Deciphering the functional and structural complexity of the Solar Lake flat mat microbial benthic communities.

The Solar Lake in Taba, Egypt, encompasses one of the few modern-day microbial mats' systems metabolically analogous to Precambrian stromatolites. Solar Lake benthic communities and their adaptation to the Lake's unique limnological cycle have not been described for over two decades. In this study, we revisit the flat mat and describe the summer's shallow water versus exposed microbial community; the latter occurs in response to the seasonal partial receding of water. We employed metagenomic NovaSeq-6000 shotgun sequencing and 16S rRNA, mcrA, and dsrB quantitative PCR. A total of 292 medium-to-high-quality metagenome-assembled genomes (MAGs) were reconstructed. At the structural level, Candidatus Aenigmatarchaeota, Micrarchaeota, and Omnitrophota MAGs were exclusively detected in the shallow-water mats, whereas Halobacteria and Myxococcota MAGs were specific to the exposed microbial mat. Functionally, genes involved in reactive oxygen species (ROS) detoxification and osmotic pressure were more abundant in the exposed than in the shallow-water microbial mats, whereas genes involved in sulfate reduction/oxidation and nitrogen fixation were ubiquitously detected. Genes involved in the utilization of methylated amines for methane production were predominant when compared with genes associated with alternative methanogenesis pathways. Solar Lake methanogen MAGs belonged to Methanosarcinia, Bathyarchaeia, Candidatus Methanofastidiosales, and Archaeoglobales. The latter had the genetic capacity for anaerobic methane oxidation. Moreover, Coleofasciculus chthonoplastes, previously reported to dominate the winter shallow-water flat mat, had a substantial presence in the summer. These findings reveal the taxonomic and biochemical microbial zonation of the exposed and shallow-water Solar Lake flat mat benthic community and their capacity to ecologically adapt to the summer water recession. IMPORTANCE: Fifty-five years ago, the extremophilic "Solar Lake" was discovered on the Red Sea shores, garnering microbiologists' interest worldwide from the 1970s to 1990s. Nevertheless, research on the lake paused at the turn of the millennium. In our study, we revisited the Solar Lake benthic community using a genome-centric approach and described the distinct microbial communities in the exposed versus shallow-water mat unveiling microbial zonation in the benthic communities surrounding the Solar Lake. Our findings highlighted the unique structural and functional adaptations employed by these microbial mat communities. Moreover, we report new methanogens and phototrophs, including an intriguing methanogen from the Archaeoglobales family. We describe how the Solar Lake's flat mat microbial community adapts to stressors like oxygen intrusion and drought due to summer water level changes, which provides insights into the genomic strategies of microbial communities to cope with altered and extreme environmental conditions.

SNP (A > G - rs13057211) but not GT(n) polymorphism in HMOX-1 promotor gene is associated with COVID-19 mortality.

INTRODUCTION: COVID-19 causes severe inflammatory respiratory distress syndrome. The global pandemic caused millions of cases of morbidity and mortality worldwide. Patients may present with variable symptoms including dyspnea, fever, and GIT manifestations. The HMOX-1 gene is located on the long (q) arm of chromosome 22 at position 12.3. HMOX-1 is expressed in all mammalian tissues at basal levels and is considered as a stress response enzyme. HMOX-1 has a specific polymorphic site with variable GT(n) repeats at the promotor region. Several authors evaluated the HMOX-1 GT(n) promoter polymorphism in different inflammatory conditions. We evaluated HMOX-1 promoter polymorphism in relation to serum Hemoxygenase level and inflammatory makers (CRP, Ferritin, PCT, IL-6 and D-dimer) in patients affected by SARS-COV-2 disease. SUBJECTS AND METHODS: Ninety patients confirmed to be infected with COVID-19 were followed up till the study end point (recovery and discharge or death). HMOX-1 promotor GT(n) polymorphism was evaluated using Sanger sequencing. HMOX-1 enzyme serum level was measured by ELISA and the level of different inflammatory markers was assessed by available commercial kits. RESULTS: A novel Single nucleotide polymorphism (SNP) (A > G) - rs13057211 in the GT(n) region of HMOX-1 promoter gene was found in 40 (61.5%) COVID-19 patients out of the studied 65 patients. This (A > G) SNP was associated with higher mortality rate in COVID-19 as it was detected in 27 patients (75% of the patients who succumbed to the disease) (p = 0.021, Odds ratio = 3.7; 95% CI:1.29-10.56). Serum IL-6 (Interleuken-6) was positively correlated the length of Hospital Stay (LOHS) and procalcitonin (PCT); (p = 0.014, r: 0.651 and p < 0.001, r:0.997) respectively while negatively correlated with levels of HMOX-1 enzyme serum level (p = 0.013, r: -0.61). CRP correlated positively with LOHS (p = 0.021, r = 0.4), PCT (p = 0.044, r = 0.425) and age (p < 0.001, r = 0.685). Higher levels of D-Dimer and PCT were observed in patients with the long repeat. There was no significant difference between patients who recovered and those who died from COVID-19 as regards HMOX-1 level and GT(n) polymorphism. CONCLUSION: We report a novel SNP (A > G, rs13057211) in the GT(n) region of HMOX-1 promoter gene that was associated with mortality in COVID-19 patients, however no significant difference was found in HMOX-1 serum level or HMOX-1 (GT)n repeats within the studied groups.

