Search

The spike protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is critical for virus infection through the engagement of the human ACE2 protein1 and is a major antibody target. Here we show that chronic infection with SARS-CoV-2 leads to viral evolution and reduced sensitivity to neutralizing antibodies in an immunosuppressed individual treated with convalescent plasma, by generating whole-genome ultra-deep sequences for 23 time points that span 101 days and using in vitro techniques to characterize the mutations revealed by sequencing. There was little change in the overall structure of the viral population after two courses of remdesivir during the first 57 days. However, after convalescent plasma therapy, we observed large, dynamic shifts in the viral population, with the emergence of a dominant viral strain that contained a substitution (D796H) in the S2 subunit and a deletion (ΔH69/ΔV70) in the S1 N-terminal domain of the spike protein. As passively transferred serum antibodies diminished, viruses with the escape genotype were reduced in frequency, before returning during a final, unsuccessful course of convalescent plasma treatment. In vitro, the spike double mutant bearing both ΔH69/ΔV70 and D796H conferred modestly decreased sensitivity to convalescent plasma, while maintaining infectivity levels that were similar to the wild-type virus.The spike substitution mutant D796H appeared to be the main contributor to the decreased susceptibility to neutralizing antibodies, but this mutation resulted in an infectivity defect. The spike deletion mutant ΔH69/ΔV70 had a twofold higher level of infectivity than wild-type SARS-CoV-2, possibly compensating for the reduced infectivity of the D796H mutation. These data reveal strong selection on SARS-CoV-2 during convalescent plasma therapy, which is associated with the emergence of viral variants that show evidence of reduced susceptibility to neutralizing antibodies in immunosuppressed individuals.

Recent evidence demonstrates that novel protein-coding genes can arise de novo from non-genic loci. This evolutionary innovation is thought to be facilitated by the pervasive translation of non-genic transcripts, which exposes a reservoir of variable polypeptides to natural selection. Here, we systematically characterize how these de novo emerging coding sequences impact fitness in budding yeast. Disruption of emerging sequences is generally inconsequential for fitness in the laboratory and in natural populations. Overexpression of emerging sequences, however, is enriched in adaptive fitness effects compared to overexpression of established genes. We find that adaptive emerging sequences tend to encode putative transmembrane domains, and that thymine-rich intergenic regions harbor a widespread potential to produce transmembrane domains. These findings, together with in-depth examination of the de novo emerging YBR196C-A locus, suggest a novel evolutionary model whereby adaptive transmembrane polypeptides emerge de novo from thymine-rich non-genic regions and subsequently accumulate changes molded by natural selection., There is increasing evidence that protein-coding genes can emerge de novo from noncoding genomic regions. Vakirlis et al. propose that sequences encoding transmembrane polypeptides can emerge de novo in thymine-rich genomic regions and provide organisms with fitness benefits.

Mycobacterial Ser/Thr protein kinases (STPKs) play a critical role in signal transduction pathways that ultimately determine mycobacterial growth and metabolic adaptation. Identification of key physiological substrates of these protein kinases is, therefore, crucial to better understand how Ser/Thr phosphorylation contributes to mycobacterial environmental adaptation, including response to stress, cell division, and host-pathogen interactions. Various substrate detection methods have been employed with limited success, with direct targets of STPKs remaining elusive. Recently developed mass spectrometry (MS)-based phosphoproteomic approaches have expanded the list of potential STPK substrate identifications, yet further investigation is required to define the most functionally significant phosphosites and their physiological importance. Prior to the application of MS workflows, for instance, GarA was the only known and validated physiological substrate for protein kinase G (PknG) from pathogenic mycobacteria. A subsequent list of at least 28 candidate PknG substrates has since been reported with the use of MS-based analyses. Herein, we integrate and critically review MS-generated datasets available on novel STPK substrates and report new functional and subcellular localization enrichment analyses on novel candidate protein kinase A (PknA), protein kinase B (PknB) and PknG substrates to deduce the possible physiological roles of these kinases. In addition, we assess substrate specificity patterns across different mycobacterial STPKs by analyzing reported sets of phosphopeptides, in order to determine whether novel motifs or consensus regions exist for mycobacterial Ser/Thr phosphorylation sites. This review focuses on MS-based techniques employed for STPK substrate identification in mycobacteria, while highlighting the advantages and challenges of the various applications.

The World Health Organization (WHO) estimates that Mycobacterium tuberculosis, the most pathogenic mycobacterium species to humans, has infected up to a quarter of the world's population, with the occurrence of multidrug-resistant strains on the rise. Research into the detailed composition of the cell envelope proteome in mycobacteria over the last 20 years has formed a key part of the efforts to understand host-pathogen interactions and to control the current tuberculosis epidemic. This is due to the great importance of the cell envelope proteome during infection and during the development of antibiotic resistance as well as the search of surface-exposed proteins that could be targeted by therapeutics and vaccines. A variety of experimental approaches and mycobacterial species have been used in proteomic studies thus far. Here we provide for the first time an extensive summary of the different approaches to isolate the mycobacterial cell envelope, highlight some of the limitations of the studies performed thus far, and comment on how the recent advances in membrane proteomics in other fields might be translated into the field of mycobacteria to provide deeper coverage.

Despite the availability of effective drug treatment, Mycobacterium tuberculosis (Mtb), the causative agent of TB disease, kills ~1. 5 million people annually, and the rising prevalence of drug resistance increasingly threatens to worsen this plight. We previously showed that sublethal exposure to the frontline anti-TB drug, rifampicin, resulted in substantial adaptive remodeling of the proteome of the model organism, Mycobacterium smegmatis, in the drug-sensitive mc2155 strain [wild type (WT)]. In this study, we investigate whether these responses are conserved in an engineered, isogenic mutant harboring the clinically relevant S531L rifampicin resistance-conferring mutation (SL) and distinguish the responses that are specific to RNA polymerase β subunit- (RpoB-) binding activity of rifampicin from those that are dependent on the presence of rifampicin alone. We verified the drug resistance status of this strain and observed no phenotypic indications of rifampicin-induced stress upon treatment with the same concentration as used in WT (2.5 μg/ml). Thereafter, we used a cell wall-enrichment strategy to focus attention on the cell wall proteome and observed 253 proteins to be dysregulated in SL bacteria in comparison with 716 proteins in WT. We observed that decreased abundance of ATP-binding cassette (ABC) transporters and increased abundance of ribosomal machinery were conserved in the SL strain, whereas the upregulation of transcriptional machinery and the downregulation of numerous two-component systems were not. We conclude that the drug-resistant M. smegmatis strain displays some of the same proteomic responses observed in WT and suggest that this evidence supports the hypothesis that rifampicin exercises effects beyond RpoB-interaction alone and that mycobacteria recognise rifampicin as a signaling molecule in an RpoB-independent manner at sublethal doses. Taken together, our data indicates mixed RpoB-independent and RpoB-dependent proteomic remodeling in WT mycobacteria, with evidence for RpoB-independent ABC transporter downregulation, but drug activity-based transcriptional upregulation and two-component system downregulation.

We conducted a population genetic analysis of the stalked kelp, Pterygophora californica, in the Santa Barbara Channel, California, USA. The results were compared with previous work on the genetic differentiation of giant kelp, Macrocystis pyrifera, in the same region. These two sympatric kelps not only share many life history and dispersal characteristics but also differ in that dislodged P. californica does not produce floating rafts with buoyant fertile sporophytes, commonly observed for M. pyrifera. We used a comparative population genetic approach with these two species to test the hypothesis that the ability to produce floating rafts increases the genetic connectivity among kelp patches in the Santa Barbara Channel. We quantified the association of habitat continuity and oceanographic distance with the genetic differentiation observed in stalked kelp, like previously conducted for giant kelp. We compared both overall (across all patches) and pairwise (between patches) genetic differentiation. We found that oceanographic transit time, habitat continuity, and geographic distance were all associated with genetic connectivity in P. californica, supporting similar previous findings for M. pyrifera. Controlling for differences in heterozygosity between kelp species using Jost's DEST , we showed that global differentiation and pairwise differentiation were similar among patches between the two kelp species, indicating that they have similar dispersal capabilities despite their differences in rafting ability. These results suggest that rafting sporophytes do not play a significant role in effective dispersal of M. pyrifera at ecologically relevant spatial and temporal scales.

