Non-essential Functions Research Articles

Functional preference and spatial response of urban renewal entities in Beijing

In the process of rapid urban renewal, quantitative research on functional preferences and spatial layout norms of multi-type urban renewal entities is crucial for optimizing spatial governance and promoting orderly control of urban renewal. We investigate urban renewal in Beijing from 2006 to 2021 using directional distribution, spatial autocorrelation, and hotspot analysis. The findings are as follows: (1) The functional preference of each type of entity differs. While most private companies and other profit-oriented entities concentrate on producer services and residential functions, policy-oriented entities dominate the urban regeneration of public service activities. (2) Every entity shows a significant agglomeration characteristic, yet exhibits a differentiated spatial response pattern. Those entities with stronger leasing ability prefer the central zone while others are the opposite. (3) Diverse types of entities have differentiated spatiotemporal evolutions. The spatial directional divergence of state-owned enterprises and scientific research institutes has increased between 2006 and 2021. This is mainly influenced by the policy of relieving Beijing of nonessential functions. However, the rest entities show a balanced spatial development trend with the difference in each direction becoming smaller. Furthermore, the center of gravity of profit-oriented entities has shifted from the suburbs to the central zone due to a combination of administrative and market forces, whereas the center of gravity of policy-oriented entities tends to move outward from the central zone.

S2VSNet: Single stage V-shaped network for image deraining & dehazing

Producing high-quality, noise-free images from noisy or hazy inputs relies on essential tasks such as single image deraining and dehazing. In many advanced multi-stage networks, there is often an imbalance in contextual information, leading to increased complexity. To address these challenges, we propose a simplified method inspired by a U-Net structure, resulting in the “Single-Stage V-Shaped Network” (S2VSNet), capable of handling both deraining and dehazing tasks. A key innovation in our approach is the introduction of a Feature Fusion Module (FFM), which facilitates the sharing of information across multiple scales and hierarchical layers within the encoder-decoder structure. As the network progresses towards deeper layers, the FFM gradually integrates insights from higher levels, ensuring that spatial details are preserved while contextual feature maps are balanced. This integration enhances the image processing capability, producing noise-free, high-quality outputs. To maintain efficiency and reduce system complexity, we replaced or removed several non-essential non-linear activation functions, opting instead for simple multiplication operations. Additionally, we introduced a “Multi-Head Attention Integrated Module” (MHAIM) as an intermediary layer between encoder-decoder levels. This module addresses the limited receptive fields of traditional Convolutional Neural Networks (CNNs), allowing for the capture of more comprehensive feature-map information. Our focus on deraining and dehazing led to extensive experiments on a wide range of synthetic and real-world datasets. To further validate the robustness of our network, we implemented S2VSNet on a low-end edge device, achieving deraining in 2.46 seconds.

Bacterial plasmid-associated and chromosomal proteins have fundamentally different properties in protein interaction networks

Plasmids facilitate horizontal gene transfer, which enables the diversification of pathogens into new anatomical and environmental niches, implying that plasmid-encoded genes can cooperate well with chromosomal genes. We hypothesise that such mobile genes are functionally different to chromosomal ones due to this ability to encode proteins performing non-essential functions like antimicrobial resistance and traverse distinct host cells. The effect of plasmid-driven gene gain on protein–protein interaction network topology is an important question in this area. Moreover, the extent to which these chromosomally- and plasmid-encoded proteins interact with proteins from their own groups compared to the levels with the other group remains unclear. Here, we examined the incidence and protein–protein interactions of all known plasmid-encoded proteins across representative specimens from most bacteria using all available plasmids. We found that plasmid-encoded genes constitute ~ 0.65% of the total number of genes per bacterial sample, and that plasmid genes are preferentially associated with different species but had limited taxonomical power beyond this. Surprisingly, plasmid-encoded proteins had both more protein–protein interactions compared to chromosomal proteins, countering the hypothesis that genes with higher mobility rates should have fewer protein-level interactions. Nonetheless, topological analysis and investigation of the protein–protein interaction networks' connectivity and change in the number of independent components demonstrated that the plasmid-encoded proteins had limited overall impact in > 96% of samples. This paper assembled extensive data on plasmid-encoded proteins, their interactions and associations with diverse bacterial specimens that is available for the community to investigate in more detail.

