An apparent lack of synergy between degradative enzymes against Staphylococcus aureus biofilms.

Enzymes combat bacterial infections by degrading biomolecules to disperse Staphylococcus aureus biofilms. Commercial enzyme mixtures, like cellulase and pepsin, show concentration-dependent dispersion, but low concentrations lack synergy. Only the sequential addition of pepsin followed by Arthrobacter luteus zymolyase 20T displays synergy, effectively dispersing biofilms. Purified zymolyase 100T outperforms zymolyase 20T but lacks synergy with pepsin. This study underscores the complexity of enzymatic biofilm dispersal, highlighting the need for tailored approaches based on enzyme properties and biofilm composition.

Evolution of a Plasmid Regulatory Circuit Ameliorates Plasmid Fitness Cost.

Plasmids play a major role in rapid adaptation of bacteria by facilitating horizontal transfer of diverse genes, most notably those conferring antibiotic resistance. While most plasmids that replicate in a broad range of bacteria also persist well in diverse hosts, there are exceptions that are poorly understood. We investigated why a broad-host range plasmid, pBP136, originally found in clinical Bordetella pertussis isolates, quickly became extinct in laboratory Escherichia coli populations. Through experimental evolution we found that inactivation of a previously uncharacterized plasmid gene, upf31, drastically improved plasmid maintenance in E. coli. This gene inactivation resulted in decreased transcription of the global plasmid regulators (korA, korB, and korC) and numerous genes in their regulons. It also caused transcriptional changes in many chromosomal genes primarily related to metabolism. In silico analyses suggested that the change in plasmid transcriptome may be initiated by Upf31 interacting with the plasmid regulator KorB. Expression of upf31 in trans negatively affected persistence of pBP136Δupf31 as well as the closely related archetypal IncP-1ß plasmid R751, which is stable in E. coli and natively encodes a truncated upf31 allele. Our results demonstrate that while the upf31 allele in pBP136 might advantageously modulate gene expression in its original host, B. pertussis, it has harmful effects in E. coli. Thus, evolution of a single plasmid gene can change the range of hosts in which that plasmid persists, due to effects on the regulation of plasmid gene transcription.

Altered Iron and Microstructure in Huntington's Disease Subcortical Nuclei: Insight From 7T MRI.

BACKGROUND: Pathophysiological changes of Huntington's disease (HD) can precede symptom onset by decades. Robust imaging biomarkers are needed to monitor HD progression, especially before the clinical onset. PURPOSE: To investigate iron dysregulation and microstructure alterations in subcortical regions as HD imaging biomarkers, and to associate such alterations with motor and cognitive impairments. STUDY TYPE: Prospective. POPULATION: Fourteen individuals with premanifest HD (38.0 ± 11.0 years, 9 females; far-from-onset N = 6, near-onset N = 8), 21 manifest HD patients (49.1 ± 12.1 years, 11 females), and 33 age-matched healthy controls (43.9 ± 12.2 years, 17 females). FIELD STRENGTH/SEQUENCE: 7 T, T1 -weighted imaging, quantitative susceptibility mapping, and diffusion tensor imaging. ASSESSMENT: Volume, susceptibility, fractional anisotropy (FA), and mean diffusivity (MD) within subcortical brain structures were compared across groups, used to establish HD classification models, and correlated to clinical measures and cognitive assessments. STATISTICAL TESTS: Generalized linear model, multivariate logistic regression, receiver operating characteristics with the area under the curve (AUC), and likelihood ratio test comparing a volumetric model to one that also includes susceptibility and diffusion metrics, Wilcoxon paired signed-rank test, and Pearson's correlation. A P-value <0.05 after Benjamini-Hochberg correction was considered statistically significant. RESULTS: Significantly higher striatal susceptibility and FA were found in premanifest and manifest HD preceding atrophy, even in far-from-onset premanifest HD compared to controls (putamen susceptibility: 0.027 ± 0.022 vs. 0.018 ± 0.013 ppm; FA: 0.358 ± 0.048 vs. 0.313 ± 0.039). The model with additional susceptibility, FA, and MD features showed higher AUC compared to volume features alone when differentiating premanifest HD from HC (0.83 vs. 0.66), and manifest from premanifest HD (0.94 vs. 0.83). Higher striatal susceptibility significantly correlated with cognitive deterioration in HD (executive function: r = -0.600; socioemotional function: r = -0.486). DATA CONCLUSION: 7 T MRI revealed iron dysregulation and microstructure alterations with HD progression, which could precede volume loss, provide added value to HD differentiation, and might be associated with cognitive changes. EVIDENCE LEVEL: 2 TECHNICAL EFFICACY: Stage 2.

yEvo: A modular eukaryotic genetics and evolution research experience for high school students.