Serotonin (5-HT) plays a regulatory role in coordinating the neural circuits regulating energy balance, with differences in both 5-HT availability at the synapse and the activity of 5-HT receptors mediating anorectic (via POMC/CART activation) and orexigenic (via NPY/AgRP activation) responses. In conditions of overweight and obesity the control of energy balance is clearly deregulated, and serotonergic modulation appears to make a significant contribution to weight gain. Fluoxetine (FLX), a selective serotonin reuptake inhibitor (SSRI) that increases 5-HT availability in the synaptic cleft may thus have potential effects on energy balance. Our aim was to use an overfeeding model to investigate the effects of chronic FLX treatment on energy balance-related parameters regulated by hypothalamic neuropeptides. Nursing male Wistar rats were assigned to normofed (9 pups/dam) or overfed (3 pups/dam) groups beginning at 3 days of age and continuing until 21 days of age, when commercial chow and water were made available ad libitum until experimental treatments were begun. From 39 through 59 days of age groups were divided according to pharmacological treatment: 1) NV group, normofed + vehicle solution (NaCl 0.9%, 10 ml/kg b.w.), 2) NF group, normofed + FLX (10 mg/kg b.w., in vehicle solution, 10 ml/kg b.w.) 3), OV, overfed + vehicle solution and 4) OF, overfed + FLX. At 60 days of age, body weight, white and brown adipose tissue content, and food intake were determined, and serum biochemical parameters and hypothalamic neuropeptide gene expression were measured. Results showed that FLX induced reductions in several murinometric indices, improvement of adipose profile, hypophagic behavior, reduction in serum parameters, and positive modulation of hypophagia-related genes. These data suggest that the beneficial effects of FLX-treatment on overfeeding-induced physical and behavioral effects in rats was due to hypothalamic alterations that led to improvement in energy balance in animals with a compromised metabolism.

Knock-in of large transgenes by Cas9-mediated homology-directed repair (HDR) is an extremely inefficient process. Although the use of single-stranded oligonucleotides (ssODN) as an HDR donor has improved the integration of smaller transgenes, they do not support efficient insertion of large DNA sequences. In an effort to gain insights into the mechanism(s) governing the HDR-mediated integration of larger transgenes and to improve the technology, we conducted knock-in experiments targeting the human EMX1 locus and applied rigorous genomic PCR analyses in the human HEK293 cell line. This exercise revealed an unexpected molecular complication arising from the transgene HDR being initiated at the single homology arm and the subsequent genomic integration of plasmid backbone sequences. To pivot around this problem, we devised a novel PCR-constructed template containing blocked long 3' single-stranded overhangs (BL3SSO) that greatly improved the efficiency of bona fide Cas9-stimulated HDR at the EMX1 locus. We further refined BL3SSO technology and successfully used it to insert GFP transgenes into two important interferon-stimulated genes (ISGs) loci, Viperin/RSAD2, and ISG15. This study demonstrates the utility of the BL3SSO platform for inserting long DNA sequences into both constitutive and inducible endogenous loci to generate novel human cell lines for the study of important biological processes.

[Image: see text] Here, it is proposed that the crocodile gut microbiome is a valuable biomaterials resource to counter threats posed by environmental and infectious hazards to the existence of Homo sapiens.

Although mosquitoes are major transmission vectors for pathogenic arboviruses, viral infection has little impact on mosquito health. This immunity is caused in part by mosquito RNA interference (RNAi) pathways that generate antiviral small interfering RNAs (siRNAs) and Piwi-interacting RNAs (piRNAs). RNAi also maintains genome integrity by potently repressing mosquito transposon activity in the germline and soma. However, viral and transposon small RNA regulatory pathways have not been systematically examined together in mosquitoes. Therefore, we developed an integrated mosquito small RNA genomics (MSRG) resource that analyzes the transposon and virus small RNA profiles in mosquito cell cultures and somatic and gonadal tissues across four medically important mosquito species. Our resource captures both somatic and gonadal small RNA expression profiles within mosquito cell cultures, and we report the evolutionary dynamics of a novel Mosquito-Conserved piRNA Cluster Locus (MCpiRCL) made up of satellite DNA repeats. In the larger culicine mosquito genomes we detected highly regular periodicity in piRNA biogenesis patterns coinciding with the expansion of Piwi pathway genes. Finally, our resource enables detection of cross talk between piRNA and siRNA populations in mosquito cells during a response to virus infection. The MSRG resource will aid efforts to dissect and combat the capacity of mosquitoes to tolerate and spread arboviruses.

Transmission of SARS-CoV-2 is uncontrolled in many parts of the world; control is compounded in some areas by the higher transmission potential of the B.1.1.7 variant1, which has now been reported in 94 countries. It is unclear whether the response of the virus to vaccines against SARS-CoV-2 on the basis of the prototypic strain will be affected by the mutations found in B.1.1.7. Here we assess the immune responses of individuals after vaccination with the mRNA-based vaccine BNT162b22. We measured neutralizing antibody responses after the first and second immunizations using pseudoviruses that expressed the wild-type spike protein or a mutated spike protein that contained the eight amino acid changes found in the B.1.1.7 variant. The sera from individuals who received the vaccine exhibited a broad range of neutralizing titres against the wild-type pseudoviruses that were modestly reduced against the B.1.1.7 variant. This reduction was also evident in sera from some patients who had recovered from COVID-19. Decreased neutralization of the B.1.1.7 variant was also observed for monoclonal antibodies that target the N-terminal domain (9 out of 10) and the receptor-binding motif (5 out of 31), but not for monoclonal antibodies that recognize the receptor-binding domain that bind outside the receptor-binding motif. Introduction of the mutation that encodes the E484K substitution in the B.1.1.7 background to reflect a newly emerged variant of concern (VOC 202102/02) led to a more-substantial loss of neutralizing activity by vaccine-elicited antibodies and monoclonal antibodies (19 out of 31) compared with the loss of neutralizing activity conferred by the mutations in B.1.1.7 alone. The emergence of the E484K substitution in a B.1.1.7 background represents a threat to the efficacy of the BNT162b2 vaccine.

Pathogenic mycobacteria, such as Mycobacterium tuberculosis, modulate the host immune system to evade clearance and promote long-term persistence, resulting in disease progression or latent infection. Understanding the mechanisms pathogenic mycobacteria use to escape elimination by the host immune system is critical to better understanding the molecular mechanisms of mycobacterial infection. Protein kinase G (PknG) in pathogenic mycobacteria has been shown to play an important role in avoiding clearance by macrophages through blocking phagosome-lysosome fusion; however, the exact mechanism is not completely understood. Here, to further investigate the role of mycobacterial PknG during early events of macrophage infection, RAW 264.7 macrophage cell lines were infected with M. bovis BCG wild-type and PknG knock-out mutant strains. After proteolysis, phosphopeptides were enriched via TiO2 columns and subjected to LC-MS/MS to identify differentially phosphorylated peptides between the wild-type and PknG mutant infected macrophages. A total of 1401 phosphosites on 914 unique proteins were identified. Following phosphoproteome normalisation and differential expression analysis, a total of 149 phosphosites were differentially phosphorylated in the wild-type infected RAW 264.7 macrophages versus the PknG knock-out mutant. A subset of 95 phosphosites was differentially up-regulated in the presence of PknG. Functional analysis of our data revealed that PknG kinase activity reprograms normal macrophage function through interfering with host cytoskeletal organisation, spliceosomal machinery, translational initiation, and programmed cell death. Differentially phosphorylated proteins in this study serve as a foundation for further validation and PknG host substrate assignment.ImportanceTuberculosis (TB) remains one of the leading causes of death from infection worldwide, due to the ability of Mycobacterium tuberculosis (Mtb) to survive and replicate within the host, establishing reservoirs of live bacteria that promote persistence and recurrence of disease. Understanding the mechanisms that Mtb uses to evade the host immune system is thus a major goal in the TB field. Protein kinase G is thought to play an important role in Mtb avoiding clearance by the host through disruption of macrophage function, but the underlying molecular mechanisms of this are not well understood. Here, our new phosphoproteomic data reveals that mycobacterial PknG substantially reprograms normal macrophage function through extensive PknG-mediated post-translational control of critical host cellular processes. These novel findings therefore considerably increase our knowledge of mycobacterial pathogenicity, including specific host cellular pathways that might be re-activatable through host-directed therapy, thereby restoring macrophage ability to eliminate Mtb.