Older adults' experiences using Alexa

The number of adults in the United States age 65 and older is expected to increase to 81 million in 2040 and 95 million in 2060. Voice-activated personal assistants (VAPAs), such as Amazon's Alexa, have the potential to ensure that older adults can successfully age in place using smart home technology to support health, wellness, social engagement, and daily functioning at home. To further explore this potential, twelve home-dwelling older adults aged 65 and older who had used the Amazon Alexa VAPA device for over six months were interviewed to learn about their perceptions and experiences with the use of the device and to gain insights into how this technology has shaped their aging experience. Findings suggest that the older adults did not fully understand the full spectrum of functionality and features of the Alexa device, as they received relatively little initial and ongoing training and support in using it. Participants enjoyed using the Alexa device for selective non-essential features and functions that were more convenient to use on the Alexa device than through alternative methods, but they did not see the use of the Alexa device as essential to their successfully aging in place.CCS CONCEPTS • Accessibility • Ubiquitous and mobile computing • Human-computer interaction

Lost in promiscuity? An evolutionary and biochemical evaluation of HSD10 function in cardiolipin metabolism

Multifunctional proteins are challenging as it can be difficult to confirm pathomechanisms associated with disease-causing genetic variants. The human 17β-hydroxysteroid dehydrogenase 10 (HSD10) is a moonlighting enzyme with at least two structurally and catalytically unrelated functions. HSD10 disease was originally described as a disorder of isoleucine metabolism, but the clinical manifestations were subsequently shown to be linked to impaired mtDNA transcript processing due to deficient function of HSD10 in the mtRNase P complex. A surprisingly large number of other, mostly enzymatic and potentially clinically relevant functions have been attributed to HSD10. Recently, HSD10 was reported to exhibit phospholipase C-like activity towards cardiolipins (CL), important mitochondrial phospholipids. To assess the physiological role of the proposed CL-cleaving function, we studied CL architectures in living cells and patient fibroblasts in different genetic backgrounds and lipid environments using our well-established LC–MS/MS cardiolipidomic pipeline. These experiments revealed no measurable effect on CLs, indicating that HSD10 does not have a physiologically relevant function towards CL metabolism. Evolutionary constraints could explain the broad range of reported substrates for HSD10 in vitro. The combination of an essential structural with a non-essential enzymatic function in the same protein could direct the evolutionary trajectory towards improvement of the former, thereby increasing the flexibility of the binding pocket, which is consistent with the results presented here.

Idiosyncratic Purifying Selection on Metabolic Enzymes in the Long-Term Evolution Experiment with Escherichia coli.

Bacteria, Archaea, and Eukarya all share a common set of metabolic reactions. This implies that the function and topology of central metabolism has been evolving under purifying selection over deep time. Central metabolism may similarly evolve under purifying selection during long-term evolution experiments, although it is unclear how long such experiments would have to run (decades, centuries, millennia) before signs of purifying selection on metabolism appear. I hypothesized that central and superessential metabolic enzymes would show evidence of purifying selection in the long-term evolution experiment with Escherichia coli (LTEE). I also hypothesized that enzymes that specialize on single substrates would show stronger evidence of purifying selection in the LTEE than generalist enzymes that catalyze multiple reactions. I tested these hypotheses by analyzing metagenomic time series covering 62,750 generations of the LTEE. I find mixed support for these hypotheses, because the observed patterns of purifying selection are idiosyncratic and population-specific. To explain this finding, I propose the Jenga hypothesis, named after a children's game in which blocks are removed from a tower until it falls. The Jenga hypothesis postulates that loss-of-function mutations degrade costly, redundant, and non-essential metabolic functions. Replicate populations can therefore follow idiosyncratic trajectories of lost redundancies, despite purifying selection on overall function. I tested the Jenga hypothesis by simulating the evolution of 1,000 minimal genomes under strong purifying selection. As predicted, the minimal genomes converge to different metabolic networks. Strikingly, the core genes common to all 1,000 minimal genomes show consistent signatures of purifying selection in the LTEE.

The Response and Survival Mechanisms of Staphylococcus aureus under High Salinity Stress in Salted Foods.

