SufB intein splicing in Mycobacterium tuberculosis is influenced by two remote conserved N-extein Histidines Inteins are auto-processing domains that implement a multi-step biochemical reaction termed protein splicing, marked by cleavage and formation of peptide bonds. They excise from a precursor protein, generating a functional protein via covalent bonding of flanking exteins. We report the kinetic study of splicing and cleavage reaction in [Fe-S] cluster assembly protein SufB from Mycobacterium tuberculosis. Although it follows a canonical intein splicing pathway, distinct features are added by extein residues present in the active site. Sequence analysis identified two conserved histidines in the N-extein region; His-5 and His-38. Kinetic analyses of His-5Ala and His-38Ala SufB mutants exhibited significant reductions in splicing and cleavage rates relative to the SufB wild-type precursor protein. Structural analysis and molecular dynamics simulations suggested that Mtu SufB displays a unique mechanism where two remote histidines work concurrently to facilitate N-terminal cleavage reaction. His-38 is stabilized by the solvent-exposed His-5, and can impact N-S acyl shift by direct interaction with the catalytic Cys1. Development of inteins as biotechnological tools or as pathogen specific novel antimicrobial targets requires a more complete understanding of such unexpected roles of conserved extein residues in protein splicing.
RSC and GRFs confer promoter directionality by limiting divergent noncoding transcription The directionality of gene promoters - the ratio of protein-coding over divergent noncoding transcription - is highly variable and regulated. How promoter directionality is controlled remains poorly understood. Here, we show that the chromatin remodelling complex RSC and general regulatory factors (GRFs) dictate promoter directionality by attenuating divergent transcription. At gene promoters that are highly directional, depletion of RSC leads to a relative increase in divergent noncoding transcription and thus a decrease in promoter directionality. We find that RSC facilitates nucleosome positioning upstream in promoters at the sites of divergent transcription. These highly directional promoters are also enriched for the binding of GRFs such as Reb1 and Abf1. Ectopic targeting of divergent transcription initiation sites with GRFs or the dCas9 protein suppresses divergent transcription. Our data suggest that RSC and GRFs play a pervasive role in limiting divergent transcription. We propose that any DNA binding factor, when stably associated with cryptic transcription start sites, form barriers for repressing divergent transcription. Our study provides an explanation as to why certain promoters are more directional than others.
TCF11 Has a Potent Tumor-Repressing Effect than Its Prototypic Nrf1α by Definition of both Similar yet Different Regulatory Profiles, with a Striking Disparity from Nrf2 Nrf1 and Nrf2, as two principal CNC-bZIP transcription factors, regulate similar but different targets involved in a variety of biological functions for maintaining cell homeostasis and organ integrity. Of note, the unique topobiological behavior of Nrf1 makes its functions more complicated than Nrf2, because it is allowed for alternatively transcribing and selectively splicing to yield multiple isoforms (e.g., TCF11, Nrf1). In order to gain a better understanding of their similarities and differences in distinct regulatory profiles, all four distinct cell models for stably expressing TCF11, TCF11{Delta}N, Nrf1 or Nrf2 have been herein established by an Flp-In T-REx-293 system and then identified by transcriptomic sequencing. Further analysis revealed that Nrf1 and TCF11 have similar yet different regulatory profiles, although both contribute basically to positive regulation of their co-targets, which are disparate from those regulated by Nrf2. Such disparity in those gene regulation by Nrf1 and Nrf2 was further corroborated by scrutinizing comprehensive functional annotation of their specific and/or common target genes. Conversely, the mutant TCF11{Delta}N, resulting from a deletion of the N-terminal amino acids 2-156 from TCF11, resembles Nrf2 with the largely consistent structure and function. Interestingly, our further experimental evidence demonstrates that TCF11 acts as a potent tumor-repressor relative to Nrf1, albeit both isoforms possess a congruous capability to prevent malignant growth of tumor and upregulate those genes critical for improving the survival of patients with hepatocellular carcinoma. The emergence of highly fit SARS-CoV-2 variants accelerated by epistasis and caused by recombination The SARS-CoV-2 pandemic recently entered an alarming new phase with the emergence of the variants of concern (VOC) and understanding their biology is paramount to predicting future ones. Current efforts mainly focus on mutations in the spike glycoprotein (S), but changes in other regions of the viral proteome are likely key. We analyzed more than 900,000 SARS-CoV-2 genomes with a computational systems biology approach including a haplotype network and protein structural analyses to reveal lineage-defining mutations and their critical functional attributes. Our results indicate that increased transmission is promoted by epistasis, i.e., combinations of mutations in S and other viral proteins. Mutations in the non-S proteins involve immune-antagonism and replication performance, suggesting convergent evolution. Furthermore, adaptive mutations appear in geographically disparate locations, suggesting that either independent, repeat mutation events or recombination among different strains are generating VOC. We demonstrate