title: A Comparison Of Fecal Glucocorticoid Metabolite Concentration And Gut Microbiota Diversity In Bonobos (Pan paniscus)

abstract:
Host sex, age, diet, stress, and social environment have all been found to influence the gut microbiota. In non-human primates (NHP), recent evidence from gorillas found fecal glucocorticoid metabolite concentration (FGMC) had no significant role in structuring their gut microbiota, but there was a significant differential abundance between family Anaerolineaceae and gorilla FGMC. This pattern has yet to be examined in other NHP, like bonobos (Pan paniscus). We compared FGMC to 16S rRNA amplicons for 201 bonobo fecal samples collected in the wild across five months to evaluate the impact of stress, measured with FGMC, on the gut microbiota. Simpsons index was the only alpha diversity index to have a significant linear relationship with FGMC [R2 = 0.9643, F(4, 210) = 28.56, p = 0.0023]. FGMC level explained 1.63% of the variation in beta diversity for Jensen-Shannon Distance, 2.49% for Weighted UniFrac, and 3.53% for Unweighted UniFrac using PERMANOVAs. Differential abundance models showed seventeen taxa that were significantly correlated with FGMC. We found that genus SHD-231 in the family Anaerolinaceae was significant in our differential abundance model results, similar to western lowland gorilla abundance model results. These results suggest bonobos exhibit different patterns than gorillas in alpha and beta diversity measures and that members of the family Anaerolinaceae may be differentially affected by host stress across great apes. Incorporating FGMC into gut microbiota research can provide a more robust understanding of how stress impacts the gut microbiota of primates and humans and has important ties to overall host health.

Graphical AbstractSee Powerpoint

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title: Multivalent interactions drive the Toxoplasma AC9:AC10:ERK7 complex to concentrate ERK7 in the apical cap

abstract:
The Toxoplasma inner membrane complex (IMC) is a specialized organelle that is crucial for the parasite to establish an intracellular lifestyle and ultimately cause disease. The IMC is composed of both membrane and cytoskeletal components, further delineated into the apical cap, body, and basal subcompartments. The apical cap cytoskeleton was recently demonstrated to govern the stability of the apical complex, which controls parasite motility, invasion, and egress. While this role was determined by individually assessing the apical cap proteins AC9, AC10, and the MAP kinase ERK7, how the three proteins collaborate to stabilize the apical complex is unknown. In this study, we use a combination of deletion analyses and yeast-2-hybrid experiments to establish that these proteins form an essential complex in the apical cap. We show that AC10 is a foundational component of the AC10:AC9:ERK7 complex and demonstrate that the interactions among them are critical to maintain the apical complex. Importantly, we identify multiple independent regions of pairwise interaction between each of the three proteins, suggesting that the AC9:AC10:ERK7 complex is organized by multivalent interactions. Together, these data support a model in which multiple interacting domains enable the oligomerization of the AC9:AC10:ERK7 complex and its assembly into the cytoskeletal IMC, which serves as a structural scaffold that concentrates ERK7 kinase activity in the apical cap.

IMPORTANCEThe phylum Apicomplexa consists of obligate, intracellular parasites including the causative agents of toxoplasmosis, malaria, and cryptosporidiosis. Hallmarks of these parasites are the IMC and the apical complex, both of which are unique structures that are conserved throughout the phylum and required for parasite survival. The apical cap portion of the IMC has previously been shown to stabilize the apical complex. Here, we expand on those studies to determine the precise protein-protein interactions of the apical cap complex that confer this essential function. We describe the multivalent nature of these interactions and show that the resulting protein oligomers likely tether ERK7 in the apical cap. This study represents the first description of the architecture of the apical cap at a molecular level, expanding our understanding of the unique cell biology that drives Toxoplasma infections.

category: microbiology


title: Exploring the onset of B12-based mutualisms using a recently evolved Chlamydomonas auxotroph and B12-producing bacteria

abstract:
Cobalamin (vitamin B12), is a cofactor for crucial metabolic reactions in multiple eukaryotic taxa, including major primary producers such as algae, and yet only prokaryotes can produce it. Many bacteria can colonise the algal phycosphere, forming stable communities that gain preferential access to exudates and in return provide compounds, such as B12. Extended coexistence can then drive gene loss, leading to greater algal-bacterial interdependence. In this study, we investigate how a recently evolved B12-dependent strain of Chlamydomonas reinhardtii, metE7, forms a mutualism with certain bacteria, including the rhizobium Mesorhizobium loti and even a strain of the gut bacterium E. coli engineered to produce cobalamin. Although metE7 was supported by B12 producers, its growth in co-culture was slower than the B12-independent wild-type, suggesting that high bacterial B12 provision may be necessary to favour B12 auxotrophs and their evolution. Moreover, we found that an E. coli strain that releases more B12 makes a better mutualistic partner, and although this trait may be more costly in isolation, greater B12 release provided an advantage in co-cultures. We hypothesise that, given the right conditions, bacteria that release more B12 may be selected for, particularly if they form close interactions with B12-dependent algae.

Originality-Significance statementMicroalgae are fundamental to the global carbon cycle, and yet despite being photosynthetic they often rely on other organisms for micronutrients. One of these micronutrients is vitamin B12 (cobalamin), which they receive from bacteria. Many environmental studies support the widespread role of B12 in algal-bacterial mutualisms, so here we wished to investigate how these mutualisms may arise evolutionarily by using an experimentally evolved B12-dependent alga and various B12-producers. A B12-producing rhizobium, Mesorhizobium loti, could stably support the B12-dependent Chlamydomonas reinhardtii metE7 strain, and vice versa, but nutrient supplementation increased growth of both species further. metE7 could also be supported by E. coli strains engineered to produce B12, and engineering a strain to release a higher proportion of B12 led to better algal growth, which increased bacterial growth in turn. We suggest that as B12-based mutualisms develop, increased B12 release may be selected for and therefore lead to more productive symbioses.


title: Human land-use impacts viral diversity and abundance in a New Zealand river

abstract: Although water-borne viruses have important implications for the health of humans and other animals, little is known about the impact of human land-use on viral diversity and evolution in water systems such as rivers. We used metagenomic next-generation sequencing to compare the diversity and abundance of viruses at sampling sites along a single river in New Zealand that differed in human land use impact, ranging from pristine to urban. From this we identified 504 putative virus species, of which 97% were novel. Many of the novel viruses were highly divergent, and likely included a new subfamily within the Parvoviridae. We identified at least 63 virus species that may infect vertebrates - most likely fish and water birds - from the Astroviridae, Birnaviridae, Parvoviridae and Picornaviridae. No putative human viruses were detected. Importantly, we observed differences in the composition of viral communities at sites impacted by human land-use (farming and urban) compared to native forest sites (pristine). At the viral species level, the urban sites had higher diversity (327 virus species) than the farming (n=150) and pristine sites (n=119), and more viruses were shared between the urban and farming sites (n=76) than between the pristine and farming or urban sites (n=24). The two farming sites had a lower viral abundance across all host types, while the pristine sites had a higher abundance of viruses associated with animals, plants and fungi. We also identified viruses linked to agriculture and human impact at the river sampling sites in farming and urban areas that were not present at the native forest sites. Overall, our study shows that human land-use can impact viral communities in rivers, such that further work is needed to reduce the impact of intensive farming and urbanization on water systems.


title: Core Microbiota Drive the Prevalence of Extracellular Antibiotic Resistome in the Water Compartments

abstract:
Unlike intracellular chromosome, extracellular DNA (eDNA) may accelerate the spreading of antibiotic resistance genes (ARGs) through natural transformation, but one of the core issues regarding to the bacterial taxonomic characterization of eDNA in the complex water environments is largely unknown. Hence, Illumina Miseq sequencing was used to identify the genotype of eDNA from wastewater (WW), river water (RW) and stormwater (SW) runoff. High-throughput qPCR targeting 384 genes was implemented to detect extracellular ARGs (eARGs) and mobile genetic elements (eMGEs). We obtained 2,708,291 high quality sequences from 66 eDNA samples. The SW exhibited the significant higher Shannon Index. Subsequently, we identified 34 core bacteria sources of eDNA widely distributed in the three water compartments. Among which, Pseudomonas, Flavobacterium, Limnohabitans, Burkholderiaceae_unclassified, Methylotenera and Acinetobacter were the most prevalent. A total of 302 eARGs and eMGEs were detected, suggesting that eDNA is an important antibiotic resistance reservoir. Among the 127 shared genes of the three groups, 15 core resistance genes were filtered, including IS6100, sul1 NEW, intI1, ISPps1-pseud, aac3-Via, qacH_351 and ISSm2-Xanthob. The Procrustes analysis and Variance Partitioning Analysis (VPA) demonstrated that core bacteria and MGEs were significantly correlated with eARGs. These results suggested that the occurrence and changes of eARGs in the water compartments may be largely attributed to the core microbiota and eMGEs.

SynopsisThe identification of bacterial taxa of extracellular DNA is the preliminary issue for the natural transformation of extracellular antibiotic resistome.


title: Enhanced loss of retinoic acid network genes in Xenopus laevis achieves a tighter signal regulation

abstract: Retinoic acid (RA) is a major regulatory signal during embryogenesis produced from vitamin A (retinol) by an extensive, autoregulating metabolic and signaling network to prevent fluctuations that result in developmental malformations. Xenopus laevis is an allotetraploid hybrid frog species whose genome includes L (long) and S (short) chromosomes from the originating species. Evolutionarily, the X. laevis subgenomes have been losing either L or S homoeologs in about 43% of genes to generate singletons. In the RA network, out of the 47 genes, about 46% have lost one of the homoeologs, like the genome average. In contrast, RA metabolism genes from storage (retinyl esters) to retinaldehyde production exhibit enhanced gene loss with 75% singletons out of 28 genes. The effect of this gene loss on RA signaling autoregulation was studied. Employing transient RA manipulations, homoeolog gene pairs were identified in which one homeolog exhibits enhanced responses or looser regulation than the other, while in other pairs both homoeologs exhibit similar RA responses. CRISPR /Cas9 targeting of individual homoeologs to reduce their activity supports the hypothesis where the RA metabolic network gene loss results in tighter network regulation and more efficient RA robustness responses to overcome complex regulation conditions.

