Miracle-dz/UniFAR-FTUs huggingface dataset:
Efficient precision genome editing of Chlamydomonas reinhardtii with CRISPR/Cas

CRISPR/Cas genome engineering in the unicellular green algal model Chlamydomonas reinhardtii has until now only been applied to targeted gene disruption, whereas scar-less knock-in transgenesis has generally been considered infeasible. We have developed highly efficient homology-directed knock-in mutagenesis in cell-walled strains of Chlamydomonas. Our method allows scarless integration of fusion tags and sequence modifications of near arbitrary proteins without need for a preceding mutant line.
Specific recognition and ubiquitination of slow-moving ribosomes by human CCR4-NOT

Eukaryotic messenger RNA (mRNA) decay is generally initiated by removal of the 3’ polyadenosine (poly(A)) tail by the CCR4-NOT complex. Yeast Ccr4-Not binds and ubiquitinates ribosomes stalled on mRNAs with sub-optimal codons to trigger deadenylation and decay of the associated transcript. However, the mammalian ortholog of the E3 ubiquitin ligase subunit, CNOT4, is not a constitutive component of human CCR4-NOT. It therefore remains unclear how the mammalian deadenylation machinery targets stalled ribosomes. Here, we reconstitute translational stalling on non-optimal codons. We find that human CCR4-NOT recognizes translating mammalian ribosomes and is required for stable CNOT4 association. Our cryoEM structure reveals that the CNOT3 subunit detects slow translation and locks the L1 stalk of the ribosome in an open conformation to impede further elongation. Using crosslinking mass spectrometry, we show that CNOT4 and CNOT11 also bind in the vicinity of the E site. Overall, our work defines how CCR4-NOT enforces ribosomal stalling in response to low codon optimality.
Identification of a microbial sub-community from the feral chicken gut that reduces Salmonella colonization and improves gut health in a gnotobiotic chicken model

A complex microbial community in the gut generally prevent the colonization of enteric pathogens such as Salmonella. Because of the high complexity, several species or combination of species in the gut can confer colonization resistance. To gain a better understanding of the colonization resistance against Salmonella enterica, we isolated a library of 1,300 bacterial strains from feral chicken gut microbiota which represented a total of 51 species. Using a co-culture assay, we screened the representative species from this library and identified 30 species that inhibited Salmonella enterica Typhimurium. To improve the Salmonella inhibition capacity, from a pool of fast-growing species, we formulated 66 bacterial blends, each of which composed of 10 species. Bacterial blends were more efficient in inhibiting Salmonella as compared to individual species. The blend that showed maximum inhibition (Mix10) also inhibited other serotypes of Salmonella frequently found in poultry. The in vivo effect of Mix10 was examined in a gnotobiotic and conventional chicken model. The Mix10 consortium reduced Salmonella colonization, intestinal tissue damage and inflammation in both models. Cell free supernatant of Mix10 did not show Salmonella inhibition, indicating that Mix10 inhibits Salmonella through either nutritional competition or reinforcement of host immunity. Out of ten species, three species in Mix10 did not colonize while three species constituted more than 70% of the community. Two of these species represents previously uncultured bacteria. Our approach could be used as a high-throughput screening system to identify additional bacterial sub-communities that confer colonization resistance against enteric pathogens and its effect on the host. Importance Salmonella colonization in chicken and human infections originating from Salmonella- contaminated poultry is a significant problem. Poultry has been identified as the most common food linked to enteric pathogen outbreaks in the United States. Since multi-drug resistant Salmonella often colonize chicken and cause human infections, methods to control Salmonella colonization in poultry are needed. The method we describe here could form the basis of developing gut microbiota-derived bacterial blends as a microbial ecosystem therapeutic against Salmonella.
From vacuole to cytosol – Disruptive invasion triggers cytosolic release of Salmonella Paratyphi A and subsequent cytosolic motility favors evasion of xenophagy

