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NOD.Cg- Prkdc scid Il2rg tm1Wjl /SzJ (NSG) mice, lacking many components of a mature immune system, are at increased risk of disease. General understanding of potential pathogens of these mice is limited. We describe a high mortality disease outbreak caused by an opportunistic bacterial infection in NSG mice. Affected animals exhibited perianal fecal staining, dehydration, and wasting. Histopathologic lesions included a primary necrotizing enterocolitis, with inflammatory and necrotizing lesions also occurring in the liver, kidneys, heart, and brain of some mice. All affected individuals tested negative for known opportunistic pathogens of immunodeficient mice. We initially identified a member of Enterobacter cloacae complex (ECC) in association with the outbreak by traditional diagnostics. ECC was cultured from extraintestinal organs, both with and without histopathologic lesions, suggesting bacteremia. Infrared spectroscopy and MALDI-TOF mass spectrometry demonstrated that isolates from the outbreak shared molecular features and likely a common origin. We subsequently hypothesized that advanced sequencing methods would identify a single species of ECC associated with clinical disease. Using a novel targeted amplicon-based next-generation sequencing assay, we identified Enterobacter hormaechei in association with this outbreak. Knowledge of this organism as a potential opportunistic pathogen in NSG mice is critical for preclinical studies to prevent loss of animals and confounding of research., Competing Interests: Declaration of Conflicting InterestsThe author(s) declared the following potential conflicts of interest with respect to the research, authorship, and/or publication of this article: KN, CW, and KSH are employees of Charles River Laboratories.

COVID-19 is a global health problem caused by SARS-CoV-2, which has led to over 600 million infections and 6 million deaths. Developing novel antiviral drugs is of pivotal importance to slow down the epidemic swiftly. In this study, we identified five azo compounds as effective antiviral drugs to SARS-CoV-2, and mechanism study revealed their targets for impeding viral particles' ability to bind to host receptors. Direct Blue 53, which displayed the strongest inhibitory impact, inhibited five mutant strains at micromole. In vitro, mechanism study demonstrated Direct Blue 53 inhibited viral infection through interaction with the spike of SARS-CoV-2. And 25 mg/kg/d compound treatment showed 50% or 60% survival protection against lethal Delta or Omicron BA.2 infection in vivo. Taken together, our results demonstrate that azo compounds with dimethyl-biphenyl-diyl-bis(azo)bis structure may be promising anti-SARS-CoV-2 drug candidates, which provide practicable therapies with the aid of structural optimizations and further research., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023. Published by Elsevier Inc.)

The spike protein of SARS-CoV-2 mediates receptor binding and cell entry and is the key immunogenic target for virus neutralization and the present attention of many vaccine layouts. It exhibits significant conformational flexibility. We study the structural fluctuations of spike protein among the most common mutations that appeared in the variant of concerns (VOC). We report the thermodynamics of conformational changes in mutant spike protein with respect to the wild-type from the distributions of the dihedral angles obtained from the equilibrium configurations generated via all-atom molecular dynamics simulations. We find that the mutation causes the increase in distance between the N-terminal domain and receptor binding domain, leading to an obtuse angle cosine θ distribution in the trimeric structure in spike protein. Thus, an increase in open state is conferred to the more infectious variants of SARS-CoV-2. The thermodynamically destabilized and disordered residues of receptor binding motif among the mutant variants of spike protein are proposed to serve as better binding sites for the host factor. We identify a short stretch of region connecting the N-terminal domain and receptor binding domain forming a linker loop where many residues undergo stabilization in the open state compared to the closed one., (© 2022 International Union of Biochemistry and Molecular Biology, Inc.)

