Composition, Abundance, and Diversity of the Soil Microbiome Associated with the Halophytic Plants Tamarix aphylla and Halopeplis perfoliata on Jeddah Seacoast, Saudi Arabia.

The coast of the Red Sea in Jeddah City is home to a unique microbial community that has adapted to extreme environmental conditions. Therefore, it is essential to characterize the microbial community in this unique microbiome to predict how environmental changes will affect it. The aim of this study was to conduct metagenomic sequencing of 16S rRNA and ITS rRNA genes for the taxonomic classification of the microbial community in soil samples associated with the halophytic plants Tamarix aphylla and Halopeplis perfoliata. Fifteen soil samples were collected in triplicate to enhance robustness and minimize sampling bias. Firstly, to identify novel microbial candidates, the gDNAs were isolated from the saline soil samples surrounding each plant, and then bacterial 16S (V3-V4) and fungal ITS1 regions were sequenced utilizing a high-throughput approach (next-generation sequencing; NGS) on an Illumina MiSeq platform. Quality assessment of the constructed amplicon libraries was conducted using Agilent Bioanalyzer and fluorometric quantification methods. The raw data were processed and analyzed using the Pipeline (Nova Lifetech, Singapore) for bioinformatics analysis. Based on the total number of readings, it was determined that the phylum Actinobacteriota was the most prevalent in the soil samples examined, followed by the phylum Proteobacteria. Based on ITS rRNA gene analysis, the alpha and beta fungal diversity in the studied soil samples revealed that the fungal population is structured into various groups according to the crust (c) and/or rhizosphere (r) plant parts. Fungal communities in the soil samples indicated that Ascomycota and Basidiomycota were the two most abundant phyla based on the total amount of sequence reads. Secondly, heat-map analysis of the diversity indices showed that the bacterial alpha diversity, as measured by Shannon, Simpson, and InvSimpson, was associated with soil crust (Hc and Tc enclosing H. perfoliata and T. aphylla, respectively) and that the soil rhizosphere (Hr and Tr) was strongly correlated with bacterial beta diversity. Finally, fungal-associated Tc and Hc samples clustered together, according to observations made using the Fisher and Chao1 methods, and Hr and Tr samples clustered together according to Shannon, Simpson, and InvSimpson analyses. As a result of the soil investigation, potential agents that have been identified could lead to innovative agricultural, medical, and industrial applications.

GC-MS Analysis of Bioactive Compounds Extracted from Plant Rhazya stricta Using Various Solvents.

Worldwide, human beings have traditionally employed many folkloric herbal resources as complementary and alternative remedies, and these remedies have played a pivotal role in modern medicines for many decades, as scientists have used them to develop drugs. We studied the effects of employing solvents with varying polarity on the yields of phytochemical components extracted from the plant Rhazya stricta. We used chloroform-methanol (1:1), methanol, ethanol, diethyl ether, and ethyl acetate as extraction solvents. The results showed that the efficiencies of the solvents at extracting phytochemical compounds were in this order: chloroform-methanol < ethanol < methanol < diethyl ether < ethyl acetate extract. The chloroform-methanol extract produced the highest concentration of phenolic and flavonoid contents among the five solvents tested (13.3 mg GAE/g DM and 5.43 CE/g DM). The yields of the extracted phytochemical compounds ranged from 47.55 to 6.05%. The results revealed that the properties of the extraction solvents considerably impacted the extraction yield and the phytochemical components of the R. stricta extract. Furthermore, compared with the other solvents, the chloroform-methanol extraction led to the highest yield (47.55%) and to more phytochemical substances being extracted. The aim of this study is to investigate the phytochemical compounds extracted from R. stricta with different solvents that have different polarities.

Exploring the taxonomic and functional diversity of marine benthic micro-Eukaryotes along the Red Sea coast of Jeddah city.