Salmonella enterica is a common foodborne, facultative intracellular enteropathogen. Typhoidal S. enterica serovars like Paratyphi A (SPA) are human restricted and cause a severe systemic disease, while many S. enterica serovars like Typhimurium (STM) have broad host range, and in human hosts usually lead to self-limiting gastroenteritis. There are key differences between typhoidal and non-typhoidal Salmonella in pathogenesis, but underlying mechanisms remain largely unknown. Several genes encoding Salmonella pathogenicity island (SPI) effector proteins are absent or pseudogenes in SPA. Expression of virulence and metabolism genes show differential expression compared to STM. The intracellular transcriptomic architecture and phenotypes during presence in epithelial cells were recently described. Surprisingly, induction of motility, flagella and chemotaxis genes showed distinct expression patterns in intracellular SPA vs. STM and led to cytosolic motility of SPA. This study applies single cell microscopic analyses approaches to investigate the triggers and cellular consequences of cytosolic motility. Live cell imaging (LCI) revealed that SPA invades host cells in a highly cooperative manner. Extensive membrane ruffling at the invasion site leads to increased membrane damage in the nascent SCV with subsequent cytosolic release. After release into the cytosol, motile bacteria showed same velocity as under culture conditions used for infection. Reduced capture of SPA by autophagosomal membranes was observed by LCI and electron microscopy. Our results reveal flagella-mediated cytosolic motility as possible xenophagy evasion mechanism that could drive disease progression and contributes to dissemination of invasion-primed SPA during systemic infection. Importance Intracellular pathogens are commonly adapted to life in host cells either in a pathogen-containing vacuole, or free in host cell cytosol. However, transitions between these lifestyles are possible and demand specific adaptations, especially to avoid recognition and killing by host cell-autonomous immune defense. Salmonella enterica serovar Paratyphi A (SPA) belongs to typhoidal Salmonella able to cause live-threatening systemic infections in human hosts. We observed that SPA invades host cells in a way that often results in damage of the nascent vacuole and release of SPA in host cell cytosol. Here, SPA deploy flagella-mediated motility for rapid locomotion within infected cells. We demonstrate on single cell level that flagella-mediated motility enables evasion of xenophagic capture and control by the host cells. SPA uses a novel form of intracellular motility to successfully colonize human host cells.
Identification of pre-existing microbiome and metabolic vulnerabilities to escalation of oxycodone self-administration and identification of a causal role of short-chain fatty acids in addiction-like behaviors

The gut brain axis is thought to play a role in behavior and physiological responses through chemical, immunological, and metabolite signaling. Antibiotics, diet, and drugs can alter the transit time of gut contents as well as the makeup of the microbiome. Heterogeneity in genetics and environment are also well-known factors involved in the initiation and perpetuation of substance use disorders. Few viable genetic or biological markers are available to identify individuals who are at risk of escalating opioid intake. Primarily, the addiction field has focused on the nervous system, limiting the discovery of peripheral factors that contribute to addiction. To address this gap, we characterized the microbiome before and after drug exposure, and after antibiotics depletion in male and female heterogenous stock rats to determine if microbiome constituents are protective of escalation. We hypothesized that individuals that are prone to escalation of opioid self-administration will have distinct microbial and metabolic profiles. The fecal microbiome and behavioral responses were measured over several weeks of oxycodone self-administration and after antibiotic treatment. Antibiotic treatment reduces circulating short-chain fatty acids (SCFA) by depleting microbes that ferment fiber into these essential signaling molecules for the gut-brain axis. Depletion of the microbiome increased oxycodone self-administration in a subpopulation of animals (Responders). Supplementation of SCFAs in antibiotic depleted animals decreased elevated oxycodone self-administration. Phylogenetic functional analysis reveals distinct metabolic differences in the subpopulations of animals that are sensitive to antibiotic depletion and animals rescued by SCFA supplementation. In conclusion, this study identifies pre-existing microbiome and metabolic vulnerabilities to escalation of oxycodone self-administration, demonstrates that escalation of oxycodone self-administration dysregulates the microbiome and metabolic landscape, and identifies a causal role of short-chain fatty acids in addiction-like behaviors.
Design of A 1:12 Power Divider at 5 GHz for Ground Surveillance Radar Application