that recombination is a stronger hypothesis, and may be accelerating the emergence of VOC by bringing together cooperative mutations. This emphasizes the importance of a global response to stop the COVID-19 pandemic. Intermittent compression increases flow through a gel: a mechanical model perhaps relevant to flow of cerebrospinal fluid Exchange of molecules between cerebrospinal fluid (CSF) and brain cells contributes to brain function and protection from dementia. Despite widespread acceptance of the  glymphatic theory, the route by which CSF is brought close enough to the neural tissue for solutes to be exchanged by extracellular diffusion is not entirely clear. Exogenous molecules injected into CSF are observed to reach the basement lamina that surrounds the dense capillary network. Transport of solutes by diffusion along the basement lamina, a gel of macromolcules about 100 nm thick, would be too slow; bulk flow in a static geometry would require unphysiologically high pressures. However, it is known that the pulsation of blood aids transport of CSF, and we hypothesized that this is because the pulsation intermittently squeezes the pericapillary lamina. In a primitive mimicry, we have tested whether intermittent squeezing increases flow through an agar gel. In all but one of 216 tests, pulsation caused a reversible increase, sometimes by a factor of 100 or more. The enhancement was greatest for frequencies 5-11 Hz and, over the tested range of pressure heads (20 - 50 cmH2O), was greatest for the lowest pressure. The results suggest a reason why exercise slows the aging of the brain. Naming Human Diseases: Ethical Principles of Curating Exclusive Substitute for Inopportune Nosology BackgroundIn the medical sphere, understanding naming conventions strengthen the integrity of naming human diseases remains nominal rather than substantial yet. Since the current nosology-based standard for human diseases could not offer a one-size-fits-all corrective mechanism, many idiomatic but flawed names frequently appear in scientific literature and news outlets at the cost of sociocultural impacts.

ObjectiveWe attempt to examine the ethical oversights of current naming practices and propose heuristic rationales and approaches to determine a pithy name instead of an inopportune nosology.

MethodsFirst, we examined the compiled global online news volumes and emotional tones on some inopportune nosology like German measles, Middle Eastern Respiratory Syndrome, Spanish flu, Hong Kong flu, and Huntingtons disease in the wake of COVID-19. Second, we prototypically scrutinize the lexical dynamics and pathological differentials of German measles and common synonyms by leveraging the capacity of the Google Books Ngram Corpus. Third, we demonstrated the empirical approaches to curate an exclusive substitute for an anachronistic nosology German measles based on deep learning models and post-hoc explanations.

ResultsThe infodemiological study shows that the public informed the offensive names with extremely negative tones in textual and visual narratives. The findings of the historiographical study indicate that many synonyms of German measles did not survive, while German measles became an anachronistic usage, and rubella has taken the dominant place since 1994. The PubMedBERT model could identify rubella as a potential substitution for German measles with the highest semantic similarity. The results of the semantic drift experiments further indicate that rubella tends to survive during the ebb and flow of semantic drift.

ConclusionsOur findings indicate that the nosological evolution of anachronistic names could result in sociocultural impacts without a corrective mechanism. To mitigate such impacts, we introduce some ethical principles for formulating an improved naming scheme. Based on deep learning models and post-hoc explanations, our illustrated experiments could provide hallmark references to the remedial mechanism of naming practices and pertinent credit allocations. Genome streamlining, proteorhodopsin, and organic nitrogen metabolism in freshwater nitrifiers Microbial nitrification is a critical process governing nitrogen availability in aquatic systems. Freshwater nitrifiers have received little attention, leaving many unanswered questions about their taxonomic distribution, functional potential, and ecological interactions. Here, we reconstructed genomes to infer the metabolism and ecology of free-living picoplanktonic nitrifiers across the Laurentian Great Lakes, a connected series of five of Earths largest lakes. Surprisingly, ammonia oxidizing Bacteria (AOB) related to Nitrosospira dominated over ammonia oxidizing Archaea (AOA) at nearly all stations, with distinct ecotypes prevailing in the transparent, oligotrophic upper lakes compared to Lakes Erie and Ontario. Unexpectedly, one ecotype of Nitrosospira encodes proteorhodopsin, which could enhance survival in conditions where ammonia oxidation is inhibited or substrate limited. Nitrite oxidizing Bacteria (NOB) Ca. Nitrotoga and Nitrospira fluctuated in dominance, with the latter prevailing in deeper, less productive basins. Genome reconstructions reveal highly reduced genomes and features consistent with genome streamlining, along with diverse adaptations to sunlight and oxidative stress and widespread capacity for organic nitrogen use. Our findings expand the known functional diversity of nitrifiers and establish their ecological genomics in large lake ecosystems. By elucidating links between microbial biodiversity and biogeochemical cycling, our work also informs ecosystem models of the Laurentian Great Lakes, a critical freshwater resource experiencing rapid environmental change.