Backgrounds: Diverse marine habitats along Jeddah's Red Sea coast support rich biodiversity. Few studies have been done on its diverse communities, especially its microbial counterparts. Metagenomic analysis of marine benthic micro-eukaryotic communities was performed for the first time on the Red Sea coast of Jeddah. This research looks into their community structure and metabolic potential. Methods: Next-generation sequencing was used to examine the micro-eukaryotic communities of seven sedimentary soil samples from four Jeddah coast locations. After isolating DNA from seven benthic sedimentary soil samples, the 18S rDNA V4 regions were amplified and sequenced on the Illumina MiSeq. It was also verified using an Agilent Technologies 2100 Bioanalyzer with a DNA 1000 chip (Agilent Technologies, Fisher Scientific). A standard curve of fluorescence readings generated by qPCR quantification using the Illumina library was achieved using the GS FLX library. Metagenomic data analysis was used to evaluate the microbial communities' biochemical and enzymatic allocations in studied samples. Results: Blast analysis showed that the top ten phyla were Annelida, Eukaryota, Diatomea, Porifera, Phragmoplastophyta, Arthropoda, Dinoflagellata, Xenacoelomorpha Nematoda, and uncultured. Annelida was also found in the highest percentage (93%), in the sample M followed by Porifera (64%), the most abundant in the control sample then Eukaryotes (61%), Phragmatoplastophyta (55%), Arthropoda, and Diatomea (the least common) (32%). community diversity analysis: using Shannon and inverse Simpson indices showed sediment composition to be effective. Also, PICRUST2 indicated that the most abundant pathways were pyruvate fermentation to isobutanol, pyrimidine deoxyribonucleotide phosphorylation, adenosine ribonucleotide de novo biosynthesis, guanosine ribonucleotide de novo biosynthesis, NAD salvage pathway I, the super pathway of glyoxylate bypass and aerobic respiration I (cytochrome c). Conclusion: Results showed that high throughput metagenomics could reveal species diversity and estimate gene profiles. Environmental factors appear to be more important than geographic variation in determining the structure of these microbial communities. This study provides the first report of marine benthic micro-eukaryotic communities found on the Red Sea coast of Jeddah and will serve as a good platform for future research.

In silico screening of some compounds derived from the desert medicinal plant Rhazya stricta for the potential treatment of COVID-19.

The latest coronavirus pandemic (SARS-CoV-2) poses an exceptional threat to human health and society worldwide. The coronavirus (SARS-CoV-2) spike (S) protein, which is required for viral-host cell penetration, might be considered a promising and suitable target for treatment. In this study, we utilized the nonalkaloid fraction of the medicinal plant Rhazya stricta to computationally investigate its antiviral activity against SARS-CoV-2. Molecular docking and molecular dynamics simulations were the main tools used to examine the binding interactions of the compounds isolated by HPLC analysis. Ceftazidime was utilized as a reference control, which showed high potency against the SARS-CoV-2 receptor binding domain (RBD) in an in vitro study. The five compounds (CID:1, CID:2, CID:3, CID:4, and CID:5) exhibited remarkable binding affinities (CID:1, - 8.9; CID:2, - 8.7; and CID:3, 4, and 5, - 8.5 kcal/mol) compared to the control compound (- 6.2 kcal/mol). MD simulations over a period of 200 ns further corroborated that certain interactions occurred with the five compounds and the nonalkaloidal compounds retained their positions within the RBD active site. CID:2, CID:4, and CID:5 demonstrated high stability and less variance, while CID:1 and CID:3 were less stable than ceftazidime. The average number of hydrogen bonds formed per timeframe by CID:1, CID:2, CID:3, and CID:5 (0.914, 0.451, 1.566, and 1.755, respectively) were greater than that formed by ceftazidime (0.317). The total binding free energy calculations revealed that the five compounds interacted more strongly within RBD residues (CID:1 = - 68.8, CID:2 = - 71.6, CID:3 = - 74.9, CID:4 = - 75.4, CID:5 = - 60.9 kJ/mol) than ceftazidime (- 34.5 kJ/mol). The drug-like properties of the selected compounds were relatively similar to those of ceftazidime, and the toxicity predictions categorized these compounds into less toxic classes. Structural similarity and functional group analyses suggested that the presence of more H-acceptor atoms, electronegative atoms, acidic oxygen groups, and nitrogen atoms in amide or aromatic groups were common among the compounds with the lowest binding affinities. In conclusion, this in silico work predicts for the first time the potential of using five R. stricta nonalkaloid compounds as a treatment strategy to control SARS-CoV-2 viral entry.