Introduction: We examined the neutralizing antibody production efficiency of the second and third severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccine doses (2 nd - and 3 rd -dose) and neutralizing activity on mutant strains, including, the Ancestral, Beta and Omicron strains using green fluorescent protein-carrying recombinant SARS-CoV-2, in living-donor liver transplantation (LDLT) recipients., Methods: The patients who were administered vaccines other than Pfizer- BioNTechBNT162b2 and who had coronavirus disease 2019 in this study period were excluded. We enrolled 154 LDLT recipients and 50 healthy controls., Result: The median time were 21 days (between 1 st and 2 nd vaccination) and 244 days (between 2 nd and 3 rd vaccination). The median neutralizing antibody titer after 2 nd -dose was lower in LDLT recipients than in controls (0.46 vs 1.00, P<0.0001). All controls had SARS-CoV-2 neutralizing antibodies, whereas 39 LDLT recipients (25.3%) had no neutralizing antibodies after 2 nd -dose; age at vaccination, presence of ascites, multiple immunosuppressive treatments, and mycophenolate mofetil treatment were significant risk factors for nonresponder. The neutralizing activities of recipient sera were approximately 3-fold and 5-fold lower than those of control sera against the Ancestral and Beta strains, respectively. The median antibody titer after 3 rd -dose was not significantly different between recipients and controls (1.02 vs 1.22, p=0.0758); only 5% recipients was non-responder. The neutralizing activity after third dose to Omicron strains were enhanced and had no significant difference between two groups., Conclusion: Only the 2nd-dose was not sufficiently effective in recipients; however, 3rd-dose had sufficient neutralizing activity against the mutant strain and was as effective as that in healthy controls., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Tomiyama, Suzuki, Harada, Tamura, Toshida, Kosai-Fujimoto, Tomino, Yoshiya, Nagao, Takeishi, Itoh, Kobayashi, Ito, Yoshio, Kanto, Yoshizumi and Fukuhara.)

The cell surface of lactic acid bacteria (LAB) plays an essential role in cell-cell and cell-host interactions. Exopolysaccharides (EPSs) are produced on the cell surface of LAB or in the surrounding medium and are considered to be in favor of the strain- specific probiotic surface characteristics. In this work, the structure features of EPS from Lacticaseibacillus paracasei S-NB were analyzed preliminarily, and the genes involved in EPS biosynthesis of S-NB strain were hypothesized and annotated, and their role in phenotypic characteristics were demonstrated by gene deletion analysis. Four mutant strains with deletion of crucial genes involved in EPS synthesis were analyzed for strain characteristics that are closely related to their ability to interact with the host intestinal epithelium cells, including strain surface characteristics and viability under the gastrointestinal stress conditions (both acid and bile stress). Furthermore, the adherence and immunomodulatory properties of wild-type S-NB and its mutant strains were compared using Caco-2 and RAW 264.7 cell lines, respectively. Taken together, the results indicated the importance of genes associated with EPS biosynthesis in L. paracasei S-NB as a determinant in strain surface characteristics and cell-host interaction, especially for S-NB&#95;2176 (responsible for EPS polymerization) and S-NB&#95;2175 (responsible for CpsD/CapB family tyrosine-protein kinase)., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Background and Objectives: Remarkable infectivity of severe acute respiratory syndrome-coronavirus 2 (SARS-CoV2) is due to the rapid emergence of various strains which enable the virus to ruling the world. Over the course of SARS-CoV2 pandemic, the scientific communities worldwide are responding to newly emerging genetic variants. However, mechanism behind the persistent infection of these variants is still not known due to the paucity of study of these variants at molecular level. In this scenario, computational methods have immense utility in understanding the molecular and functional properties of different variants., Methods: The various mutants (MTs) of SpikeS1 receptor binding domain (RBD) of highly infectious SARS-CoV2 strains were manifested and elucidated the protein structure and binding strength using molecular dynamics (MD) simulation and protein-protein docking approaches., Results: MD simulation study showed that all MTs exhibited stable structures with altered functional properties. Furthermore, the binding strength of different MTs along with WT (wildtype) was revealed that MTs showed differential binding affinities to host protein with high binding strength exhibited by V367F and V483A MTs., Conclusion: Hence, this study shed light on the molecular basis of infection caused by different variants of SARS-CoV2, which might play an important role in to cease the transmission and pathogenesis of virus and also implicate in rational designing of a specific drug., Competing Interests: Declaration of Competing Interest The authors have no conflict of interest to declare, (Copyright © 2021 Elsevier B.V. All rights reserved.)

The COVID-19 disease caused by severe acute respiratory syndrome coronavirus -2 (SARS-CoV-2) has posed as a major health concern for people all across the globe. Along with the increasing confirmed patients being readmitted with complaints for fever, cough, cold, the effective monitoring of 'relapse' of the SARS-CoV-2 virus in the previously discharged patients have become the next area of focus. However, availability of limited data on reactivation of SARS-CoV-2 makes the disease prognosis as well as the effective control of re-infection an immense challenge. Prompted by these challenges, we assessed the possibility of re-infection in discharged patients and the risk of the transmission, proficiency of RT-PCR results and approximate period required for the quarantine, and the real challenges for the development of vaccine. In the present review, the published literature on all the possible cases of re-infection from February to July were reported, thereby selected 142 studies from a hub of overall 669 studies after full text screening. The incomplete virus clearance, poor sensitivity of the present diagnostic testing, emergence of mutant strains, insufficient mucus collection from the throat swab etc., are some of the possible causes of re-infection. The new protocols for management of COVID-19 discharged patients should be revised in the guidelines., (© 2022 Published by Elsevier Ltd.)