Genetic mechanisms that repress transposable elements (TEs) in young animals decline during aging, as reflected by increased TE expression in aged animals. Does increased TE expression during aging lead to more genomic TE copies in older animals? To answer this question, we quantified TE Landscapes (TLs) via whole genome sequencing of young and aged Drosophila strains of wild-type and mutant backgrounds. We quantified TLs in whole flies and dissected brains and validated the feasibility of our approach in detecting new TE insertions in aging Drosophila genomes when natural defenses like RNA interference (RNAi) pathways are compromised. By also incorporating droplet digital PCR to validate genomic TE loads, we confirm TL changes can occur in a single lifespan of Drosophila when TEs are not suppressed. We also describe improved sequencing methods to quantify extra-chromosomal DNA circles (eccDNAs) in Drosophila as an additional source of TE copies that accumulate during aging. Lastly, to combat the natural progression of aging-associated TE expression, we show that knocking down PAF1, a conserved transcription elongation factor that antagonizes RNAi pathways, may bolster suppression of TEs during aging and extend lifespan. Our study suggests that RNAi mechanisms generally mitigate genomic TL expansion despite the increase in TE transcripts during aging.

Mayaro virus (MAYV) is an arboviral pathogen in the genus Alphavirus that is circulating in South America with potential to spread to naïve regions. MAYV is also one of the few viruses with the ability to be transmitted by mosquitoes in the genus Anopheles, as well as the typical arboviral transmitting mosquitoes in the genus Aedes. Few studies have investigated the infection response of Anopheles mosquitoes. In this study we detail the transcriptomic and small RNA responses of An. stephensi to infection with MAYV via infectious bloodmeal at 2, 7, and 14 days post infection (dpi). 487 unique transcripts were significantly regulated, 78 putative novel miRNAs were identified, and an siRNA response is observed targeting the MAYV genome. Gene ontology analysis of transcripts regulated at each timepoint suggested activation of the Toll pathway at 7 dpi and repression of pathways related to autophagy and apoptosis at 14 dpi. These findings provide a basic understanding of the infection response of An. stephensi to MAYV and help to identify host factors which might be useful to target to inhibit viral replication in Anopheles mosquitoes.AUTHOR SUMMARYMayaro virus (MAYV) is a mosquito-borne Alphavirus responsible for outbreaks in South America and the Caribbean. In this study we infected Anopheles stephensi with MAYV and sequenced mRNA and small RNA to understand how MAYV infection impacts gene transcription and the expression of small RNAs in the mosquito vector. Genes involved with innate immunity and signaling pathways related to cell death are regulated in response to MAYV infection of An. stephensi, we also discover novel miRNAs and describe the expression patterns of miRNAs, siRNAs, and piRNAs following bloodmeal ingestion. These results suggest that MAYV does induce a molecular response to infection in its mosquito vector species.

Culicine mosquitoes are vectors of human disease-causing pathogens like filarial worms and several arthropod-borne viruses (arboviruses). Currently, there has been an increase in emerging and re-emerging vector-borne diseases along coastal Kenya, which has been of major concern in public health. This study aimed at determining culicine mosquito species abundance, diversity and their host feeding preferences in Taita-Taveta County, Coastal Kenya. Entomological sampling was done during the long-wet season (March and May) and long dry season (June to October) 2016–2018. Mosquito sampling was done using CDC light traps and Backpack aspiration for indoor and outdoor environments. All culicine mosquitoes collected were identified morphologically and categorized according to their physiological status. Blood fed culicine mosquitoes were tested for bloodmeal sources using ELISA. In total, 3,278 culicine mosquitoes were collected, of which 738 (22.5 %) were found indoors and 2,540, (77.5 %) outdoors. The mosquitoes consisted of 18 species belonging to four genera: Aedes (7), Culex (8), Mansonia (2), and Coquillettidia (1). Overall, there was high mosquito species diversity (H) in outdoors (H = 2.4339) than in indoors (H = 2.2523), whereas even distribution (EH) was higher in indoors (EH = 0.9064) than outdoors (EH = 0.8266). Majorly the bloodmeals identified were from multiple host sources with (51.6%), single hosts (41.3%), and unidentified (7.2%). This study has demonstrated a high diversity of culicine mosquitoes with relaxed feeding tendencies. These mosquitoes are contributing to mosquito biting nuisance and the likelihood of exposure of populations to diseases of public health.

Animals face the dual threat of virus infections hijacking cellular function and transposons proliferating in germline genomes. For insects, the deeply conserved RNA interference (RNAi) pathways and other chromatin regulators provide an important line of defense against both viruses and transposons. For example, this innate immune system displays adaptiveness to new invasions by generating cognate small RNAs for targeting gene silencing measures against the viral and genomic intruders. However, within the Dipteran clade of insects, Drosophilid fruit flies and Culicids mosquitoes have evolved several unique mechanistic aspects of their RNAi defenses to combat invading transposons and viruses, with the Piwi-piRNA arm of the RNAi pathways showing the greatest degree of novel evolution. Whereas central features of Piwi-piRNA pathways are conserved between Drosophilids and Culicids, multiple lineage-specific innovations have arisen that may reflect distinct genome composition differences and specific ecological and physiological features dividing these two branches of Dipterans. This perspective review focuses on the most recent findings illuminating the Piwi/piRNA pathway distinctions between fruit flies and mosquitoes, and raises open questions that need to be addressed in order to ameliorate human diseases caused by pathogenic viruses that mosquitoes transmit as vectors.

Although mosquitoes are major transmission vectors for pathogenic arboviruses, viral infection has little impact on mosquito health. This immunity is due in part to mosquito RNA interference (RNAi) pathways that generate antiviral small interfering RNAs (siRNAs) and Piwi-interacting RNAs (piRNAs). RNAi also maintains genome integrity by potently repressing mosquito transposon activity in the germline and soma. However, viral and transposon small RNA regulatory pathways have not been systematically examined together in mosquitoes. Therefore, we developed an integrated Mosquito Small RNA Genomics (MSRG) resource that analyzes the transposon and virus small RNA profiles in mosquito cell cultures and somatic and gonadal tissues across four medically important mosquito species. Our resource captures both somatic and gonadal small RNA expression profiles within mosquito cell cultures, and we report the evolutionary dynamics of a novel Mosquito-Conserved piRNA Cluster Locus (MCpiRCL) composed of satellite DNA repeats. In the larger culicine mosquito genomes we detected highly regular periodicity in piRNA biogenesis patterns coinciding with the expansion of Piwi pathway genes. Finally, our resource enables detection of crosstalk between piRNA and siRNA populations in mosquito cells during a response to virus infection. The MSRG resource will aid efforts to dissect and combat the capacity of mosquitoes to tolerate and spread arboviruses.