Radio detection and ranging (radar) is a system that can be used to detect and map an object by using electromagnetic waves and the echoes. Radar technology is a system that can help state institutions and apparatus in mastering and securing a country. One of the most important components in a radar is an antenna. The function of antenna is to transmit and receive the signals that will detect the presence of objects around the radar. The antenna commonly used in a radar usually has the high gain, and one of the techniques to enhance the antenna gain is using an antenna array. An antenna array has an important part that is the power divider. In this paper, we will discuss regarding the 1:12 power divider for ground surveillance radar at C-band frequency. We use CST Studio Suite to design and simulate the power divider. In this design and simulation, we use FR-4 with a dielectric constant of 4.5, and the thickness is 1.6 as a substrate. According to the simulation results, it can be seen that this power divider has the bandwidth 200 MHz with the center frequency 5 GHz, VSWR is less than 1.5, and all the output ports are in phase.
Hydrogel Assisted Double Molding of 3D-Print Enables Prestress Regulation of Micro-Heart Muscle Physiology

Tissue engineered in-vitro models are an essential tool in biomedical research. Tissue geometry is a key determinant of function, but controlling geometry of micro-scale tissues remains a challenge. We developed a new double molding approach that allows precise replication of high-resolution stereolithographic prints into poly(dimethylsiloxane), facilitating rapid design iterations and highly parallelized sample production. Hydrogels are used as an intermediary mold, and gel mechanical properties including crosslink density predict replication fidelity. We leveraged this approach to study the effects of geometry on the electrophysiology of miniaturized heart muscles engineered from human induced pluripotent stem cells (iPSC). Geometries predicted to increase tissue prestress globally affected cardiomyocyte structure and tissue electrophysiology. Strikingly, pharmacologic studies revealed a prestress threshold is required for sodium channel function. Analysis of RNA and protein levels suggest electrophysiology changes were related to post-transcriptional and potentially post-translational changes of the gap-junction protein Connexin 43 and the sodium channel Nav1.5.
Decreased Ventral Tegmental Area CB1R Signaling Reduces Sign Tracking and Shifts Cue–Outcome Dynamics in Rat Nucleus Accumbens

Sign-tracking (ST) rats show enhanced cue sensitivity before drug experience that predicts greater discrete cue-induced drug seeking compared with goal-tracking or intermediate rats. Cue-evoked dopamine in the nucleus accumbens (NAc) is a neurobiological signature of sign-tracking behaviors. Here, we examine a critical regulator of the dopamine system, endocannabinoids, which bind the cannabinoid receptor-1 (CB1R) in the ventral tegmental area (VTA) to control cue-evoked striatal dopamine levels. We use cell type-specific optogenetics, intra-VTA pharmacology, and fiber photometry to test the hypothesis that VTA CB1R receptor signaling regulates NAc dopamine levels to control sign tracking. We trained male and female rats in a Pavlovian lever autoshaping (PLA) task to determine their tracking groups before testing the effect of VTA → NAc dopamine inhibition. We found that this circuit is critical for mediating the vigor of the ST response. Upstream of this circuit, intra-VTA infusions of rimonabant, a CB1R inverse agonist, during PLA decrease lever and increase food cup approach in sign-trackers. Using fiber photometry to measure fluorescent signals from a dopamine sensor, GRABDA (AAV9-hSyn-DA2m), we tested the effects of intra-VTA rimonabant on NAc dopamine dynamics during autoshaping in female rats. We found that intra-VTA rimonabant decreased sign-tracking behaviors, which was associated with increases in NAc shell, but not core, dopamine levels during reward delivery [unconditioned stimulus (US)]. Our results suggest that CB1R signaling in the VTA influences the balance between the conditioned stimulus-evoked and US-evoked dopamine responses in the NAc shell and biases behavioral responding to cues in sign-tracking rats. SIGNIFICANCE STATEMENT Substance use disorder (SUD) is a chronically relapsing psychological disorder that affects a subset of individuals who engage in drug use. Recent research suggests that there are individual behavioral and neurobiological differences before drug experience that predict SUD and relapse vulnerabilities. Here, we investigate how midbrain endocannabinoids regulate a brain pathway that is exclusively involved in driving cue-motivated behaviors of sign-tracking rats. This work contributes to our mechanistic understanding of individual vulnerabilities to cue-triggered natural reward seeking that have relevance for drug-motivated behaviors.
EvoBind: in silico directed evolution of peptide binders with AlphaFold