Infections represent highly dynamic processes, characterized by evolutionary changes and events that involve both the pathogen and the host. Among infectious agents, viruses, such as Severe Acute Respiratory Syndrome-related Coronavirus type 2 (SARS-CoV-2), the infectious agent responsible for the currently ongoing Coronavirus disease 2019 (COVID-2019) pandemic, have a particularly high mutation rate. Taking into account the mutational landscape of an infectious agent, it is important to shed light on its evolution capability over time. As new, more infectious strains of COVID-19 emerge around the world, it is imperative to estimate when these new strains may overtake the wild-type strain in different populations. Therefore, we developed a general-purpose framework to estimate the time at which a mutant variant is able to take over a wild-type strain during an emerging infectious disease outbreak. In this study, we used COVID-19 as a case-study; however, the model is adaptable to any emerging pathogen. We devised a two-strain mathematical framework to model a wild- and a mutant-type viral population and fit cumulative case data to parameterize the model, using Ontario as a case study. We found that, in the context of under-reporting and the current case levels, a variant strain was unlikely to dominate until March/April 2021. The current non-pharmaceutical interventions in Ontario need to be kept in place longer even with vaccination in order to prevent another outbreak. The spread of a variant strain in Ontario will likely be observed by a widened peak of the daily reported cases. If vaccine efficacy is maintained across strains, then it is still possible to achieve high levels of immunity in the population by the end of 2021. Our findings have important practical implications in terms of public health as policy- and decision-makers are equipped with a mathematical tool that can enable the estimation of the take-over of a mutant strain of an emerging infectious disease.

Under nitrogen-limiting conditions, symbiotic nodulation promotes the growth of legume plants via the fixation of atmospheric nitrogen to ammonia by rhizobia in root nodules. The rhizobial Nod factor (NF) and type III secretion system (T3SS) are two key signaling pathways for establishing the legume-rhizobium symbiosis. However, whether NF signaling is involved in the nodulation of Robinia pseudoacacia and Mesorhizobium amorphae CCNWGS0123, and its symbiotic differences compared with T3SS signaling remain unclear. Therefore, to elucidate the function of NF signaling in nodulation, we mutated nodC in M. amorphae CCNWGS0123, which aborted NF synthesis. Compared with the plants inoculated with the wild type strain, the plants inoculated with the NF-deficient strain exhibited shorter shoots with etiolated leaves. These phenotypic characteristics were similar to those of the plants inoculated with the T3SS-deficient strain, which served as a Nod- (non-effective nodulation) control. The plants inoculated with both the NF- and T3SS-deficient strains formed massive root hair swellings, but no normal infection threads were detected. Sections of the nodules showed that inoculation with the NF- and T3SS-deficient strains induced small, white bumps without any rhizobia inside. Analyzing the accumulation of 6 plant hormones and the expression of 10 plant genes indicated that the NF- and T3SS-deficient strains activated plant defense reactions while suppressing plant symbiotic signaling during the perception and nodulation processes. The requirement for NF signaling appeared to be conserved in two other leguminous trees that can establish symbiosis with M. amorphae CCNWGS0123. In contrast, the function of the T3SS might differ among species, even within the same subfamily (Faboideae). Overall, this work demonstrated that nodulation of R. pseudoacacia and M. amorphae CCNWGS0123 was both NF and T3SS dependent., (© The Author(s) 2020. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permission@oup.com.)

Background: Using Brucella abortus Strain 19 (S19) to control bovine brucellosis is restricted due to induce antibodies to the O-side chain of the smooth lipopolysaccharide (LPS) which may be difficult to differentiate vaccinated and infected animals. Furthermore, it is virulent for humans and can induce abortion to cattle., Objectives: The aim of this study was to employ gene knockout B. abortus S19 for the first time to eliminate diagnostic defects and obtain the attenuated mutant strain., Material and Methods: The wbkA gene, which is one of the LPS O-chain coding genes, was knocked out in vaccinal Brucella abortus S19. The proliferative response and immunoglobulin M production were analyzed in wbkA deletion strain-infected BALB/c mice., Results: The loss of wbkA gene function resulted in induction of the splenocyte proliferative response in mice infected by the mutant S19 strain compare to those induced by parental S19 and RB51 strains. Moreover, wbkA mutant did not induce any IgM antibody response using the enzyme-linked immunosorbent assay., Conclusions: As a result, the new mutant S19 strain had deficiency in its LPS O-chain structure, besides cannot induce IgM response then, reduce mistakes to discriminate between vaccinated and infected animal, and also can be considered as a new vaccine candidate., (Copyright: © 2019 The Author(s); Published by National Institute of Genetic Engineering and Biotechnology.)