category: molecular biology


title: Thiamine metabolism genes in diatoms are not regulated by thiamine despite the presence of predicted riboswitches

abstract:
O_LIThiamine pyrophosphate (TPP), an essential co-factor for all species, is biosynthesised through a metabolically expensive pathway regulated by TPP riboswitches in bacteria, fungi, plants and green algae. Diatoms are microalgae responsible for approximately 20% of global primary production. They have been predicted to contain TPP aptamers in the 3UTR of some thiamine metabolism-related genes, but little is known about their function and regulation.
C_LIO_LIWe used bioinformatics, antimetabolite growth assays, RT-qPCR, targeted mutagenesis and reporter constructs to test whether the predicted TPP riboswitches respond to thiamine supplementation in diatoms. Gene editing was used to investigate the functions of the genes with associated TPP riboswitches in Phaeodactylum tricornutum.
C_LIO_LIWe found that thiamine-related genes with putative TPP aptamers are not responsive to thiamine or its precursor 4-amino-5-hydroxymethyl-2-methylpyrimidine (HMP), and the targeted mutation of the TPP aptamer in the HMP-P synthase (THIC) does not deregulate thiamine biosynthesis in P. tricornutum. Through genome editing we established that PtSSSP is necessary for thiamine uptake and that PtTHIC is essential for thiamine biosynthesis.
C_LIO_LIOur results highlight the importance of experimentally testing bioinformatic aptamer predictions and provide new insights into the thiamine metabolism shaping the structure of marine microbial communities with global biogeochemical importance.
C_LI


title: Histone deacetylase inhibitor overrides the effect of soft hydrogel on the mechanoresponse of human mesenchymal stem cells

abstract:
Human Mesenchymal stem cells (hMSCs) are promising in regenerative medicine for their multi-lineage differentiation capability. It has been demonstrated that lineage specification is governed by both chemical and mechanical cues. Among all the different mechanical cues known to control hMSCs fate, substrate stiffness is the most well-studied. It has been shown that the naive mesenchymal stem cells when cultured on soft gel, they commit towards adipogenic lineage while on stiff gel they become osteogenic. Soft substrates also cause less cell spreading, less traction, less focal adhesion assembly and stress fibre formation. Furthermore, chromatin condensation increases when cells are cultured on soft substrates. As the nucleus has been postulated to be mechanosensor and mechanotransducer, in this paper we asked the question how mechanosensing and mechanoresponse process will be influenced if we reduce the chromatin condensation in hMSCs cultured on soft substrates by using an external reagent. To address this question, we treated hMSCs cultured on soft polyacrylamide (PA) gels with a histone deacetylase inhibitor (HDACi) called Valproic Acid (VA) which decondense the chromatin by hyperacetylation of histone proteins. We found that the treatment with VA overrides the effect of soft substrates on hMSCs morphology, cellular traction, nuclear localization of mechnosensory protein YAP (yes-associated protein), and differentiation. VA treated cells behaved as if they are on stiff substrates in all aspects tested here. In contrast, when we increase chromatin condensation using histone acetyl transferase inhibitor (HATi), adipogenic differentiation on soft gel increases. Furthermore, we have shown that VA controls hMSCs differentiation via activation of ERK/MAPK pathway by increasing the p-ERK expression. Collectively, these findings demonstrate for the first time that inhibiting histone deacetylase can override the mechanoresponse of hMSCs. This work will help us to fundamentally understand the mechanosignalling process and to control the hMSCs differentiation in tissue engineering and regenerative medicine.

SignificanceTo harness the clinical potential of Human mesenchymal stem cells (hMSCs), it is important to understand the working mechanism of various signals in deciding cell fate. It has been shown in the recent past that the substrate rigidity influence hMSCs differentiation. Though the mechanism is not fully understood, the mechanosignals are known to alter the cell fate via reorganizing the chromatin packing. In this work, we ask if altering chromatin packing can change the cell fate as dictated by the mechanical properties of the substrates. We have shown that almost all the known effector of mechanosignals get modified when we tweak the chromatin packing via histone deacetylase inhibitor (HDACi). This study is helpful for developing a better understanding of the mechano-signalling process. It gives us a tool to override the effect of the substrates, particularly when the mechanical properties of biomaterials are not conducive for the desired differentiation outcome. Additionally, as HDACi are being used in the therapy of cancer and different neurodegenerative diseases, our understanding of their crosstalk with tissue stiffness in controlling cell fate will help to understand any unknown and undesirable side effects of using HDACi as therapeutic agent.

category: cell biology


title: Guardian crypt-base-goblet-cells protect the human colonic stem cell niche by triggering cholinergic calcium signal-dependent MUC2 secretion and luminal flushing

abstract: Mucus secreting goblet cells play a vital role in the maintenance of tissue homeostasis. Here we report the discovery of an enigmatic mechanism for the generation of calcium signals that couple cholinergic input to secretion of hydrated mucus in the human colonic stem cell niche. Mechanistic insights for this study were derived from native human colonic crypts and crypt-like organoids expressing MUC2-mNEON. Importantly, we demonstrate that the human colonic stem cell niche is also a cholinergic niche, and that activation of muscarinic receptors initiates calcium signals at the apical pole of intestinal stem cells and neighbouring crypt-base-goblet-cells. The calcium signal  trigger zone is defined by a microdomain of juxtaposed calcium stores expressing TPC1 and InsP3R3 calcium channels. Co-activation of TPC1 and InsP3R3 is required for generation of cholinergic calcium signals and downstream secretion of hydrated mucus, which culminates in the flushing of the colonic stem cell niche.


title: Gene expression profiles of some prognostic markers in a human breast cell line (MCF7) exposed to Curcumin nanoparticles and nanocapsules

abstract:
1.IntroductionWorldwide, cancer is a significant public health problem. Curcumin exhibits anti-inflammatory, antiproliferative, and anticancer properties when used in medicine. Investigated study for Curcumins chemopreventive mechanism against human malignancies, this research examined the cellular and molecular alterations generated by curcumin modified compound in breast cancer (MCF-7) cell lines. Oncogenic EGFR and VEGFR2 mutations lead to the formation, invasion, and maintenance of malignant phenotypes in humans, including breast cancer. Studied prognostic markers such as C-myc and Ki67 in breast cancer, and the apoptotic gene as Caspase-3 have been done.

Aim of the workThe purpose of this study is to determine the therapeutic efficacy of curcumin nanoparticles and nanocapsules in breast cancer cell lines (MCF7).

Materials and methodsWe used real-time PCR to assess the expression of the C-myc, Ki67, EGFR, VEGFR2, and Caspase-3 genes in MCF7 cells treated with Curcumin nanoparticles and nanocapsules.

ResultsCurcumin nanoparticles and nanocapsules boosted apoptotic cell populations considerably regardless of the nanotechnology used. Additionally, the mRNA expression analysis results indicated that the mechanism activated by curcumin nanocapsules involved the upregulation of the oncogenes EGFR and VEGFR2. In comparison to curcumin nanoparticles, curcumin nanocapsules significantly reduced the expression of Ki67 and c-myc mRNAs in breast cancer cells. The mRNA expression study revealed that curcumin nanocapsules produce an increase in the apoptotic Caspase-3 gene production compared to cells treated with curcumin nanoparticles.

ConclusionThis work demonstrates that curcumin nanoparticles created using a novel mechanical process can be employed successfully as an anticancer agent. These findings add to our understanding of the molecular mechanisms behind curcumin nanocapsules anticancer activity in breast cancer.

category: genetics


title: New genomic signals underlying the emergence of human proto-genes

abstract: De novo genes are novel genes which emerge from non-coding DNA. Until now, little is known about de novo genes properties, correlated to their age and mechanisms of emergence. In this study, we investigate four properties: introns, upstream regulatory motifs, 5 UTRs and protein domains, in 23135 human proto-genes. We found that proto-genes contain introns, whose number and position correlates with the genomic position of proto-gene emergence. The origin of these introns is debated, as our result suggest that 41% proto-genes might have captured existing introns, as well as the fact that 13.7% of them do not splice the ORF. We show that proto-genes which emerged via overprinting tend to be more enriched in core promotor motifs, while intergenic and intronic ones are more enriched in enhancers, even if the motif TATA is most expressed upstream these genes. Intergenic and intronic 5 UTRs of protogenes have a lower potential to stabilise mRNA structures than exonic proto-genes and established human genes. Finally, we confirm that proto-genes gain new putative domains with age. Overall, we find that regulatory motifs inducing transcription and translation of previously non-coding sequences may facilitate proto-gene emergence. Our paper demonstrates that introns, 5UTRs, and domains have specific properties in proto-genes. We also show the importance of studying proto-genes in relation to their genomic position, as it strongly impacts these properties.

category: genomics


title: Understanding host response to infectious salmon anaemia virus in an Atlantic salmon cell line using single-cell RNA sequencing

abstract:
BackgroundInfectious Salmon Anaemia Virus (ISAV) is an Orthomixovirus that currently represents a large problem for salmonid aquaculture worldwide. Prevention and treatment methods are only partially effective. Genetic selection and genome engineering strategies have potential to develop ISAV resistant salmon stocks. However, this requires a detailed understanding of the genomic regulation of ISAV pathogenesis. Here, we used single cell RNA sequencing on a salmonid cell line to provide a high dimensional insight into the transcriptional landscape that underpin host-virus interactions during ISAV infection at the single cell level.

ResultsSalmon head kidney 1 (SHK-1) cells were single-cell RNA sequenced before challenge, and at 24h, 48h, and 96h post-ISAV challenge. The results revealed marked changes in the host transcriptome at 48h and 96h post-infection, even in uninfected cells, potentially suggesting paracrine signalling. This paracrine activation of uninfected cells seemed to be unspecific, involving pathways such as mRNA sensing, ubiquitination or proteasome, and also the up-regulation of the mitochondrial ribosome genes. At 24h post infection, cells showed expression signatures consistent with viral entry, with up-regulation of genes such as PI3K, FAK or JNK. At 48h and 96h, infected cells showed a clear anti-viral response, characterised by the expression of IFNA2 or IRF2.

