Developing Wolbachia-based disease interventions for an extreme environment.

Aedes aegypti mosquitoes carrying self-spreading, virus-blocking Wolbachia bacteria are being deployed to suppress dengue transmission. However, there are challenges in applying this technology in extreme environments. We introduced two Wolbachia strains into Ae. aegypti from Saudi Arabia for a release program in the hot coastal city of Jeddah. Wolbachia reduced infection and dissemination of dengue virus (DENV2) in Saudi Arabian mosquitoes and showed complete maternal transmission and cytoplasmic incompatibility. Wolbachia reduced egg hatch under a range of environmental conditions, with the Wolbachia strains showing differential thermal stability. Wolbachia effects were similar across mosquito genetic backgrounds but we found evidence of local adaptation, with Saudi Arabian mosquitoes having lower egg viability but higher adult desiccation tolerance than Australian mosquitoes. Genetic background effects will influence Wolbachia invasion dynamics, reinforcing the need to use local genotypes for mosquito release programs, particularly in extreme environments like Jeddah. Our comprehensive characterization of Wolbachia strains provides a foundation for Wolbachia-based disease interventions in harsh climates.

Close Kin Dyads Indicate Intergenerational Dispersal and Barriers.

AbstractThe movement of individuals through continuous space is typically constrained by dispersal ability and dispersal barriers. A range of approaches have been developed to investigate these. Kindisperse is a new approach that infers recent intergenerational dispersal (σ) from close kin dyads and appears particularly useful for investigating taxa that are difficult to observe individually. This study, focusing on the mosquito Aedes aegypti, shows how the same close kin data can also be used for barrier detection. We empirically demonstrate this new extension of the method using genome-wide sequence data from 266 Ae. aegypti. First, we use the spatial distribution of full-sib dyads collected within one generation to infer past movements of ovipositing female mosquitoes. These dyads indicated the relative barrier strengths of two roads and performed favorably against alternative genetic methods for detecting barriers. We then use Kindisperse to quantify recent intergenerational dispersal (σ=81.5-197.1 m generation-1/2) from the difference in variance between the sib and the first cousin spatial distributions and, from this, estimate effective population density (ρ=833-4,864 km-2). Dispersal estimates showed general agreement with those from mark-release-recapture studies. Barriers, σ, ρ, and neighborhood size (331-526) can inform forthcoming releases of dengue-suppressing Wolbachia bacteria into this mosquito population.

Spatio-Temporal Modelling Informing Wolbachia Replacement Releases in a Low Rainfall Climate.

Releases of Aedes aegypti carrying Wolbachia bacteria are known to suppress arbovirus transmission and reduce the incidence of vector-borne diseases. In planning for Wolbachia releases in the arid environment of Jeddah, Saudi Arabia, we collected entomological data with ovitraps across a 7-month period in four locations. Herein, we show that mosquito presence in basements does not differ from that of non-basement areas of buildings. In modelling mosquito presence across the study sites, we found the spatial structure to be statistically significant in one of the four sites, while a significant spatial structure was found for egg production data across three of the four sites. The length scales of the spatial covariance functions fitted to the egg production data ranged from 143 m to 574 m, indicating that high productivity regions can be extensive in size. Rank-correlation analyses indicated that mosquito presence tended to persist from the dry to wet season, but that egg production ranks at locations could reverse. The data suggest that, in Jeddah, the quality of the local environment for breeding can vary over time. The data support the feasibility of dry season releases but with release numbers needing to be flexible depending on local rates of invasion.

Comparative analysis of transposable elements provides insights into genome evolution in the genus Camelus.