Single Cell Oil (SCO)-Based Bioactive Compounds: I-Enzymatic Synthesis of Fatty Acid Amides Using SCOs as Acyl Group Donors and Their Biological Activities.

Fatty acid amides (FAAs) are of great interest due to their broad industrial applications. They can be synthesized enzymatically with many advantages over chemical synthesis. In this study, the fatty acid moieties of lipids of Cunninghamella echinulata ATHUM 4411, Umbelopsis isabellina ATHUM 2935, Nannochloropsis gaditana CCAP 849/5, olive oil, and an eicosapentaenoic acid (EPA) concentrate were converted into their fatty acid methyl esters and used in the FAA (i.e., ethylene diamine amides) enzymatic synthesis, using lipases as biocatalysts. The FAA synthesis, monitored using in situ NMR, FT-IR, and thin-layer chromatography, was catalyzed efficiently by the immobilized Candida rugosa lipase. The synthesized FAAs exhibited a significant antimicrobial activity, especially those containing oleic acid in high proportions (i.e., derived from olive oil and U. isabellina oil), against several human pathogenic microorganisms, insecticidal activity against yellow fever mosquito, especially those of C. echinulata containing gamma-linolenic acid, and anticancer properties against SKOV-3 ovarian cancer cell line, especially those containing EPA in their structures (i.e., EPA concentrate and N. gaditana oil). We conclude that FAAs can be efficiently synthesized using microbial oils of different fatty acid composition and used in specific biological applications.

Assessment of fungal diversity in soil rhizosphere associated with Rhazya stricta and some desert plants using metagenomics.

This study aimed to compare the fungal rhizosphere communities of Rhazya stricta, Enneapogon desvauxii, Citrullus colocynthis, Senna italica, and Zygophyllum simplex, and the gut mycobiota of Poekilocerus bufonius (Orthoptera, Pyrgomorphidae, "Usherhopper"). A total of 164,485 fungal reads were observed from the five plant rhizospheres and Usherhopper gut. The highest reads were in S. italica rhizosphere (29,883 reads). Species richness in the P. bufonius gut was the highest among the six samples. Ascomycota was dominant in all samples, with the highest reads in E. desvauxii (26,734 reads) rhizosphere. Sordariomycetes and Dothideomycetes were the dominant classes detected with the highest abundance in C. colocynthis and E. desvauxii rhizospheres. Aspergillus and Ceratobasidium were the most abundant genera in the R. stricta rhizosphere, Fusarium and Penicillium in the E. desvauxii rhizosphere and P. bufonius gut, Ceratobasidium and Myrothecium in the C. colocynthis rhizosphere, Aspergillus and Fusarium in the S. italica rhizosphere, and Cochliobolus in the Z. simplex rhizosphere. Aspergillus terreus was the most abundant species in the R. stricta and S. italica rhizospheres, Fusarium sp. in E. desvauxii rhizosphere, Ceratobasidium sp. in C. colocynthis rhizosphere, Cochliobolus sp. in Z. simplex rhizosphere, and Penicillium sp. in P. bufonius gut. The phylogenetic results revealed the unclassified species were related closely to Ascomycota and the species in E. desvauxii, S. italica and Z. simplex rhizospheres were closely related, where the species in the P. bufonius gut, were closely related to the species in the R. stricta, and C. colocynthis rhizospheres.

Analysis of sugar composition and pesticides using HPLC and GC-MS techniques in honey samples collected from Saudi Arabian markets.