ImportanceMicroorganisms play critical roles in Earths nitrogen cycle. In lakes, microorganisms called nitrifiers derive energy from reduced nitrogen compounds. In doing so, they transform nitrogen into a form that can ultimately be lost to the atmosphere by a process called denitrification, which helps mitigate nitrogen pollution from fertilizer runoff and sewage. Despite their importance, freshwater nitrifiers are virtually unexplored. To understand their diversity and function, we reconstructed genomes of freshwater nitrifiers across some of Earths largest freshwater lakes, the Laurentian Great Lakes. We discovered several new species of nitrifiers specialized for clear low nutrient waters, and distinct species in comparatively turbid Lake Erie. Surprisingly, one species may be able to harness light energy using a protein called proteorhodopsin, despite the fact that nitrifiers typically live in deep dark water. Our work reveals unique biodiversity of the Great Lakes and fills key gaps in our knowledge of an important microbial group, the nitrifiers. Myoparr-associated and -independent multiple roles of heterogeneous nuclear ribonucleoprotein K during skeletal muscle cell differentiation RNA-binding proteins (RBPs) regulate cell physiology via the formation of ribonucleic-protein complexes with coding and non-coding RNAs. RBPs have multiple functions in the same cells; however, the precise mechanism through which their pleiotropic functions are determined remains unknown. In this study, we revealed the multiple inhibitory functions of heterogeneous nuclear ribonucleoprotein K (hnRNPK) for myogenic differentiation. We first identified hnRNPK as a lncRNA Myoparr binding protein. Gain- and loss-of-function experiments showed that hnRNPK repressed the expression of myogenin at the transcriptional level. The hnRNPK-binding region of Myoparr was required to repress myogenin expression. Moreover, hnRNPK repressed the expression of a set of genes coding for aminoacyl-tRNA synthetases in a Myoparr-independent manner. Mechanistically, hnRNPK regulated the eIF2/Atf4 pathway, one branch of the intrinsic pathways of the endoplasmic reticulum sensors, in differentiating myoblasts. Thus, our findings demonstrate that hnRNPK plays lncRNA-associated and - independent multiple roles during myogenic differentiation, indicating that the analysis of lncRNA-binding proteins will be useful for elucidating both the physiological functions of lncRNAs and the multiple functions of RBPs. Cell-specific Bioorthogonal Tagging of Glycoproteins Altered glycosylation is an undisputed corollary of cancer development. Understanding these alterations is paramount but hampered by limitations underlying cellular model systems. For instance, the intricate interactions between tumour and host cannot be adequately recapitulated in monoculture of tumour-derived cell lines. More complex co-culture models usually rely on sorting procedures for proteome analyses and rarely capture the details of protein glycosylation. Here, we report a strategy termed Bio-Orthogonal Cell line-specific Tagging of Glycoproteins (BOCTAG). Cells are equipped by transfection with an artificial biosynthetic pathway that transforms bioorthogonally tagged sugars into the corresponding nucleotide-sugars. Only transfected cells incorporate bioorthogonal tags into glycoproteins in the presence of non-transfected cells. We employ BOCTAG as an imaging technique and to annotate cell-specific glycosylation sites in mass spectrometry-glycoproteomics. We demonstrate application in co-culture and mouse models, allowing for profiling of the glycoproteome as an important modulator of cellular function. Living with high potassium: a balance between nutrient acquisition and stress signaling during K-induced salt stress O_LIHigh potassium (K) in the growth medium is more toxic to plants than Na at similar concentrations. However, the molecular mechanisms underlying plant responses to K-induced salt stress are virtually unknown.
C_LIO_LIWe examined Arabidopsis thaliana and its extremophyte relative Schrenkiella parvula, using a comparative multi-omics approach to identify cellular processes affected by excess K and understand which deterministic regulatory pathways are active to avoid tissue damage while sustaining growth.