BACKGROUND: Transposable elements (TEs) are common features in eukaryotic genomes that are known to affect genome evolution critically and to play roles in gene regulation. Vertebrate genomes are dominated by TEs, which can reach copy numbers in the hundreds of thousands. To date, details regarding the presence and characteristics of TEs in camelid genomes have not been made available. RESULTS: We conducted a genome-wide comparative analysis of camelid TEs, focusing on the identification of TEs and elucidation of transposition histories in four species: Camelus dromedarius, C. bactrianus, C. ferus, and Vicugna pacos. Our TE library was created using both de novo structure-based and homology-based searching strategies ( https://github.com/kacst-bioinfo-lab/TE_ideintification_pipeline ). Annotation results indicated a similar proportion of each genomes comprising TEs (35-36%). Class I LTR retrotransposons comprised 16-20% of genomes, and mostly consisted of the endogenous retroviruses (ERVs) groups ERVL, ERVL-MaLR, ERV_classI, and ERV_classII. Non-LTR elements comprised about 12% of genomes and consisted of SINEs (MIRs) and the LINE superfamilies LINE1, LINE2, L3/CR1, and RTE clades. Least represented were the Class II DNA transposons (2%), consisting of hAT-Charlie, TcMar-Tigger, and Helitron elements and comprising about 1-2% of each genome. CONCLUSIONS: The findings of the present study revealed that the distribution of transposable elements across camelid genomes is approximately similar. This investigation presents a characterization of TE content in four camelid to contribute to developing a better understanding of camelid genome architecture and evolution.

Complete mitochondrial genome of the longhorn date palm stem borer Jebusaea hammerschmidtii (Reiche, 1878).

The 15,619 bp mitochondrial genome of Jebusaea hammerschmidtii was assembled from short reads, annotated, and compared to the genomes of other longhorn beetles (Cerambycidae). Gene content was typical of animal mitochondrial genomes and contained 13 protein-coding, 22 tRNA, and 2 rRNA genes. Gene organization was identical to that of other longhorn beetles. Phylogenetic analysis placed J. hammerschmidtii within the subfamily Cerambycinae, and strongly supported the monophyly of the Cerambycinae, Lamiinae, and Prioninae subfamilies.

Targeting allosteric sites of human aromatase: a comprehensive in-silico and in-vitro workflow to find potential plant-based anti-breast cancer therapeutics.

Recent findings suggested several allosteric pockets on human aromatase that could be utilised for the development of new modulators able to inhibit this enzyme in a new mechanism. Herein, we applied an integrated in-silico-based approach supported by in-vitro enzyme-based and cell-based validation assays to select the best leads able to target these allosteric binding sites from a small library of plant-derived natural products. Chrysin, apigenin, and resveratrol were found to be the best inhibitors targeting the enzyme's substrate access channel and were able to produce a competitive inhibition with IC50 values ranged from 1.7 to 15.8 µM. Moreover, they showed a more potent antiproliferative effect against ER+ (MCF-7) than ER- one (MDA-MB-231) cell lines. On the other hand, both pomiferin and berberine were the best hits for the enzyme's haem-proximal cavity producing a non-competitive inhibition (IC50 15.1 and 21.4 µM, respectively) and showed selective antiproliferative activity towards MCF-7 cell lines.

Haplotype-resolved genome assembly enables gene discovery in the red palm weevil Rhynchophorus ferrugineus.

The red palm weevil Rhynchophorus ferrugineus (Coleoptera: Curculionidae) is an economically-important invasive species that attacks multiple species of palm trees around the world. A better understanding of gene content and function in R. ferrugineus has the potential to inform pest control strategies and thereby mitigate economic and biodiversity losses caused by this species. Using 10x Genomics linked-read sequencing, we produced a haplotype-resolved diploid genome assembly for R. ferrugineus from a single heterozygous individual with modest sequencing coverage ([Formula: see text] 62x). Benchmarking against conserved single-copy Arthropod orthologs suggests both pseudo-haplotypes in our R. ferrugineus genome assembly are highly complete with respect to gene content, and do not suffer from haplotype-induced duplication artifacts present in a recently published hybrid assembly for this species. Annotation of the larger pseudo-haplotype in our assembly provides evidence for 23,413 protein-coding loci in R. ferrugineus, including over 13,000 predicted proteins annotated with Gene Ontology terms and over 6000 loci independently supported by high-quality Iso-Seq transcriptomic data. Our assembly also includes 95% of R. ferrugineus chemosensory, detoxification and neuropeptide-related transcripts identified previously using RNA-seq transcriptomic data, and provides a platform for the molecular analysis of these and other functionally-relevant genes that can help guide management of this widespread insect pest.