Honey is a complex foodstuff found in nature which is used without any processing. Honey has been in use in medicine as well as raw food since ancient times. Essentially, it is a blend of sugars especially fructose and glucose. The objectives of the study were to determine major sugar composition as well as pesticides contamination in honey samples. Further, Hydroxy-methyl-furfuraldehyde (HMF) level was also determined to ascertain the freshness of honey samples. A total of 14 samples were collected from local market and tested for fructose, glucose, sucrose, HMF and organochlorine pesticides using HPLC and GC-MS techniques respectively. The total sugars in the 14 honey samples were found ranging between 50.26 and 74.74 g/100 g of honey. The chromatographic results showed the presence of the sugars like fructose and glucose in all honey samples. The honey sample SH-11 was found to contain the highest amount of fructose (40.63%). On the other hand, the lowest amount of fructose with 29.08% was observed in SH-7. The HPLC analysis also revealed the presence of sucrose in two samples but under the permissible limit. The average ratio of fructose to glucose in these honey samples was 1.3. None of the sample has ratio below 1.0 indicating lesser chances for honey to crystallize on storage. Out of 14 honey samples, 13 samples were found negative for the presence of any of the 63 pesticides tested. Only sample No. 13, was found to contain 15.95 ppb hexachlorobenzene per kilogram of honey. The HMF was not detected in four samples but in remaining samples it was well below the maximum permissible limit. No pesticide and sugar adulteration was observed in any of the honey samples. The honeys collected from Saudi Arabian markets were found to confirm the standards set by the regional and international standardization organization, the GSO and Codex Alimentarius Commission respectively.

Comparative Study of Gene Expression Profiling Unravels Functions Associated with Pathogenesis of Dengue Infection.

BACKGROUND: Dengue virus is a potential source of propagating dengue hemorrhagic fever. This virus leads to dengue hemorrhagic fever/dengue shock syndrome, benign syndrome, and severe syndrome and due to its infection, there occurs alterations at multiple levels such as gene expression and pathway levels. So, it is critical to understand the pathogenesis of dengue infection in terms of gene expression and the associated functions. METHODS: For this purpose, here, we have analyzed the temporal gene expression profiling for the dengue hemorrhagic fever dataset at 12, 24, and 48 hours. RESULTS: The outcome appears that the dengue hemorrhagic fever evolves differently at different time periods or stages. CONCLUSION: The change in the gene expression pattern increases exponentially from 12 hours to 48 hours and the number of altered functions (pathways) also increases. Wnt, apoptosis, and transcription signaling are among the critical pathways which are dominantly altered. In the initial phase (first 12 hours), only two pathways are altered due to dengue infection, while in the next 12 hours, eight pathways are altered, and finally, in the next 24 hours, 11 pathways are altered and most of these 11 pathways are very critical in terms of biological pathways and functions.

Investigating the pathogenic SNPs in BLM helicase and their biological consequences by computational approach.

The BLM helicase protein plays a vital role in DNA replication and the maintenance of genomic integrity. Variation in the BLM helicase gene resulted in defects in the DNA repair mechanism and was reported to be associated with Bloom syndrome (BS) and cancer. Despite extensive investigation of helicase proteins in humans, no attempt has previously been made to comprehensively analyse the single nucleotide polymorphism (SNPs) of the BLM gene. In this study, a comprehensive analysis of SNPs on the BLM gene was performed to identify, characterize and validate the pathogenic SNPs using computational approaches. We obtained SNP data from the dbSNP database version 150 and mapped these data to the genomic coordinates of the "NM_000057.3" transcript expressing BLM helicase (P54132). There were 607 SNPs mapped to missense, 29 SNPs mapped to nonsense, and 19 SNPs mapped to 3'-UTR regions. Initially, we used many consensus tools of SIFT, PROVEAN, Condel, and PolyPhen-2, which together increased the accuracy of prediction and identified 18 highly pathogenic non-synonymous SNPs (nsSNPs) out of 607 SNPs. Subsequently, these 18 high-confidence pathogenic nsSNPs were analysed for BLM protein stability, structure-function relationships and disease associations using various bioinformatics tools. These 18 mutants of the BLM protein along with the native protein were further investigated using molecular dynamics simulations to examine the structural consequences of the mutations, which might reveal their malfunction and contribution to disease. In addition, 28 SNPs were predicted as "stop gained" nonsense SNPs and one SNP was predicted as "start lost". Two SNPs in the 3'UTR were found to abolish miRNA binding and thus may enhance the expression of BLM. Interestingly, we found that BLM mRNA overexpression is associated with different types of cancers. Further investigation showed that the dysregulation of BLM is associated with poor overall survival (OS) for lung and gastric cancer patients and hence led to the conclusion that BLM has the potential to be used as an important prognostic marker for the detection of lung and gastric cancer.

