Rapid and Reliable Detection of SARS-CoV-2 Using Direct RT-LAMP.

Background: The global pandemic coronavirus SARS-CoV-2 has a healthcare, social and economic burden. To limit the spread of the virus, the World Health Organization (WHO) urgently called for extensive screening of suspected individuals; thus, a quick, simple, and sensitive diagnostic assay is always in need. Methods: We applied reverse transcription-loop-mediated isothermal amplification (RT-LAMP) for the detection of SARS-CoV-2. The RT-LAMP method was optimized by evaluating two fluorescence amplification mixes and several reaction times, and results were compared to the standard real-time RT-PCR (rtRT-PCR). The assay was validated using 200 nasopharyngeal swabs collected in viral transport media (62 positive for SARS-CoV-2, and 138 negative for SARS-CoV-2 detected by the rtRT-PCR method). The samples were diluted 1:4 in diethylpyrocarbonate (DEPC)-treated water, utilized for RT-LAMP using different singleplex and multiplex sets of LAMP primers (N gene, S gene, and orf1ab gene), and incubated at 65 °C using real-time PCR 7500. Results: Our direct detection with the RT-LAMP protocol showed 100% concordance (sensitivity and specificity) with the standard protocol used for the detection of SARS-CoV-2 nucleic acid. Conclusions: In this study, we set up a rapid, simple, and sensitive RT-LAMP assay for the detection of SARS-CoV-2 in clinical samples. The assay is suitable for point of care detection in public hospitals, medical centers in rural areas, and in transportation hubs.

The influence of antioxidant dietary-derived polyphenolic combination on breast cancer: Molecular study.

Breast cancer remains a leading cause of female mortality worldwide. Therefore, novel complementary treatments have been sought. Recently, there has been a growing interest in investigating the possible complementary effects of polyphenolic compounds against various malignancies. In the present study, using MCF-7 and MDA-MB-231 human breast adenocarcinoma cells, the anticancer efficacy of a polyphenolic mixture (PFM) was investigated. PFM is composed of curcumin, resveratrol, epigallocatechin gallate, and quercetin. PFM treatment led to a dose-dependent inhibition of cell proliferation, with IC50 values of 25.9 ± 3 µg/ml and 29.4 ± 0.9 µg/ml for MCF-7 and MDA-MB-231 cells, respectively. In addition, PFM induced apoptosis in MDA-MB-231 cells and cell cycle arrest at the S phase in MCF-7 cells. Using RT-qPCR, PFM treatment was observed to result in significant downregulation of the oncogenic miR-155 (P < 0.05), as well as significant downregulation of the rate-limiting glycolytic enzyme, hexokinase 2 (HK2) (P < 0.05), while upregulating the expression of the zinc finger E-box binding homeobox 2 gene (P < 0.01). PFM was also found to exert an anti-migration effect in breast cancer cells using the wound healing assay, as well as significantly (P < 0.05) increasing the median survival of Ehrlich ascites carcinoma (EAC) tumor-bearing mice. These results suggest that PFM possesses potential antitumor effects against breast cancer. A possible mechanism of action could be due to PFM's effect in modulating the expression of the glycolytic enzyme HK2 through suppression of miR-155 in MCF-7 cells. Combining polyphenolic compounds that interact with one another could result in synergistic effects that potentially target various tumour hallmarks.

Analysis of RAS gene mutations in cytogenetically normal de novo acute myeloid leukemia patients reveals some novel alterations.