Staphylococcus aureus (S. aureus) has a strong tolerance to high salt stress. It is a major reason as to why the contamination of S. aureus in salted food cannot be eradicated. To elucidate its response and survival mechanisms, changes in the morphology, biofilm formation, virulence, transcriptome, and metabolome of S. aureus were investigated. IsaA positively regulates and participates in the formation of biofilm. Virulence was downregulated to reduce the depletion of nonessential cellular functions. Inositol phosphate metabolism was downregulated to reduce the conversion of functional molecules. The MtsABC transport system was downregulated to reduce ion transport and signaling. Aminoacyl-tRNA biosynthesis was upregulated to improve cellular homeostasis. The betaine biosynthesis pathway was upregulated to protect the active structure of proteins and nucleic acids. Within a 10% NaCl concentration, the L-proline content was upregulated to increase osmotic stability. In addition, 20 hub genes were identified through an interaction analysis. The findings provide theoretical support for the prevention and control of salt-tolerant bacteria in salted foods.

Identification of construction and demolition waste based on change detection and deep learning

ABSTRACT With the gradual adjustment of urban expansion and discontinuation of non-essential functions in Beijing, many decades-old buildings have been demolished. Thus, construction and demolition waste (CDW) has become the focus of urban and dust pollution management. However, CDW piles are volatile and present irregular boundaries. Therefore, it is essential to map CDW regions in a timely and accurate manner to achieve urban development while protecting the environment. To address this issue, we proposed a method of CDW identification based on change detection and deep learning. First, ZY-3 multispectral images from 2016 and 2019 and their difference images were used for initial sample preparation. We expanded the samples using the post-classification comparison method of change detection, resulting in a 25.4% increase in the valid sample set. The expanded samples were then used as an input to the DeepLabV3+ for training. Thereafter, combined with the change information from the digital elevation model, specific forms, such as demolition remains, landfill CDW, and large-scale dump, were extracted using spatial analysis methods. The overall accuracy of CDW recognition was 91.67%, with a Kappa coefficient of 0.8642. In addition, we calculated the accuracy indices using only the initial samples, obtaining a mean Intersection-over-Union value that was 0.086 lower than that obtained using the expanded sample set. Similar results were obtained in PSPNet and UNet. This suggests that change detection is useful in improving the accuracy of the deep learning models. This study is the first to identify three existing forms of CDW and can effectively address the misclassification between CDW and bare land to identify CDW efficiently.

Keep Teaching: Leveraging Disruption as a Catalyst for Change

In response to the COVID-19 global pandemic, the University of Maryland (UMD) Libraries quickly switched to online teaching and learning. This disruption created a chance for innovation, allowing the UMD Libraries to scale back nonessential functions and focus on improving mission-critical work. The authors present the teaching program at UMD Libraries as a case study for innovation under pressure, highlighting three areas: (1) redevelopment of the Fearless Teaching Institute, an online professional development program for library teachers; (2) transition of a fundamental program—the Academic Writing Program—from an in-person to an online learning environment; and (3) redesign of the Research and Teaching Fellowship, a teacher training program for master of library and information science (MLIS) students, all to better support online learning and pedagogy.

Role of MicroRNAs in Extreme Animal Survival Strategies.

The critical role microRNAs play in modulating global functions is emerging, both in the maintenance of homeostatic mechanisms and in the adaptation to diverse environmental stresses. When stressed, cells must divert metabolic requirements toward immediate survival and eventual recovery and the unique features of miRNAs, such as their relatively ATP-inexpensive biogenesis costs, and the quick and reversible nature of their action, renders them excellent "master controllers" for rapid responses. Many animal survival strategies for dealing with extreme environmental pressures involve prolonged retreats into states of suspended animation to extend the time that they can survive on their limited internal fuel reserves until conditions improve. The ability to retreat into such hypometabolic states is only possible by coupling the global suppression of nonessential energy-expensive functions with an activation of prosurvival networks, a process in which miRNAs are now known to play a major role. In this chapter, we discuss the activation, expression, biogenesis, and unique attributes of miRNA regulation required to facilitate profound metabolic rate depression and implement stress-specific metabolic adaptations. We examine the role of miRNA in strategies of biochemical adaptation including mammalian hibernation, freeze tolerance, freeze avoidance, anoxia and hypoxia survival, estivation, and dehydration tolerance. By comparing these seemingly different adaptive programs in traditional and exotic animal models, we highlight both unique and conserved miRNA-meditated mechanisms for survival. Additional topics discussed include transcription factor networks, temperature dependent miRNA-targeting, and novel species-specific and stress-specific miRNAs.