Neurotoxic, Hepatotoxic and Nephrotoxic Effects of Tramadol Administration in Rats.

The current study was performed to study the tramadol HCL toxic effects on the brain, liver, and kidney of adult male rats. Forty male adult albino rats were divided into 4 groups; the first one was considered as a control group, the others were orally administrated with 25, 50, and 100 b.wt. representing therapeutic, double therapeutic, and 4 times therapeutic doses, respectively, of tramadol HCL daily for 1 month. Serum and brain, hepatic, and renal tissues were collected for biochemical and molecular investigations. Tramadol HCL resulted in a significant increase in the brain serotonin, 8-hydroxy-2'-deoxyguanosine (8-OHdG), and malonyldialdehyde (MDA) levels with a significant decrease in the reduced glutathione (GSH), catalase (CAT), and superoxide dismutase (SOD) activities. At the same line, hepatic and renal 8-OHdG and MDA levels showed a significant increase with a significant decrease in reduced glutathione (GSH), CAT, and SOD activities. In addition, hepatic and renal function parameters including serum alanine amino transferase (ALT), aspartate amino transferase (AST), urea, and creatinine were increased in a dose-dependent manner. At the molecular levels, hepatic cytochrome P5402E1 (CYP2E1), renal Kidney Injury Molecule-1 (KIM-1), and tissue inhibitor of metalloproteinase-1 (TIMP-1) showed also a significant increase in the expression levels. Histopathological evaluation of the brain confirmed the above biochemical results. In conclusion, tramadol HCL induced neurotoxic, hepatotoxic, and nephrotoxic effects in a manner relative to its concentration by affecting brain serotonin levels and hepatic and renal function, with the production of DNA damage and oxidative stress.

Shelf life extension of wheat bread by alhydwan flour and Carboxymethylcellulose and improvement of their quality characteristics, dough rheological and microstructure.

Alhydwan, has been used in bakery products for many years in South of Yemen. Alhydwan primary function in bakery products is to inhibit moisture content during storage, leading to improved shelf life. However, as a fresh strategy to bread staling, no extensive study has been conducted to evaluate its potential. The present study therefore examined the alhydwan as a comparison with Carboxymethylcellulose (CMC) at 0.5% (w/w) level in dough rheology improvement, microstructure, quality parameters and delayed wheat bread staling. The CMC or alhydwan for farinograph characteristics comprising the dough's portrayed showed greater water absorption, while growth and stability time was significantly decreased. Staling of bread, the findings showed that in both alhydwan and CMC minimized crumb hardening frequency and enhanced freshness, quality and retention ability for moisture, making the bread softer and postponed staling. The microstructure of CMC or alhydwan supplemented formulation showed the distinguishable characteristics and constituents that could explain, to some degree, that the CMC and alhydwan had antistaling effect. The incorporation of alhydwan such as CMC into the formulation of bread could thus play a sustainable role in improving the quality of bread by having an extended shelf life.

A Comprehensive Analysis of cis-Acting RNA Elements in the SARS-CoV-2 Genome by a Bioinformatics Approach.