Metabolically engineered recombinant Saccharomyces cerevisiae for the production of 2-Deoxy-scyllo-inosose (2-DOI).

Saccharomyces cerevisiae is a versatile industrial host for chemical production and has been engineered to produce efficiently many valuable compounds. 2-Deoxy-scyllo-inosose (2-DOI) is an important precursor for the biosynthesis of 2-deoxystreptamine-containing aminoglycosides antibiotics and benzenoid metabolites. Bacterial and cyanobacterial strains have been metabolically engineered to generate 2-DOI; nevertheless, the production of 2-DOI using a yeast host has not been reported. Here, we have metabolically engineered a series of CEN.PK yeast strains to produce 2-DOI using a synthetically yeast codon-optimized btrC gene from Bacillus circulans. The expression of the 2-Deoxy-scyllo-inosose synthase (2-DOIS) gene was successfully achieved via an expression vector and through chromosomal integration at a high-expression locus. In addition, the production of 2-DOI was further investigated for the CEN.PK knockout strains of phosphoglucose isomerase (Δpgi1), D-glucose-6-phosphate dehydrogenase (Δzwf1) and a double mutant (Δpgi1, Δzwf1) in a medium consisting of 2% fructose and 0.05% glucose as a carbon source. We have found that all the recombinant strains are capable of producing 2-DOI and reducing it into scyllo-quercitol and (-)-vibo-quercitol. Comparatively, the high production of 2-DOI and its analogs was observed for the recombinant CEN.PK-btrC carrying the multicopy btrC-expression vector. GC/MS analysis of culture filtrates of this strain showed 11 times higher response in EIC for the m/z 479 (methyloxime-tetra-TMS derivative of 2-DOI) than the YP-btrC recombinant that has only a single copy of btrC expression cassette integrated into the genomic DNA of the CEN.PK strain. The knockout strains namely Δpgi1-btrC and Δpgi1Δzwf1-btrC, that are transformed with the btrC-expression plasmids, have inactive Pgi1 and produced only traces of the compounds. In contrast, Δzwf1-btrC recombinant which has intact pgi1 yielded relatively higher amount of the carbocyclic compounds. Additionally, 1H-NMR analysis of samples showed slow consumption of fructose and no accumulation of 2-DOI and the quercitols in the culture broth of the recombinant CEN.PK-btrC suggesting that S. cerevisiae is capable of assimilating 2-DOI.

Genomic Analysis of Intrinsically Disordered Proteins in the Genus Camelus.

Intrinsically disordered proteins/regions (IDPs/IDRs) fail to fold completely into 3D structures, but have major roles in determining protein function. While natively disordered proteins/regions have been found to fulfill a wide variety of primary cellular roles, the functions of many disordered proteins in numerous species remain to be uncovered. Here, we perform the first large-scale study of IDPs/IDRs in the genus Camelus, one of the most important mammalians in Asia and North Africa, in order to explore the biological roles of these proteins. The study includes the prediction of disordered proteins/regions in Camelus species and in humans using multiple state-of-the-art prediction tools. Additionally, we provide a comparative analysis of Camelus and Homo sapiens IDPs/IDRs for the sake of highlighting the distinctive use of disorder in each genus. Our findings indicate that the human proteome is more disordered than the Camelus proteome. Gene Ontology analysis also revealed that Camelus IDPs are enriched in glutathione catabolism and lactose biosynthesis.

Genomic Sequencing and Analysis of Eight Camel-Derived Middle East Respiratory Syndrome Coronavirus (MERS-CoV) Isolates in Saudi Arabia.