Rat sarcoma gene (RAS) holds great importance in pathogenesis of acute myeloid leukemia (AML). The activated mutations in Neuroblastoma rat sarcoma viral oncogene homolog (NRAS) and Kirsten rat sarcoma viral oncogene homolog (KRAS) confers proliferative and survival signals, deliberating numerous effects on overall survival and progression free survival in AML patients. In this study thirty one (31) blood samples of adult newly diagnosed AML patients were collected to identify possible incidence of mutations through amplification of KRAS (exon 1 and 2) and NRAS gene (exon 1 and 2) using polymerase chain reaction (PCR). Amplicons were then subjected to sequencing and were analyzed through Geneious Prime 2019. Five of thirty one (16.12%) patients had altered sites in either NRAS or KRAS. The NRAS mutations were observed in three AML patients (N = 3, 9.67%). A novel missense mutation NRAS-I36R (239 T > G) representing a substitution of single nucleotide basepair found in NRAS exon 1 while exon 2 was detected with heterozygous mutation NRAS-E63X (318G > T) and insertion (A), resulting in frameshift of the amino acid sequence and insertion of two nucleotide basepairs (TA) in two of the patients. KRAS mutations (N = 2, 6.45%) were found in exon 1 whereas no mutations in KRAS exon 2 were detected in our patient cohort. Mutation in KRAS Exon 1, KRAS-D30N (280G > A) was observed in two patients and one of them also had a novel heterozygous mutation KRAS-L16N (240G > C). In addition there was no statistically significant association of mutRAS gene of AML patients with several prognostic markers including age, gender, karyotyping, CD34 positivity, cytogenetic abnormalities, total leukocyte count, white blood cell count and French-American-British (FAB) classification. However, the presence of mutRAS gene were strongly associated (p = 0.001) with increased percentage of bone marrow blasts. The prevalence of mutations in correlation with clinical and hematological parameter is useful for risk stratification in AML patients.

Cross-sectional prevalence study of MERS-CoV in local and imported dromedary camels in Saudi Arabia, 2016-2018.

The Middle East Respiratory Syndrome-Coronavirus (MERS-CoV) is an endemic virus in dromedaries. Annually, Saudi Arabia imports thousands of camels from the Horn of Africa, yet the epidemiology of MERS-CoV in these animals is largely unknown. Here, MERS-CoV prevalence was compared in imported African camels and their local counterparts. A total of 1399 paired sera and nasal swabs were collected from camels between 2016 and 2018. Imported animals from Sudan (n = 829) and Djibouti (n = 328) were sampled on incoming ships at Jeddah Islamic seaport before unloading, and local camels were sampled from Jeddah (n = 242). Samples were screened for neutralizing antibodies (nAbs) and MERS-CoV viral RNA. The overall seroprevalence was 92.7% and RNA detection rate was 17.2%. Imported camels had higher seroprevalence compared to resident herds (93.8% vs 87.6%, p <0.01) in contrast to RNA detection (13.3% vs 35.5%, p <0.0001). Seroprevalence significantly increased with age (p<0.0001) and viral RNA detection rate was ~2-folds higher in camels <2-year-old compared to older animals. RNA detection was higher in males verses females (24.3% vs 12.6%, p<0.0001) but seroprevalence was similar. Concurrent positivity for viral RNA and nAbs was found in >87% of the RNA positive animals, increased with age and was sex-dependent. Importantly, reduced viral RNA load was positively correlated with nAb titers. Our data confirm the widespread of MERS-CoV in imported and domestic camels in Saudi Arabia and highlight the need for continuous active surveillance and better prevention measures. Further studies are also warranted to understand camels correlates of protection for proper vaccine development.

Enzootic patterns of Middle East respiratory syndrome coronavirus in imported African and local Arabian dromedary camels: a prospective genomic study.