The emergence of a new coronavirus (CoV), severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), responsible for severe respiratory disease in humans termed coronavirus disease of 2019 (COVID-19), became a new global threat for health and the economy. The SARS-CoV-2 genome is about a 29,800-nucleotide-long plus-strand RNA that can form functionally important secondary and higher-order structures called cis-acting RNA elements. These elements can interact with viral proteins, host proteins, or other RNAs and be involved in regulating translation and replication processes of the viral genome and encapsidation of the virus. However, the cis-acting RNA elements and their biological roles in SARS-CoV-2 as well as their comparative analysis in the closely related viral genome have not been well explored, which is very important to understand the molecular mechanism of viral infection and pathogenies. In this study, we used a bioinformatics approach to identify the cis-acting RNA elements in the SARS-CoV-2 genome. Initially, we aligned the full genomic sequence of six different CoVs, and a phylogenetic analysis was performed to understand their evolutionary relationship. Next, we predicted the cis-acting RNA elements in the SARS-CoV-2 genome using the structRNAfinder tool. Then, we annotated the location of these cis-acting RNA elements in different genomic regions of SARS-CoV-2. After that, we analyzed the sequence conservation patterns of each cis-acting RNA element among the six CoVs. Finally, the presence of cis-acting RNA elements across different CoV genomes and their comparative analysis was performed. Our study identified 12 important cis-acting RNA elements in the SARS-CoV-2 genome; among them, Corona_FSE, Corona_pk3, and s2m are highly conserved across most of the studied CoVs, and Thr_leader, MAT2A_D, and MS2 are uniquely present in SARS-CoV-2. These RNA structure elements can be involved in viral translation, replication, and encapsidation and, therefore, can be potential targets for better treatment of COVID-19. It is imperative to further characterize these cis-acting RNA elements experimentally for a better mechanistic understanding of SARS-CoV-2 infection and therapeutic intervention.

Immobilisation of α-amylase on activated amidrazone acrylic fabric: a new approach for the enhancement of enzyme stability and reusability.

In this study, amidrazone acrylic fabric was applied as an immobilising support for α-amylase. The immobilised α-amylase was characterised by Fourier transform infrared spectroscopy and scanning electron microscopy. Furthermore, the optimum conditions for immobilisation efficiency, immobilisation time, reusability, kinetic parameters and pH, for the immobilisation process were examined. The study demonstrated that with 4% cyanuric chloride, and a pH of 7.0, the highest immobilization efficiency of 81% was obtained. Around 65% of the initial activity was maintained after storage at 4 °C for 8 weeks. The immobilised enzyme retained 53% of its original activity after being reused 15 times and exhibited improved stability compared with the free enzyme in relation to heavy metal ions, pH, temperature and inhibitors. The immobilised enzyme presented kinetic parameters of 2.6 mg starch and 0.65 µmol maltose/mL for Km and Vmax respectively, compared with 3.7 mg starch and 0.83 µmol maltose/ mL for the free enzyme. The improvements in the enzyme's catalytic properties, stability and reusability obtained from immobilisation make amidrazone acrylic fabric support a good promising candidate for bio-industrial applications.

Immune-protective, antioxidant and relative genes expression impacts of ß-glucan against fipronil toxicity in Nile tilapia, Oreochromis niloticus.

Our study is considered to attempt reducing the immune-toxic and antioxidant impacts of exposure to fipronil (FP) on Nile tilapia, Oreochromis niloticus using the ß-glucan (ßG). Two hundred and seventy fingerlings of Nile tilapia were divided randomly into six groups (45 tilapias of each, in 3 replicates): group I control (CT) group nourished on a basal diet. Group II (ßG) nourished a basal diet supplemented with 0.4% ßG. Groups III (1/20 FP) and V (1/10 FP) was exposed to 1/20 and 1/10 of the 96 h LC50 of FP in water and nourished the basal diet respectively. Groups IV (1/20 FP+ ßG) and VI (1/10 FP+ ßG) were exposed to 1/20 and 1/10 FP concomitantly with 0.4% ßG supplementation for 90 successive days. Growth performance metrics were higher in ßG group than CT. While those metrics were fallen at exposure to 1/20 or 1/10 FP. Supplementation with ßG elevated the IgM and lysozyme levels.Whereas, tilapias exposed to FP only at different concentration showed lowering of those compared to CT. Supplementation with ßG was effectively augmented IgM and lysozyme in 1/20 FP exposed tilapias. Furthermore, in a minor grade at 1/10 FP exposed tilapias. Exposure to FP increased the activities of hepatic markers chiefly at 1/10, however the ßG supplementation was successfully improved these markers. There was imbalance of cortisol level at FP exposure where, ßG combining to FP alleviate this disparity. There was fallen in LDH, MDH and FDPase in ßG tilapias where continuing raise in 1/10 FP followed by 1/20 FP. ßG supplementation raise the level of GSH, without significant variations in MDA conversely occurs in FP alone. Genes expression of ßG caused raise of both GPx and GR, without fluctuations in CAT and SOD. Exposure to FP diminishes all evaluated antioxidant genes. It could fulfilled that supplementation with ßG successfully alleviated the immune-toxic and antioxidant impact of FP in tilapias.