Without transposon-silencing Piwi-interacting RNAs (piRNAs), transposition causes an ovarian atrophy syndrome in Drosophila called gonadal dysgenesis (GD). Harwich (Har) strains with P-elements cause severe GD in F1 daughters when Har fathers mate with mothers lacking P-element-piRNAs (i.e. ISO1 strain). To address the mystery of why Har induces severe GD, we bred hybrid Drosophila with Har genomic fragments into the ISO1 background to create HISR-D or HISR-N lines that still cause Dysgenesis or are Non-dysgenic, respectively. In these lines, we discovered a highly truncated P-element variant we named ‘Har-P’ as the most frequent de novo insertion. Although HISR-D lines still contain full-length P-elements, HISR-N lines lost functional P-transposase but retained Har-P’s that when crossed back to P-transposase restores GD induction. Finally, we uncovered P-element-piRNA-directed repression on Har-P’s transmitted paternally to suppress somatic transposition. The Drosophila short Har-P’s and full-length P-elements relationship parallels the MITEs/DNA-transposase in plants and SINEs/LINEs in mammals., eLife digest DNA provides the instructions needed for life, a role that relies on it being a very stable and organized molecule. However, some sections of DNA are able to move from one place in the genome to another. When these “mobile genetic elements” move they may disrupt other genes and cause disease. For example, a mobile section of DNA known as the P-element causes a condition called gonadal dysgenesis in female fruit flies, leading to infertility. Only certain strains of fruit flies carry P-elements and the severity of gonadal dysgenesis in their daughters varies. For example, when male fruit flies of a strain known as Harwich (or Har for short) is crossed with female fruit flies that do not contain P-elements, all of their daughters develop severe gonadal dysgenesis and are infertile. However, if the cross is done the other way around, and female Har flies mate with males that do not contain P-elements, the daughters are fertile because the Har mothers provide their daughters with protective molecules that silence the P-elements. But it was a mystery as to why the P-elements from the Har fathers always caused such severe gonadal dysgenesis in all the daughters. Here, Srivastav et al. bred fruit flies to create offspring that had different pieces of Har DNA in a genetic background that was normally free from P-elements; they then analyzed the ‘hybrid’ offspring to identify which pieces of the Har genome caused gonadal dysgenesis in the daughter flies. These experiments showed that Har flies possess a very short variant of the P-element (named “Har-P”) that is more mobile than other variants. However, the Har-P variants still depended on an enzyme known as P-transposase encoded by the full-length P-elements to move around the genome. Further experiments showed that other strains of fruit flies that cause severe gonadal dysgenesis also had very short P-element variants that were almost identical to Har-P. These findings may explain why Har and some other strains of fruit flies drive severe gonadal dysgenesis. In the future, it may be possible to transfer P-transposase and Har-P into mosquitoes, ticks and other biting insects to make them infertile and help reduce the spread of certain diseases in humans.

Aim Drivers of intraspecific biodiversity include past climate-driven range shifts and contemporary ecological conditions mediating connectivity, but these are rarely integrated in a common comprehensive approach. This is particularly relevant for marine organisms, as ocean currents strongly influence population isolation or connectivity, keeping or diluting the signatures left by past climates. Here we ask whether the coupling between past range shifts and contemporary connectivity explain the extant gene pools of Laminaria ochroleuca, a large brown alga structuring important marine forests from shallow to deep infralittoral grounds. Location Northeastern Atlantic Ocean. Taxon Laminaria ochroleuca. Methods We estimated population genetic diversity and structure of L. ochroleuca across its entire distribution range using 15 polymorphic microsatellite markers. This was compared with the outcomes of a palaeoclimatic model predicting latitudinal and depth range shifts from the Last Glacial Maximum (LGM) to the present. Genetic differentiation was further compared with potential connectivity inferred with a biophysical model developed with high-resolution data from HYCOM (Hybrid Coordinate Ocean Model). Results The biogeographical distribution of genetic variability showed overall agreement with the predictions from independently inferred past range shifts. Multiple regions of persistence were identified in deep and upwelling settings at the lowest latitudes of the current species distribution, where higher and unique genetic diversity was retained. The biophysical model revealed that despite the possibility of long-distance migration, contemporary oceanographic barriers strongly restrict connectivity of isolated genetic lineages. Main conclusions Integrating different processes at biogeographical scales explained the extant gene pools of marine forests of L. ochroleuca. Low-latitude genetic relics harbour a disproportional evolutionary significance, persisting as ancient populations in isolated deep and upwelling climate refugia. Their inferred rates of dispersal may be insufficient to accommodate anticipated climate warming.

Graphical abstract, The comprehensive characterisation of the cell wall proteome of mycobacteria is of considerable relevance to both the discovery of new drug targets as well as to the design of new vaccines against Mycobacterium tuberculosis. However, due to its extremely hydrophobic nature, the coverage of proteomic studies of this subcellular compartment is still far from complete. Here, we report novel gel-free cell wall sample preparation procedures and quantitative LC–MS/MS measurements on a Q Exactive mass spectrometer. We combine these with a novel post-measurement bioinformatic analysis to filter out likely cytosolic contaminants. This reveals a subset of proteins that are highly enriched for cell wall proteins. The success of this approach is verified by peptide-centric measurement of the abundance of known subcellular markers, as well as analysis of the percentage of predicted membrane proteins within the purified fraction. While M. smegmatis was used during this study to establish and optimise the sample preparation procedures, these can easily be applied to other mycobacterial species, such as M. bovis BCG or M. tuberculosis. • Improved gel-free cell wall sample preparation gives higher yields of tryptic peptides for LC–MS/MS measurement. • Higher yields of tryptic peptides provide better quantitation and coverage of cell wall proteome. • Post-measurement enrichment analysis filters out high abundance cytosolic contaminants that have carried through the experimental analysis.

The Coronavirus Disease 2019 (COVID-19) Genomics UK Consortium (COG-UK) was launched in March, 2020, with £20 million support from UK Research and Innovation, the UK Department of Health and Social Care, and Wellcome Trust. The goal of this consortium is to sequence severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) for up to 230 000 patients, health-care workers, and other essential workers in the UK with COVID-19, which will help to enable the tracking of SARS-CoV-2 transmission, identify viral mutations, and integrate with health data to assess how the viral genome interacts with cofactors and consequences of COVID-19.

The bulk of the human genome (~98%) is comprised of non-coding sequences. Cis-regulatory elements (CREs) are non-coding DNA sequences that contain binding sites for transcriptional regulators to modulate gene expression. Alterations of CREs have been implicated in various diseases including cancer. While promoters and enhancers have been the primary CREs for studying gene regulation, very little is known about the role of silencer, which is another type of CRE that mediates gene repression. Originally identified as an adaptive immunity system in prokaryotes, CRISPR/Cas9 has been exploited to be a powerful tool for eukaryotic genome editing. Here, we present the use of this technique to delete an intronic silencer in the human RUNX1 gene and investigate the impacts on gene expression in OCI-AML3 leukemic cells. Our approach relies on electroporation-mediated delivery of two preassembled Cas9/guide RNA (gRNA) ribonucleoprotein (RNP) complexes to create two double-strand breaks (DSBs) that flank the silencer. Deletions can be readily screened by fragment analysis. Expression analyses of different mRNAs transcribed from alternative promoters help evaluate promoter-dependent effects. This strategy can be used to study other CREs and is particularly suitable for hematopoietic cells, which are often difficult to transfect with plasmid-based methods. The use of a plasmid- and virus-free strategy allows simple and fast assessments of gene regulatory functions.

Without transposon-silencing Piwi-interacting RNAs (piRNAs), transposition causes an ovarian atrophy syndrome inDrosophilacalled gonadal dysgenesis (GD).Harwich(Har) strains withP-elements cause severe GD in F1 daughters whenHarfathers mate with mothers lackingP-element-piRNAs (i.e.ISO1strain). To address the mystery of whyHarinduces severe GD, we bred hybridDrosophilawithHargenomic fragments into theISO1background to createHISR-D or HISR-Nlines that still causeDysgenesis or areNon-dysgenic, respectively. In these lines, we discovered a highly truncatedP-element variant we named “Har-P” as the most frequentde novoinsertion. AlthoughHISR-Dlines still contain full-lengthP-elements,HISR-Nlines lost functionalP-transposase but retainedHar-P’s that when crossed back toP-transposase restores GD induction. Finally, we uncoveredP-element-piRNA-directed repression onHar-P’stransmitted paternally to suppress somatic transposition. TheDrosophilashortHar-P’sand full-lengthP-elements relationship parallels the MITEs/DNA-transposase in plants and SINEs/LINEs in mammals.