Power Consumption Analysis of Optical Repeater Subsystem in Multicore Fiber Link

The power consumption of optical repeater subsystems in a multicore fiber (MCF) link is analyzed via the simulation of an erbium-doped fiber amplifier (EDFA) considering the power consumption of an ancillary function and an EDFA. Ancillary power consumption is attributed to nonessential functions that are required only for the subsystem to operate. In the analysis, the ancillary power consumption of optical repeater subsystems that employ multicore (MC) EDFAs is compared with subsystems that employ multiple conventional single-core (SC) EDFAs to determine whether an MC-EDFA has an advantage over the use of the number of cores ( <inline-formula xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink"><tex-math notation="LaTeX">$N_c$</tex-math></inline-formula> ) of SC-EDFAs in an MCF link. In a subsystem with a cladding-pumped MC-EDFA, the required sharable ancillary power consumption such that the total power consumption of the repeater subsystem with a cladding-pumped MC-EDFA is smaller than that with <inline-formula xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink"><tex-math notation="LaTeX">$N_c$</tex-math></inline-formula> of SC-EDFAs, is several tens of watts in the C-band, whereas it is less than 10 W in the L-band. This required sharable ancillary power consumption is converted to a ratio of required sharable ancillary power consumption to ancillary power consumption of a conventional subsystem. The ratios for the C-band is 30–100 %, and most of the ratio values for the L-band are less than 40 % with minimum values of 0 %. However, the L-band cladding-pumped MC-EDFA has a 3-dB poorer noise figure compared to that for SC-EDFA, which makes the L-band cladding-pumped MC-EDFA achieve a higher output power to maintain the optical signal to noise ratio and may introduce the degradation caused by nonlinear optical effects. In contrast, the required sharable ancillary power consumption for a subsystem with a core-pumped MC-EDFA is a few watts and less than 10 W for the C- and L-bands, respectively. This implies that most of the required sharable ancillary power consumption ratio values for the C- and L-bands are less than 10 % and 40 %, respectively. In terms of power consumption, a cladding-pumped MC-EDFA has less advantage over the <inline-formula xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink"><tex-math notation="LaTeX">$N_c$</tex-math></inline-formula> of SC-EDFAs, and a core-pumped MC-EDFA may have an advantage over the <inline-formula xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink"><tex-math notation="LaTeX">$N_c$</tex-math></inline-formula> of SC-EDFA if the size of a core-pumped MC-EDFA is reduced to a value smaller than the <inline-formula xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink"><tex-math notation="LaTeX">$N_c$</tex-math></inline-formula> of the SC-EDFA by integrating MCF amplifier components.

Patients With Common Variable Immunodeficiency (CVID) Show Higher Gut Bacterial Diversity and Levels of Low-Abundance Genes Than the Healthy Housemates.

Common variable immunodeficiency (CVID) is a clinically and genetically heterogeneous disorder with inadequate antibody responses and low levels of immunoglobulins including IgA that is involved in the maintenance of the intestinal homeostasis. In this study, we analyzed the taxonomical and functional metagenome of the fecal microbiota and stool metabolome in a cohort of six CVID patients without gastroenterological symptomatology and their healthy housemates. The fecal microbiome of CVID patients contained higher numbers of bacterial species and altered abundance of thirty-four species. Hungatella hathewayi was frequent in CVID microbiome and absent in controls. Moreover, the CVID metagenome was enriched for low-abundance genes likely encoding nonessential functions, such as bacterial motility and metabolism of aromatic compounds. Metabolomics revealed dysregulation in several metabolic pathways, mostly associated with decreased levels of adenosine in CVID patients. Identified features have been consistently associated with CVID diagnosis across the patients with various immunological characteristics, length of treatment, and age. Taken together, this initial study revealed expansion of bacterial diversity in the host immunodeficient conditions and suggested several bacterial species and metabolites, which have potential to be diagnostic and/or prognostic CVID markers in the future.