α-Amylase immobilization on amidoximated acrylic microfibres activated by cyanuric chloride.

Enzyme immobilization is one of the most important techniques for industrial applications. It makes the immobilized enzyme more stable and advantageous than the free form in different aspects. α-Amylase was immobilized on 4% cyanuric chloride-activated amidoximated acrylic fabric at pH 7.0 with (79%) maximum efficiency. A field emission scanning electron microscope and Fourier transform infrared were used to confirm the immobilization process. Even after being recycled 10 times, the immobilized enzyme lost just 28% of its initial activity. Owing to immobilization, the pH of the soluble α-amylase was shifted from 6.0 to 6.5. The immobilized α-amylases showed thermal stability at 60°C, and became more resistant to heavy metal ions. The k m values of the immobilized and soluble α-amylases were 9.6 and 3.8 mg starch ml-1, respectively. In conclusion, this method shows that the immobilized α-amylase proved to be more efficient than its soluble form, and hence could be used during saccharification of starch.

Expression and Purification of C-Peptide Containing Insulin Using Pichia pastoris Expression System.

Increase in the incidence of Insulin Dependent Diabetes Mellitus (IDDM) among people from developed and developing countries has created a large global market for insulin. Moreover, exploration of new methods for insulin delivery including oral or inhalation route which require very high doses would further increase the demand of cost-effective recombinant insulin. Various bacterial and yeast strains have been optimized to overproduce important biopharmaceuticals. One of the approaches we have taken is the production of recombinant human insulin along with C-peptide in yeast Pichia pastoris. We procured a cDNA clone of insulin from Origene Inc., USA. Insulin cDNA was PCR amplified and cloned into yeast vector pPICZ-α. Cloned insulin cDNA was confirmed by restriction analysis and DNA sequencing. pPICZ-α-insulin clone was transformed into Pichia pastoris SuperMan 5 strain. Several Zeocin resistant clones were obtained and integration of insulin cDNA in Pichia genome was confirmed by PCR using insulin specific primers. Expression of insulin in Pichia clones was confirmed by ELISA, SDS-PAGE, and Western blot analysis. In vivo efficacy studies in streptozotocin induced diabetic mice confirmed the activity of recombinant insulin. In conclusion, a biologically active human proinsulin along with C-peptide was expressed at high level using Pichia pastoris expression system.

The nuclear genome of Rhazya stricta and the evolution of alkaloid diversity in a medically relevant clade of Apocynaceae.

Alkaloid accumulation in plants is activated in response to stress, is limited in distribution and specific alkaloid repertoires are variable across taxa. Rauvolfioideae (Apocynaceae, Gentianales) represents a major center of structural expansion in the monoterpenoid indole alkaloids (MIAs) yielding thousands of unique molecules including highly valuable chemotherapeutics. The paucity of genome-level data for Apocynaceae precludes a deeper understanding of MIA pathway evolution hindering the elucidation of remaining pathway enzymes and the improvement of MIA availability in planta or in vitro. We sequenced the nuclear genome of Rhazya stricta (Apocynaceae, Rauvolfioideae) and present this high quality assembly in comparison with that of coffee (Rubiaceae, Coffea canephora, Gentianales) and others to investigate the evolution of genome-scale features. The annotated Rhazya genome was used to develop the community resource, RhaCyc, a metabolic pathway database. Gene family trees were constructed to identify homologs of MIA pathway genes and to examine their evolutionary history. We found that, unlike Coffea, the Rhazya lineage has experienced many structural rearrangements. Gene tree analyses suggest recent, lineage-specific expansion and diversification among homologs encoding MIA pathway genes in Gentianales and provide candidate sequences with the potential to close gaps in characterized pathways and support prospecting for new MIA production avenues.