BACKGROUND: The Middle East respiratory syndrome coronavirus (MERS-CoV) is a lethal zoonotic pathogen endemic to the Arabian Peninsula. Dromedary camels are a likely source of infection and the virus probably originated in Africa. We studied the genetic diversity, geographical structure, infection prevalence, and age-associated prevalence among camels at the largest entry port of camels from Africa into the Arabian Peninsula. METHODS: In this prospective genomic study, we took nasal samples from camels imported from Sudan and Djibouti into the Port of Jeddah in Jeddah, Saudi Arabia, over an almost 2-year period and local Arabian camels over 2 months in the year after surveillance of the port. We determined the prevalence of MERS-CoV infection, age-associated patterns of infection, and undertook phylogeographical and migration analyses to determine intercountry virus transmission after local lineage establishment. We compared all virological characteristics between the local and imported cohorts. We compared major gene deletions between African and Arabian strains of the virus. Reproductive numbers were inferred with Bayesian birth death skyline analyses. FINDINGS: Between Aug 10, 2016, and May 3, 2018, we collected samples from 1196 imported camels, of which 868 originated from Sudan and 328 from Djibouti, and between May 1, and June 25, 2018, we collected samples from 472 local camels, of which 189 were from Riyadh and 283 were from Jeddah, Saudi Arabia. Virus prevalence was higher in local camels than in imported camels (224 [47·5%] of 472 vs 157 [13·1%] of 1196; p<0·0001). Infection prevalence peaked among camels older than 1 year and aged up to 2 years in both groups, with 255 (66·9%) of 381 positive cases in this age group. Although the overall geographical distribution of the virus corresponded with the phylogenetic tree topology, some virus exchange was observed between countries corresponding with trade routes in the region. East and west African strains of the virus appear to be geographically separated, with an origin of west African strains in east Africa. African strains of the virus were not re-sampled in Saudi Arabia despite sampling approximately 1 year after importation from Africa. All local Arabian samples contained strains of the virus that belong to a novel recombinant clade (NRC) first detected in 2014 in Saudi Arabia. Reproduction number estimates informed by the sequences suggest sustained endemicity of NRC, with a mean Re of 1·16. INTERPRETATION: Despite frequent imports of MERS-CoV with camels from Africa, African lineages of MERS-CoV do not establish themselves in Saudi Arabia. Arabian strains of the virus should be tested for changes in virulence and transmissibility. FUNDING: German Ministry of Research and Education, EU Horizon 2020, and Emerging Diseases Clinical Trials Partnership.

Bioactive Diketopiperazines and Nucleoside Derivatives from a Sponge-Derived Streptomyces Species.

Fractionation and purification of the ethyl acetate extract of the culture of a sponge-derived actinomycete, Streptomyces species Call-36, resulted in the isolation and identification of a new diketopiperazine, actinozine A (1), cyclo(2-OH-d-Pro-l-Leu) (2), two new nucleosides, thymidine-3-mercaptocarbamic acid (3) and thymidine-3-thioamine (4), together with cyclo(d-Pro-l-Phe) (5) and cyclo(l-Pro-l-Phe) (6). The structure assignments of the compounds were carried out by interpretation of 1D and 2D NMR data and mass spectral determinations. The absolute configurations of 1 and 2 were determined by Marfey's method and by comparison of the experimental and TDDFT-calculated ECD spectra. Actinozine A possesses an unprecedented hydroperoxy moiety at C-2 of the proline moiety, while 3 and 4 possess unusual mercaptocarbamic acid and thiohydroxylamine functionalities at N-3 of the thymine moiety. The isolated compounds displayed variable cytotoxic and antimicrobial activities.

Floral markers and biological activity of Saudi honey.

The objectives of this research were to identify certain chemical compounds that may be used as fingerprints of Saudi honey and to evaluate their antioxidant and antibacterial activities. Eleven Saudi 'monofloral' honey samples were analyzed and evaluated. Non-phenolic compounds, such as 2,3-dihydro-3,5-dihydroxy-6-methyl-4H-pyran-4-one, methyl 3-hydroxyhexanaote and 5-hydroxymethyl-2-furancarboxaldehyde were present in different types of tested honey samples. Glyceraldehyde was only detected in five of the honey samples tested. The most promising result was the detection of an alkaloid (by using GC-MS) in only two types of Saudi honey samples. This alkaloid may be of great importance and has the potential to be used as a fingerprint marker for the botanical sources of the various honey samples tested. This alkaloid was present in Toran and Saha. The detected compound is 2-amino-4-hydroxypteridine-6-carboxylic acid, which may originate from the degradation of folic acid as identified by previous studies. These findings can be used as a gateway to obtain a fingerprint for these two types of honey samples and can potentially be used to track any impurities in honey sold on the market. All of the tested honey samples showed antioxidant and antibacterial activities. The highly effective activity was in Toran honey against Staphylococcus aureus and Methicillin resistant Staphylococcus aureus (MRSA). Shafalah honey was effective against MRSA and Acinetobacter baumannii which showed bactericidal effects at concentrations 70-100%. This study also examined the antioxidant activity of honey samples using the DPPH assay. DPPH values of tested honey samples varied between 53.93 ±â€¯0.21%, as the highest value and 5.89 ±â€¯0.125%, as the lowest value. Significant correlations between the antibacterial and the antioxidant activities of the tested honey samples were noticed. The corresponding total phenolic contents (TPC) values supported the fact that phenolic compounds enhanced the antibacterial activity. The study revealed that some of the locally produced honey samples, specifically Zaitoon, Shaflah, Saha, Rabea Aja and Bareq contained the monosaccharides called glyceraldehydes which was the precursor to produce methylglyoxal (MGO) compound, which has antibacterial effects as documented in several previous studies. There was no clear relationship between these activities and the sum total of phenolic compounds present in Saudi honey.