The resources for carrying out and analyzing microbial evolution experiments have become more accessible, making it possible to expand these studies beyond the research laboratory and into the classroom. We developed five connected, standards-aligned yeast evolution laboratory modules, called "yEvo," for high school students. The modules enable students to take agency in answering open-ended research questions. In Module 1, students evolve baker's yeast to tolerate an antifungal drug, and in subsequent modules, investigate how evolved yeasts adapted to this stressful condition at both the phenotype and genotype levels. We used pre- and post-surveys from 72 students at two different schools and post-interviews with students and teachers to assess our program goals and guide module improvement over 3 years. We measured changes in student conceptions, confidence in scientific practices, and interest in STEM careers. Students who participated in yEvo showed improvements in understanding of activity-specific concepts and reported increased confidence in designing a valid biology experiment. Student experimental data replicated literature findings and has led to new insights into antifungal resistance. The modules and provided materials, alongside "proof of concept" evaluation metrics, will serve as a model for other university researchers and K - 16 classrooms interested in engaging in open-ended research questions using yeast as a model system.

The prevalence of killer yeasts and double-stranded RNAs in the budding yeast Saccharomyces cerevisiae.

Killer toxins are antifungal proteins produced by many species of "killer" yeasts, including the brewer's and baker's yeast Saccharomyces cerevisiae. Screening 1270 strains of S. cerevisiae for killer toxin production found that 50% are killer yeasts, with a higher prevalence of yeasts isolated from human clinical samples and winemaking processes. Since many killer toxins are encoded by satellite double-stranded RNAs (dsRNAs) associated with mycoviruses, S. cerevisiae strains were also assayed for the presence of dsRNAs. This screen identified that 51% of strains contained dsRNAs from the mycovirus families Totiviridae and Partitiviridae, as well as satellite dsRNAs. Killer toxin production was correlated with the presence of satellite dsRNAs but not mycoviruses. However, in most killer yeasts, whole genome analysis identified the killer toxin gene KHS1 as significantly associated with killer toxin production. Most killer yeasts had unique spectrums of antifungal activities compared to canonical killer toxins, and sequence analysis identified mutations that altered their antifungal activities. The prevalence of mycoviruses and killer toxins in S. cerevisiae is important because of their known impact on yeast fitness, with implications for academic research and industrial application of this yeast species.

An apparent lack of synergy between degradative enzymes against Staphylococcus aureus biofilms.

The use of enzymes represents an approach to combat bacterial infections by degrading extracellular biomolecules to disperse Staphylococcus aureus biofilms. Commercial enzyme preparations, including cellulase, amylase, pectinase, zymolyase, and pepsin, exhibit concentration-dependent dispersion of S. aureus biofilms. Here, we report that low concentrations of these enzymes generally lack synergy when combined or added together sequentially to biofilms. Only the addition of a protease (pepsin) followed by a commercial mixture of degradative enzymes from Arthrobacter luteus (zymolyase 20T), demonstrated synergy and was effective at dispersing S. aureus biofilms. A more purified mixture of Arthrobacter luteus enzymes (zymolyase 100T) showed improved dispersal of S. aureus biofilms compared to zymolyase 20T but lacked synergy with pepsin. This study emphasizes the complexity of enzymatic biofilm dispersal and the need for tailored approaches based on the properties of degradative enzymes and biofilm composition.

Novel viruses of the family Partitiviridae discovered in Saccharomyces cerevisiae.