ConclusionsThis study has increased our understanding of the cellular response of Atlantic salmon during ISAV infection, and revealed potential host-virus interactions at the cellular level. The results highlight the value of single-cell sequencing to characterise cell culture models of viral infection, and the results can be exploited in future functional studies to increase the resistance of Atlantic salmon to ISAV.


title: The crystal structure of TRPM2 MHR1/2 domain reveals a conserved Zn2+-binding domain essential for ligand binding and activity

abstract: Transient receptor potential melastatin 2 (TRPM2) is a Ca2+-permeable, non-selective cation channel involved in diverse physiological processes such as immune response, apoptosis and body temperature sensing. TRPM2 is activated by ADP-ribose (ADPR) and 2'-deoxy-ADPR in a Ca2+-dependent manner. While two species-specific binding sites exist for ADPR, a binding site for 2'-deoxy-ADPR is not known yet. Here, we report the crystal structure of the MHR1/2 domain of TRPM2 from zebrafish (Danio rerio) and show binding of both ligands to this domain. We identified a so-far unrecognized Zn2+-binding domain that was not resolved in previous cryo-EM structures and that is conserved in most TRPM channels. In combination with patch clamp experiments, we comprehensively characterize the effect of the Zn2+-binding domain on TRPM2 activation. Our results provide insight into a conserved structural element essential for channel activity.

category: biochemistry


title: Rotavirus RNA chaperone mediates global transcriptome-wide increase in RNA backbone flexibility

abstract: Due to genome segmentation, rotaviruses must co-package a set of eleven distinct genomic RNAs. The packaging is mediated by the RNA chaperone NSP2. While the activities of RNA chaperones are well studied with short RNAs, little is known about their global effect on the entire viral transcriptome. Here we used Selective 2'-hydroxyl Acylation Analyzed by Primer Extension and Mutational Profiling (SHAPE-MaP) to systematically examine the secondary structure of the rotavirus transcriptome alone and in the presence of NSP2. Surprisingly, SHAPE-MaP data reveal that despite the well-characterized helix-unwinding activity of NSP2 in vitro, its incubation with rotavirus transcripts does not induce a significant change in the SHAPE reactivities. However, a quantitative analysis of the per nucleotide mutation rate, from which SHAPE reactivities are derived, reveals a global five-fold rate increase in the presence of molar excess of NSP2. Further analysis of the mutation rate in the context of structural classification reveals a larger effect on stems rather than loop elements. Together, these data provide the first experimentally derived secondary structure model of the rotavirus transcriptome and reveal that NSP2 exerts a larger effect on stems, while acting by globally increasing RNA backbone flexibility in a protein concentration-dependent manner.


title: A Low-Cost, 3D-Printed Biosensor for Rapid Detection of Escherichia coli

abstract: Detection of bacterial pathogens is significant in the fields of food safety, medicine, public health, etc. If bacterial pathogens are not treated promptly, antimicrobial resistance is possible and can lead to morbidity and mortality. Current bacterial detection methodologies rely on laboratory-based techniques that pose limitations such as long turnaround detection times, expensive costs, inadequate accuracy, and required trained specialists. Here, we describe a cost-effective and portable 3D-printed electrochemical biosensor that facilitates rapid detection of certain Escherichia coli (E. coli) strains (DH5, BL21, TOP10, and JM109) within 15 minutes using 500 L of sample and costs $2.50 per test. The sensor displayed an excellent limit of detection (LOD) of 53 cfu, limit of quantification (LOQ) of 270 cfu, and showed cross-reactivity with strains BL21 and JM109 due to shared epitopes. This advantageous diagnostic device is a potential candidate for high-frequency testing at point of care as well as applicable to various fields where pathogen detection is of interest.

category: bioengineering


title: Embracing enzyme promiscuity with activity-based compressed biosensing

abstract: Genome-scale activity-based profiling of proteases requires identifying substrates that are specific to each individual protease. However, this process becomes increasingly difficult as the number of target proteases increases because most substrates are promiscuously cleaved by multiple proteases. We introduce a method - Substrate Libraries for Compressed sensing of Enzymes (SLICE) - for selecting complementary sets of promiscuous substrates to compile libraries that classify complex protease samples (1) without requiring deconvolution of the compressed signals and (2) without the use of highly specific substrates. SLICE ranks substrate libraries according to two features: substrate orthogonality and protease coverage. To quantify these features, we design a compression score that was predictive of classification accuracy across 140 in silico libraries (Pearson r = 0.71) and 55 in vitro libraries (Pearson r = 0.55) of protease substrates. We demonstrate that a library comprising only two protease substrates selected with SLICE can accurately classify twenty complex mixtures of 11 enzymes with perfect accuracy. We envision that SLICE will enable the selection of peptide libraries that capture information from hundreds of enzymes while using fewer substrates for applications such as the design of activity-based sensors for imaging and diagnostics.


title: Reduced local mechanical stimuli in spaceflight diminishes osteocyte lacunar morphometry and spatial heterogeneity in mouse cortical bone

abstract:
Three-dimensional (3D) imaging of osteocyte lacunae has recently substantiated the connection between lacunar shape and size, and osteocyte age, viability, and mechanotransduction. Yet it remains unclear why individual osteocytes reshape their lacunae and how networks of osteocytes change in response to local alterations in mechanical loads. We evaluated the effects of local mechanical stimuli on osteocyte lacunar morphometrics in tibial cortical bone from young female mice flown on the Space Shuttle for [~]13 days. We optimized scan parameters, using a laboratory-based submicrometer-resolution X-Ray Microscope, to achieve large [~] 0.3 mm3 fields of view with sufficient resolution ([≥] 0.3 m) to visualize and measure thousands of lacunae per scan. Our novel approach avoids large measurement errors that are inherent in 2D and enables a facile 3D solution as compared to the lower resolution from benchtop micro-computed tomography (CT) systems or the cost and inaccessibility of synchrotron-based CT. Osteocyte lacunae were altered following microgravity exposure in a region-specific manner: more elongated (+7.0% Stretch) in predominately tensile-loaded bone as compared to those in compressively-loaded regions. In compressively-loaded bone, lacunae formed in microgravity were significantly larger (+6.9% Volume) than in the same region formed on Earth. We also evaluated lacunar heterogeneity (i.e., spatial autocorrelation of lacunar morphometric parameters) via kriging models. These statistical models demonstrated that heterogeneity varied with underlying spatial contributors, i.e. the local mechanical and biological environment. Yet in the absence of gravitational loading, osteocyte lacunae in newly formed bone were larger and were collectively more homogenous than in bone formed on Earth. Overall, this study shows that osteocyte reshape their lacunae in response to changes, or absence, in local mechanical stimuli and different biological environments. Additionally, spatial relationships among osteocytes are complex and necessitate evaluation in carefully selected regions of interest and of large cell populations.

GRAPHICAL ABSTRACT

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title: Identification of bacteria strains using the Recombinase Polymerase Amplification assay on a miniaturized solid-state pH sensor

abstract: Rapid identification of bacteria based on nucleic acid amplification allows dealing with the detection of pathogens in clinical, food, and environmental samples. Amplification product must be detected and analyzed by external devices or integrated complicated optical systems. Here, we developed a solid-state pH electrode based on iridium oxide (IrO2) films to measure released hydrogen ions (H+) from isothermal nucleic acid (NA) amplification of bacterial samples. By recombinase polymerase amplification (RPA), we achieved rapid (< 15 min) and sensitive (<30 copies) detection with an accuracy of about 0.03 pH. The RPA-based hydrogen ion sensing assay shows higher specificity, sensitivity, and efficiency as the same polymerase chain reaction (PCR) methods. We initially used the RPA-based sensor to detect E. coli species in laboratory samples. Among, 27 random laboratory samples of E. coli samples, 6 were found to be DH5alpha, 9 BL21, 3 HB101, 6 TOP10, and 3 JM109. The electrical detection of amplification provides generally applicable techniques for the detection of nucleic acid amplification, enabling molecular diagnostic tests in the field and integrating data transmission to the mobile device. These results can be future developed into an efficient tool for rapid on-site detection of bacterial pathogens in clinical samples.


title: Control of bacterial anti-phage signaling by a WYL domain transcription factor

abstract: Bacteria use diverse immune systems to defend themselves from ubiquitous viruses termed bacteriophages (phages). Many anti-phage systems function by abortive infection to kill a phage-infected cell, raising the question of how they are regulated to avoid cell killing outside the context of infection. Here, we identify a transcription factor associated with the widespread CBASS bacterial immune system, that we term CapW. CapW forms a homodimer and binds a palindromic DNA sequence in the CBASS promoter region. Two crystal structures of CapW suggest that the protein switches from an unliganded, DNA binding-competent state to a ligand-bound state unable to bind DNA. We show that CapW strongly represses CBASS gene expression in uninfected cells, and that phage infection causes increased CBASS expression in a CapW-dependent manner. Unexpectedly, this CapW-dependent increase in CBASS expression is not required for robust anti-phage activity, suggesting that CapW may mediate CBASS activation and cell death in response to a signal other than phage infection. Our results parallel concurrent reports on the structure and activity of BrxR, a transcription factor associated with the BREX anti-phage system, suggesting that CapW and BrxR are members of a family of universal defense signaling proteins.

category: biophysics


title: Control of bacterial immune signaling by a WYL domain transcription factor


title: Mobility of nucleosomes is regulated by their binding partners

abstract: Chromatin accessibility is modulated in a variety of ways, both to create open and closed chromatin states which are critical for eukaryotic gene regulation. At the mechanistic single molecule level, how accessibility is regulated remains a fundamental question in the field. Here, we use single molecule tracking by high-speed atomic force microscopy to investigate this question using chromatin arrays and extend our findings into the nucleus. By high-speed atomic force microscopy, we tracked chromatin dynamics in real time and observed that the essential kinetochore protein CENP-C reduces the diffusion constant of CENP-A nucleosomes and the linker H1.5 protein restricts H3 nucleosome mobility. We subsequently interrogated how CENP-C modulates CENP-A chromatin dynamics in vivo. Overexpressing CENP-C resulted in reduced centromeric transcription and impaired loading of new CENP-A molecules. These data suggest a model in which inner kinetochore proteins are critically involved in modulating chromatin accessibility and consequently, noncoding transcription at human centromeres.


title: Structure-function-dynamics relationships in the peculiar Planktothrix PCC7805 OCP1: impact of his-tagging and carotenoid type.

abstract:
The orange carotenoid protein (OCP) is a photoactive protein involved in cyanobacterial photoprotection. Here, we report on the functional, spectral and structural characteristics of the peculiar Planktothrix PCC7805 OCP (Plankto-OCP). We show that this OCP variant is characterized by higher photoactivation and recovery rates, and a stronger energy-quenching activity, compared to other OCPs studied thus far. We characterize the effect of the functionalizing carotenoid and of his-tagging on these reactions, and the time scales on which these modifications affect photoactivation. The presence of a His-tag at the C-terminus has a large influence on photoactivation, thermal recovery and PBS-fluorescence quenching, and likewise for the nature of the carotenoid that additionally affects the yield and characteristics of excited states and the ns-s dynamics of photoactivated OCP. By solving the structures of Plankto-OCP in the ECN- and CAN-functionalized states, each in two closely-related crystal forms, we further unveil the molecular breathing motions that animate Plankto-OCP at the monomer and dimer levels. We finally discuss the structural changes that could explain the peculiar properties of Plankto-OCP.

