce spontaneous revertants with the ability to grow on medium containing a non-fermentable carbon source. Each revertant is shown to harbor a secondary intragenic suppressor mutation within the COQ8 gene. The intragenic suppressors restore the synthesis of coenzyme Q. One class of the suppressors fully restores the levels of coenzyme Q and key Coq polypeptides necessary for the maintenance and integrity of the high-molecular mass CoQ synthome (also termed complex Q), while the other class provides only a partial rescue. Mutants harboring the first class of suppressors grow robustly under respiratory conditions, while mutants containing the second class grow more slowly under these conditions. Our work provides insight into the function of this important yet still enigmatic Coq8 family

Intragenic suppressor mutations of the COQ8 protein kinase homolog restore coenzyme Q biosynthesis and function in Saccharomyces cerevisiae.

Saccharomyces cerevisiae Coq8 is a member of the ancient UbiB atypical protein kinase family. Coq8, and its orthologs UbiB, ABC1, ADCK3, and ADCK4, are required for the biosynthesis of coenzyme Q in yeast, E. coli, A. thaliana, and humans. Each Coq8 ortholog retains nine highly conserved protein kinase-like motifs, yet its functional role in coenzyme Q biosynthesis remains mysterious. Coq8 may function as an ATPase whose activity is stimulated by coenzyme Q intermediates and phospholipids. A key yeast point mutant expressing Coq8-A197V was previously shown to result in a coenzyme Q-less, respiratory deficient phenotype. The A197V substitution occurs in the crucial Ala-rich protein kinase-like motif I of yeast Coq8. Here we show that long-term cultures of mutants expressing Coq8-A197V produce spontaneous revertants with the ability to grow on medium containing a non-fermentable carbon source. Each revertant is shown to harbor a secondary intragenic suppressor mutation within the COQ8 gene. The intragenic suppressors restore the synthesis of coenzyme Q. One class of the suppressors fully restores the levels of coenzyme Q and key Coq polypeptides necessary for the maintenance and integrity of the high-molecular mass CoQ synthome (also termed complex Q), while the other class provides only a partial rescue. Mutants harboring the first class of suppressors grow robustly under respiratory conditions, while mutants containing the second class grow more slowly under these conditions. Our work provides insight into the function of this important yet still enigmatic Coq8 family

Characterization of an AGAMOUS-like MADS Box Protein, a Probable Constituent of Flowering and Fruit Ripening Regulatory System in Banana

<div><p>The MADS-box family of genes has been shown to play a significant role in the development of reproductive organs, including dry and fleshy fruits. In this study, the molecular properties of an AGAMOUS like MADS box transcription factor in banana cultivar Giant governor <em>(Musa sp</em>, AAA group, subgroup Cavendish) has been elucidated. We have detected a CArG-box sequence binding AGAMOUS MADS-box protein in banana flower and fruit nuclear extracts in DNA-protein interaction assays. The protein fraction in the DNA-protein complex was analyzed by mass spectrometry and using this information we have obtained the full length cDNA of the corresponding protein. The deduced protein sequence showed ∼95% amino acid sequence homology with MA-MADS5, a MADS-box protein described previously from banana. We have characterized the domains of the identified AGAMOUS MADS-box protein involved in DNA binding and homodimer formation <em>in vitro</em> using full-length and truncated versions of affinity purified recombinant proteins. Furthermore, in order to gain insight about how DNA bending is achieved by this MADS-box factor, we performed circular permutation and phasing analysis using the wild type recombinant protein. The AGAMOUS MADS-box protein identified in this study has been found to predominantly accumulate in the climacteric fruit pulp and also in female flower ovary. <em>In vivo</em> and <em>in vitro</em> assays have revealed specific binding of the identified AGAMOUS MADS-box protein to CArG-box sequence in the promoters of major ripening genes in banana fruit. Overall, the expression patterns of this MADS-box protein in banana female flower ovary and during various phases of fruit ripening along with the interaction of the protein to the CArG-box sequence in the promoters of major ripening genes lead to interesting assumption about the possible involvement of this AGAMOUS MADS-box factor in banana fruit ripening and floral reproductive organ development.</p> </div