It has been 49 years since the last discovery of a new virus family in the model yeast Saccharomyces cerevisiae. A large-scale screen to determine the diversity of double-stranded RNA (dsRNA) viruses in S. cerevisiae has identified multiple novel viruses from the family Partitiviridae that have been previously shown to infect plants, fungi, protozoans, and insects. Most S. cerevisiae partitiviruses (ScPVs) are associated with strains of yeasts isolated from coffee and cacao beans. The presence of partitiviruses was confirmed by sequencing the viral dsRNAs and purifying and visualizing isometric, non-enveloped viral particles. ScPVs have a typical bipartite genome encoding an RNA-dependent RNA polymerase (RdRP) and a coat protein (CP). Phylogenetic analysis of ScPVs identified three species of ScPV, which are most closely related to viruses of the genus Cryspovirus from the mammalian pathogenic protozoan Cryptosporidium parvum. Molecular modeling of the ScPV RdRP revealed a conserved tertiary structure and catalytic site organization when compared to the RdRPs of the Picornaviridae. The ScPV CP is the smallest so far identified in the Partitiviridae and has structural homology with the CP of other partitiviruses but likely lacks a protrusion domain that is a conspicuous feature of other partitivirus particles. ScPVs were stably maintained during laboratory growth and were successfully transferred to haploid progeny after sporulation, which provides future opportunities to study partitivirus-host interactions using the powerful genetic tools available for the model organism S. cerevisiae.

Fungal Glycoside Hydrolases Display Unique Specificities for Polysaccharides and Staphylococcus aureus Biofilms.

Commercially available cellulases and amylases can disperse the pathogenic bacteria embedded in biofilms. This suggests that polysaccharide-degrading enzymes would be useful as antibacterial therapies to aid the treatment of biofilm-associated bacteria, e.g., in chronic wounds. Using a published enzyme library, we explored the capacity of 76 diverse recombinant glycoside hydrolases to disperse Staphylococcus aureus biofilms. Four of the 76 recombinant glycoside hydrolases digested purified cellulose, amylose, or pectin. However, these enzymes did not disperse biofilms, indicating that anti-biofilm activity is not general to all glycoside hydrolases and that biofilm activity cannot be predicted from the activity on pure substrates. Only one of the 76 recombinant enzymes was detectably active in biofilm dispersion, an α-xylosidase from Aspergillus nidulans. An α-xylosidase cloned subsequently from Aspergillus thermomutatus likewise demonstrated antibiofilm activity, suggesting that α-xylosidases, in general, can disperse Staphylococcus biofilms. Surprisingly, neither of the two ß-xylosidases in the library degraded biofilms. Commercial preparations of amylase and cellulase that are known to be effective in the dispersion of Staphylococcus biofilms were also analyzed. The commercial cellulase contained contaminating proteins with multiple enzymes exhibiting biofilm-dispersing activity. Successfully prospecting for additional antibiofilm enzymes may thus require large libraries and may benefit from purified enzymes. The complexity of biofilms and the diversity of glycoside hydrolases continue to make it difficult to predict or understand the enzymes that could have future therapeutic applications.

yEvo: experimental evolution in high school classrooms selects for novel mutations that impact clotrimazole resistance in Saccharomyces cerevisiae.

Antifungal resistance in pathogenic fungi is a growing global health concern. Nonpathogenic laboratory strains of Saccharomyces cerevisiae are an important model for studying mechanisms of antifungal resistance that are relevant to understanding the same processes in pathogenic fungi. We have developed a series of laboratory modules in which high school students used experimental evolution to study antifungal resistance by isolating azole-resistant S. cerevisiae mutants and examining the genetic basis of resistance. We have sequenced 99 clones from these experiments and found that all possessed mutations previously shown to impact azole resistance, validating our approach. We additionally found recurrent mutations in an mRNA degradation pathway and an uncharacterized mitochondrial protein (Csf1) that have possible mechanistic connections to azole resistance. The scale of replication in this initiative allowed us to identify candidate epistatic interactions, as evidenced by pairs of mutations that occur in the same clone more frequently than expected by chance (positive epistasis) or less frequently (negative epistasis). We validated one of these pairs, a negative epistatic interaction between gain-of-function mutations in the multidrug resistance transcription factors Pdr1 and Pdr3. This high school-university collaboration can serve as a model for involving members of the broader public in the scientific process to make meaningful discoveries in biomedical research.

Defining the HIV Capsid Binding Site of Nucleoporin 153.