C_LIO_LIA. thaliana showed limited capacity to curb excess K accumulation and prevent nutrient depletion contrasting to S. parvula which could limit excess K accumulation without restricting nutrient uptake. Facilitated by a targeted transcriptomic response, promoting nitrogen uptake along with other key nutrients and uninterrupted N assimilation into primary metabolites during excess K-stress allowed S. parvula to boost its antioxidant and osmolyte pools concurrently leading to sustained growth. Antithetically, A. thaliana showed transcriptional responses indicative of a poor balance between stress signaling, increased ROS levels, and reduced photosynthesis, subsequently leading to inhibited growth.
C_LIO_LIThe ability to regulate independent nutrient uptake and a coordinated transcriptomic response to avoid non-specific stress signaling are two main deterministic steps towards building stress resilience to excess K+-induced salt stress.
C_LI SERCA function is impaired in skeletal and cardiac muscles from young DBA/2J mdx mice The DBA/2J (D2) mdx mouse has emerged as a more severe model of Duchenne muscular dystrophy when compared to the traditional C57BL/10 (C57) mdx mouse. Here, we questioned whether sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA) function would differ in muscles from young D2 and C57 mdx mice. In gastrocnemius muscles, both D2- and C57 mdx mice exhibited signs of impaired Ca2+ uptake, however, this was more severe in D2 mdx mice. Maximal SERCA activity was lowered only in D2 mdx gastrocnemius muscles and not C57 mdx muscles. Furthermore, in the left ventricle and diaphragm, Ca2+ uptake was impaired in C57 mdx muscles with lowered rates of Ca2+ uptake compared with C57 WT mice, whereas in muscles from D2 mdx mice, rates of Ca2+ uptake were unattainable due to the severe impairments in their ability to transport Ca2+. Overall, our study demonstrates that SERCA function is drastically impaired in young D2 mdx mice. The compound of baicalein, wogonin, and oroxylin-A inhibiting EMT in A549 cell line via PI3K/AKT-TWIST1-glycolysis pathway Non-small cell lung cancer (NSCLC) is a worldwide disease with high morbidity and mortality, which is most derived from its metastasis. Some researches show that the epithelial-mesenchymal transition (EMT) process promotes lung cancer cells migration and invasion, leading to NSCLC metastasis. Total Flavonoid Aglycones Extract (TFAE) isolated from Scutellaria baicalensis was reported to inhibit tumor growth and induce apoptosis. In this study, we found that baicalein, wogonin, and oroxylin-A were the active compounds of TFAE. After reconstructing with these three compounds (baicalein (65.8%), wogonin (21.2%), and oroxylin-A (13.0%)), the reconstructed TFAE (reTFAE) present inhibitory effect on the EMT process of A549 cells. Then, bioinformatic technology was employed to elucidate the potential pharmacodynamic mechanism network of reTFAE. We identified the relationship between reTFAE and PI3K/Akt signaling pathways, with TWIST1 as the key protein. LY294002, the inhibitor of PI3K/Akt signaling pathway, and knock-down TWIST1 could significantly enhance the efficacy of reTFAE, with increasing expression of epithelial markers and decreasing expression of mesenchymal markers in A549 cells at the same time. Furthermore, stable isotope dimethyl-labeled proteomics technology was conducted to complement the follow-up mechanism that the EMT-inhibition process may be realized through glycolysis pathway. In conclusion, we claim that TWIST1-targeted flavonoids could provide a new strategy to inhibit EMT progress for the treatment of NSCLC. Modeling miRNA-driven post-transcriptional regulation by using exon-intron split analysis (EISA) in pigs The contribution of microRNAs (miRNAs) to mRNA regulation has often been explored by post hoc selection of downregulated genes and determining whether they harbor binding sites for miRNAs of interest. This approach, however, does not discriminate whether these mRNAs are also downregulated at the transcriptional level. Here, we have characterized the transcriptional and post-transcriptional changes of mRNA expression in two porcine tissues: gluteus medius muscle of fasted and fed Duroc gilts and adipose tissue of lean and obese Duroc-Gottingen minipigs. Exon-intron split analysis (EISA) of RNA-seq data allowed us to identify downregulated mRNAs with high post-transcriptional signals in fed or obese states, and we assessed whether they harbor binding sites for upregulated miRNAs in any of these two physiological states. We found 26 downregulated mRNAs with high post-transcriptional signals in the muscle of fed gilts and 21 of these were predicted targets of upregulated miRNAs also in the fed state. For adipose tissue, 44 downregulated mRNAs in obese minipigs displayed high post-transcriptional signals, and 25 of these were predicted targets of miRNAs upregulated in the obese state. These results suggest that the contribution of miRNAs to mRNA repression is more prominent in the skeletal muscle system. Finally, we identified several genes that may play relevant roles in the energy homeostasis of the pig skeletal muscle (DKK2 and PDK4) and adipose (SESN3 and ESRRG) tissues. By differentiating transcriptional from post-transcriptional changes in mRNA expression, EISA provides a valuable view about the regulation of gene expression, complementary to canonical differential expression analyses. Task-independent metrics of computational hardness predict human cognitive performance The survival of human organisms depends on our ability to solve complex tasks in the face of limited cognitive resources. However, little is known about the factors that drive the complexity of those tasks. Here, building on insights from computational complexity theory, we quantify the computational hardness of cognitive tasks using a set of task-independent metrics related to the computational resource requirements of individual instances of a task. We then examine the relation between those metrics and human behavior and find that they predict both time spent on a task as well as accuracy in three canonical cognitive tasks. Our findings demonstrate that performance in cognitive tasks can be predicted based on generic metrics of their inherent computational hardness.