Molecular and functional characterization of unique thermo-halophilic thioredoxin from the metagenome of an exotic environment.

The lower convective layer (LCL) at Atlantis II brine pool of the Red sea represents one of the exceptional, unique ecosystems. Thioredoxin is a multi-functional antioxidant redox protein that has a crucial role in various vital cellular processes. In the current study, a functional metagenomics approach was used to isolate and characterize thioredoxin from the LCL of Atlantis II Deep brine pool (Trx-ATII). From the metagenomic DNA of the LCL, the thioredoxin gene was directly retrieved and sequenced. Sequence analysis showed that the gene belonged to thioredoxin-like superfamily with classical Trx motif (-CXXC-). Phylogenetic analysis revealed that Trx-ATII was closely related to Trx of Prochlorococcus marinus with a maximum identity of 86%. Successfully, Trx-ATII was cloned and expressed in E. coli, where the purified protein had M.wt of 16 kDa. Characterization studies revealed that Trx-ATII protein is halophilic; can tolerate up to 2.5 M NaCl and thermostable, where 90% of its activity was retained at 60 °C. Trx-ATII can reduce both DTNB and insulin disulfide- containing substrates. In conclusion, a unique thioredoxin protein was isolated from a harsh environment that can maintain its activity under extreme conditions of salinity and temperature as a promising redox protein for biotechnological applications.

Fabrication of Poly(vinyl alcohol)/Chitosan/Bidens pilosa Composite Electrospun Nanofibers with Enhanced Antibacterial Activities.

Due to the current challenges faced by the increasing rate of drug-resistant bacteria, attention is gradually shifting from synthetic antimicrobial chemical compounds to natural products that are ecofriendly with a wide spectrum of properties. The aim of this research was to successfully fabricate electrospun nanofibers from poly(vinyl alcohol) (PVA), PVA blended with Bidens pilosa and chitosan composite blends and investigate their potential antibacterial activities against Escherichia coli and Staphylococcus aureus. Fabrication of nanofibers was performed by the electrospinning technique, which applies high voltage on the polymer, forcing it to spin off as a jet onto a plate collector. Characterization of the nanofibers was successfully performed by scanning electron microscopy and Fourier transform infrared spectroscopy. Antibacterial assessment was carried out by colony forming unit enumeration. The results obtained revealed a 12% increase in growth inhibition of bacteria in composite nanofibers as compared with their parental forms, which were >91 and 79%, respectively. Chitosan nanofibers have been extensively researched, and their antibacterial properties have been studied. However B. pilosa antibacterial properties in a nanofiber form have not been previously reported. These composite nanofibers open new avenues toward using natural materials as potent antibacterial agents.

Antibacterial and anticancer activities of orphan biosynthetic gene clusters from Atlantis II Red Sea brine pool.