Isolation and molecular characterization of mycotoxigenic fungi in agarwood.

Agarwood (Oudh), is often used by people in the Kingdom of Saudi Arabia. The Oudh has been mentioned in the Hadith and is traditionally used for its aroma (perfuming smell) and potential medicinal applications. The aim of the study was to isolate mycotoxigenic fungi that grow on agarwood and the factors and storage conditions that enhance their growth potential. In addition to the detection of associated mycotoxins like: Aflatoxin B1 (AFB1) and ochratoxin A (OTA) from agarwood. Agarwood samples were collected from local markets of Jeddah governorate, Kingdom of Saudi Arabia. Standard dilution plate method was used for the isolation of fungi. Isolated fungi were identified based on morphological characteristics and confirmed using molecular biology techniques. AFB1 and OTA were detected by High Performance Liquid Chromatography (HLPC). The results indicated that the most commonly isolated fungal genera were in the following descending order: Aspergillus, Penicillium, Fusarium and Rhizopus. Among Aspergillus genera, A. flavus and A. ochraceus were detected based on their morphology and confirmed by PCR using specific primers. It was also noted that AFB1 was released by 15.3 and 55.0% of A. flavus and A. parasiticus isolates respectively with levels reaching up to 14.60 µg/L. The moisture content in the samples ranged from 3% to 10% affected fungal growth. AFB1 was detected in 22 out of 50 of the samples. The maximum level of AFB1 (50.7 µg/kg) was detected in samples with higher moisture content (12%) stored at a temperature of 32 °C. Aspergillus fungi were found to be the most predominant fungal genera found on agarwood. Moisture content (9-10%) and storage temperature (32 °C) stimulated fungal growth and their ability to produce mycotoxins. For this reason, storage conditions at the marketing place should be adequate in order not to provide a conducive environment for fungal growth which is associated with the mycotoxin production. In order to prevent fungal growth and mycotoxin production, it would be recommended to store agarwood at temperatures not exceeding 25 °C and moisture content of up to a maximum of 5-6%.

Isolation, Molecular Characterization and Probiotic Potential of Lactic Acid Bacteria in Saudi Raw and Fermented Milk.

Probiotic bacteria can confer health benefits to the human gastrointestinal tract. Lactic acid bacteria (LAB) are candidate probiotic bacteria that are widely distributed in nature and can be used in the food industry. The objective of this study is to isolate and characterize LAB present in raw and fermented milk in Saudi Arabia. Ninety-three suspected LAB were isolated from thirteen different types of raw and fermented milk from indigenous animals in Saudi Arabia. The identification of forty-six selected LAB strains and their genetic relatedness was performed based on 16S rDNA gene sequence comparisons. None of the strains exhibited resistance to clinically relevant antibiotics or had any undesirable hemolytic activity, but they differed in their other probiotic characteristics, that is, tolerance to acidic pH, resistance to bile, and antibacterial activity. In conclusion, the isolates Lactobacillus casei MSJ1, Lactobacillus casei Dwan5, Lactobacillus plantarum EyLan2, and Enterococcus faecium Gail-BawZir8 are most likely the best with probiotic potentials. We speculate that studying the synergistic effects of bacterial combinations might result in a more effective probiotic potential. We suspect that raw and fermented milk products from animals in Saudi Arabia, especially Laban made from camel milk, are rich in LAB and have promising probiotic potential.