HighlightsO_LIComplete functional characterization of Synechocystis and Planktothrix OCPs
C_LIO_LIHitherto unknown structures of ECN- and CAN-functionalized Planktothrix OCP
C_LI


title: Siglec-5 is an inhibitory immune checkpoint molecule for human T cells

abstract:
Sialic acid-binding immunoglobulin-type lectins (Siglecs) are a family of immunoglobulin-type lectins that mediate protein-carbohydrate interactions via sialic acids attached to glycoproteins or glycolipids. Most of the CD33-related Siglecs (CD33rSiglecs), a major subfamily of rapidly evolving Siglecs, contain a cytoplasmic signaling domain consisting of the immunoreceptor tyrosine-based inhibitory motif (ITIM) and immunoreceptor tyrosine-based switch motif (ITSM) and mediate suppressive signals for lymphoid and myeloid cells. While most CD33rSiglecs are expressed by innate immune cells, such as monocytes and neutrophils, to date, the expression of Siglecs in human T cells has not been well appreciated. In this study, we found that Siglec-5, a member of the CD33rSiglecs, is expressed by most activated T cells upon antigen receptor stimulation. Functionally, Siglec-5 suppresses T cell activation. In support of these findings, we found that Siglec-5 overexpression abrogates antigen receptor induced activation of Nuclear factor of activated T cells (NFAT) and Activator protein 1 (AP-1). Furthermore, we show that GBS {beta}-protein, a known bacterial ligand of Siglec-5, reduces the production of cytokines and cytolytic molecules by activated primary T cells in a Siglec-5 dependent manner. Our data also show that some cancer cell lines express a putative Siglec-5 ligand(s), and that the presence of soluble Siglec-5 enhances tumor-cell specific T cell activation, suggesting that some tumor cells inhibit T cell activation via Siglec-5. Together, our data demonstrate that Siglec-5 is a previously unrecognized inhibitory T cell immune checkpoint molecule and suggests that blockade of Siglec-5 could serve as a new strategy to enhance anti-tumor T cell functions.

One Sentence SummarySiglec-5 is a novel checkpoint receptor expressed by activated human T cells that antagonizes TCR mediated activation and suppresses T cell effector functions such as cytokine production.

category: immunology


title: The AMA1-RON complex drives Plasmodium sporozoite invasion in the mosquito and mammalian hosts

abstract: Plasmodium sporozoites that are transmitted by blood-feeding female Anopheles mosquitoes invade hepatocytes for an initial round of intracellular replication, leading to the release of merozoites that invade and multiply within red blood cells. Sporozoites and merozoites share a number of proteins that are expressed by both stages, including the Apical Membrane Antigen 1 (AMA1) and the Rhoptry Neck Proteins (RONs). Although AMA1 and RONs are essential for merozoite invasion of erythrocytes during asexual blood stage replication of the parasite, their function in sporozoites is still unclear. Here we show that AMA1 interacts with RONs in mature sporozoites. By using DiCre-mediated conditional gene deletion in P. berghei, we demonstrate that loss of AMA1, RON2 or RON4 in sporozoites impairs colonization of the mosquito salivary glands and invasion of mammalian hepatocytes, without affecting transcellular parasite migration. Our data establish that AMA1 and RONs facilitate host cell invasion across Plasmodium invasive stages, and suggest that sporozoites use the AMA1-RON complex to safely enter the mosquito salivary glands without causing cell damage, to ensure successful parasite transmission. These results open up the possibility of targeting the AMA1-RON complex for transmission-blocking antimalarial strategies.


title: Dissociating encoding of memory and salience by manipulating long-term synaptic potentiation

abstract:
Neural codes are thought to be reorganized during memory formation by long-term potentiation (LTP) of synapses. Here, using a novel approach for selectively blocking LTP, we found that eliminating LTP in hippocampal or striatal circuits only produces limited effects on learning and memory. To reconcile the discrepancy between the large physiological effect of blocking LTP and the absent effect on learning, we studied how LTP impacts neuronal computations in the hippocampus using in-vivo Ca2+-imaging. Contrary to current conceptual frameworks, we found that hippocampal CA1-region LTP is not required for accurate representations of space in hippocampal neurons, but rather endows these neurons with reward- and novelty-coding properties. Thus, instead of driving formation of cognitive maps and memory engrams, CA1-region LTP incorporates salience information into cognitive representations.

One-Sentence SummaryA novel approach for studying long-term potentiation reveals its surprising and selective role in salience encoding

category: neuroscience


title: Proteomic identification of phosphorylation dependent Septin 7 interactors that drive dendritic spine formation

abstract: Septins are a family of cytoskeletal proteins that regulate several important aspects of neuronal development. Septin 7 (Sept7) is enriched at the base of dendritic spines in excitatory neurons and mediates both spine formation and spine-synapse maturation. Phosphorylation at a conserved C-terminal tail residue of Sept7 mediates its translocation into the dendritic spine head to allow spine-synapse maturation. The mechanistic basis for postsynaptic stability and compartmentalization conferred by phosphorylated Sept7, however, is not known. We report herein the proteomic identification of Sept7 phosphorylation dependent neuronal interactors. Using Sept7 C-terminal phosphopeptide pulldown and biochemical assays, we show that the 14-3-3 family of proteins specifically interact with Sept7 when phosphorylated at the T426 residue. Biochemically, we validate the interaction between Sept7 and 14-3-3 isoform gamma, and show that 14-3-3 gamma is also enriched in mature dendritic spine head. Further, we demonstrate that interaction of phosphorylated Sept7 with 14-3-3 protects it from dephosphorylation, as expression of a 14-3-3 antagonist significantly decreases phosphorylated Sept7 in neurons. This study identifies 14-3-3 proteins as an important physiological regulator of Sept7 function in neuronal development.


title: Heterogeneity of social cognition in temporo-parietal junction: Overlapping yet distinct representation between visual perspective-taking and theory of mind

abstract:
Visual perspective taking (VPT), particularly level 2 VPT (VPT2), which allows an individual to understand that the same object can be seen differently by others, is related to the theory of mind (ToM), because both functions require a decoupled representation from oneself. Although previous neuroimaging studies have shown that VPT and ToM activate the temporo-parietal junction (TPJ), it is unclear whether common neural substrates are involved in VPT and ToM. To clarify this point, the present study directly compared the TPJ activation patterns of individual participants performing VPT2 and ToM tasks using functional magnetic resonance imaging and within-subjects design. VPT2-induced activations were compared with activations observed during a mental rotation task as a control task, whereas ToM-related activities were identified with a standard ToM localizer using false-belief stories. A whole-brain analysis revealed that VPT2 and ToM activated overlapping areas in the posterior part of the TPJ. By comparing the activations induced by VPT2 and ToM in individual participants, we found that the peak voxels induced by ToM were located significantly more anteriorly and dorsally within the bilateral TPJ than those measured during the VPT2 task. We further confirmed that these activity areas were spatially distinct from the nearby extrastriate body area (EBA), visual motion area (MT+), and the posterior superior temporal sulcus (pSTS) using independent localizer scans. Our findings revealed that VPT2 and ToM have distinct representations, albeit partially overlapping, indicating the functional heterogeneity of social cognition within the TPJ.

Significance StatementThe temporo-parietal junction (TPJ) is consistently activated by social cognitive tasks such as visual perspective taking (VPT) and theory of mind (ToM) tasks. The present study investigated whether VPT and ToM have the same neural substrates within the TPJ using functional magnetic resonance imaging (fMRI) with a within-subjects design. While VPT and ToM tasks activated overlapping areas in the posterior part of the TPJ, the individual peak voxels induced by ToM were located significantly more anteriorly and dorsally compared with those observed during the VPT task. Moreover, they were spatially distinct from the nearby functional modules, such as the extrastriate body area, visual motion area, and posterior superior temporal sulcus. Our findings reveal the heterogeneity of social cognition representation within the TPJ.


title: The runaway evolution of SARS-CoV-2 leading to the highly evolved Delta strain

abstract: In new epidemics after the host shift, the pathogens may experience accelerated evolution driven by novel selective pressures. When the accelerated evolution enters a positive feedback loop with the expanding epidemics, the pathogens runaway evolution may be triggered. To test this possibility in COVID-19, we analyze the extensive databases and identify 5 major waves of strains, one replacing the previous one in 2020 - 2021. The mutations differ entirely between waves and the number of mutations continues to increase, from 3-4 to 21-31. The latest wave is the Delta strain which accrues 31 new mutations to become highly prevalent. Interestingly, these new mutations in Delta strain emerge in multiple stages with each stage driven by 6 - 12 coding mutations that form a fitness group. In short, the evolution of SARS-CoV-2 from the oldest to the youngest wave, and from the earlier to the later stages of the Delta wave, is a process of acceleration with more and more mutations. The global increase in the viral population size (M(t), at time t) and the mutation accumulation (R(t)) may have indeed triggered the runaway evolution in late 2020, leading to the highly evolved Alpha and then Delta strain. To suppress the pandemic, it is crucial to break the positive feedback loop between M(t) and R(t), neither of which has yet to be effectively dampened by late 2021. New waves beyond Delta, hence, should not be surprising.

category: evolutionary biology


title: Cucumeropsis mannii seed oil (CMSO) ameliorates adipokines dysfunction and dyslipidemia in male Wistar rats exposed to Bisphenol-A

abstract: Bisphenol-A (BPA) and its analog are extensively utilized in the production of plastics which are rather ubiquitous in our environment. At high temperatures, BPA is leached into water and food packed in plastic containers. This research investigated the ameliorative effects of CMSO on adipokines dysfunction and dyslipidemia in male Wistar rats exposed to Bisphenol-A. thirty-six (36) albino rats weighing 100 - 200 g were randomly assigned into six (6) different experimental groups of controls (1, 2, and 3) and the tests (4, 5, and 6). Group 1 was given only 1 ml of olive oil, group 2 received 100 mg/Kg body weight (b.w) of BPA, group 3 was given 7.5 ml/Kg b.w of CMSO, groups 4, 5, and 6 received 100 mg/Kg b.w of BPA and 7.5, 5 and 2.5 mg/Kg b.w of CMSO respectively. CMSO and BPA were concurrently administered via oral intubation for periods of 42 days. Lipid profile and adipokines levels were determined in plasma and adipose tissue. BPA in male rats significantly (p<0.05) elevated the levels of cholesterol, triglycerides, LDL-C, liptin, and coronary and atherogenic risk indices in plasma and adipose tissue with reductions in HDL-C and adiponectin levels. BPA plus CMSO in male rats significantly (p<0.05) decreased the levels of cholesterol, triglycerides, LDL-C, liptin, and coronary and atherogenic risk indices with an elevation of HDL-C and adiponectin levels in both plasma and adipose tissue. These results suggest that CMSO could be useful in the management of cardiovascular-related diseases induced by BPA.