BACKGROUND: Cancer and infectious diseases are problematic because of continuous emergence of drug resistance. One way to address this enormous global health threat is bioprospecting the unlikeliest environments, such as extreme marine niches, which have tremendous biodiversity that is barely explored. One such environment is the Red Sea brine pool, Atlantis II Deep (ATII). Here, we functionally screened a fosmid library of metagenomic DNA isolated from the ATII lower convective layer (LCL) for antibacterial and anticancer activities. RESULTS: Selected clones, 14-7E and 10-2G, displayed antibacterial effects on the marine strain Bacillus sp. Cc6. Moreover, whole cell lysates from 14-7E and 10-2G exhibited decreased cell viability against MCF-7 (39.1% ± 6.6, 42% ± 8.1 at 50% v/v) and U2OS cells (35.7% ± 1.9, 79.9% ± 5.9 at 50% v/v), respectively. By sequencing the insert DNA from 14-7E and 10-2G, we identified two putative orphan biosynthetic gene clusters. Both clusters harbored putative ATP-binding cassette (ABC) transporter permeases and S-adenosylmethionine-related genes. Interestingly, the biosynthetic gene cluster identified on 14-7E is of archaeal origin and harbors a putative transcription factor. Several identified genes may be responsible for the observed antibacterial and anticancer activities. The 14-7E biosynthetic gene cluster may be encoding enzymes producing a specialized metabolite (effect of detected genes involved in C-C bond formation and glycosylation). The bioactivity may also be due to predicted subtilases encoded by this cluster. The 10-2G cluster harbored putative glycosyltransferase and non-ribosomal peptide synthase genes; thus the observed activity of this clone could be caused by a bioactive peptide. CONCLUSIONS: The ATII LCL prokaryotic metagenome hosts putative orphan biosynthetic gene clusters that confer antibiotic and anticancer effects. Further biochemical studies should characterize the detected bioactive components, and the potential use of 14-7E metabolite for antibiosis and 10-2G metabolite as a selective anti-breast cancer drug.

miRNA-221 and miRNA-222 are promising biomarkers for progression of liver fibrosis in HCV Egyptian patients.

BACKGROUND: Current methodologies used to determine the progression of hepatic fibrosis rely heavily on liver biopsy, a dangerous and invasive procedure, with semi-subjective analysis of the results of the biopsy. Thus, a new approach is immensely needed for monitoring the progression of liver fibrosis in Hepatitis C virus (HCV) patients. AIM OF WORK: The purpose of this study was to find highly specific and sensitive miRNA biomarkers that can be used to detect different stages of liver fibrosis. METHODOLOGY: The study consisted of 42 cases of chronic hepatitis C (CHC) with early-stage fibrosis, 45 cases of CHC with late-stage fibrosis, and 40 healthy subjects with no CHC or fibrosis as controls. Expression patterns of 5 miRNAs (miR-16, miR-146a, miR-214-5p, miR-221, and miR-222) were analyzed in each group using TaqMan real-time PCR. RESULTS: Serum levels of miRNA-16, miRNA-146a, miRNA-221, and miRNA-222 were all significantly up-regulated in early and late stages of liver fibrosis. miRNA-222 had the highest sensitivity and specificity values in early and late fibrosis. miRNA-221 had the second highest sensitivity and specificity with the late-stage fibrosis group. Furthermore, miRNA-221 showed significant positive correlations with both miRNA-16 and miRNA-146a in the early- and late-stage fibrosis groups, with the early stage having a stronger correlation. CONCLUSIONS: The results indicated that miRNA-16, miRNA-146a, miRNA-221, and miRNA-222 can be used to detect the presence of liver fibrosis. The high sensitivity and specificity of miRNA-222 and miRNA-221 in late-stage fibrosis indicate promising prognostic biomarkers for HCV-induced liver fibrosis.

Expression analysis of liver-specific circulating microRNAs in HCV-induced hepatocellular Carcinoma in Egyptian patients.

OBJECTIVES: Due to the absence of reliable and accurate biomarkers for the early detection of liver malignancy, circulating microRNAs have recently emerged as great candidates for prompt cancer identification. Therefore, the aim of this study was to investigate the potential of liver-specific circulating microRNAs as an accurate non-invasive diagnostic tool for early diagnosis of hepatitis C virus (HCV)-induced hepatocellular carcinoma (HCC). METHODOLOGY: A total of 165 patients were enrolled in this study and categorized into four main groups: 42 chronic hepatitis C (CHC) without cirrhosis, 45 CHC with cirrhosis (LC), 38 HCC with HCV patients, and 40 healthy controls. The expression profiles of seven miRNAs (miR-16, miR-34a, miR-125a, miR-139, miR-145, miR-199a, and miR-221) were analyzed using real-time PCR. RESULTS: Serum levels of miRNA-125a, miRNA-139, miRNA-145, and miRNA199a were significantly lower (p < 0.01) in HCC than in both CHC and LC groups. On the other hand, no significant difference was shown in the expression of miR-16, miR-34a, and miR-221 between the CHC, LC, and HCC groups. MiR-16, miR-34a, and miR-221 were significantly elevated in the HCC group compared to the control group. MiR-34a showed the highest specificity and sensitivity. CONCLUSIONS: The results indicated that the measurement of serum levels of miR-125a, miR-139, miR-145, and miR-199a can help to differentiate HCC from CHC and LC. Also, miR-16, miR-34a, and miR-221 serum levels would have a prognostic value. MiR-34a had the highest specificity and sensitivity, indicating that it might serve as a novel and potential non-invasive biomarker for HCV-induced HCC.