Manuka Honey Exerts Antioxidant and Anti-Inflammatory Activities That Promote Healing of Acetic Acid-Induced Gastric Ulcer in Rats.

Gastric ulcers are a major problem worldwide with no effective treatment. The objective of this study was to evaluate the use of manuka honey in the treatment of acetic acid-induced chronic gastric ulcers in rats. Different groups of rats were treated with three different concentrations of honey. Stomachs were checked macroscopically for ulcerative lesions in the glandular mucosa and microscopically for histopathological alterations. Treatment with manuka honey significantly reduced the ulcer index and maintained the glycoprotein content. It also reduced the mucosal myeloperoxidase activity, lipid peroxidation (MDA), and the inflammatory cytokines (TNF-α, IL-1ß, and IL-6) as compared to untreated control group. In addition, honey-treated groups showed significant increase in enzymatic (GPx and SOD) and nonenzymatic (GSH) antioxidants besides levels of the anti-inflammatory cytokine IL-10. Flow cytometry studies showed that treatment of animals with manuka honey has normalized cell cycle distribution and significantly lowered apoptosis in gastric mucosa. In conclusion, the results indicated that manuka honey is effective in the treatment of chronic ulcer and preservation of mucosal glycoproteins. Its effects are due to its antioxidant and anti-inflammatory properties that resulted in a significant reduction of the gastric mucosal MDA, TNF-α, IL-1ß, and IL-6 and caused an elevation in IL-10 levels.

Gut Microbiota: A Contributing Factor to Obesity.

Obesity, a global epidemic of the modern era, is a risk factor for cardiovascular diseases (CVD) and diabetes. The pervasiveness of obesity and overweight in both developed as well as developing populations is on the rise and placing a huge burden on health and economic resources. Consequently, research to control this emerging epidemic is of utmost importance. Recently, host interactions with their resident gut microbiota (GM) have been reported to be involved in the pathogenesis of many metabolic diseases, including obesity, diabetes, and CVD. Around 10(14) microorganisms reside within the lower human intestine and many of these 10(14) microorganisms have developed mutualistic or commensal associations with the host and actively involved in many physiological processes of the host. However, dysbiosis (altered gut microbial composition) with other predisposing genetic and environmental factors, may contribute to host metabolic disorders resulting in many ailments. Therefore, delineating the role of GM as a contributing factor to obesity is the main objective of this review. Obesity research, as a field is expanding rapidly due to major advances in nutrigenomics, metabolomics, RNA silencing, epigenetics, and other disciplines that may result in the emergence of new technologies and methods to better interpret causal relationships between microbiota and obesity.

Bioactive 2(1H)-Pyrazinones and Diketopiperazine Alkaloids from a Tunicate-Derived Actinomycete Streptomyces sp.

As a part of our ongoing effort to allocate marine microbial bioactive leads, a tunicate-derived actinomycete, Streptomyces sp. Did-27, was investigated. Three new 2(1H)-pyrazinones derivatives, (S)-6-(sec-butyl)-3-isopropylpyrazin-2(1H)-one (1), (S)-3-(sec-butyl)-6-isopropylpyrazin-2(1H)-one (2) and (S)-6-(sec-butyl)-3-isobutylpyrazin-2(1H)-one (3), together with the known (1H)-pyrazinones analogues deoxymutaaspergillic acid (4), 3,6-diisobutyl-2(1H)-pyrazinone (5) and 3,6-di-sec-butyl-2(1H)-pyrazinone (6), and the diketopiperazine alkaloids cyclo(6-OH-d-Pro-l-Phe) (7), bacillusamide B (8), cyclo(l-Pro-l-Leu) and cyclo(l-Pro-l-Ile) (10) were isolated from this strain. The structures of the compounds were determined by study of their one- and two-dimensional NMR spectra as well as high-resolution mass spectral determinations. Compound 4 was reported previously as a synthetic product, while compound 6 was reported as 2-hydroxy-3,6-di-sec-butylpyrazine. Herein, we report the complete NMR data for compounds 4 and 6. The compounds were evaluated for their cytotoxic activities against three cell lines. Compound 5 showed potent and selective activity against HCT-116 cell line with IC50 of 1.5 µg/mL, while 1-10 showed variable cytotoxic activities against these cancer cell lines. These results provide further understanding about the chemistry and bioactivities of the alkylated 2(1H)-pyrazinone derivatives.