TeaserThe ability of humans to solve cognitive tasks is affected by generic mathematical properties of problems related to their computational complexity. Can double PPO mutations exist in the same allele and are such mutants functional? BackgroundResistance to PPO-inhibiting herbicides is primarily endowed by target site mutations at the PPO2 gene that compromise binding of the herbicide to the catalytic domain. In Amaranthus ssp. PPO2, the most prevalent target mutations are deletion of the G210 codon and the R128G and G339A substitutions. These mutations strongly affect the dynamic of the PPO2 binding pocket resulting in reduced affinity with the ligand. Here we investigated the likelihood of co-occurrence of the most widespread target site mutations in the same ppo2 allele.

ResultsPlants carrying R128G+/+ {Delta}G210+/-, where + indicates presence of the mutation, were crossed with each other. The ppo2 of the offspring was subjected to pyrosequencing and E. coli- based Sanger sequencing to determine mutation frequencies and allele co-occurrence. The data show that R128G {Delta}G210 can occur only in one allele; the second allele carries only one mutation. Double mutation in both alleles is less likely because of significant loss of enzyme activity. The segregation of offspring populations derived from a cross between heterozygous plants carrying {Delta}G210 G399A also showed no co-occurrence in the same allele. The offspring exhibited the expected mutation distribution patterns with few exceptions.

ConclusionsHomozygous double-mutants are not physiologically viable. Double-mutant plants can only exist in a heterozygous state. Alternatively, if two mutations are detected in one plant, each mutation would occur in a separate allele.

NomenclaturePalmer amaranth, Amaranthus palmeri S. Wats.; protoporphyrinogen oxidase, PPO; tall waterhemp, Amaranthus tuberculatus (Moq.) J.D.Sauer First evidence of in vitro cytotoxic effects of marine microlitter on Merluccius merluccius and Mullus barbatus, two Mediterranean commercial fish species Marine litter is composed mainly of plastics and is recognized as a serious threats to marine ecosystems. Ecotoxicological approaches have started elucidating the potential severity of microplastics (MPs) in controlled laboratory studies with pristine materials but no information exist on marine environmental microlitter as a whole. Here, we characterized the litter in the coastal Northern Tyrrhenian sea and in the stomach of two fish species of socio-economic importance, and exposed primary cell cultures of mucosal and lymphoid organs to marine microlitter for evaluating possible cytotoxic effects. An average of 0.30 {+/-} 0.02 microlitter items m-3 was found in water samples. FT-IR analysis revealed that plastic particles, namely HDPE, polyamide and polypropylene were present in 100% and 83.3% of Merluccius merluccius and Mullus barbatus analyzed, which overall ingested 14.67 {+/-} 4.10 and 5.50 {+/-} 1.97 items/individual, respectively. Moreover, microlitter was confirmed as a vector of microorganisms. Lastly, the apical end-point of viability was found to be significantly reduced in splenic cells exposed in vitro to two microlitter conditions. Considering the role of the spleen in the mounting of adaptive immune responses, our results warrant more in-depth investigations for clarifying the actual susceptibility of these two species to anthropogenic microlitter.