Alterocin, an Antibiofilm Protein Secreted by Pseudoalteromonas sp. Strain 3J6

We sought to identify and study the antibiofilm protein secreted by the marine bacterium Pseudoalteromonas sp. strain 3J6. The latter is active against marine and terrestrial bacteria, including Pseudomonas aeruginosa clinical strains forming different biofilm types. Several amino acid sequences were obtained from the partially purified antibiofilm protein, named alterocin. The Pseudoalteromonas sp. 3J6 genome was sequenced, and a candidate alt gene was identified by comparing the genome-encoded proteins to the sequences from purified alterocin. Expressing the alt gene in another nonactive Pseudoalteromonas sp. strain, 3J3, demonstrated that it is responsible for the antibiofilm activity. Alterocin is a 139-residue protein that includes a predicted 20-residue signal sequence, which would be cleaved off upon export by the general secretion system. No sequence homology was found between alterocin and proteins of known functions. The alt gene is not part of an operon and adjacent genes do not seem related to alterocin production, immunity, or regulation, suggesting that these functions are not fulfilled by devoted proteins. During growth in liquid medium, the alt mRNA level peaked during the stationary phase. A single promoter was experimentally identified, and several inverted repeats could be binding sites for regulators. alt genes were found in about 30% of the Pseudoalteromonas genomes and in only a few instances of other marine bacteria of the Hahella and Paraglaciecola genera. Comparative genomics yielded the hypothesis that alt gene losses occurred within the Pseudoalteromonas genus. Overall, alterocin is a novel kind of antibiofilm protein of ecological and biotechnological interest.IMPORTANCE Biofilms are microbial communities that develop on solid surfaces or interfaces and are detrimental in a number of fields, including for example food industry, aquaculture, and medicine. In the latter, antibiotics are insufficient to clear biofilm infections, leading to chronic infections such as in the case of infection by Pseudomonas aeruginosa of the lungs of cystic fibrosis patients. Antibiofilm molecules are thus urgently needed to be used in conjunction with conventional antibiotics, as well as in other fields of application, especially if they are environmentally friendly molecules. Here, we describe alterocin, a novel antibiofilm protein secreted by a marine bacterium belonging to the Pseudoalteromonas genus, and its gene. Alterocin homologs were found in about 30% of Pseudoalteromonas strains, indicating that this new family of antibiofilm proteins likely plays an important albeit nonessential function in the biology of these bacteria. This study opens up the possibility of a variety of applications.

Correcting an instance of synthetic lethality with a pro-survival sequence

A human cDNA encoding the LIM domain containing 194 amino acid cysteine and glycine rich protein 3 (CSRP3) was identified as a BAX suppressor in yeast and a pro-survival sequence that abrogated copper mediated regulated cell death (RCD). Yeast lacks a CSRP3 orthologue but it has four LIM sequences, namely RGA1, RGA2, LRG1 and PXL1. These are known regulators of stress responses yet their roles in RCD remain unknown. Given that LIMs interact with other LIMs, we ruled out the possibility that overexpressed yeast LIMs alone could prevent RCD and that CSRP3 functions by acting as a dominant regulator of yeast LIMs. Of interest was the discovery that even though yeast cells lacking the LIM encoding PXL1 had no overt growth defect, it was nevertheless supersensitive to the effects of sublethal levels of copper. Heterologous expression of human CSPR3 as well as the pro-survival 14-3-3 sequence corrected this copper supersensitivity. These results show that the pxl1∆-copper synthetic lethality is likely due to the induction of RCD. This differs from the prevailing model in which synthetic lethality occurs because of specific defects generated by the combined loss of two overlapping but non-essential functions.

Small and mighty: adaptation of superphylum Patescibacteria to groundwater environment drives their genome simplicity

BackgroundThe newly defined superphylum Patescibacteria such as Parcubacteria (OD1) and Microgenomates (OP11) has been found to be prevalent in groundwater, sediment, lake, and other aquifer environments. Recently increasing attention has been paid to this diverse superphylum including > 20 candidate phyla (a large part of the candidate phylum radiation, CPR) because it refreshed our view of the tree of life. However, adaptive traits contributing to its prevalence are still not well known.ResultsHere, we investigated the genomic features and metabolic pathways of Patescibacteria in groundwater through genome-resolved metagenomics analysis of > 600 Gbp sequence data. We observed that, while the members of Patescibacteria have reduced genomes (~ 1 Mbp) exclusively, functions essential to growth and reproduction such as genetic information processing were retained. Surprisingly, they have sharply reduced redundant and nonessential functions, including specific metabolic activities and stress response systems. The Patescibacteria have ultra-small cells and simplified membrane structures, including flagellar assembly, transporters, and two-component systems. Despite the lack of CRISPR viral defense, the bacteria may evade predation through deletion of common membrane phage receptors and other alternative strategies, which may explain the low representation of prophage proteins in their genomes and lack of CRISPR. By establishing the linkages between bacterial features and the groundwater environmental conditions, our results provide important insights into the functions and evolution of this CPR group.ConclusionsWe found that Patescibacteria has streamlined many functions while acquiring advantages such as avoiding phage invasion, to adapt to the groundwater environment. The unique features of small genome size, ultra-small cell size, and lacking CRISPR of this large lineage are bringing new understandings on life of Bacteria. Our results provide important insights into the mechanisms for adaptation of the superphylum in the groundwater environments, and demonstrate a case where less is more, and small is mighty.1EX9Jugkb3j7HLsLG9gAj8Video abstract.