SummaryRecent evidence demonstrates that novel protein-coding genes can arisede novofrom intergenic loci. This evolutionary innovation is thought to be facilitated by the pervasive translation of intergenic transcripts, which exposes a reservoir of variable polypeptides to natural selection. Do intergenic translation events yield polypeptides with useful biochemical capacities? The answer to this question remains controversial. Here, we systematically characterized howde novoemerging coding sequences impact fitness. In budding yeast, overexpression of these sequences was enriched in beneficial effects, while their disruption was generally inconsequential. We found that beneficial emerging sequences have a strong tendency to encode putative transmembrane proteins, which appears to stem from a cryptic propensity for transmembrane signals throughout thymine-rich intergenic regions of the genome. These findings suggest that novel genes with useful biochemical capacities, such as transmembrane domains, tend to evolvede novowithin intergenic loci that already harbored a blueprint for these capacities.

Ink disease (caused by the pathogen Phytophthora cinnamomi) and chestnut blight (caused by the fungus Cryphonectria parasitica) are the two most destructive diseases affecting European chestnut (Castanea sativa) and American chestnut (C. dentata). Therefore, breeding for resistance to both pathogens is essential for the chestnut sustainability in Europe and the United States of America. Several genomic approaches have been implemented in order to map the resistance first to ink disease and then to blight chestnut using a hybrid population: C. sativa crossed with resistant Asian species (C. crenata and C. mollissima). The transcriptome of C. sativa and C. crenata inoculated and not with P. cinnamomi was recently sequenced and allowed the identification of genes putatively involved in ink disease resistance. Taking advantage of molecular markers (microsatellite and SNPs), developed from the referred tanscriptomes and from C. mollissima transcriptome, the first C. sativa x C. crenata genetic map was constructed. Additionally, a reliable phenotyping method was established to evaluate the level of P. cinnamomi resistance of each hybrid allowing the identification of two Quantitative Trait Loci (QTLs). These results are the first step for understanding the resistance to ink disease in chestnut., Revista de Ciências Agrárias, vol. 39 n.º 2 (2016)

When Drosophila mothers lacking P-element-piRNAs (i.e. ISO1) mate with certain fathers harboring active P-elements (i.e. Harwich, Har), hybrid daughters suffer from gonadal dysgenesis (GD), where ovaries collapse from uncontrolled P-element transposition. To discover the P-element driving GD, we selected hybrid D. melanogaster lines with portions of Har DNA within the ISO1 genome. We generated HISR-D and HISR-N lines retaining or losing GD induction, respectively. Despite greatly-reduced numbers of P-elements, HISR-D lines produced as many P-element piRNAs as parental Har. In these lines, we discovered a highly-truncated P-element variant: “Har-P”, which mobilized de novo in all HISR lines. Crossing P-transposase with Har-Ps in HISR-N lines restored GD and revealed a paternal P-element-piRNA-directed imprint on Har-Ps to suppress lethal somatic transposition. Har-P may weaponize P-transposase during catastrophic transposition.

The Escherichia coli proteome is the most extensively characterized and studied of all prokaryotic proteomes. Despite this, large scale bacterial proteomics experiments performed on E. coli cells grown in liquid cultures have failed to identify key virulence factors thought to be important determinants in establishing bacterial infections. It seems likely that many important determinants associated with virulence and host cell adhesion are exclusively expressed during growth in biofilms, which can be crudely mimicked on solid media. This method describes a simple workflow to characterize the unique proteome signature of individual, isolated single colonies, using E. coli K12 strain grown on solid media as a model system. The workflow thus provides a means to explore the proteomes of minimally passaged clinical isolates of bacteria grown on primary culture plates and to identify both unique and differentially expressed proteins contained therein. Value of the method: - Simple mass spectrometry-based proteomics workflow to characterise the proteome of single colony forming units - Enables exploration of the proteomes of minimally passaged clinical isolates from primary culture plates - Identification of virulence factors expressed in true or mimicked biofilms that may be missed in liquid cultures Method name: E. coli single colony proteome analysis

Most of the largest vertebrate genomes are found in salamanders, a clade of amphibians that includes 686 species. Salamander genomes range in size from 14 to 120 Gb, reflecting the accumulation of large numbers of transposable element (TE) sequences from all three TE classes. Although DNA loss rates are slow in salamanders relative to other vertebrates, high levels of TE insertion are also likely required to explain such high TE loads. Across the Tree of Life, novel TE insertions are suppressed by several pathways involving small RNA molecules. In most known animals, TE activity in the germline is primarily regulated by the Piwi-interacting RNA (piRNA) pathway. In this study, we test the hypothesis that salamanders' unusually high TE loads reflect the loss of the ancestral piRNA-mediated TE-silencing machinery. We characterized the small RNA pool in the female and male adult gonads, testing for the presence of small RNA molecules that bear the characteristics of TE-targeting piRNAs. We also analyzed the amino acid sequences of piRNA pathway proteins from salamanders and other vertebrates, testing whether the overall patterns of sequence divergence are consistent with conserved pathway function across the vertebrate clade. Our results do not support the hypothesis of piRNA pathway loss; instead, they suggest that the piRNA pathway is expressed in salamanders. Given these results, we propose hypotheses to explain how the extraordinary TE loads in salamander genomes could have accumulated, despite the expression of TE-silencing machinery.

International audience; The copepod Calanus glacialis is endemic to the Arctic Ocean and peripheral seas and forms a key component of the Arctic marine ecosystems. It is the major contributor to zooplankton biomass, a predominant grazer, and an important prey for seabirds, and fish. As for a planktonic species, its dispersal is expected to be widespread and mediated by ocean currents. However, complex circulation patterns and the existence of semi-enclosed fjords and seas in the Arctic can be hypothesized to influence the population genetic structure of this species. In this study, we aimed to infer patterns of connectivity between populations of C. glacialis distributed around the Arctic and across putative barriers formed by oceanographic currents and semi-enclosed fjords and seas. To achieve this, we used 11 polymorphic microsatellite loci to genotype 189 individuals from 7 locations: Svalbard fjords (Kongsfjorden, Hornsund, Isfjorden, Rijpfjorden, and Storfjorden), White Sea, and Amundsen Gulf, thus providing greater genetic resolution over a larger biogeographical scale than in previous studies. The results revealed a lack of structure among all seven locations around the Arctic, indicating a panmictic population with large-scale gene flow. This study also supports the hypothesis that the planktonic fauna of the White Sea is not isolated from that of the other Arctic regions.