HighlightsO_LI0.30 {+/-} 0.02 microlitter items m-3 were found at the surface of coastal Northern Tyrrhenian sea
C_LIO_LI14.67 {+/-} 4.10 and 5.50 {+/-} 1.97 items/individual were retrieved from the stomach of hakes and mullets
C_LIO_LIThe ingested microlitter contained plastic items
C_LIO_LIMicrolitter was validated as a carrier of bacteria, fungi and flagellates
C_LIO_LISplenic cells exposed to two microlitter conditions for 72 hours suffered cytotoxicity
C_LI Interfacial tension driven open droplet microfluidics We developed an open channel droplet microfluidic system that autonomously generates droplets at low Ca (~10-4-10-3) by leveraging competing hydrostatic and capillary pressure. With only our open channel polytetrafluoroethylene (PTFE) device, pipettes, and commercially available carrier fluid, we produce hundreds of microliter droplets; tubing, electronics, or pumps are not required, making droplet technology feasible for research labs without external flow generators. Furthermore, we demonstrated conceptual applications that showcase the process of droplet generation, splitting, transport, incubation, mixing, and sorting in our system. Unlike conventional droplet microfluidics, the open nature of the device enables the use of physical tools such as tweezers and styli to directly access the system; with this, we developed a new method of droplet sorting and transfer that capitalizes on the Cheerios effect, the aggregation of buoyant objects along a liquid interface. Our platform offers enhanced usability, direct access to the droplet contents, easy manufacturability, compact footprint, and high customizability. This design is a first step in exploring the space of power-free open droplet microfluidic systems and provide design rules for similar channel designs. High-throughput characterization of photocrosslinker-bearing ion channel variants to map residues critical for function and pharmacology Incorporation of non-canonical amino acids (ncAAs) can endow proteins with novel functionalities, such as crosslinking or fluorescence. In ion channels, the function of these variants can be studied with great precision using standard electrophysiology, but this approach is typically labor intensive and low throughput. Here, we establish a high-throughput protocol to conduct functional and pharmacological investigations of ncAA-containing hASIC1a (human acid-sensing ion channel 1a) variants in transiently transfected mammalian cells. We introduce three different photocrosslinking ncAAs into 103 positions and assess the function of the resulting 309 variants with automated patch-clamp (APC). We demonstrate that the approach is efficient and versatile, as it is amenable to assessing even complex pharmacological modulation by peptides. The data show that the acidic pocket is a major determinant for current decay and live-cell crosslinking provides insight into the hASIC1a-psalmotoxin-1 interaction. Further, we provide evidence that the protocol can be applied to other ion channels, such as P2X2 and GluA2 receptors. We therefore anticipate the approach to enable future APC-based studies of ncAA-containing ion channels in mammalian cells. Simultaneous suppression of ribosome biogenesis and Tor activation by TRIM-NHL proteins promotes terminal differentiation Proper tissue development and homeostasis depends on the balance between growth and terminal differentiation, but the mechanisms coordinating these processes remain elusive. Accumulating evidence indicates that ribosome biogenesis (RiBi) and protein synthesis, two of the most energy-consuming cellular processes sustaining growth, are tightly regulated and yet can be uncoupled during stem cell fate transitions. Here, using the Drosophila adult female germline stem cell (GSC) and larval neuroblast (NB) systems, we show that Mei-P26 and Brat, two Drosophila TRIM-NHL paralogs of the mammalian TRIM32 protein family, are responsible for uncoupling RiBi and protein synthesis during GSC and NB differentiation, respectively. Mei-P26 and Brat modify the metabolism of differentiating cells by activating the Target of rapamycin (Tor) kinase to promote translation, while concomitantly repressing RiBi. Depletion of Mei-P26 or Brat results in excessive cellular growth and defective terminal differentiation, which can be rescued by ectopic activation of Tor together with suppression of RiBi. Our results indicate that the anabolic reprogramming established by TRIM-NHL activity by uncoupling RiBi and translation activities creates the conditions required for terminal differentiation. Identifying complex sequence patterns in massive omics data with a variable-convolutional layer in deep neural network Motif identification is among the most common and essential computational tasks for bioinformatics and genomics. Here we proposed a novel convolutional layer for deep neural network, named Variable Convolutional (vConv) layer, for effective motif identification in high-throughput omics data by learning kernel length from data adaptively. Empirical evaluations on DNA-protein binding and DNase footprinting cases well demonstrated that vConv-based networks have superior performance to their convolutional counterparts regardless of model complexity. Meanwhile, vConv could be readily integrated into multi-layer neural networks as an "in-place replacement" of canonical convolutional layer. All source codes are freely available on GitHub for academic usage. From adverse to beneficial - contrasting dietary effects of freshwater mixotrophs on zooplankton O_LIThe importance of mixotrophic algae as key bacterivores in microbial food webs is increasingly acknowledged, but their effects on the next trophic level remain poorly understood. Their high stoichiometric food quality is contrasted by anti-grazing strategies.
C_LIO_LIWe tested the quality of freshwater mixotrophs as prey for zooplankton, using four non-colonial chrysophyte species and a cryptophyte as a high quality reference food. We (1) analyzed the stoichiometric and biochemical (fatty acid) composition of the mixotrophs, and (2) quantified their dietary effects on Daphnia longispina survival.