In this study, a novel monomer aspartokinase (AK) from Corynebacterium pekinense was identified, and its monomer model was constructed. Site 380 was identified by homologous sequencing and monomer model comparison as the key site which was conserved and located around the binding site of the inhibitor Lys. Furthermore, the mutant A380I with enzyme activity 11.32-fold higher than wild type AK (WT-AK), was obtained by site-directed mutagenesis and high throughput screening. In the mutant A380I, the optimal temperature was raised from 26 °C (WT-AK) to 28 °C, the optimal pH remained unchanged at 8.0, and the half-life was prolonged from 4.5 h (WT-AK) to 6.0 h, indicating enhanced thermal stability. The inhibition of A380I was weakened at various inhibitor concentrations and even activated at certain inhibitor concentrations (10 mM of Lys, 5 mM or 10 mM of Lys + Thr, 10 mM of Lys + Met, 5 mM of Lys + Thr + Met). Molecular dynamics simulation results indicated that the occupancy rate of hydrogen bond between A380I and ATP was enhanced, the effect of Lys (inhibitor) on the protein was weakened, and the angle between Ser281-Tyre358 and Asp359-Gly427 was increased after mutation, leading to an open conformation (R-state) that favored the binding of substrate.

Purpose: Diffusional kurtosis imaging (DKI) enables sensitive measurement of tissue microstructure by quantifying the non-Gaussian diffusion of water. Although DKI is widely applied in many situations, histological correlation with DKI analysis is lacking. The purpose of this study was to determine the relationship between DKI metrics and neurite density measured using confocal microscopy of a cleared mouse brain., Methods: One thy-1 yellow fluorescent protein 16 mouse was deeply anesthetized and perfusion fixation was performed. The brain was carefully dissected out and whole-brain MRI was performed using a 7T animal MRI system. DKI and diffusion tensor imaging (DTI) data were obtained. After the MRI scan, brain sections were prepared and then cleared using aminoalcohols (CUBIC). Confocal microscopy was performed using a two-photon confocal microscope with a laser. Forty-eight ROIs were set on the caudate putamen, seven ROIs on the anterior commissure, and seven ROIs on the ventral hippocampal commissure on the confocal microscopic image and a corresponding MR image. In each ROI, histological neurite density and the metrics of DKI and DTI were calculated. The correlations between diffusion metrics and neurite density were analyzed using Pearson correlation coefficient analysis., Results: Mean kurtosis (MK) (P = 5.2 × 10 -9 , r = 0.73) and radial kurtosis (P = 2.3 × 10 -9 , r = 0.74) strongly correlated with neurite density in the caudate putamen. The correlation between fractional anisotropy (FA) and neurite density was moderate (P = 0.0030, r = 0.42). In the anterior commissure and the ventral hippocampal commissure, neurite density and FA are very strongly correlated (P = 1.3 × 10 -5 , r = 0.90). MK in these areas were very high value and showed no significant correlation (P = 0.48)., Conclusion: DKI accurately reflected neurite density in the area with crossing fibers, potentially allowing evaluation of complex microstructures.

The fungus Candida albicans can grow as either yeast or filaments, which include hyphae and pseudohyphae, depending on environmental conditions. Filamentous growth is of particular interest because it is required for biofilm formation and for pathogenesis. Environmentally induced filamentous growth is associated with expression of filamentation-associated genes, and both filamentous growth and associated gene expression depend upon several well-characterized transcription factors. Surprisingly, strains with reduced expression of many essential genes display filamentous growth under non-inducing conditions-those in which the wild type grows as yeast. We found recently that diminished expression of several essential protein kinase genes leads to both filamentous cell morphology and filamentation-associated gene expression under non-inducing conditions. Reduced expression of the essential protein kinase gene CAK1 promoted filamentation-associated gene expression and biofilm formation in strains that lacked key transcriptional activators of these processes, thus indicating that CAK1 expression is critical for both environmental and genetic control of filamentation. In this study, we extend our genetic interaction analysis to a second essential protein kinase gene, KIN28. Reduced expression of KIN28 also permits filamentation-associated gene expression, though not biofilm formation, in the absence of several key transcriptional activators. Our results argue that impairment of several essential cellular processes can alter the regulatory requirements for filamentation-associated gene expression. Our results also indicate that levels of filamentation-associated gene expression are not fully predictive of biofilm formation ability.