Currently, there is no accurate method to computationally design peptide binders towards a specific protein interface using only a target structure. Experimental methods such as phage display can produce strong binders, but it is impossible to know where these bind without solving the structures. Using AlphaFold2 (AF) and other AI methods to distinguish true binders has proven highly successful but relies on the availability of binding scaffolds. Here, we develop EvoBind, an in silico directed-evolution platform based on AF that designs peptide binders towards an interface using only sequence information. We show that AF can distinguish between native and mutated peptide binders using the plDDT score and find that AF adapts the receptor interface structure to the binders during optimisation. We analyse previously designed minibinder proteins and show that AF can distinguish designed binders from non-binders. We compare ELISA ratios of different peptide binders and find the affinity can not be distinguished among binders, possibly due to varying binding sites and low AF confidence. We test the recovery of binding motifs and find that up to 75% of motifs are recovered. In principle, EvoBind can be used to design binders towards any interface conditioned on if AF can predict these. We expect that EvoBind will aid experimentalists substantially, providing a starting point for further laboratory analysis and optimisation. We hope that the use of AI-based methods will come to make binder design significantly cheaper and more accurate in tackling unmet clinical needs. EvoBind is freely available at: https://colab.research. google.com/github/patrickbryant1/EvoBind/blob/master/EvoBind.ipynb
A Prime/Pull RR2/CXCL11 Therapeutic Vaccine that Bolsters the Number and Function of Dorsal Root Ganglia Tissue-Resident HSV-Specific CD8+ TRM Cells Protects Latently Infected Guinea Pigs from Recurrent Genital Herpes

Reactivation of herpes simplex virus type 2 (HSV-2) from latently infected dorsal root ganglia (DRG) and subsequent virus shedding in the genital tract trigger recurrent genital herpes. Memory CD8+ T cells play a critical role in preventing HSV-2 reactivation from latently infected DRG, thus reducing recurrent genital lesions. The role of T-cell attracting chemokines in promoting CD8+ T cell protective immunity in recurrent genital herpes remains to be fully elucidated. In this study, we investigated whether and how the CXCL11/CXCR3 pathway affects the frequency and function of DRG-resident CD8+ T cells and the severity of recurrent genital herpes. Latently infected guinea pigs were primed with the HSV-1 RR2 protein, delivered intramuscularly with CpG/Alum adjuvants, and the induced T cells were “pulled” from the periphery into the latently infected DRG using T-cell attracting CXCL11 chemokine, delivered to DRG. In the guinea pigs that received the prime/pull vaccine, we detected a significant increase in both the number and function of tissue-resident IFN-γ+CD103+CD44+CXCR3+CD8+ TRM cells that infiltrated healed sites of the vaginal mucosa (VM) and DRG tissues. This was associated with a significant decrease in virus shedding and a reduction in both the severity and frequency of recurrent genital herpes lesions. In contrast, in the guinea pigs that received the RR2 vaccine alone, we detected fewer functional CD8+ TRM cells and no reduction in the severity of recurrent genital herpes. These findings highlight the role of the CXCL11/CXCR3 chemokine pathway in shaping tissue-resident CD8+ TRM cell protective immunity against recurrent genital herpes. IMPORTANCE Recurrent genital herpes is a common sexually transmitted disease worldwide. Currently, no FDA-approved therapeutic vaccines are available. In the present study, we used HSV-2 latently infected guinea pig to investigate a novel therapeutic prime/pull vaccine strategy based on priming T cells systemically, with a recombinantly expressed herpes envelope and tegument protein RR2 and “pulling” primed T cells into the tissues of latently infected ganglia with the T-cell-attracting chemokine, CXCL11. We discovered that this RR2/CXCL11 prime/pull vaccine elicited a significant reduction in virus shedding and a decrease in both the severity and frequency of recurrent genital herpes sores. This protection correlated with increased numbers of functional tissue-resident IFN-γ+CD103+CD44+CXCR3+CD8+ TRM cells that infiltrate healed sites of the VM tissues and DRG. Our findings shed light on the role of TRM cells in protection against recurrent genital herpes and propose the prime/pull therapeutic vaccine as a new strategy against genital herpes. TWEET The present study presents a novel RR2/CXCL11 prime/pull therapeutic vaccine that elicited a significant reduction in virus shedding and a decrease in both the severity and frequency of recurrent genital herpes sores.
Predicting the specific substrate for transmembrane transport proteins using BERT language model