Nanomaterial Induced Immune Responses and Cytotoxicity.

Nanomaterials are utilized in a wide array of end user products such as pharmaceuticals, electronics, clothes and cosmetic products. Due to its size (< 100 nm), nanoparticles have the propensity to enter through the airway and skin, making its path perilous with the potential to cause damages of varying severity. Once within the body, these particles have unconstrained access to different tissues and organs including the brain, liver, and kidney. As a result, nanomaterials may cause the perturbation of the immune system eliciting an inflammatory response and cytotoxicity. This potential role is dependent on many factors such as the characteristics of the nanomaterials, presence or absence of diseases, and genetic predisposition. Cobalt and nickel nanoparticles, for example, were shown to have inflammogenic properties, while silver nanoparticles were shown to reduce allergic inflammation. Just as asbestos fibers, carbon nanotubes were shown to cause lungs damage. Some nanomaterials were shown, based on animal studies, to result in cell damage, leading to the formation of pre-cancerous lesions. This review highlights the impact of nanomaterials on immune system and its effect on human health with toxicity consideration. It recommends the development of suitable animal models to study the toxicity and bio-clearance of nanomaterials and propose safety guidelines.

Review-Therapeutic implications of Nigella sativa against cancer metastasis.

Nigella sativa (N. sativa), remedial usage against different diseases associated with skeleton, cardiovascular, digestive and urinary systems has a long-standing history. At present, efforts are underway to study its effects against various cancers at both the cellular and molecular levels. In this review, the role of active constituents like thymoquinone (TQ) on different types of cancer has been explored. TQ putative involvement in metastasis has been assessed by elucidating its effects on cell proliferation, adhesion, invasion and angiogenesis. Up regulation of caspase 3, Smac and down regulation of p-AKT, p65, XIAP, Bcl-2, COX-2 is also influenced by N. sativa. These findings prove a significant positive correlation between TQ concentrations and induction of apoptosis, decrease in motility and a reduction in invasion and angiogenesis in cancerous cells. However, there are still quite a few unaddressed domains, which need to be understood. One of these may include target specificity of N. sativa against cancerous tissues, mode of administration, dosage and downstream regulators in mediating these effects. In reference to earlier findings and low cost availability, N. sativa may, also, be suggested as either a suitable sole remedy for cancer or as a complementary to ongoing conventional therapy based extensive and rigorous in vivo optimization and validation.

Antimicrobial activities of chicken ß-defensin (4 and 10) peptides against pathogenic bacteria and fungi.

Host Defense Peptides (HDPs) are small cationic peptides found in several organisms. They play a vital role in innate immunity response and immunomodulatory stimulation. This investigation was designed to study the antimicrobial activities of ß-defensin peptide-4 (sAvBD-4) and 10 (sAvBD-4) derived from chickens against pathogenic organisms including bacteria and fungi. Ten bacterial strains and three fungal species were used in investigation. The results showed that the sAvBD-10 displayed a higher bactericidal potency against all the tested bacterial strains than that of sAvBD-4. The exhibited bactericidal activity was significant against almost the different bacterial strains at different peptide concentrations except for that of Pseudomonas aeruginosa (P. aeruginosa) and Streptococcus bovis (Str. bovis) strains where a moderate effect was noted. Both peptides were effective in the inactivation of fungal species tested yielding a killing rate of up to 95%. The results revealed that the synthetic peptides were resistant to salt at a concentration of 50 mM NaCl. However, they lost antimicrobial potency when applied in the presence of high salt concentrations. Based on blood hemolysis studies, a little hemolytic effect was showed in the case of both peptides even when applied at high concentrations. The data obtained from this study indicated that synthetic avian peptides exhibit strong antibacterial and antifungal activity. In conclusion, future work and research should be tailored to a better understanding of the mechanisms of action of those peptides and their potential use in the pharmaceutical industry to help reduce the incidence and impact of infectious agent and be marketed as a naturally occurring antibiotic.