title: Influence of sequencing depth on bacterial classification and abundance in bacterial communities

abstract: Microbial diversity is the most abundant form of life. Next Generation Sequencing technologies provide the capacity to study complex bacterial communities, in which the depth and the bioinformatic tools can influence the results. In this work we explored two different protocols for bacterial classification and abundance evaluation, using 10 bacterial genomes in a simulated sample at different sequencing. Protocol A consisted of metagenome assembly with Megahit and Ray Meta and taxonomic classification with Kraken2 and Centrifuge. In protocol B only taxonomic classification. In both protocols, rarefaction, relative abundance and beta diversity were analyzed. In the protocol A, Megahit had a mean contig length of 1,128 and Ray Meta de 8,893 nucleotides. The number of species correctly classified in all depth assays were 6 out of 10 for protocol A, and 9 out of 10 using protocol B. The rarefaction analysis showed an overestimation of the number of species in almost all assays regardless of the protocol, and the beta diversity analysis results indicated significant differences in all comparisons. Protocol A was more efficient for diversity analysis, while protocol B estimated a more precise relative abundance. Our results do not allow us to suggest an optimal sequencing depth at specie level.

category: bioinformatics


title: An Antioxidant Polysaccharide from Ganoderma lucidum Induces Apoptotic Activity in Breast Cancer Cell Line

abstract: The purpose of this study is to elucidate the apoptotic activity of Ganoderma lucidum polysaccharide (GLP) in a human breast cancer cell line MCF-7 in vitro. According to DPPH assay, GLP showed a good antioxidant (IC50 value is 202.4 g/mL). Based on MTT assay, the results showed that GLP inhibits MCF-7 cells proliferation in a dose- and time-dependent manner (p<0.001). IC50 values of the cytotoxicity of GLP and doxorubin were 110.907 g/mL and 58.206 g/mL respectively. The results from the flow cytometry indicated that GLP could induce apoptotic activity through inducing the up-regulation of the Bax and Caspase-9 and the down-regulation of the BcL-2 in MCF-7 cells. At 2xIC50, GLP increased the early-apoptotic and dead cells of MCF-7 from 18.23% to 34.76% and 8.45% to16.34% respectively. In conclusion, the GLP shows anticancer activity against MCF-7 through preventing the proliferation and inducing the apoptosis of MCF-7 cells. Our data provide the potential molecular targets in cancer prevention and reveal the key barriers in the current anticancer drugs development.

category: pharmacology and toxicology


title: Cost-effectiveness of quadrivalent versus trivalent inactivated influenza vaccines for the Portuguese elderly population

abstract:
BackgroundQuadrivalent Inactivated Vaccine (QIV) is expected to replace Trivalent Inactivated Vaccine (TIV) over time. In Portugal, TIV is free of charge for risk groups, which include older adults. In its turn, QIV - which provides broader protection as it includes an additional lineage B strain - was introduced in Portugal in October 2018, but only from the influenza season 2019/20 was provided free of charge for the risk groups. This study evaluates the cost-effectiveness of switching from TIV to QIV from the National Health Service perspective in the Portuguese elderly mainland population ([≥] 65 years old).

MethodsA decision tree model was developed to compare TIV and QIV, based on Portuguese hospitalization data for the 2015/16 influenza season. The primary health economic outcome under consideration was the Incremental Cost-Effectiveness Ratio (ICER). In addition, a one-Way Sensitivity Analysis was performed to evaluate the impact of model parameters on the ICER; Probabilistic Sensitivity Analysis was also carried out to analyze the robustness of the base case results.

ResultsThe high cost of QIV (approximately three times the cost of TIV) would lead to a total increment of 5,283,047 {euro}, and the resulting ICER would be 26,403,007{euro}/QALY, mainly above the usual willingness-to-pay threshold.

ConclusionsFrom the National Health Service perspective, our findings reveal that QIV is not cost-effective for the Portuguese elderly population due to the high cost. If the QIV costs were the same as the TIV, then QIV would be cost-effective.

category: scientific communication and education


title: Isolation and Identification of taxonomically diverse bacterial endophytes from citrus in Punjab Pakistan

abstract: Citrus is an economically important fruit crop grown in all provinces of Pakistan, while Punjab accounting for 95 percent of total production due to its favorable climate for citrus production. Commercially grown varieties in Pakistan include sweet oranges, grapefruits, Mandarine, Lime, and lemon. The goal of this research was to see how diverse the cultivable bacterial populations are found in citrus cultivars. Out of 90 isolated cultures, 37 endophytic bacterial species and 15 different genera of bacteria were characterized based on morphological, biochemical, and molecular methods from citrus leaves. All the isolated bacteria were subjected to PCR amplification through 16S rDNA followed by sequencing. RDP base classification revealed that class Bacilli has the largest percentage of isolates, whereas class Alpha, Beta, and Gamma Proteobacteria have the lowest percentage among all genotypes used. According to the findings, the phylum Firmicutes contains a common genus (Brevibacterium, 1%; Enterococcus, 6%; Staphylococcus, 7% and Bacillus, 60%). Alpha (Rhizobium) beta (Burkholderia cepacia; Comamonas terigena) gamma Proteobacteria (Enterobacter hermachei (1%), Klebsiella pneumoniae (1%), Proteus mirabilis (8%), Pseudomonas aeruginosa (5%), Psychrobacter pulmonis and Yersinia molalretti (1%) respectively. These results revealed that cultivars of the plants might contribute to the structure and endophytic bacterial communities associated with citrus. Endophytes extracted from leaf samples of different citrus cultivars in Pakistan are reported for the first time. The idea of employing endophytes bacteria to produce enzymes stimulate plant growth, and its purpose as a biological control agent will be investigated in the future.


title: Pathogens in exacerbations of chronic rhinosinusitis differ in sensitivity to silver nanoparticles

abstract:
IntroductionThe significance of the microbiome in chronic rhinosinusitis (CRS) is not clear. Antimicrobials are recommended in acute exacerbations of the disease (AECRS). Increasing rates of antibiotic resistance stimulate research on alternative therapeutic options including silver nanoparticles (AgNPs), sometimes referred to as "colloidal silver". However, there are concerns regarding the safety of silver administration and the emergence of silver resistance. In this cross-sectional observational study, we assessed the sensitivity of sinonasal pathogens to AgNPs and compared it with the toxicity of AgNPs for nasal epithelial cells.

MethodNegatively charged AgNPs (13{+/-}5 nm) were obtained with the use of tannic acid. Minimal inhibitory concentrations (MIC) of the AgNPs were determined for pathogens isolated from patients with AECRS. Cytotoxicity was tested on human nasal epithelial cells line in vitro.

Results48 clinical isolates and 4 reference strains were included in the study (Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli, Klebsiella pneumoniae, Klebsiella oxytoca, Acinetobacter baumanii, Serratia marcescens, Enterobacter cloacae). The MIC values differed between isolates, even within the same species. All of the strains were sensitive to AgNPs in concentrations nontoxic to human cells during 24 hours exposition. However, 48h exposition to AgNPs increased toxicity to human cells, narrowing their therapeutic window and enabling 19% of pathogens to resist the AgNPs biocidal action.

ConclusionsAgNPs can potentially be used in intranasal drugs to treat most episodes of AECRS. Sensitivity testing may be necessary before application. Results of sensitivity testing for reference strains cannot be extrapolated to other strains of the same species.

ImportanceChronic rhinosinusitis is characterized by nasal congestion, discharge and facial pain. Bacterial infections cause acute exacerbations of the disease when the symptoms temporarily get worse. Exacerbations are usually treated with antibiotics. Increasing rates of antibiotic resistance stimulate research on alternative antibacterial agents. Various forms of silver, including silver nanoparticles, are suggested as intranasal antibacterial drugs. However, there are concerns regarding the safety of silver administration and reports of silver-resistant bacteria in various clinical settings. In our study, we attempted to assess whether silver nanoparticles could be used to treat patients with exacerbations of chronic rhinosinusitis. We found out that most of the bacteria isolated from the patients were sensitive to silver nanoparticles at concentrations that were still safe for the nasal epithelium. It suggests that silver nanoparticles could potentially be used as intranasal drugs but sensitivity testing should be obligatory before their administration to prevent the development of silver resistance.


title: Cross-species analysis of LZTR1 loss-of-function mutants demonstrates dependency to RIT1 orthologs

abstract: RAS GTPases are highly conserved proteins involved in the regulation of mitogenic signaling. We have previously described a novel Cullin 3 RING E3 ubiquitin ligase complex formed by the substrate adaptor protein LZTR1 that binds, ubiquitinates, and promotes proteasomal degradation of the RAS GTPase RIT1. In addition, others have described that this complex is also responsible for the ubiquitination of canonical RAS GTPases. Here, we have analyzed the phenotypes of LZTR1 loss-of-function mutants in both fruit flies and mice and have demonstrated biochemical dependency on their RIT1 orthologs. Moreover, we show that LZTR1 is haplosufficient in mice and that embryonic lethality of the homozygous null allele can be rescued by deletion of RIT1.


title: ifCNV: a novel isolation-forest-based package to detect copy number variations from NGS datasets

abstract:
Copy number variations (CNVs) are an essential component of genetic variation distributed across large parts of the human genome. CNV detection from next-generation sequencing data and artificial intelligence algorithms has progressed in recent years. However, only a few tools have taken advantage of machine learning algorithms for CNV detection, and none propose using artificial intelligence to automatically detect probable CNV-positive samples. Furthermore, in general, most CNV software that is developed for specific data types has sub-optimal reliability for routine practice. In addition, the most developed approach is to use a reference or normal dataset to compare with the samples of interest, and it is well known that selecting appropriate normal samples represents a challenging task which dramatically influences the precision of results in all CNV-detecting tools. With careful consideration of these issues, we propose here ifCNV, a new software based on isolation forests that creates its own reference, available in R and python with customisable parameters. ifCNV combines artificial intelligence using two isolation forests and a comprehensive scoring method to faithfully detect CNVs among various samples. It was validated using datasets from diverse origins (capture and amplicon, germline and somatic), and it exhibits high sensitivity, specificity and accuracy. ifCNV is a publicly available open-source software that allows the detection of CNVs in many clinical situations.