Universal highly efficient conditional knockout system in Leishmania, with a focus on untranscribed region preservation.

Trypanosomatids are divergent eukaryotes of high medical and economical relevance. Their biology exhibits original features that remain poorly understood; particularly, Leishmania is known for its high degree of genomic plasticity that makes genomic manipulation challenging. CRISPR-Cas9 has been applied successfully to these parasites providing a robust tool to study non-essential gene functions. Here, we have developed a versatile inducible system combining Di-Cre recombinase and CRISPR-Cas9 advantages. Cas9 is used to integrate the LoxP sequences, and the Cre-recombinase catalyses the recombination between LoxP sites, thereby excising the target gene. We used a Leishmania mexicana cell line expressing Di-Cre, Cas9, and T7 polymerase and then transfected donor DNAs and single guide RNAs as polymerase chain reaction (PCR) products. Because the location of LoxP sequences in the genomic DNA can interfere with the function and localisation of certain proteins of interest, we proposed to target the least transcribed regions upstream and/or downstream the gene of interest. To do so, we developed "universal" template plasmids for donor DNA cassettes with or without a tag, where LoxP sequences may be located either immediately upstream the ATG and downstream the stop codon of the gene of interest, or in the least transcribed areas of intergenic regions. Our methodology is fast, PCR-based (molecular cloning-free), highly efficient, versatile, and able to overcome the problems posed by genomic plasticity in Leishmania.

Functional Domains of the Herpes Simplex Virus Type 1 Tegument Protein pUL37: The Amino Terminus is Dispensable for Virus Replication in Tissue Culture.

The herpes simplex virus type 1 (HSV-1) UL37 gene encodes for a multifunctional component of the virion tegument, which is necessary for secondary envelopment in the cytoplasm of infected cells, for motility of the viral particle, and for the first steps in the initiation of virus infection. This 120 kDa protein has several known viral interacting partners, including pUL36, gK/pUL20, pUS10, and VP26, and cellular interacting proteins which include TRAF6, RIG-I, and dystonin. These interactions are likely important for the functions of pUL37 at both early and late stages of infection. We employed a genetic approach to determine essential domains and amino acid residues of pUL37 and their associated functions in cellular localization and virion morphogenesis. Using marker-rescue/marker-transfer methods, we generated a library of GFP-tagged pUL37 mutations in the HSV-1 strain KOS genome. Through viral growth and ultra-structural analysis, we discovered that the C-terminus is essential for replication. The N-terminal 480 amino acids are dispensable for replication in cell culture, although serve some non-essential function as viral titers are reduced in the presence of this truncation. Furthermore, the C-terminal 133 amino acids are important in so much that their absence leads to a lethal phenotype. We further probed the carboxy terminal half of pUL37 by alanine scanning mutagenesis of conserved residues among alphaherpesviruses. Mutant viruses were screened for the inability to form plaques—or greatly reduced plaque size—on Vero cells, of which 22 mutations were chosen for additional analysis. Viruses discovered to have the greatest reduction in viral titers on Vero cells were examined by electron microscopy (EM) and by confocal light microscopy for pUL37–EGFP cellular localization. This genetic approach identified both essential and non-essential domains and residues of the HSV-1 UL37 gene product. The mutations identified in this study are recognized as significant candidates for further analysis of the pUL37 function and may unveil previously undiscovered roles and interactions of this essential tegument gene.

Harnessing Biological Insight to Accelerate Tuberculosis Drug Discovery.