The interaction between the HIV-1 capsid and human nucleoporin 153 (NUP153) is vital for delivering the HIV-1 preintegration complex into the nucleus via the nuclear pore complex. The interaction with the capsid requires a phenylalanine/glycine-containing motif in the C-terminus of NUP153 (NUP153C). This study used molecular modeling and biochemical assays to comprehensively determine the amino acids in NUP153 that are important for capsid interaction. Molecular dynamics, FoldX, and PyRosetta simulations delineated the minimal capsid binding motif of NUP153 based on the known structure of NUP153 bound to the HIV-1 capsid hexamer. Computational predictions were experimentally validated by testing the interaction of NUP153 with capsid using an in vitro binding assay and a cell-based TRIM-NUP153C restriction assay. This work identified eight amino acids from P1411 to G1418 that stably engage with capsid, with significant correlations between the interactions predicted by molecular models and empirical experiments. This validated the usefulness of this multidisciplinary approach to rapidly characterize the interaction between human proteins and the HIV-1 capsid. IMPORTANCE The human immunodeficiency virus (HIV) can infect nondividing cells by interacting with the host nuclear pore complex. The host nuclear pore protein NUP153 directly interacts with the HIV capsid to promote viral nuclear entry. This study used a multidisciplinary approach combining computational and experimental techniques to comprehensively map the effect of mutating the amino acids of NUP153 on HIV capsid interaction. This work showed a significant correlation between computational and empirical data sets, revealing that the HIV capsid interacted specifically with only six amino acids of NUP153. The simplicity of the interaction motif suggested other FG-containing motifs could also interact with the HIV-1 capsid. Furthermore, it was predicted that naturally occurring polymorphisms in human and nonhuman primates would disrupt NUP153 interaction with capsid, potentially protecting certain populations from HIV-1 infection.

Physiological constraints dictate toxin spatial heterogeneity in snake venom glands.

BACKGROUND: Venoms are ecological innovations that have evolved numerous times, on each occasion accompanied by the co-evolution of specialised morphological and behavioural characters for venom production and delivery. The close evolutionary interdependence between these characters is exemplified by animals that control the composition of their secreted venom. This ability depends in part on the production of different toxins in different locations of the venom gland, which was recently documented in venomous snakes. Here, we test the hypothesis that the distinct spatial distributions of toxins in snake venom glands are an adaptation that enables the secretion of venoms with distinct ecological functions. RESULTS: We show that the main defensive and predatory peptide toxins are produced in distinct regions of the venom glands of the black-necked spitting cobra (Naja nigricollis), but these distributions likely reflect developmental effects. Indeed, we detected no significant differences in venom collected via defensive 'spitting' or predatory 'biting' events from the same specimens representing multiple lineages of spitting cobra. We also found the same spatial distribution of toxins in a non-spitting cobra and show that heterogeneous toxin distribution is a feature shared with a viper with primarily predatory venom. CONCLUSIONS: Our findings suggest that heterogeneous distributions of toxins are not an adaptation to controlling venom composition in snakes. Instead, it likely reflects physiological constraints on toxin production by the venom glands, opening avenues for future research on the mechanisms of functional differentiation of populations of protein-secreting cells within adaptive contexts.

The Characterization of a Novel Virus Discovered in the Yeast Pichia membranifaciens.

Mycoviruses are widely distributed across fungi, including the yeasts of the Saccharomycotina subphylum. This manuscript reports the first double-stranded RNA (dsRNA) virus isolated from Pichia membranifaciens. This novel virus has been named Pichia membranifaciens virus L-A (PmV-L-A) and is a member of the Totiviridae. PmV-L-A is 4579 bp in length, with RNA secondary structures similar to the packaging, replication, and frameshift signals of totiviruses that infect Saccharomycotina yeasts. PmV-L-A was found to be part of a monophyletic group within the I-A totiviruses, implying a shared ancestry between mycoviruses isolated from the Pichiaceae and Saccharomycetaceae yeasts. Energy-minimized AlphaFold2 molecular models of the PmV-L-A Gag protein revealed structural conservation with the Gag protein of Saccharomyces cerevisiae virus L-A (ScV-L-A). The predicted tertiary structure of the PmV-L-A Pol and other homologs provided a possible mechanism for totivirus RNA replication due to structural similarities with the RNA-dependent RNA polymerases of mammalian dsRNA viruses. Insights into the structure, function, and evolution of totiviruses gained from yeasts are essential because of their emerging role in animal disease and their parallels with mammalian viruses.

Nuku, a family of primate retrocopies derived from KU70.