Key pointsO_LICopy number variation detection
C_LIO_LIMachine learning
C_LIO_LILocalisation scoring
C_LIO_LIBenchmark on various clinical situations and on various datasets
C_LIO_LIEasy-to-use R and Python open-source Package
C_LI


title: A multiwell-plate Caenorhabditis elegans assay for assessing the therapeutic potential of Bacteriophages against Clinical Pathogens

abstract:
In order to establish phage therapy as a standard clinical treatment for bacterial infections, testing of every phage to ensure the suitability and safety of the biological compound is required. While some issues have been addressed over recent years, standard and easy-to-use animal models to test phages are still rare. Testing of phages in highly suitable mammalian models such as mice is subjected to strict ethical regulations, while insect larvae such as the Galleria mellonella model suffers from batch-to-batch variations and requires manual operator skills to inject bacteria, resulting in unreliable experimental outcomes. A much simpler model is the nematode Caenorhabditis elegans which feeds on bacteria, a fast growing and easy to handle organism which can be used in high-throughput screening. In this study, two clinical bacterial strains of Escherichia coli, one Klebsiella pneumoniae and one Enterobacter cloacae strain were tested on the model system together with lytic bacteriophages that we isolated previously. We developed a liquid-based assay, in which the efficiency of phage treatment was evaluated using a scoring system based on microscopy and counting of the nematodes, allowing increasing statistical significance compared to other assays such as larvae or mice. Our work demonstrates the potential to use Caenorhabditis elegans to test the virulence of strains of Klebsiella pneumoniae, Enterobacter cloacae and EHEC/ EPEC as well as the efficacy of bacteriophages to treat or prevent infections, allowing a more reliable evaluation for the clinical therapeutic potential of lytic phages.

ImportanceValidating the efficacy and safety of phages prior to clinical application is crucial to see phage therapy in practice. Current animal models include mice and insect larvae, which pose ethical or technical challenges. This study examined the use of the nematode model organism, C. elegans as a quick, reliable and simple alternative for testing phages. The data shows that all the four tested bacteriophages can eliminate bacterial pathogens and protect the nematode from infections. Survival rates of the nematodes increased from <20% in the infection group to >90% in the phage treatment group. Even the nematodes with poly-microbial infections recovered during phage cocktail treatment. The use of C. elegans as a simple whole-animal infection model is a rapid and robust way to study the efficacy of phages before testing them on more complex model animals such as mice.


title: Structure of a bacterial ribonucleoprotein complex central to the control of cell envelope biogenesis

abstract: The biogenesis of the essential precursor of the bacterial cell envelope, glucosamine-6-phosphate (GlcN6P), is controlled through intricate post-transcription networks mediated by GlmZ, a small regulatory RNA (sRNA). GlmZ stimulates translation of the mRNA encoding GlcN6P synthetase in Escherichia coli, but when bound by the protein RapZ, it becomes inactivated through cleavage by the endoribonuclease RNase E. Here we report the cryoEM structure of the RapZ:GlmZ complex, revealing a complementary match of the protein tetrameric quaternary structure to an imperfect structural repeat in the RNA. The RNA is contacted mostly through a highly conserved domain of RapZ that shares deep evolutionary relationship with phosphofructokinase and suggests links between metabolism and riboregulation. We also present the structure of a pre-cleavage encounter intermediate formed between the binary RapZ:GlmZ complex and RNase E that reveals how GlmZ is presented and recognised for cleavage. The structures suggest how other encounter complexes might guide recognition and action of endoribonucleases on target transcripts, and how structured substrates in polycistronic precursors are recognised for processing.


title: Plant speciation in the Namib Desert: origin of a widespread derivative species from a narrow endemic

abstract:
BackgroundParapatric (or  budding) speciation is increasingly recognized as an important phenomenon in plant evolution but its role in extreme (e.g. desert) environments is poorly documented.

AimsTo test this speciation model in a hypothesized sister pair, the Southwest-North African disjunct Senecio flavus and its putative progenitor, the Namibian Desert endemic S. englerianus.

MethodsPhylogenetic inferences were combined with niche divergence tests, morphometrics, and experimental-genetic approaches. We also evaluated the potential role of an African Dry-Corridor (ADC) in promoting the hypothesized northward expansion of S. flavus (from Namibia), using palaeodistribution models.

ResultsBelonging to an isolated (potential  relict) clade, the two morphologically distinct species show pronounced niche divergence in Namibia and signs of digenic-epistatic hybrid incompatibility (based on F2 pollen fertility). The presence of  connate-fluked pappus hairs in S. flavus, likely increasing dispersal ability, is controlled by a single gene locus.

ConclusionsOur results provide support for a rare example of  budding speciation in which a wider-ranged derivative (S. flavus) originated at the periphery of a smaller-ranged progenitor (S. englerianus) in the Namib Desert region. The Southwest-North African disjunction of S. flavus could have been established by dispersal across intermediate ADC areas during periods of (Late) Pleistocene aridification.


title: Comparative plant transcriptome profiling of Arabidopsis and Camelina infested with Myzus persicae aphids acquiring circulative and non-circulative viruses reveals virus- and plant-specific alterations relevant to aphid feeding behavior and transmission

abstract:
BackgroundEvidence accumulates that plant viruses alter host-plant traits in ways that modify their insect vectors behavior. These alterations often enhance virus transmission, which has led to the hypothesis that these effects are manipulations caused by viral adaptation. However, the genetic basis of these indirect, plant-mediated effects on vectors and their dependence on the plant host and the mode of virus transmission is hardly known.

ResultsTranscriptome profiling of Arabidopsis thaliana and Camelina sativa plants infected with turnip yellows virus (TuYV) or cauliflower mosaic virus (CaMV) and infested with the common aphid vector Myzus persicae revealed strong virus- and host-specific differences in the gene expression patterns. CaMV infection caused more severe effects on the phenotype of both plant hosts than did TuYV infection, and the severity of symptoms correlated strongly with the proportion of differentially expressed genes, especially photosynthesis genes. Accordingly, CaMV infection modified aphid behavior and fecundity stronger than did infection with TuYV.

ConclusionsOverall, infection with CaMV - relying on the non-circulative transmission mode - tends to have effects on metabolic pathways with strong potential implications for insect-vector / plant-host interactions (e.g. photosynthesis, jasmonic acid, ethylene and glucosinolate biosynthetic processes), while TuYV - using the circulative transmission mode - alters these pathways only weakly. These virus-induced deregulations of genes that are related to plant physiology and defense responses might impact aphid probing and feeding behavior on both infected host plants, with potentially distinct effects on virus transmission.

category: plant biology


title: Asymmetric wall ingrowth deposition in Arabidopsis phloem parenchyma transfer cells is tightly associated with sieve elements

abstract:
In Arabidopsis, polarized deposition of wall ingrowths in phloem parenchyma (PP) transfer cells (TCs) occurs adjacent to cells of the sieve element/companion cell (SE/CC) complex. However, the spatial relationships between these different cell types in minor veins, where phloem loading occurs, are poorly understood. PP TC development and wall ingrowth localization were compared to other phloem cells in leaves of Col-0 and the transgenic lines AtSUC2::AtSTP9-GFP and AtSWEET11::AtSWEET11-GFP that identify CCs and PP respectively. The development of PP TCs in minor veins, indicated by deposition of wall ingrowths, proceeded basipetally in leaves. However, not all PP develop ingrowths and higher levels of wall ingrowth deposition occur in abaxial-compared to adaxial-positioned PP TCs. Furthermore, the deposition of wall ingrowths was exclusively initiated on and preferentially covered the PP TC/SE interface, rather than the PP TC/CC interface, and only occurred in PP cells that were adjacent to SEs. Collectively, these results demonstrate the dominant impact of SEs on wall ingrowth deposition in PP TCs and suggest the existence of two sub-types of PP cells in leaf minor veins. Compared to PP cells, PP TCs showed more abundant accumulation of AtSWEET11-GFP, indicating functional differences in phloem loading between PP and PP TCs.

HighlightWall ingrowth deposition in phloem parenchyma transfer cells of Arabidopsis leaf minor veins is initiated adjacent to sieve elements, rather than companion cells, and expands to preferentially cover this interface.


title: Hypoxia-Induced Ethanol Production in Arabidopsis Root Apex Cells Affects Endocytic Vesicle Recycling and F-Actin Organisation

abstract: Ethanol (EtOH) is a short-chain alcohol that is abundant in nature. EtOH is endogenously produced by plants under hypoxic conditions, and exogenously applied EtOH improves plant stress tolerance at low concentrations (<1%). However, no direct observations have shown how EtOH affects cellular events in plants. In intact Arabidopsis roots, 0.1% EtOH promoted reactive oxygen species production in root apex cells. EtOH also accelerated exocytic vesicle recycling and altered F-actin organisation, both of which are closely related to cell membrane properties. In addition to exogenous EtOH application, hypoxic treatment resulted in EtOH production in roots and degradation of the cross-wall actin cytoskeleton in root epidermal cells. We conclude that hypoxia-induced EtOH production affects endocytic vesicle recycling and associated signalling pathways.


title: Tobacco HY5, NtHY5, positively regulates flavonoid biosynthesis and enhances salt stress tolerance

abstract:
Plants have evolved complex signaling networks to regulate their growth and development. Some of these signaling components also play a crucial role in secondary metabolite biosynthesis. Among the signaling components identified to date, ELONGATED HYPOCOTYL 5 (HY5), a bZIP family transcription factor is the most investigated and known as the center of transcriptional network hub. However, HY5 has not been characterized from plants known to synthesize important secondary metabolites. In this study, based on homology search and phylogenetic analysis, HY5 has been identified from Nicotiana tobaccum, and characterized for its role in secondary plant product biosynthesis and stress response through developing overexpressing lines and CRISPR/Cas9-based knockout mutant plants. NtHY5 was able to complement the Arabidopsis thaliana hy5 mutant at molecular, morphological and biochemical levels. Overexpression of NtHY5 in tobacco led to the up-regulation of the phenylpropanoid pathway genes and enhanced the flavonoid content, whereas mutant plants had the opposite effect. Electrophoretic Mobility Shift Assay (EMSA) suggested that NtHY5 interacts with the promoter of NtMYB12, a transcription factor known to regulate flavonoid biosynthesis. In addition, NtHY5 enhanced the abiotic stress tolerance as evident by the salt tolerance ability of HY5 overexpressing lines by diminishing the ROS accumulation after salt treatment. These data provide credible evidence about the potential role of NtHY5 in light-mediated flavonoid biosynthesis, plant growth and abiotic stress tolerance in tobacco. The photomorphogenic mutant, Nthy5, developed in this study, will help in elucidating the role of the HY5 in different biological processes in tobacco.