Background: The functional interplay between tumor cells and their adjacent stroma has been suggested to play crucial roles in the initiation and progression of tumors and the effectiveness of chemotherapy. The extracellular matrix (ECM), a complex network of extracellular proteins, provides both physical and chemicals cues necessary for cell proliferation, survival, and migration. Understanding how ECM composition and biomechanical properties affect cancer progression and response to chemotherapeutic drugs is vital to the development of targeted treatments. Methods: 3D cell-derived-ECMs and esophageal cancer cell lines were used as a model to investigate the effect of ECM proteins on esophageal cancer cell lines response to chemotherapeutics. Immunohistochemical and qRT-PCR evaluation of ECM proteins and integrin gene expression was done on clinical esophageal squamous cell carcinoma biopsies. Esophageal cancer cell lines (WHCO1, WHCO5, WHCO6, KYSE180, KYSE 450 and KYSE 520) were cultured on decellularised ECMs (fibroblasts-derived ECM, cancer cell-derived ECM, combinatorial-ECM) and treated with 0.1% Dimethyl sulfoxide (DMSO), 4.2 &micro, M cisplatin, 3.5 &micro, M 5-fluorouracil and 2.5 &micro, M epirubicin for 24 h. Cell proliferation, cell cycle progression, colony formation, apoptosis, migration and activation of signaling pathways were used as our study endpoints. Results: The expression of collagens, fibronectin and laminins was significantly increased in esophageal squamous cell carcinomas (ESCC) tumor samples compared to the corresponding normal tissue. Decellularised ECMs abrogated the effect of drugs on cancer cell cycling, proliferation and reduced drug induced apoptosis by 20&ndash, 60% that of those plated on plastic. The mitogen-activated protein kinase-extracellular signal-regulated kinase (MEK-ERK) and phosphoinositide 3-kinase-protein kinase B (PI3K/Akt) signaling pathways were upregulated in the presence of the ECMs. Furthermore, our data show that concomitant addition of chemotherapeutic drugs and the use of collagen- and fibronectin-deficient ECMs through siRNA inhibition synergistically increased cancer cell sensitivity to drugs by 30&ndash, 50%, and reduced colony formation and cancer cell migration. Conclusion: Our study shows that ECM proteins play a key role in the response of cancer cells to chemotherapy and suggest that targeting ECM proteins can be an effective therapeutic strategy against chemoresistant tumors.

RUNX1 encodes a Runt-related transcription factor that is critical for hematopoiesis. In this study, through a combinatorial molecular approach, we characterized a novel t(5;21)(q13;q22) translocation involving RUNX1 that was acquired during the progression of myelodysplastic syndrome to acute myeloid leukemia (AML) in a pediatric patient. We found that this translocation did not generate RUNX1 fusion but aberrantly upregulated RUNX1. This upregulation was attributed to the disruption of long-range chromatin interactions between the RUNX1 P2 promoter and a silencer in the first intron of the gene. Characterization of the silencer revealed a role of SNAG repressors and their corepressor LSD1/KDM1A in mediating the effect. Our findings suggest that chromosomal rearrangements may activate RUNX1 by perturbing its transcriptional control to contribute to AML pathogenesis, in keeping with an emerging oncogenic role of RUNX1 in leukemia. Electronic supplementary material The online version of this article (10.1186/s12943-018-0881-2) contains supplementary material, which is available to authorized users.

Dispersal beyond the local patch in clonal plants was typically thought to result from sexual reproduction via seed dispersal. However, evidence for the separation, transport by water, and re-establishment of asexual propagules (asexual hydrochory) is mounting suggesting other important means of dispersal in aquatic plants. Using an unprecedented sampling size and microsatellite genetic identification, we describe the distribution of seagrass clones along tens of km within a coastal lagoon in Southern Portugal. Our spatially explicit individual-based sampling design covered 84 km(2) and collected 3 185 Zostera noltei ramets from 803 sites. We estimated clone age, assuming rhizome elongation as the only mechanism of clone spread, and contrasted it with paleo-oceanographic sea level change. We also studied the association between a source of disturbance and the location of large clones. A total of 16 clones were sampled more than 10 times and the most abundant one was sampled 59 times. The largest distance between two samples from the same clone was 26.4 km and a total of 58 and 10 clones were sampled across more than 2 and 10 km, respectively. The number of extremely large clone sizes, and their old ages when assuming the rhizome elongation as the single causal mechanism, suggests other processes are behind the span of these clones. We discuss how the dispersal of vegetative fragments in a stepping-stone manner might have produced this pattern. We found higher probabilities to sample large clones away from the lagoon inlet, considered a source of disturbance. This study corroborates previous experiments on the success of transport and re-establishment of asexual fragments and supports the hypothesis that asexual hydrochory is responsible for the extent of these clones. Fundacao para a Ciencia e a Tecnologia, Portugal [PTDC/MAR/099887/2008]; Fundacao para a Ciencia e Tecnologia [SFRH/BD/68570/2010]

Summary The execution of shock following high dose E. coli lipopolysaccharide (LPS) or bacterial sepsis in mice required pro-apoptotic caspase-8 in addition to pro-pyroptotic caspase-11 and gasdermin D. Hematopoietic cells produced MyD88- and TRIF-dependent inflammatory cytokines sufficient to initiate shock without any contribution from caspase-8 or caspase-11. Both proteases had to be present to support tumor necrosis factor- and interferon-β-dependent tissue injury first observed in the small intestine and later in spleen and thymus. Caspase-11 enhanced the activation of caspase-8 and extrinsic cell death machinery within the lower small intestine. Neither caspase-8 nor caspase-11 was individually sufficient for shock. Both caspases collaborated to amplify inflammatory signals associated with tissue damage. Therefore, combined pyroptotic and apoptotic signaling mediated endotoxemia independently of RIPK1 kinase activity and RIPK3 function. These observations bring to light the relevance of tissue compartmentalization to disease processes in vivo where cytokines act in parallel to execute diverse cell death pathways.

To understand how transposon landscapes (TLs) vary across animal genomes, we describe a new method called the Transposon Insertion and Depletion AnaLyzer (TIDAL) and a database of >300 TLs in Drosophila melanogaster (TIDAL-Fly). Our analysis reveals pervasive TL diversity across cell lines and fly strains, even for identically named sub-strains from different laboratories such as the ISO1 strain used for the reference genome sequence. On average, >500 novel insertions exist in every lab strain, inbred strains of the Drosophila Genetic Reference Panel (DGRP), and fly isolates in the Drosophila Genome Nexus (DGN). A minority (70%) of TL diversity across fly strains. A sharp contrast between insertion and depletion patterns indicates that many transposons are unique to the ISO1 reference genome sequence. Although TL diversity from fly strains reaches asymptotic limits with increasing sequencing depth, rampant TL diversity causes unsaturated detection of TLs in pools of flies. Finally, we show novel transposon insertions negatively correlate with Piwi-interacting RNA (piRNA) levels for most transposon families, except for the highly-abundant roo retrotransposon. Our study provides a useful resource for Drosophila geneticists to understand how transposons create extensive genomic diversity in fly cell lines and strains.

Busia and Kisii Central districts are areas in western Kenya that have repeatedly reported high levels of stunting growth in children and an increase in hepatocellular carcinoma (HCC); an aspect often positively associated with chronic exposure to aflatoxins especially through consumption of foods such as peanuts. The objectives of the study were to determine the incidence, types and levels of aflatoxin in different varieties of peanuts produced in Busia and Kisii Central districts. One hundred and two (102) peanuts samples were collected from farmers’ in each district. Aflatoxin types and levels of aflatoxins were analyzed using high performance liquid chromatography (HPLC) technique. All the peanuts samples from Kisii Central and 97.06% samples from Busia were contaminated with aflatoxins. However, aflatoxin was not detected in 2.94% of samples from Busia district. The levels of total aflatoxin ranges were 0.1 to 268 μg/kg and 1.63 to 591.1 μg/kg in peanuts from Busia and Kisii Central respectively. Majority of peanuts samples had levels within Kenya Bureau of Standards (KEBS) and European Union (EU) regulatory limits for total aflatoxins. Improved variety (Valencia red) had significantly lower aflatoxin contamination compared to local varieties (Uganda local red, Homabay local and Local red). Aflatoxins B1, B2, G1 and G2 were found in peanuts; B1 was the most predominant in both districts (t = 12.4, df = 3, P = 0.034). The levels of aflatoxins especially in peanuts from Kisii Central district were high (591.1 μg/kg) where 44.6% of samples analyzed were unfit for even animal feed (USFDA regulatory limit). An assessment on the levels of aflatoxins should be done by the relevant stakeholders in other key foods in the areas for example maize. The most lethal aflatoxin type B1 was found to be the most predominant peanuts from both districts of study. This calls for frequent aflatoxin screening of peanuts from the districts particularly aflatoxin type B.