The Egyptian Red Sea coastal microbiome: A study revealing differential microbial responses to diverse anthropogenic pollutants.

The Red Sea is considered one of the youngest oceanic systems, with unique physical, geochemical and biological characteristics. Tourism, industrialization, extensive fishing, oil processing and shipping are extensive sources of pollution in the Red Sea. We analyzed the geochemical characteristics and microbial community of sediments along the Egyptian coast of the Red Sea. Our sites mainly included 1) four ports used for shipping aluminum, ilmenite and phosphate; 2) a site previously reported to have suffered extensive oil spills; and 3) a site impacted by tourism. Two major datasets for the sediment of ten Red Sea coastal sites were generated; i) a chemical dataset included measurements of carbon, hydrogen, nitrogen and sulfur, metals and selected semi-volatile oil; and ii) a 16S rRNA Pyrotags bacterial metagenomic dataset. Based on the taxonomic assignments of the 16S rRNA Pyrotags to major bacterial groups, we report 30 taxa constituting an Egyptian Red Sea Coastal Microbiome. Bacteria that degrade hydrocarbons were predominant in the majority of the sites, particularly in two ports where they reached up to 76% of the total identified genera. In contrast, sulfate-reducing and sulfate-oxidizing bacteria dominated two lakes at the expense of other hydrocarbon metabolizers. Despite the reported "Egyptian Red Sea Coastal Microbiome," sites with similar anthropogenic pollutants showed unique microbial community abundances. This suggests that the abundance of a specific bacterial community is an evolutionary mechanism induced in response to selected anthropogenic pollutants.

A localized PCR inhibitor in a porcelain crab suggests a protective role.

A number of polymerase chain reaction (PCR) inhibitors have been identified from biological and environmental samples. By and large, such substances are treated as random nuisances and contaminants with alternate functions; their inhibitory effects on DNA replication being a coincidental property of their molecular structure. Here, we demonstrate the presence of a localized PCR inhibitor in the foregut of the porcelain crab Petrolisthes rufescens (Anomura: Porcellanidae) from the Red Sea. The inhibitor precluded amplification of 28s, 16s and 18s gene sequences effectively but lost activity at 10(-2) dilutions from initial concentration. Heat treatment was ineffective in arresting inhibition and spectrophotometric techniques suggested that the inhibitor was not a melanin-type compound. The compound was not detected from midgut, hindgut, or gills of the crab. Activity of the inhibitor was precluded when samples were treated with suspensions from the midgut, suggesting that enzymatic degradation of the inhibitor likely happens at that part of the gut. As many microbial pathogens invade their hosts via ingestion, we suggest the presence of the localized inhibitor could carry a defensive or immunological role for P. rufescens. The identity of the inhibitory molecule remains unknown.

Aerobic methanotrophic communities at the Red Sea brine-seawater interface.

The central rift of the Red Sea contains 25 brine pools with different physicochemical conditions, dictating the diversity and abundance of the microbial community. Three of these pools, the Atlantis II, Kebrit and Discovery Deeps, are uniquely characterized by a high concentration of hydrocarbons. The brine-seawater interface, described as an anoxic-oxic (brine-seawater) boundary, is characterized by a high methane concentration, thus favoring aerobic methane oxidation. The current study analyzed the aerobic free-living methane-oxidizing bacterial communities that potentially contribute to methane oxidation at the brine-seawater interfaces of the three aforementioned brine pools, using metagenomic pyrosequencing, 16S rRNA pyrotags and pmoA library constructs. The sequencing of 16S rRNA pyrotags revealed that these interfaces are characterized by high microbial community diversity. Signatures of aerobic methane-oxidizing bacteria were detected in the Atlantis II Interface (ATII-I) and the Kebrit Deep Upper (KB-U) and Lower (KB-L) brine-seawater interfaces. Through phylogenetic analysis of pmoA, we further demonstrated that the ATII-I aerobic methanotroph community is highly diverse. We propose four ATII-I pmoA clusters. Most importantly, cluster 2 groups with marine methane seep methanotrophs, and cluster 4 represent a unique lineage of an uncultured bacterium with divergent alkane monooxygenases. Moreover, non-metric multidimensional scaling (NMDS) based on the ordination of putative enzymes involved in methane metabolism showed that the Kebrit interface layers were distinct from the ATII-I and DD-I brine-seawater interfaces.