One sentence summaryAnalysis of NtHY5 overexpressing and mutant plants suggests role of NtHY5 in flavonoid biosynthesis and salt stress tolerance.


title: The evolution of partner specificity in mutualisms

abstract: Mutualistic species vary in their level of partner specificity, which has important evolutionary, ecological, and management implications. Yet, the evolutionary mechanisms which underpin partner specificity are not fully understood. Most work on specialization focuses on the trade-off between generalism and specialism, where specialists receive more benefits from preferred partners at the expense of benefits from non-preferred partners, while generalists receive similar benefits from all partners. Because all mutualisms involve some degree of both cooperation and conflict between partners, we highlight that specialization to a mutualistic partner can be cooperative, increasing benefit to a focal species and a partner, or antagonistic, increasing resource extraction by a focal species from a partner. We devise an evolutionary game theoretic model to assess the evolutionary dynamics of cooperative specialization, antagonistic specialization, and generalism. Our model shows that cooperative specialization leads to bistability: stable equilibria with a specialist host and its preferred partner excluding all others. We also show that under cooperative specialization with spatial effects, generalists can thrive at the boundaries between differing specialist patches. Under antagonistic specialization, generalism is evolutionarily stable. We provide predictions for how a cooperation-antagonism continuum may determine the patterns of partner specificity that develop within mutualistic relationships.


title: An elite broadly neutralizing antibody protects SARS-CoV-2 Omicron variant challenge

abstract: The strikingly high transmissibility and antibody evasion of SARS-CoV-2 Omicron variant have posted great challenges on the efficacy of current vaccines and antibody immunotherapy.Here, we screened 34 BNT162b2-vaccinees and cloned a public broadly neutralizing antibody (bNAb) ZCB11 from an elite vaccinee. ZCB11 neutralized all authentic SARS-CoV-2 variants of concern (VOCs), including Omicron and OmicronR346K with potent IC50 concentrations of 36.8 and 11.7 ng/mL, respectively. Functional analysis demonstrated that ZCB11 targeted viral receptor-binding domain (RBD) and competed strongly with ZB8, a known RBD-specific class II NAb. Pseudovirus-based mapping of 57 naturally occurred single mutations or deletions revealed that only S371L resulted in 11-fold neutralization resistance, but this phenotype was not observed in the Omicron variant. Furthermore,prophylactic ZCB11 administration protected lung infection against both the circulating pandemic Delta and Omicron variants in golden Syrian hamsters. These results demonstrated that vaccine-induced ZCB11 is a promising bNAb for immunotherapy against pandemic SARS-CoV-2 VOCs.


title: Sequence assignment validation in cryo-EM models with checkMySequence

abstract:
The availability of new AI-based protein structure prediction tools radically changed the way cryo-EM maps are interpreted, but it has not eliminated the challenges of map interpretation faced by a microscopist. Models will continue to be locally rebuilt and refined using interactive tools. This inevitably results in occasional errors, among which register-shifts remain one of the most difficult to identify and correct. Here we introduce checkMySequence; a fast, fully automated and parameter-free method for detecting register-shifts in protein models built into cryo-EM maps. We show that the method can assist model building in cases where poorer map resolution hinders visual interpretation. We also show that checkMySequence could have helped avoid a widely discussed sequence register error in a model of SARS-CoV-2 RNA-dependent RNA polymerase that was originally detected thanks to a visual residue-by-residue inspection by members of the structural biology community.

SynopsisWe present a new method, checkMySequence, for fast and automated detection of register errors in protein models built into cryo-EM reconstructions.


title: Genomic perspectives on the emerging SARS-CoV-2 Omicron variant

abstract: A new variant of concern for SARS-CoV-2, Omicron (B.1.1.529), was designated by the World Health Organization on November 26, 2021. This study analyzed the viral genome sequencing data of 108 samples collected from patients infected with Omicron. First, we found that the enrichment efficiency of viral nucleic acids was reduced due to mutations in the region where the primers anneal to. Second, the Omicron variant possesses an excessive number of mutations compared to other variants circulating at the same time (62 vs. 45), especially in the Spike gene. Mutations in the Spike gene confer alterations in 32 amino acid residues, which was more than those observed in other SARS-CoV-2 variants. Moreover, a large number of nonsynonymous mutations occur in the codons for the amino acid residues located on the surface of the Spike protein, which could potentially affect the replication, infectivity, and antigenicity of SARS-CoV-2. Third, there are 53 mutations between the Omicron variant and its closest sequences available in public databases. Many of those mutations were rarely observed in the public database and had a low mutation rate. In addition, the linkage disequilibrium between these mutations were low, with a limited number of mutations (6) concurrently observed in the same genome, suggesting that the Omicron variant would be in a different evolutionary branch from the currently prevalent variants. To improve our ability to detect and track the source of new variants rapidly, it is imperative to further strengthen genomic surveillance and data sharing globally in a timely manner.


title: Computational study of the furin cleavage domain of SARS-CoV-2: delta binds strongest of extant variants

abstract: We demonstrate that AlphaFold and AlphaFold Multimer, implemented within the ColabFold suite, can accurately predict the structures of the furin enzyme with known six residue inhibitory peptides. Noting the similarity of the peptide inhibitors to polybasic furin cleavage domain insertion region of the SARS-CoV-2, which begins at P681, we implement this approach to study the wild type furin cleavage domain for the virus and several mutants. We introduce mutations in silico for alpha, omicron, and delta variants, for several sequences which have been rarely observed, for sequences which have not yet been observed, for other coronaviruses (NL63, OC43, HUK1a, HUK1b, MERS, and 229E), and for the H5N1 flu. We show that interfacial hydrogen bonds between the furin cleavage domain and furin are a good measure of binding strength that correlate well with endpoint binding free energy estimates, and conclude that among all candidate viral sequences studied, delta is near the very top binding strength within statistical accuracy. However, the binding strength of several rare sequences match delta within statistical accuracy. We find that the furin S1 pocket is optimized for binding arginine as opposed to lysine. This residue, typically at sequence position five, contains the most hydrogen bonds to the furin, and hydrogen bond count for just this residue shows a strong positive correlation with the overall hydrogen bond count. We demonstrate that the root mean square backbone C-alpha fluctuation of the first residue in the furin cleavage domain has a strong negative correlation with the interfacial hydrogen bond count. We show by considering the variation with the number of basic residues that the maximum mean number of interfacial hydrogen bonds expected is 15.7 at 4 basic residues.


title: Towards an optimal monoclonal antibody with higher binding affinity to the receptor-binding domain of SARS-CoV-2 spike proteins from different variants

abstract: A highly efficient and robust multiple scales in silico protocol, consisting of atomistic constant charge Molecular Dynamics (MD), constant-charge coarse-grain (CG) MD and constant-pH CG Monte Carlo (MC), has been used to study the binding affinities, the free energy of complexation of selected antigen-binding fragments of the monoclonal antibody (mAbs) CR3022 (originally derived from SARS-CoV-1 patients almost two decades ago) and 11 SARS-CoV-2 variants including the wild type. CR3022 binds strongly to the receptor-binding domain (RBD) of SARS-CoV-2 spike protein, but chooses a different site rather than the receptor-binding motif (RBM) of RBD, allowing its combined use with other mAbs against new emerging virus variants. Totally 235,000 mAbs structures were generated using the RosettaAntibodyDesign software, resulting in top 10 scored CR3022-RBD complexes with critical mutations and compared to the native one, all having the potential to block virus-host cell interaction. Of these 10 finalists, two candidates were further identified in the CG simulations to be clearly best against all virus variants, and surprisingly, all 10 candidates and the native CR3022 did exhibit a higher affinity for the Omicron variant with its highest number of mutations (15) of them all considered in this study. The multiscale protocol gives us a powerful rational tool to design efficient mAbs. The electrostatic interactions play a crucial role and appear to be controlling the affinity and complex building. Clearly, mAbs carrying a lower net charge show a higher affinity. Structural determinants could be identified in atomistic simulations and their roles are discussed in detail to further hint at a strategy towards designing the best RBD binder. Although the SARS-CoV-2 was specifically targeted in this work, our approach is generally suitable for many diseases and viral and bacterial pathogens, leukemia, cancer, multiple sclerosis, rheumatoid, arthritis, lupus, and more.


title: Virtually the same? Evaluating the effectiveness of remote undergraduate research experiences

abstract: In-person undergraduate research experiences (UREs) promote students integration into careers in life science research. In 2020, the COVID-19 pandemic prompted institutions hosting summer URE programs to offer them remotely, raising questions about whether undergraduates who participate in remote research can experience scientific integration. To address this, we investigated indicators of scientific integration for students who participated in remote life science URE programs in summer 2020. We found that these students experienced gains in their scientific self-efficacy and scientific identity similar to results reported for in-person UREs. We also found that these students perceived high benefits and low costs of doing research at the outset of their programs, and their perceptions did not change despite the remote circumstances. Yet, their perceptions differed by program, indicating that programs differentially affected students perceptions of the costs of doing research. Finally, we observed that students with prior research experience made greater gains in self-efficacy and identity, as well as in their perceptions of the alignment of their values with those of the scientific community, in comparison to students with no prior research experience. This finding suggests that additional programming may be needed for undergraduates with no prior experience to benefit from remote research.


title: SARS-CoV-2 Infection of Microglia Elicits Pro-inflammatory Activation and Apoptotic Cell Death

abstract:
Accumulating evidence suggests that severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection causes various neurological symptoms in coronavirus disease 2019 (COVID-19) patients. The most dominant immune cells in the brain are microglia. Yet, the relationship between neurological manifestations, neuroinflammation, and host immune response of microglia to SARS-CoV-2 has not been well characterized. Here, we report that SARS-CoV-2 can directly infect human microglia, eliciting M1-like pro-inflammatory responses, followed by cytopathic effects. Specifically, SARS-CoV-2 infected human microglial clone 3 (HMC3), leading to inflammatory activation and cell death. RNA-seq analysis also revealed that ER stress and immune responses were induced in the early and apoptotic processes in the late phase of viral infection. SARS-CoV-2-infected HMC3 showed the M1 phenotype and produced pro-inflammatory cytokines such as interleukin (IL)-1{beta}, IL-6, and tumour necrosis factor  (TNF-), but not the anti-inflammatory cytokine IL-10. After this pro-inflammatory activation, SARS-CoV-2 infection promoted both intrinsic and extrinsic death receptor-mediated apoptosis in HMC3. Using K18-hACE2 transgenic mice, murine microglia were also infected by intranasal inoculation of SARS-CoV-2. This infection induced the acute production of pro-inflammatory microglial IL-6 and TNF- and provoked a chronic loss of microglia. Our findings suggest that microglia are potential mediators of SARS-CoV-2-induced neurological problems and, consequently, can be targets of therapeutic strategies against neurological diseases in COVID-19 patients.