ConspectusTuberculosis (TB) is the leading cause of mortality globally resulting from an infectious disease, killing almost 1.6 million people annually and accounting for approximately 30% of deaths attributed to antimicrobial resistance (AMR). This despite the widespread administration of a neonatal vaccine, and the availability of an effective combination drug therapy against the causative agent, Mycobacterium tuberculosis (Mtb). Instead, TB prevalence worldwide is characterized by high-burden regions in which co-epidemics, such as HIV, and social and economic factors, undermine efforts to control TB. These elements additionally ensure conditions that favor the emergence of drug-resistant Mtb strains, which further threaten prospects for future TB control.To address this challenge, significant resources have been invested in developing a TB drug pipeline, an initiative given impetus by the recent regulatory approval of two new anti-TB drugs. However, both drugs have been reserved for drug-resistant disease, and the seeming inevitability of new resistance plus the recognized need to shorten the duration of chemotherapy demands continual replenishment of the pipeline with high-quality “hits” with novel mechanisms of action. This represents a massive challenge, which has been undermined by key gaps in our understanding of Mtb physiology and metabolism, especially during host infection. Whereas drug discovery for other bacterial infections can rely on predictive in vitro assays and animal models, for Mtb, inherent metabolic flexibility and uncertainties about the nutrients available to infecting bacilli in different host (micro)environments instead requires educated predictions or demonstrations of efficacy in animal models of arguable relevance to human disease. Even microbiological methods for enumeration of viable mycobacterial cells are fraught with complication.Our research has focused on elucidating those aspects of mycobacterial metabolism that contribute to the robustness of the bacillus to host immunological defenses and applied antibiotics and that, possibly, drive the emergence of drug resistance. This work has identified a handful of metabolic pathways that appear vulnerable to antibiotic targeting. Those highlighted, here, include the inter-related functions of pantothenate and coenzyme A biosynthesis and recycling and nucleotide metabolism—the last of which reinforces our view that DNA metabolism constitutes an under-explored area for new TB drug development. Although nonessential functions have traditionally been deprioritized for antibiotic development, a common theme emerging from this work is that these very functions might represent attractive targets because of the potential to cripple mechanisms critical to bacillary survival under stress (for example, the RelMtb-dependent stringent response) or to adaptability under unfavorable, potentially lethal, conditions including antibiotic therapy (for example, DnaE2-dependent SOS mutagenesis). The bar, however, is high: demonstrating convincingly the likely efficacy of this strategy will require innovative models of human TB disease.In the concluding section, we focus on the need for improved techniques to elucidate mycobacterial metabolism during infection and its impact on disease outcomes. Here, we argue that developments in other fields suggest the potential to break through this barrier by harnessing chemical-biology approaches in tandem with the most advanced technologies. As researchers based in a high-burden country, we are impelled to continue participating in this important endeavor.

SCAF4 and SCAF8, mRNA Anti-Terminator Proteins

SummaryAccurate regulation of mRNA termination is required for correct gene expression. Here, we describe a role for SCAF4 and SCAF8 as anti-terminators, suppressing the use of early, alternative polyadenylation (polyA) sites. The SCAF4/8 proteins bind the hyper-phosphorylated RNAPII C-terminal repeat domain (CTD) phosphorylated on both Ser2 and Ser5 and are detected at early, alternative polyA sites. Concomitant knockout of human SCAF4 and SCAF8 results in altered polyA selection and subsequent early termination, leading to expression of truncated mRNAs and proteins lacking functional domains and is cell lethal. While SCAF4 and SCAF8 work redundantly to suppress early mRNA termination, they also have independent, non-essential functions. SCAF8 is an RNAPII elongation factor, whereas SCAF4 is required for correct termination at canonical, distal transcription termination sites in the presence of SCAF8. Together, SCAF4 and SCAF8 coordinate the transition between elongation and termination, ensuring correct polyA site selection and RNAPII transcriptional termination in human cells.

Analysis and Design of Course Website for Software Testing Based on SPOC

Technologies of the architecture on SSH(Struts-Spring-Hibernate), database interface access, and web data mining are used to analyse and design of a lightweight course website for software testing which is based on SPOC(Small Private Online Course) for blended teaching on campus. Functions of learning topics releasing on the course, organizing and coordinating learners’ discussion to promote the learning process can be achieved through the website for teachers, and the website can also provide learners with selecting some social learning tools(BBS, QQ, WeChat) to complete online communication for the course learning. Finally, further work is expected in the paper. Firstly, the targeted and distinctive mobile learning resources and smart education mode should be constructed which is based on SPOC. Secondly, the visualized, diversified third-party platforms and tools in the course website should be integrated effectively which are enable learners to complete the process of online exercising or examining for some types of subjective testing items. Thirdly, how to ensure the reliability, toughness, running performance, safety, stability for the course website which is formed by cutting out some non-essential functions from existed MOOC(Massive Open Online Course) platforms, and improve the functions of multiple data statistics with web data mining technology and motivational observation for the website development.