Middle East respiratory syndrome coronavirus (MERS-CoV) causes severe respiratory illness in humans; the second-largest and most deadly outbreak to date occurred in Saudi Arabia. The dromedary camel is considered a possible host of the virus and also to act as a reservoir, transmitting the virus to humans. Here, we studied evolutionary relationships for 31 complete genomes of betacoronaviruses, including eight newly sequenced MERS-CoV genomes isolated from dromedary camels in Saudi Arabia. Through bioinformatics tools, we also used available sequences and 3D structure of MERS-CoV spike glycoprotein to predict MERS-CoV epitopes and assess antibody binding affinity. Phylogenetic analysis showed the eight new sequences have close relationships with existing strains detected in camels and humans in Arabian Gulf countries. The 2019-nCov strain appears to have higher homology to both bat coronavirus and SARS-CoV than to MERS-CoV strains. The spike protein tree exhibited clustering of MERS-CoV sequences similar to the complete genome tree, except for one sequence from Qatar (KF961222). B cell epitope analysis determined that the MERS-CoV spike protein has 24 total discontinuous regions from which just six epitopes were selected with score values of >80%. Our results suggest that the virus circulates by way of camels crossing the borders of Arabian Gulf countries. This study contributes to finding more effective vaccines in order to provide long-term protection against MERS-CoV and identifying neutralizing antibodies.

Genome-wide characterization of simple sequence repeats in Palmae genomes.

BACKGROUND: Microsatellites or simple sequence repeats (SSRs) have become the most significant DNA marker technology used in genetic research. The availability of complete draft genomes for a number of Palmae species has made it possible to perform genome-wide analysis of SSRs in these species. Palm trees are tropical and subtropical plants with agricultural and economic importance due to the nutritional value of their fruit cultivars. OBJECTIVE: This is the first comprehensive study examining and comparing microsatellites in completely-sequenced draft genomes of Palmae species. METHODS: We identified and compared perfect SSRs with 1-6 bp nucleotide motifs to characterize microsatellites in Palmae species using PERF v0.2.5. We analyzed their relative abundance, relative density, and GC content in five palm species: Phoenix dactylifera, Cocos nucifera, Calamus simplicifolius, Elaeis oleifera, and Elaeis guineensis. RESULTS: A total of 118241, 328189, 450753, 176608, and 70694 SSRs were identified, respectively. The six repeat types were not evenly distributed across the five genomes. Mono- and dinucleotide SSRs were the most abundant, and GC content was highest in tri- and hexanucleotide SSRs. CONCLUSION: We envisage that this analysis would further substantiate more in-depth computational, biochemical, and molecular studies on the roles SSRs may play in the genome organization of the palm species. The current study contributes a detailed characterization of simple sequence repeats in palm genomes.

Comparative analysis of camelid mitochondrial genomes.

Camelus dromedarius has played a pivotal role in both culture and way of life in the Arabian peninsula, particularly in arid regions where other domestic animals cannot be easily domesticated. Although, the mitochondrial genomes have recently been sequenced for several camelid species, wider phylogenetic studies are yet to be performed. The features of conserved gene elements, rapid evolutionary rate, and rare recombination make the mitochondrial genome a useful molecular marker for phylogenetic studies of closely related species. Here we carried out a comparative analysis of previously sequenced mitochondrial genomes of camelids with an emphasis on C. dromedarius, revealing a number of noticeable findings. First, the arrangement of mitochondrial genes in C. dromedarius is similar to those of the other camelids. Second, multiple sequence alignment of intergenic regions shows up to 90% similarity across different kinds of camels, with dromedary camels to reach 99%. Third, we successfully identified the three domains (termination-associated sequence, conserved domain and conserved sequence block) of the control region structure. The phylogenetic tree analysis showed that C. dromedarius mitogenomes were significantly clustered in the same clade with Lama pacos mitogenome. These findings will enhance our understanding of the nucleotide composition and molecular evolution of the mitogenomes of the genus Camelus, and provide more data for comparative mitogenomics in the family Camelidae.

Effect of RNA Interference on the Hepatitis C Virus Core Expression in HepG2/C3A Cells Using Genotype 4 Isolates from Saudi Patients.