Soil compartment is a major determinant of the impact of simulated rainfall on desert microbiota.

Although desert soils support functionally important microbial communities that affect plant growth and influence many biogeochemical processes, the impact of future changes in precipitation patterns on the microbiota and their activities is largely unknown. We performed in-situ experiments to investigate the effect of simulated rainfall on bacterial communities associated with the widespread perennial shrub, Rhazya stricta in Arabian desert soils. The bacterial community composition was distinct between three different soil compartments: surface biological crust, root-attached, and the broader rhizosphere. Simulated rainfall had no significant effect on the overall bacterial community composition, but some population-level responses were observed, especially in soil crusts where Betaproteobacteria, Sphingobacteria, and Bacilli became more abundant. Bacterial biomass in the nutrient-rich crust increased three-fold one week after watering, whereas it did not change in the rhizosphere, despite its much higher water retention. These findings indicate that between rainfall events, desert-soil microbial communities enter into stasis, with limited species turnover, and reactivate rapidly and relatively uniformly when water becomes available. However, microbiota in the crust, which was relatively enriched in nutrients and organic matter, were primarily water-limited, compared with the rhizosphere microbiota that were co-limited by nutrients and water.

High-quality draft genome sequence of Flavobacterium suncheonense GH29-5(T) (DSM 17707(T)) isolated from greenhouse soil in South Korea, and emended description of Flavobacterium suncheonense GH29-5(T).

Flavobacterium suncheonense is a member of the family Flavobacteriaceae in the phylum Bacteroidetes. Strain GH29-5(T) (DSM 17707(T)) was isolated from greenhouse soil in Suncheon, South Korea. F. suncheonense GH29-5(T) is part of the G enomic E ncyclopedia of B acteria and A rchaea project. The 2,880,663 bp long draft genome consists of 54 scaffolds with 2739 protein-coding genes and 82 RNA genes. The genome of strain GH29-5(T) has 117 genes encoding peptidases but a small number of genes encoding carbohydrate active enzymes (51 CAZymes). Metallo and serine peptidases were found most frequently. Among CAZymes, eight glycoside hydrolase families, nine glycosyl transferase families, two carbohydrate binding module families and four carbohydrate esterase families were identified. Suprisingly, polysaccharides utilization loci (PULs) were not found in strain GH29-5(T). Based on the coherent physiological and genomic characteristics we suggest that F. suncheonense GH29-5(T) feeds rather on proteins than saccharides and lipids.

High-quality permanent draft genome sequence of Ensifer sp. PC2, isolated from a nitrogen-fixing root nodule of the legume tree (Khejri) native to the Thar Desert of India.

Ensifer sp. PC2 is an aerobic, motile, Gram-negative, non-spore-forming rod that was isolated from a nitrogen-fixing nodule of the tree legume P. cineraria (L.) Druce (Khejri), which is a keystone species that grows in arid and semi-arid regions of the Indian Thar desert. Strain PC2 exists as a dominant saprophyte in alkaline soils of Western Rajasthan. It is fast growing, well-adapted to arid conditions and is able to form an effective symbiosis with several annual crop legumes as well as species of mimosoid trees and shrubs. Here we describe the features of Ensifer sp. PC2, together with genome sequence information and its annotation. The 8,458,965 bp high-quality permanent draft genome is arranged into 171 scaffolds of 171 contigs containing 8,344 protein-coding genes and 139 RNA-only encoding genes, and is one of the rhizobial genomes sequenced as part of the DOE Joint Genome Institute 2010 Genomic Encyclopedia for Bacteria and Archaea-Root Nodule Bacteria (GEBA-RNB) project proposal.