At present, Brazil imports approximately 11 billion liters/year of diesel. With the interruption of the works in the new Petrobras refineries, the projection is that by 2025 this volume will increase to 24.2 billion liters of diesel/year. In this sense, the biodiesel factory Grand Valle Bio Energy Ltda., located in the state of Rio de Janeiro, in conjunction with the FAPERJ makes some investments in technology development for the cultivation and use of microalgae as an alternative raw material in the production of biodiesel. Based on arguments previously said, this work presents the results of the microalgae cultivation Monoraphidium sp. in photobioreactors the pilot plant of the company. The installation with an area of 120 m2 is included with 2 open photobioreactors of type falling film (20 m × 1 m), with a cascade of 18mm and capacity of 4000 L. The lineage cultivated is selected from previous ecophysiological studies that are identified as promising for biodiesel production by having a high potential for the production of lipids. This lineage is maintained at collection of the stock of cultures Laboratory of Green Technologies of the School of Chemistry/ UFRJ. The cultivation was performed in means ASM-1 (Gorham et al., 1964), initial pH 8.0, with aeration and circulation average of 8 hours a day during 19 days. The culture was started with an inoculum of 1 × 107 cel/ml. The lipid production was determined in two phases of growth: on day 4 (exponential phase) and 15 day (stationary phase). For the determination and quantification of lipid content, two different methods were assessed for a sample of biomass, submitted to the same processes the separation and drying. The results showed the methodology of Bligh & Dyer with modifications as the most efficient in extracting lipids. The total lipid content of the biomass Monoraphidium sp. was 30.58%. The growth rate varied between 0.74 ± 0.01 and 0.68 ± 0.02.

Understanding the cell wall of mycobacteria is crucial for improving drug design or identifying new antigens suitable to vaccination. Yet this remains problematic due to the complexity of the cell wall composition. In this study, we successfully developed gel-free approaches to study cell wall proteins in Mycobacterium smegmatis. The cell wall was subjected to differential centrifugation, differential detergent solubilisation and phase separation to yield the genuine cell wall proteome. Next, protein extracts were digested by filter-assisted sample preparation for LC-MS/MS analysis on a Q Exactive mass spectrometer, and identified proteins filtered through a stringent bioinformatics pipeline. This yielded the unprecedented coverage of 96 lipoproteins, 475 membrane proteins and 73 secreted proteins. Employing this approach, we next quantified changes in the cell wall proteome during exposure of M. smegmatis to sub-lethal concentration of rifampicin. This facilitated detailed characterisation of the dysregulation of ABC transporters, virulence factors such as Mce proteins and PknG, and proteins involved in cell wall and lipid synthesis. Crucially, these cell wall proteins are under-represented in previous proteome analysis of M. smegmatis. This approach enables further quantitative proteomic studies of the role of the cell wall proteome of mycobacteria in virulence or during drug exposure. SIGNIFICANCE: We developed novel gel-free sample preparation workflows for the cell wall fraction of mycobacteria that significantly increase the coverage of the cell wall proteome compared to previous studies. We then provide a data analysis workflow that enables the removal of likely cytosolic contaminants in the cell wall fraction post-measurement. Combined, these approaches increase the coverage of the cell wall proteome while ensuring that the identified proteins are true cell wall proteins and not carry-over of high-abundance contaminants from the cytosol. We have applied these approaches to quantify the dysregulation of cell wall proteins during exposure of M. smegmatis to rifampicin, which has shed new light on the coordinated down-regulation of ABC transporters as well as virulence factors present in the cell wall proteome.

We report a novel HBB: c.114G>C mutation in a Chinese family. This mutation resulted in a β37(C3)Trp→Cys amino acid substitution and was synonymous with Hb Kent, a hemoglobin (Hb) variant that was reported exclusively in patients of European descent. Though Hb Kent has a normal oxygen affinity and molecular stability, it has a characteristic dual variant appearance on cellulose acetate electrophoresis (CAE) and high performance liquid chromatography (HPLC) caused by the posttranslational modification of cysteine. We also report the phenotypic expression of this variant when coinherited with the Southeast Asian (- -SEA) double α-globin gene deletion.

A significant proportion of human immunodeficiency virus type 1 (HIV)-positive individuals are affected by the cognitive, motor and behavioral dysfunction that characterizes HIV-associated neurocognitive disorders (HAND). While the molecular etiology of HAND remains largely uncharacterized, HIV transactivator of transcription (HIV-Tat) is thought to be an important etiological cause. Here we have used mass spectrometry (MS)-based discovery proteomics to identify the quantitative, cell-wide changes that occur when non-transformed, differentiated human neurons are treated with HIV-Tat over time. We identified over 4000 protein groups (false discovery rate

Vitamin C has been found to affect mycobacteria in multiple ways, including increasing susceptibility to antimicrobial drugs, inducing dormancy, and having a bactericidal effect. However, the regulatory events mediating vitamin C related adaptations remain largely elusive. Ser/Thr/Tyr protein phosphorylation plays an important regulatory role in mycobacteria, contributing to environmental adaptation, including dormancy and drug resistance. This study utilised the model organism, Mycobacterium smegmatis, and TiO2 phosphopeptide enrichment combined with mass spectrometry-based proteomics methods to elucidate the mycobacterial signalling and regulatory response to sub-lethal concentrations of vitamin C. After initial validation of peptide spectra, 224 non-redundant phosphosites in 154 proteins were retained with high confidence. Data analysis revealed that 30 peptides were differentially phosphorylated with Vitamin C treatment, including novel phosphosites found on both PknG and GarA. Of these significant proteins, we validated 11 by parallel reaction monitoring of high-confidence phosphopeptides. Interestingly, 17/30 phosphopeptides were annotated as part of transmembrane proteins, suggesting that it is likely vitamin C triggers typical signal transduction events in which the protein periplasmic domain perceives environmental signals and the cytoplasmic domain is then phosphorylated. Finally, the diverse nature of phosphorylated proteins involved in signalling, transport, and carbohydrate biosynthesis indicates the extent of such regulatory phosphorylation events. Biological significance Our findings provide new mechanistic insight into a coordinated network of signalling and regulatory responses to sub-lethal vitamin C in Mycobacterium smegmatis and provide evidence that vitamin C is able to act as a novel extracellular signalling molecule. Vitamin C treatment caused changes in both the proteome and phosphoproteome associated with response to oxidative stress, a shift in metabolic regulation and progression toward dormancy, as well as phospho-dependent activation of specific secretory pathways and activation of specific two component and Ser/Thr/Tyr protein kinase activities. This study confirms the potential of vitamin C as convenient means to study aspects of mycobacterial dormancy, including those regulated at post-translational level.

Piwi proteins utilize small RNAs (piRNAs) to recognize target transcripts such as transposable elements (TE). However, extensive piRNA sequence diversity also suggests that Piwi/piRNA complexes interact with many transcripts beyond TEs. To determine Piwi target RNAs, we used ribonucleoprotein-immunoprecipitation (RIP) and cross-linking and immunoprecipitation (CLIP) to identify thousands of transcripts associated with the Piwi proteins XIWI and XILI (Piwi-protein-associated transcripts, PATs) from early stage oocytes of X. laevis and X. tropicalis. Most PATs associate with both XIWI and XILI and include transcripts of developmentally important proteins in oogenesis and embryogenesis. Only a minor fraction of PATs in both frog species displayed near perfect matches to piRNAs. Since predicting imperfect pairing between all piRNAs and target RNAs remains intractable, we instead determined that PAT read counts correlate well with the lengths and expression levels of transcripts, features that have also been observed for oocyte mRNAs associated with Drosophila Piwi proteins. We used an in vitro assay with exogenous RNA to confirm that XIWI associates with RNAs in a length- and concentration-dependent manner. In this assay, noncoding transcripts with many perfectly matched antisense piRNAs were unstable, whereas coding transcripts with matching piRNAs were stable, consistent with emerging evidence that Piwi proteins both promote the turnover of TEs and other RNAs, and may also regulate mRNA localization and translation. Our study suggests that Piwi proteins play multiple roles in germ cells and establishes a tractable vertebrate system to study the role of Piwi proteins in transcript regulation.