OBJECTIVE: To explore the effects of two different methodologies of RNA interference, namely small interfering RNA, and vector-based short hairpin RNA, on the expression levels of hepatitis C virus core RNA and protein of Saudi genotype 4 isolates. STUDY DESIGN: An experimental study. PLACE AND DURATION OF STUDY: Laboratories of the College of Medicine Research Center, King Saud University, Saudi Arabia, from January to December 2018. METHODOLOGY: Hepatitis C virus core small interfering RNA molecule and short hairpin RNA vector were designed against core region. Viral RNA expression was tested by RT-PCR; whereas, core protein was tested by flow cytometry and immunofluorescence. Results were statistically analysed by Chi-square analysis to calculate the p-value. RESULTS: Both molecules caused a reduction in core RNA and protein expression in infected cells. The effect of 100-pmol of small interfering RNA was more evident. For the vector-based short hairpin RNA, inhibition of core RNA expression was quite evident after 96 hours (p = 0.007). The results of flow cytometry and immunofluorescence showed a decline in core protein expression. The most dramatic effect was observed with 100-pmol small interfering RNA treatment of cells for 24 and 48 hours, which resulted in 63.5% and 91.1% core RNA expression reduction, respectively. CONCLUSION: RNA interference of hepatitis C virus core gene efficiently stopped viral replication and offer a promising therapeutic alternative against virus infection.

Neuronal growth and differentiation on biodegradable membranes.

Semipermeable polymeric membranes with appropriate morphological, physicochemical and transport properties are relevant to inducing neural regeneration. We developed novel biodegradable membranes to support neuronal differentiation. In particular, we developed chitosan, polycaprolactone and polyurethane flat membranes and a biosynthetic blend between polycaprolactone and polyurethane by phase-inversion techniques. The biodegradable membranes were characterized in order to evaluate their morphological, physicochemical, mechanical and degradation properties. We investigated the efficacy of these different membranes to promote the adhesion and differentiation of neuronal cells. We employed as model cell system the human neuroblastoma cell line SHSY5Y, which is a well-established system for studying neuronal differentiation. The investigation of viability and specific neuronal marker expression allowed assessment that the correct neuronal differentiation and the formation of neuronal network had taken place in vitro in the cells seeded on different biodegradable membranes. Overall, this study provides evidence that neural cell responses depend on the nature of the biodegradable polymer used to form the membranes, as well as on the dissolution, hydrophilic and, above all, mechanical membrane properties. PCL-PU membranes exhibit mechanical properties that improve neurite outgrowth and the expression of specific neuronal markers.

Neuroprotective effect of didymin on hydrogen peroxide-induced injury in the neuronal membrane system.

In this study, the flavonoid didymin was administered in vitro in neuronal cells after hydrogen peroxide (H2O2)-induced injury (neurorescue) in order to investigate the effects of this natural molecule on cell damage in a neuronal membrane system. The results showed the effects of didymin in neuronal cells by using a polycaprolactone biodegradable membrane system as an in vitro model. Two major findings are presented in this study: first is the antioxidant property of didymin and, second, for the first time we provide evidence concerning its ability to rescue neuronal cells from oxidative damage. Didymin showed radical scavenging activities and it protected the neuronal cells against H2O2-induced neurotoxicity. Didymin increased cell viability, decreased intracellular reactive oxygen species generation, stimulated superoxide dismutase, catalase and glutathione peroxidase activity in neuronal cells which were previously insulted with H2O2. In addition, didymin strikingly inhibited H2O2-induced mitochondrial dysfunctions in terms of reduction of mitochondria membrane potential and the activation of cleaved caspase-3, and also decreased dramatically the H2O2-induced phosphorylation of c-Jun N-terminal kinase. Therefore, this molecule is capable of inducing recovery from oxidative damage, and promoting and/or restoring normal cellular conditions. Moreover, the mechanism underlying the protective effects of didymin in H2O2-injured neuronal cells might be related to the activation of antioxidant defense enzymes as well as to the inhibition of apoptotic features, such as p-JNK and caspase-3 activation. These data suggest that didymin may be a potential therapeutic molecule for the treatment of neurodegenerative disorders associated with oxidative stress.