IMPORTANCERecent studies reported neurological manifestations and complications in COVID-19 patients, which are associated with neuroinflammation. As microglia are the dominant immune cells in brains, it needs to be elucidate the relationship between neuroinflammation and host immune response of microglia to SARS-CoV-2. Here, we suggest that SARS-CoV-2 can directly infect human microglia with cytopathic effect (CPE) using human microglial clone 3 (HMC3). The infected microglia were promoted to pro-inflammatory activation following apoptotic cell death. This pro-inflammatory activation was accompanied by the high production of pro-inflammatory cytokines, and led to neurotoxic-M1 phenotype polarization. In vivo, murine microglia were infected and produced pro-inflammatory cytokines and provoked a chronic loss using K18-hACE2 mice. Thus, our data present that SARS-CoV-2-infected microglia are potential mediators of neurological problems in COVID-19 patients. In addition, HMC3 cells are susceptible to SARS-CoV-2 and exhibit the CPE, which can be further used to investigate cellular and molecular mechanisms of neuroinflammation reported in COVID-19 patients.


title: SIRT5 is a proviral factor that interacts with SARS-CoV-2 Nsp14 protein

abstract: SARS-CoV-2 non-structural protein Nsp14 is a highly conserved enzyme necessary for viral replication. Nsp14 forms a stable complex with non-structural protein Nsp10 and exhibits exoribonuclease and N7-methyltransferase activities. Protein-interactome studies identified human sirtuin 5 (SIRT5) as a putative binding partner of Nsp14. SIRT5 is an NAD-dependent protein deacylase critical for cellular metabolism that removes succinyl and malonyl groups from lysine residues. Here we investigated the nature of this interaction and the role of SIRT5 during SARS-CoV-2 infection. We showed that SIRT5 stably interacts with Nsp14, but not with Nsp10, suggesting that SIRT5 and Nsp10 are parts of separate complexes. We found that SIRT5 catalytic domain is necessary for the interaction with Nsp14, but that Nsp14 does not appear to be directly deacylated by SIRT5. Furthermore, knock-out of SIRT5 or treatment with specific SIRT5 inhibitors reduced SARS-CoV-2 viral levels in cell-culture experiments. SIRT5 knock-out cells expressed higher basal levels of innate immunity markers and mounted a stronger antiviral response. Our results indicate that SIRT5 is a proviral factor necessary for efficient viral replication, which opens novel avenues for therapeutic interventions.


title: Differential mosquito attraction to humans is associated with skin-derived carboxylic acid levels

abstract: Female Aedes aegypti mosquitoes feed on human blood, which they use to develop their eggs. It has been widely noted that some people are more attractive to mosquitoes than others, but the mechanistic basis of this phenomenon is poorly understood. Here we tested mosquito attraction to skin odor collected from human subjects and identified people who are exceptionally attractive or unattractive to mosquitoes. Notably, these preferences were stable over several years, indicating consistent longitudinal differences in skin odor between subjects. We carried out gas chromatography/quadrupole time of flight-mass spectrometry to analyze the chemical composition of human skin odor in these subjects and discovered that highly attractive people produce significantly increased levels of carboxylic acids. Mosquitoes could reliably distinguish a highly attractive human from their weakly attractive counterparts unless we substantially diluted the odor of the "mosquito magnet." This is consistent with the hypothesis that odor concentration is a major driver of differential attraction, rather than the less-favored skin odor blend containing repellent odors, although these are not mutually- exclusive. Mosquitoes detect carboxylic acids with a large family of odor-gated ion channels encoded by the Ionotropic Receptor gene superfamily. Mutant mosquitoes lacking any of the Ionotropic Receptor (IR) co-receptors Ir8a, Ir25a, and Ir76b, were severely impaired in attraction to human scent but retained the ability to differentiate highly and weakly attractive people. The link between elevated carboxylic acids in "mosquito-magnet" human skin odor and phenotypes of genetic mutations in carboxylic acid receptors suggests that such compounds contribute to differential mosquito attraction. Understanding why some humans are more attractive than others provides insights into what skin odorants are most important to the mosquito and could inform the development of more effective repellents.


title: Structure-based identification of naphthoquinones and derivatives as novel inhibitors of main protease Mpro and papain-like protease PLpro of SARS-CoV-2

abstract: The worldwide COVID-19 pandemic caused by the coronavirus SARS-CoV-2 urgently demands novel direct antiviral treatments. The main protease (Mpro) and papain-like protease (PLpro) are attractive drug targets among coronaviruses due to their essential role in processing the polyproteins translated from the viral RNA. In the present work, we virtually screened 688 naphthoquinoidal compounds and derivatives against Mpro of SARS-CoV-2. Twenty-four derivatives were selected and evaluated in biochemical assays against Mpro using a novel fluorogenic substrate. In parallel, these compounds were also assayed with SARS-CoV-2 PLpro. Four compounds inhibited Mpro with half-maximal inhibitory concentration (IC50) values between 0.41 {micro}M and 66 {micro}M. In addition, eight compounds inhibited PLpro with IC50 ranging from 1.7 {micro}M to 46 {micro}M. Molecular dynamics simulations suggest stable binding modes for Mpro inhibitors with frequent interactions with residues in the S1 and S2 pockets of the active site. For two PLpro inhibitors, interactions occur in the S3 and S4 pockets. In summary, our structure-based computational and biochemical approach identified novel naphthoquinonal scaffolds that can be further explored as SARS-CoV-2 antivirals.


title: Unraveling membrane properties at the organelle-level with LipidDyn

abstract:
Cellular membranes are formed from many different lipids in various amounts and proportions depending on the subcellular localization. The lipid composition of membranes is sensitive to changes in the cellular environment, and their alterations are linked to several diseases, including cancer. Lipids not only form lipid-lipid interactions but also interact with other biomolecules, including proteins, profoundly impacting each other.

Molecular dynamics (MD) simulations are a powerful tool to study the properties of cellular membranes and membrane-protein interactions on different timescales and at varying levels of resolution. Over the last few years, software and hardware for biomolecular simulations have been optimized to routinely run long simulations of large and complex biological systems. On the other hand, high-throughput techniques based on lipidomics provide accurate estimates of the composition of cellular membranes at the level of subcellular compartments. The community needs computational tools for lipidomics and simulation data effectively interacting to better understand how changes in lipid compositions impact membrane function and structure. Lipidomic data can be analyzed to design biologically relevant models of membranes for MD simulations. Similar applications easily result in a massive amount of simulation data where the bottleneck becomes the analysis of the data to understand how membrane properties and membrane-protein interactions are changing in the different conditions. In this context, we developed LipidDyn, an in silico pipeline to streamline the analyses of MD simulations of membranes of different compositions. Once the simulations are collected, LipidDyn provides average properties and time series for several membrane properties such as area per lipid, thickness, diffusion motions, the density of lipid bilayers, and lipid enrichment/depletion. The calculations exploit parallelization and the pipelines include graphical outputs in a publication-ready form. We applied LipidDyn to different case studies to illustrate its potential, including membranes from cellular compartments and transmembrane protein domains. LipidDyn is implemented in Python and relies on open-source libraries. LipidDyn is available free of charge under the GNU General Public License from https://github.com/ELELAB/LipidDyn.


title: PDBspheres - a method for finding 3D similarities in local regions in proteins

abstract: We present a structure-based method for finding and evaluating structural similarities in protein regions relevant to ligand binding. PDBspheres comprises an exhaustive library of protein structure regions ("spheres") adjacent to complexed ligands derived from the Protein Data Bank (PDB), along with methods to find and evaluate structural matches between a protein of interest and spheres in the library. Currently, PDBspheres library contains more than 2 million spheres, organized to facilitate searches by sequence and/or structure similarity of protein-ligand binding sites or interfaces between interacting molecules. PDBspheres uses the LGA structure alignment algorithm as the main engine for detecting structure similarities between the protein of interest and library spheres. An all-atom structure similarity metric ensures that sidechain placement is taken into account in the PDBspheres primary assessment of confidence in structural matches. In this paper, we (1) describe the PDBspheres method, (2) demonstrate how PDBspheres can be used to detect and characterize binding sites in protein structures, (3) compare PDBspheres use for binding site prediction with seven other binding site prediction methods using a curated dataset of 2,528 ligand-bound and ligand-free crystal structures, and (4) use PDBspheres to cluster pockets and assess structural similarities among protein binding sites of the 4,876 structures in the "refined set" of PDBbind 2019 dataset. The PDBspheres library is made publicly available for download at https://proteinmodel.org/AS2TS/PDBspheres


title: SPITFIR(e): A supermaneuverable algorithm for restoring 2D-3D fluorescence images and videos,and background subtraction

abstract: While fluorescent microscopy imaging has become the spearhead of modern biology as it is able to generate long-term videos depicting 4D nanoscale cell behaviors, it is still limited by the optical aberrations and the photon budget available in the specimen and to some extend to photo-toxicity. A direct consequence is the necessity to develop flexible and "off-road" algorithms in order to recover structural details and improve spatial resolution, which is critical when pushing the illumination to the low levels in order to limit photo-damages. Moreover, as the processing of very large temporal series of images considerably slows down the analysis, special attention must be paid to the feasibility and scalability of the developed restoration algorithms. To address these specifications, we present a very flexible method designed to restore 2D-3D+Time fluorescent images and subtract undesirable out-of-focus background. We assume that the images are sparse and piece-wise smooth, and are corrupted by mixed Poisson-Gaussian noise. To recover the unknown image, we consider a novel convex and non-quadratic regularizer Sparse Hessian Variation) defined as the mixed norms which gathers image intensity and spatial second-order derivatives. This resulting restoration algorithm named SPITFIR(e) (SParse fIT for Fluorescence Image Restoration) utilizes the primal-dual optimization principle for energy minimization and can be used to process large images acquired with varied fluorescence microscopy modalities. It is nearly parameter-free as the practitioner needs only to specify the amount of desired sparsity (weak, moderate, high). Experimental results in lattice light sheet, stimulated emission depletion, multifocus microscopy, spinning disk confocal, and wide-field microscopy demonstrate the generic ability of the SPITFIR(e) algorithm to efficiently reduce noise and blur, and to subtract undesirable fluorescent background, while avoiding the emergence of deconvolution artifacts.