Shiga-toxin-producing Escherichia coli (STEC) and enteropathogenic Escherichia coli (EPEC) are key diarrhoea-causing foodborne pathogens. We used proteomics to characterize the virulence and antimicrobial resistance protein profiles of three clinical pathogenic E. coli isolates (two EPEC [one resistant to ciprofloxacin] and one STEC) cultured on CHROMagar™STEC solid media after minimal laboratory passage. We identified 4767 unique peptides from 1630 protein group across all three clinical E. coli strains. Label-free proteomic analysis allowed the identification of virulence and drug resistance proteins that were unique to each of the clinical isolates compared in this study. The B subunit of Shiga toxin, ToxB, was uniquely detected in the STEC strain while several other virulence factors including SheA, OmpF, OmpC and OmpX were significantly more abundant in the STEC strain. The ciprofloxacin resistant EPEC isolate possessed reduced levels of key virulence proteins compared to the ciprofloxacin susceptible EPEC and STEC strains. Parallel reaction monitoring assays validated the presence of biologically relevant proteins across biologically-replicated cultures. Propagation of clinical isolates on a relevant solid medium followed by mass spectrometry analysis represents a convenient means to quantify virulence factors and drug resistance determinants that might otherwise be lost through extensive in vitro passage in enteropathogenic bacteria. Significance Through the use of quantitative proteomics, we have characterized the virulence and antimicrobial resistance attributes of three clinically isolated, pathogenic E. coli strains cultured on solid media. Our results provide new, quantitative data on the expressed proteomes of these tellurite-resistant, diarrhoeagenic E. coli strains and reveal a subset of antimicrobial resistance and virulence proteins that are differentially abundant between these clinical strains. Our quantitative proteomics-based approach should thus have applicability in microbiological diagnostic labs for the identification of pathogenic/drug resistant E. coli in the future.

Background Molecular markers are revealing a much more diverse and evolutionarily complex picture of marine biodiversity than previously anticipated. Cryptic and/or endemic marine species are continually being found throughout the world oceans, predominantly in inconspicuous tropical groups but also in larger, canopy-forming taxa from well studied temperate regions. Interspecific hybridization has also been found to be prevalent in many marine groups, for instance within dense congeneric assemblages, with introgressive gene-flow being the most common outcome. Here, using a congeneric phylogeographic approach, we investigated two monotypic and geographically complementary sister genera of north-east Pacific intertidal seaweeds (Hesperophycus and Pelvetiopsis), for which preliminary molecular tests revealed unexpected conflicts consistent with unrecognized cryptic diversity and hybridization. Results The three recovered mtDNA clades did not match a priori species delimitations. H. californicus was congruent, whereas widespread P. limitata encompassed two additional narrow-endemic species from California - P. arborescens (here genetically confirmed) and P. hybrida sp. nov. The congruence between the genotypic clusters and the mtDNA clades was absolute. Fixed heterozygosity was apparent in a high proportion of loci in P. limitata and P. hybrida, with genetic analyses showing that the latter was composed of both H. californicus and P. arborescens genomes. All four inferred species could be distinguished based on their general morphology. Conclusions This study confirmed additional diversity and reticulation within NE Pacific Hesperophycus/Pelvetiopsis, including the validity of the much endangered, modern climatic relict P. arborescens, and the identification of a new, stable allopolyploid species (P. hybrida) with clearly discernable ancestry (♀ H. californicus x ♂ P. arborescens), morphology, and geographical distribution. Allopolyploid speciation is otherwise completely unknown in brown seaweeds, and its unique occurrence within this genus (P. limitata possibly representing a second example) remains enigmatic. The taxonomic separation of Hesperophycus and Pelvetiopsis is not supported and the genera should be synonymized; we retain only the latter. The transitional coastline between Point Conception and Monterey Bay represented a diversity hotspot for the genus and the likely sites of extraordinary evolutionary events of allopolyploid speciation at sympatric range contact zones. This study pinpoints how much diversity (and evolutionary processes) potentially remains undiscovered even on a conspicuous seaweed genus from the well-studied Californian intertidal shores let alone in other, less studied marine groups and regions/depths. info:eu-repo/semantics/publishedVersion

Genetic diversity in clonal organisms includes two distinct components, (i) the diversity of genotypes or clones (i.e. genotypic richness) in a population and (ii) that of the alleles (i.e. allelic and gene diversity within populations, and differentiation between populations). We investigated how population differentiation and genotypic components are associated across a gradient of eutrophication in a clonal marine plant. To that end, we combined direct measurements of sexual allocation (i.e. flower and seed counts) and genotypic analyses, which are used as an estimator of effective sexual reproduction across multiple generations. Genetic differentiation across sites was also modelled according to a hypothesis here defined as isolation-by-demography, in which we use population-specific factors, genotypic richness and eutrophication that are hypothesized to affect the source-sink dynamics and thus influence the genetic differentiation between a pair of populations. Eutrophic populations exhibited lower genotypic richness, in agreement with lower direct measurements of sexual allocation and contemporaneous gene flow. Genetic differentiation, while not explained by distance, was best predicted by genotypic richness and habitat quality. A multiple regression model using these two predictors was considered the best model (R(2) = 0.43). In this study, the relationship between environment and effective sexual-asexual balance is not simply (linearly) predicted by direct measurements of sexual allocation. Our results indicate that population-specific factors and the isolation-by-demography model should be used more often to understand genetic differentiation.

Piwi proteins and Piwi-interacting RNAs (piRNAs) repress transposable elements (TEs) from mobilizing in gonadal cells. To determine the spectrum of piRNA-regulated targets that may extend beyond TEs, we conducted a genome-wide survey for transcripts associated with PIWI and for transcripts affected by PIWI knockdown in Drosophila ovarian somatic sheet (OSS) cells, a follicle cell line expressing the Piwi pathway. Despite the immense sequence diversity among OSS cell piRNAs, our analysis indicates that TE transcripts are the major transcripts associated with and directly regulated by PIWI. However, several coding genes were indirectly regulated by PIWI via an adjacent de novo TE insertion that generated a nascent TE transcript. Interestingly, we noticed that PIWI-regulated genes in OSS cells greatly differed from genes affected in a related follicle cell culture, ovarian somatic cells (OSCs). Therefore, we characterized the distinct genomic TE insertions across four OSS and OSC lines and discovered dynamic TE landscapes in gonadal cultures that were defined by a subset of active TEs. Particular de novo TEs appeared to stimulate the expression of novel candidate long noncoding RNAs (lncRNAs) in a cell lineage-specific manner, and some of these TE-associated lncRNAs were associated with PIWI and overlapped PIWI-regulated genes. Our analyses of OSCs and OSS cells demonstrate that despite having a Piwi pathway to suppress endogenous mobile elements, gonadal cell TE landscapes can still dramatically change and create transcriptome diversity.

Phenotypic differences between closely related species are thought to arise primarily from changes in gene expression due to mutations in cis-regulatory sequences (enhancers). However, it has remained unclear how frequently mutations alter enhancer activity or create functional enhancers de novo. Here we use STARR-seq, a recently developed quantitative enhancer assay, to determine genome-wide enhancer activity profiles for five Drosophila species in the constant trans-regulatory environment of Drosophila melanogaster S2 cells. We find that the functions of a large fraction of D. melanogaster enhancers are conserved for their orthologous sequences owing to selection and stabilizing turnover of transcription factor motifs. Moreover, hundreds of enhancers have been gained since the D. melanogaster–Drosophila yakuba split about 11 million years ago without apparent adaptive selection and can contribute to changes in gene expression in vivo. Our finding that enhancer activity is often deeply conserved and frequently gained provides functional insights into regulatory evolution.