Transmembrane transport proteins play a vital role in cells’ metabolism by the selective passage of substrates through the cell membrane. Metabolic network reconstruction requires transport reactions that describe the specific substrate transported as well as the metabolic reactions of enzyme catalysis. In this paper, we apply BERT (Bidirectional Encoder Representations from Transformers) language model for protein sequences to predict one of 12 specific substrates. Our UniProt-ICAT-100 dataset is automatically constructed from UniProt using the ChEBI and GO ontologies to identify 4,112 proteins transporting 12 inorganic anion or cation substrates. We classified this dataset using three different models including Logistic Regression with an MCC of 0.81 and accuracy of 97.5%; Feed-forward Neural Networks classifier with an MCC of 0.88 and accuracy of 98.5%. Our third model utilizes a Fine-tuned BERT language model to predict the specific substrate with an MCC of 0.95 and accuracy of 99.3% on an independent test set.
On improving the earthing quality in ship electric energy systems

ABSTRACT This paper presents a contribution in improving the earthing system of ship electric grids, which affects the phenomena developed in highly unbalanced operating conditions like the single – and two-phase faults. Furthering previous work, an alternative to ungrounded earthing scheme is introduced, namely the grounding via a conditionally varying resistance. Within this framework, the mathematical expressions with respect to zero sequence impedance, of voltages and currents under study are yielded. Furthermore, as a means to easily evaluate the quality of earthing, appropriate indices are introduced. Thus, it is shown that the effect of the earthing scheme in fault conditions can be adjusted based upon certain design limits.
A new method for the structural optimisation of a common type of square wing plate used in the wave glider

ABSTRACT The wave glider is widely used in marine engineering. As one of the most crucial components of the wave glider, the wing plate system significantly affects the utilisation efficiency of wave energy. The aim of this research is to optimise the structure of a kind of the square wing plate, which possesses a bad hydrodynamic performance. For the purpose, a new structural optimisation method was developed, in which the optimal sweepback angle and leading edge radius of square wing plate were calculated. Structural dynamic sensitivity analysis and matrix perturbation theory were combined to establish a proper guidance for structural modification, which could avoid blindness during structural modification. Then, dynamics characteristics of the optimised wing plate were investigated based on finite element analysis and experimental modal testing. Finally, the hydrodynamic performance of the optimised wing plate was evaluated. Results show that the newly proposed optimisation method is feasible and effective, and it also can be used for wing plates in other underwater vehicle applications.
Dynamically Positioned Ship Steering Making Use of Backstepping Method and Artificial Neural Networks

Abstract The article discusses the issue of designing a dynamic ship positioning system making use of the adaptive vectorial backstepping method and RBF type artificial neural networks. In the article, the backstepping controller is used to determine control laws and neural network weight adaptation laws. The artificial neural network is applied at each time instant to approximate nonlinear functions containing parametric uncertainties. The proposed control system does not require precise knowledge of the model of ship dynamics and external disturbances, it also eliminates the problem of analytical determination of the regression matrix when designing the control law with the aid of the adaptive backstepping procedure.