Secondary metabolites of endophytic fungi FSN002 from Juglans mandshurica Maxim have excellent liver cancer resistance. Preparation of mutant strains is an important means to study the biosynthesis mechanism of catalitaxol. Fungal spores germinating young hyphae with 4 to 6 cells after culturing for 13 hours were used as starting materials of ultraviolet (UV) mutagenesis. UV light intensity and irradiation time have a linear relationship with fungal mortality. The two factors had no obvious interactions. When UV light was 90 000 μJ/cm2 and irradiation time for 6 s, the mortality of fungi was around 95%. Under the optimization mutation condition, two mutant strains were obtained, of which one lost the synthesis ability of catalitaxol completely, and the another synthetized only 16% catalitaxol of the wild strain. Our findings may serve basis for further study on the biosynthesis mechanism and efficient production of catalitaxol.

Strigolactones (SLs) as components of root exudates induce hyphal branching of arbuscular mycorrhizal (AM) fungi which is thought to favor the establishment of the beneficial symbiosis. Little is known on how AM fungi respond to SLs. Since AM fungi are poor model systems due to their obligate biotrophism and the lack of genetic transformation protocols, we took advantage of the sensitivity of several phytopathogenic fungi to GR24, a synthetic SLs analog. With the aim to identify the molecular determinants involved in SLs response in AM fungi and assuming conserved mechanisms in the fungal kingdom, we exploited the fungal pathogens Botrytis cinerea and Cryphonectria parasitica, for which mutant collections are available. Exposure of B. cinerea and C. parasitica to GR24 embedded in solid medium led to reduction of fungal radial growth. We set up the screening of a set of well-characterized gene deletion mutants to isolate genotypes with altered responses to SLs. Two B. cinerea mutants (defective of BcTrr1, a thioredoxin reductase and BcLTF1, a GATA transcription factor) turned out to be less responsive to GR24. One feature shared by the two mutants is the overproduction of reactive oxygen species (ROS). Indeed, an oxidizing effect was observed in a B. cinerea strain expressing a redox-sensitive GFP2 in the mitochondrial intermembrane space upon exposure to GR24. ROS and mitochondria are, therefore, emerging as mediators of SLs actions.

Genome analysis of over 70 Saccharomyces strains revealed the existence of five groups of genetically diverged S. cerevisiae wild-type isolates, which feature distinct genetic backgrounds and reflect the natural diversity existing among the species. The strains originated from different geographical and ecological niches (Malaysian, West African, North American, Wine/European and Sake) and represent clean, non-mosaic lineages of S. cerevisiae, meaning that their genomes differ essentially by monomorphic and private SNPs. In this study, one representative strain for each of the five S. cerevisiae clean lineages was selected and mutated for several auxotroph genes by clean markerless deletions, so that all dominant markers remained available for further genetic manipulations. A set of 50 strains was assembled, including eight haploid and two diploid strains for each lineage. These strains carry different combinations of leu2∆0, lys2∆0, met15∆0, ura3∆0 and/or ura3∆::KanMX-barcoded deletions with marker configurations resembling that of the BY series, which will allow large-scale crossing with existing deletion collections. This new set of genetically tractable strains provides a powerful tool kit to explore the impact of natural variation on complex biological processes., (Copyright © 2013 John Wiley & Sons, Ltd.)

The fetoplacental arterial tree is critical for efficient distribution of arterial blood to capillaries throughout the placental exchange region; yet, little is known about the factors and mechanisms that control its development. Advances in micro-CT imaging and analysis, and available mutant mouse strains, are facilitating rapid progress. Indeed, micro-CT studies show that genetic differences between the CD1 and C57Bl/6 mouse strains, and between Gcm1 heterozygotes and wild-type littermates alter the developmental trajectory of the fetoplacental arterial tree as do environmental factors including maternal exposure to toxins in cigarette smoke and malarial infection. Relative to other vascular beds, the fetoplacental arterial tree is particularly tractable because veins can more easily be excluded when infusing the contrast agent and because of the placenta's small size, which means that the whole organ can be imaged (maintaining connectivity) and that the tree is simpler (fewer branching generations). Despite these differences, measured parameters were found to be similar to arterial trees in other adult rodent organs. Thus, micro-CT analysis provides a means for advancing of our understanding of the mechanisms controlling development of the fetoplacental arterial tree. Results will likely have relevance to other arterial vasculatures as well., (© 2013 John Wiley & Sons Ltd.)