C_LIO_LISurvival of D. longispina significantly depended on the identity of species provided as food, ranging from higher to lower as compared to starvation. This was not reflected in differences in cellular stoichiometry or fatty acid profiles of the mixotrophs. We suggest that toxicity may be the driver for the observed differences.
C_LIO_LIGeneralization of the dietary effects of mixotrophic chrysophytes does not appear straightforward. Besides fundamental species-specific differences, potential toxic effects may vary depending on environmental cues or physiological strategies. Notably in our study, Ochromonas tuberculata, a species previously reported to be deleterious turned out to be a beneficial food source, in terms of enabling high survival of D. longispina.
C_LIO_LIWe challenge the generality of the assumption that chrysophytes are of low value as food for zooplankton. We recommend that future studies test how environmental conditions and physiological strategies shape the quality of mixotrophs as food for consumers at higher trophic levels, specifically focusing on effects of dietary toxicity.
C_LI Design, immunogenicity and efficacy of a Pan-SARS-CoV-2 synthetic DNA vaccine Here we have employed SynCon(R) design technology to construct a DNA vaccine expressing a pan-Spike immunogen (INO-4802) to induce broad immunity across SARS-CoV-2 variants of concern (VOC). Compared to WT and VOC-matched vaccines which showed reduced cross-neutralizing activity, INO-4802 induced potent neutralizing antibodies and T cell responses against WT as well as B.1.1.7, P.1, and B.1.351 VOCs in a murine model. In addition, a hamster challenge model demonstrated that INO-4802 conferred superior protection following intranasal B.1.351 challenge. Protection against weight loss associated with WT, B.1.1.7, P.1 and B.1.617.2 challenge was also demonstrated. Vaccinated hamsters showed enhanced humoral responses against VOC in a heterologous WT vaccine prime and INO-4802 boost setting. These results demonstrate the potential of the pan-SARS-CoV-2 vaccine, INO-4802 to induce cross-reactive immune responses against emerging VOC as either a standalone vaccine, or as a potential boost for individuals previously immunized with WT-matched vaccines. Diverse stem-chondrichthyan oral structures and evidence for an independently acquired acanthodid dentition The teeth of sharks famously form a series of transversely-organised files with conveyor-belt replacement that are borne directly on the jaw cartilages, in contrast to the dermal plate-borne dentition of bony fishes that undergoes site-specific replacement. A major obstacle in understanding how this system evolved is the poorly understood relationships of the earliest chondrichthyans and the profusion of morphologically and terminologically diverse bones, cartilages, splints and whorls that they possess. Here we use tomographic methods to investigate mandibular structures in several early branching  acanthodian-grade stem-chondrichthyans. We show that the dentigerous jaw bones of disparate genera of ischnacanthids are united by a common construction, being growing bones with non-shedding dentition. Mandibular splints, which support the ventro-lateral edge of the Meckels cartilage in some taxa, are formed from dermal bone and may be an acanthodid synapomorphy. We demonstrate that the teeth of Acanthodopsis are borne directly on the mandibular cartilage and that this taxon is deeply nested within an edentulous radiation, representing an unexpected independent origin of teeth. Many or even all of the range of unusual oral structures may be apomorphic, but they should nonetheless be considered when building hypotheses of tooth and jaw evolution, both in chondrichthyans and more broadly. Mapping the subcortical connectome using in vivo diffusion MRI: feasibility and reliability Tractography combined with regions of interest (ROIs) has been used to non-invasively study the structural connectivity of the cortex as well as to assess the reliability of these connections. However, the subcortical connectome (subcortex to subcortex) has not been comprehensively examined, in part due to the difficulty of performing tractography in this complex and compact region. In this study, we performed an in vivo investigation using tractography to assess the feasibility and reliability of mapping known connections between structures of the subcortex using the test-retest dataset from the Human Connectome Project (HCP). We further validated our observations using a separate unrelated subjects dataset from the HCP. Quantitative assessment was performed by computing tract densities and spatial overlap of identified connections between subcortical ROIs. Further, known connections between structures of the basal ganglia and thalamus were identified and visually inspected, comparing tractography reconstructed trajectories with descriptions from tract-tracing studies. Our observations demonstrate both the feasibility and reliability of using a data-driven tractography-based approach to map the subcortical connectome in vivo. Common Dynamic Determinants Govern Quorum Quenching Activity in N-terminal Serine Hydrolases Growing concerns about microbial antibiotic resistance have motivated extensive research into ways of overcoming antibiotic resistance. Quorum quenching (QQ) processes disrupt bacterial communication via quorum sensing, which enables bacteria to sense