Core microbial functional activities in ocean environments revealed by global metagenomic profiling analyses.

Metagenomics-based functional profiling analysis is an effective means of gaining deeper insight into the composition of marine microbial populations and developing a better understanding of the interplay between the functional genome content of microbial communities and abiotic factors. Here we present a comprehensive analysis of 24 datasets covering surface and depth-related environments at 11 sites around the world's oceans. The complete datasets comprises approximately 12 million sequences, totaling 5,358 Mb. Based on profiling patterns of Clusters of Orthologous Groups (COGs) of proteins, a core set of reference photic and aphotic depth-related COGs, and a collection of COGs that are associated with extreme oxygen limitation were defined. Their inferred functions were utilized as indicators to characterize the distribution of light- and oxygen-related biological activities in marine environments. The results reveal that, while light level in the water column is a major determinant of phenotypic adaptation in marine microorganisms, oxygen concentration in the aphotic zone has a significant impact only in extremely hypoxic waters. Phylogenetic profiling of the reference photic/aphotic gene sets revealed a greater variety of source organisms in the aphotic zone, although the majority of individual photic and aphotic depth-related COGs are assigned to the same taxa across the different sites. This increase in phylogenetic and functional diversity of the core aphotic related COGs most probably reflects selection for the utilization of a broad range of alternate energy sources in the absence of light.

A novel mercuric reductase from the unique deep brine environment of Atlantis II in the Red Sea.

A unique combination of physicochemical conditions prevails in the lower convective layer (LCL) of the brine pool at Atlantis II (ATII) Deep in the Red Sea. With a maximum depth of over 2000 m, the pool is characterized by acidic pH (5.3), high temperature (68 °C), salinity (26%), low light levels, anoxia, and high concentrations of heavy metals. We have established a metagenomic dataset derived from the microbial community in the LCL, and here we describe a gene for a novel mercuric reductase, a key component of the bacterial detoxification system for mercuric and organomercurial species. The metagenome-derived gene and an ortholog from an uncultured soil bacterium were synthesized and expressed in Escherichia coli. The properties of their products show that, in contrast to the soil enzyme, the ATII-LCL mercuric reductase is functional in high salt, stable at high temperatures, resistant to high concentrations of Hg(2+), and efficiently detoxifies Hg(2+) in vivo. Interestingly, despite the marked functional differences between the orthologs, their amino acid sequences differ by less than 10%. Site-directed mutagenesis and kinetic analysis of the mutant enzymes, in conjunction with three-dimensional modeling, have identified distinct structural features that contribute to extreme halophilicity, thermostability, and high detoxification capacity, suggesting that these were acquired independently during the evolution of this enzyme. Thus, our work provides fundamental structural insights into a novel protein that has undergone multiple biochemical and biophysical adaptations to promote the survival of microorganisms that reside in the extremely demanding environment of the ATII-LCL.

Isolation and characterization of a heavy metal-resistant, thermophilic esterase from a Red Sea brine pool.

The Red Sea Atlantis II brine pool is an extreme environment that displays multiple harsh conditions such as high temperature, high salinity and high concentrations of multiple, toxic heavy metals. The survival of microbes in such an environment by utilizing resistant enzymes makes them an excellent source of extremophilic enzymes. We constructed a fosmid metagenomic library using DNA isolated from the deepest and most secluded layer of this pool. We report the isolation and biochemical characterization of an unusual esterase: EstATII. EstATII is thermophilic (optimum temperature, 65°C), halotolerant (maintains its activity in up to 4.5 M NaCl) and maintains at least 60% of its activity in the presence of a wide spectrum of heavy metals. The combination of biochemical characteristics of the Red Sea Atlantis II brine pool esterase, i.e., halotolerance, thermophilicity and resistance to heavy metals, makes it a potentially useful biocatalyst.