Camelid genomes reveal evolution and adaptation to desert environments.

Bactrian camel (Camelus bactrianus), dromedary (Camelus dromedarius) and alpaca (Vicugna pacos) are economically important livestock. Although the Bactrian camel and dromedary are large, typically arid-desert-adapted mammals, alpacas are adapted to plateaus. Here we present high-quality genome sequences of these three species. Our analysis reveals the demographic history of these species since the Tortonian Stage of the Miocene and uncovers a striking correlation between large fluctuations in population size and geological time boundaries. Comparative genomic analysis reveals complex features related to desert adaptations, including fat and water metabolism, stress responses to heat, aridity, intense ultraviolet radiation and choking dust. Transcriptomic analysis of Bactrian camels further reveals unique osmoregulation, osmoprotection and compensatory mechanisms for water reservation underpinned by high blood glucose levels. We hypothesize that these physiological mechanisms represent kidney evolutionary adaptations to the desert environment. This study advances our understanding of camelid evolution and the adaptation of camels to arid-desert environments.

Alternative promoters regulate cold inducible RNA-binding (CIRP) gene expression and enhance transgene expression in mammalian cells.

The use of a temperature shift cultivation to enhance recombinant protein yield is widely utilised in the bioprocessing industry. The responses of mammalian cells to heat stress are well characterized; however, the equivalent cold stress responses are not. In particular, the transcriptional mechanisms that lead to enhanced gene-specific expression upon cold stress have yet to be elucidated. We report here in silico and experimental identification and characterization of transcriptional control elements that regulate cold inducible RNA-binding (CIRP) gene expression and demonstrate these can be used for enhanced transgene expression. In silico analysis identified the core CIRP promoter and a number of conserved transcription factor-binding sites across mammalian species. The core promoter was confirmed by experimental studies that located the basal transcriptional regulatory elements of CIRP within 264 nucleotides upstream of the transcription start site. Deletion analysis of a fragment from -264 to -64 that contained two putative CAAT-binding sites abolished promoter activity. A second promoter was identified in the region -452 to -264 of the transcription start site which was able to drive transcription independent of the core promoter. As the two CIRP promoters were transcriptionally active and possibly cold responsive, we used electrophoretic mobility shift assays to show that both promoter regions are able to bind factors within a nuclear extract in a dose-dependent manner and that the formation of these complexes was specific to the promoter regions. Finally, we successfully demonstrate using a reporter gene approach that enhanced transgene expression can be achieved using the identified CIRP promoter.

Post-translational events of a model reporter protein proceed with higher fidelity and accuracy upon mild hypothermic culturing of Chinese hamster ovary cells.

Chinese hamster ovary cells (CHO) are routinely used in industry to produce recombinant therapeutic proteins and a number of studies have reported increased recombinant mRNA levels at temperatures <37 degrees C. Surprisingly, the effect of reduced temperature on mRNA translation in CHO cells has not been investigated despite this process being highly responsive to environmental stresses. The relationship between low temperature culturing of CHO cells and mRNA translation was therefore investigated using labeling studies and dual luciferase reporter gene technology. Global protein synthetic capacity was not greatly affected at 32 degrees C but was diminished at lower temperatures. The expression of both cap-dependent and cap-independent (IRES driven) mRNA translated luciferase reporter gene activity was highest at 32 degrees C on a per cell basis and this was partially accounted for by increased mRNA levels. Importantly, post-translational events appear to proceed with higher fidelity and accuracy at 32 than 37 degrees C resulting in increased yield of active protein as opposed to an increase in total polypeptide synthesis. Therefore at 32 degrees C recombinant cap-dependent mRNA translation appears sufficient to maintain recombinant protein yields on a per cell basis and this is associated with improved post-translational processing.