the surrounding bacterial cell density and markedly affects their virulence. Due to its indirect mode of action, QQ is believed to exert limited pressure on essential bacterial functions and may thus avoid inducing resistance. Although many enzymes display QQ activity against various bacterial signaling molecules, their mechanisms of action are poorly understood, limiting their potential optimization as QQ agents. Here we evaluate the capacity of three N-terminal serine hydrolases to degrade N-acyl homoserine lactones that serve as signaling compounds for Gram-negative bacteria. Using molecular dynamics simulations of the free enzymes and their complexes with two signaling molecules of different lengths, followed by quantum mechanics/molecular mechanics molecular dynamics simulations of their initial catalytic steps, we clarify the molecular processes underpinning their QQ activity. We conclude that all three enzymes degrade bacterial signaling molecules via similar reaction mechanisms. Moreover, we experimentally confirmed the activity of two penicillin G acylases from Escherichia coli (ecPGA) and Achromobacter spp. (aPGA), adding these biotechnologically well-optimized enzymes to the QQ toolbox. We also observed enzyme- and substrate-dependent differences in the catalytic actions of these enzymes, arising primarily from the distinct structures of their acyl-binding cavities and the dynamics of their molecular gates. As a consequence, the first reaction step catalyzed by ecPGA with a longer substrate had an elevated energy barrier because its shallow acyl binding site could not accommodate a productive substrate-binding configuration. Conversely, aPGA in complex with both substrates exhibited unfavorable energetics in both reaction steps due to the dynamics of the residues gating the acyl binding cavity entrance. Finally, the energy barriers of the second reaction step catalyzed by Pseudomonas aeruginosa acyl-homoserine lactone acylase with both substrates were higher than in the other two enzymes due to the unique positioning of Arg297{beta} in this enzyme. The discovery of these dynamic determinants will guide future efforts to design robust QQ agents capable of selectively controlling virulence in resistant bacterial species. The multi-level phenotypic impact of synonymous substitutions: heterologous gene expression in human cells. Redundancy in the genetic code allows for differences in transcription and/or translation efficiency between sequences carrying synonymous polymorphisms, potentially leading to phenotypic changes. It is commonly admitted that the evolution codon usage bias ("CUB", the over-representation of certain codons in a genome, a gene or in positions along a gene) are driven by a combination of neutral and selective processes, but their relative contribution is a matter of debate, especially in mammals. Particularly, integrative studies quantifying the phenotypic impact of CUB at different molecular and cellular levels are lacking. Here we report a multiscale analysis of the effects of synonymous codon recoding during heterologous gene expression in human cells.

Six synonymous versions of the shble antibiotic resistance gene were generated, fused to a fluorescent reporter, and independently expressed in HEK293 cells. Multiscale phenotype was assessed by means of: i) mRNA-to-DNA and protein-to-mRNA ratios for each shble version; ii) cellular fluorescence, using flow cytometry, as a proxy for single-cell level expression; and iii) real-time cell proliferation in absence or presence of antibiotic, as a proxy for the cell fitness.

We show that differences in CUB strongly impact the molecular and the cellular phenotype: i) they result in large differences in mRNA and in protein levels, as well in mRNA-to-protein ratio; ii) they introduce splicing events not predicted by current algorithms; iii) they lead to reproducible phenotypic heterogeneity; iv) they lead to a trade-off between the benefit of antibiotic resistance and the burden of heterologous expression.

We interpret that CUB modulate mRNA availability and suitability for translation in human cells, leading to differences in protein levels and eventually eliciting phenotypic differences.

AUTHOR SUMMARYThe genetic code is redundant, with several codons encoding for the same amino acid. These synonymous codons are not used with equal frequencies. Instead, codon usage bias (CUB) varies between species, genes, and even positions along a gene. At each of these levels, CUB are shaped by the overall balance between mutational biases and selection forces. To shed light on the molecular mechanisms underlying molecular and organism phenotypes, integrative studies quantifying the phenotypic impact of CUB at different levels of biological integration are necessary. Here, we monitored the multiscale changes induced by six synonymous versions of an antibiotic resistance gene independently expressed in a human cell line. We show that: 1. both mRNA levels, protein levels, and their ratios are affected by CUB; 2. potential effects on mRNA (splicing and availability for translation) seem to constitute the main level of action; 3. cell fitness is severely impacted by a trade-off between the burden of heterologous expression and the benefits of antibiotic resistance. This integrative study provides new insights on translation regulation and the associated phenotypic impact in human cells, associated to CU.