The gene encoding the ubiquitous DNA repair protein, Ku70p, has undergone extensive copy number expansion during primate evolution. Gene duplications of KU70 have the hallmark of long interspersed element-1 mediated retrotransposition with evidence of target-site duplications, the poly-A tails, and the absence of introns. Evolutionary analysis of this expanded family of KU70-derived "NUKU" retrocopies reveals that these genes are both ancient and also actively being created in extant primate species. NUKU retrocopies show evidence of functional divergence away from KU70, as evinced by their altered pattern of tissue expression and possible tissue-specific translation. Molecular modeling predicted that amino acid changes in Nuku2p at the interaction interface with Ku80p would prevent the assembly of the Ku heterodimer. The lack of Nuku2p-Ku80p interaction was confirmed by yeast two-hybrid assay, which contrasts the robust interaction of Ku70p-Ku80p. While several NUKU retrocopies appear to have been degraded by mutation, NUKU2 shows evidence of positive natural selection, suggesting that this retrocopy is undergoing neofunctionalization. Although Nuku proteins do not appear to antagonize retrovirus transduction in cell culture, the observed expansion and rapid evolution of NUKUs could be being driven by alternative selective pressures related to infectious disease or an undefined role in primate physiology.

Unexpected lack of specialisation in the flow properties of spitting cobra venom.

Venom spitting is a defence mechanism based on airborne venom delivery used by a number of different African and Asian elapid snake species ('spitting cobras'; Naja spp. and Hemachatus spp.). Adaptations underpinning venom spitting have been studied extensively at both behavioural and morphological level in cobras, but the role of the physical properties of venom itself in its effective projection remains largely unstudied. We hereby provide the first comparative study of the physical properties of venom in spitting and non-spitting cobras. We measured the viscosity, protein concentration and pH of the venom of 13 cobra species of the genus Naja from Africa and Asia, alongside the spitting elapid Hemachatus haemachatus and the non-spitting viper Bitis arietans. By using published microCT scans, we calculated the pressure required to eject venom through the fangs of a spitting and a non-spitting cobra. Despite the differences in the modes of venom delivery, we found no significant differences between spitters and non-spitters in the rheological and physical properties of the studied venoms. Furthermore, all analysed venoms showed a Newtonian flow behaviour, in contrast to previous reports. Although our results imply that the evolution of venom spitting did not significantly affect venom viscosity, our models of fang pressure suggests that the pressure requirements to eject venom are lower in spitting cobras than in non-spitting cobras.

Vaginal Isolates of Candida glabrata Are Uniquely Susceptible to Ionophoric Killer Toxins Produced by Saccharomyces cerevisiae.

Compared to other species of Candida yeasts, the growth of Candida glabrata is inhibited by many different strains of Saccharomyces killer yeasts. The ionophoric K1 and K2 killer toxins are broadly inhibitory to all clinical isolates of C. glabrata from patients with recurrent vulvovaginal candidiasis, despite high levels of resistance to clinically relevant antifungal therapeutics.

Unexpected lack of specialisation in the flow properties of spitting cobra venom.

Venom spitting is a defence mechanism based on airborne venom delivery used by a number of different African and Asian elapid snake species ('spitting cobras'; Naja spp. and Hemachatus spp.). Adaptations underpinning venom spitting have been studied extensively at both behavioural and morphological level in cobras, but the role of the physical properties of venom itself in its effective projection remains largely unstudied. We hereby provide the first comparative study of the physical properties of venom in spitting and non-spitting cobras. We measured the viscosity, protein concentration and pH of the venom of 13 cobra species of the genus Naja from Africa and Asia, alongside the spitting elapid Hemachatus haemachatus and the non-spitting viper Bitis arietans By using published microCT scans, we calculated the pressure required to eject venom through the fangs of a spitting and a non-spitting cobra. Despite the differences in the modes of venom delivery, we found no significant differences between spitters and non-spitters in the rheological and physical properties of the studied venoms. Furthermore, all analysed venoms showed a Newtonian flow behaviour, in contrast to previous reports. Although our results imply that the evolution of venom spitting did not significantly affect venom viscosity, our models of fang pressure suggests that the pressure requirements to eject venom are lower in spitting cobras than in non-spitting cobras.

The Species-Specific Acquisition and Diversification of a K1-like Family of Killer Toxins in Budding Yeasts of the Saccharomycotina.

Killer toxins are extracellular antifungal proteins that are produced by a wide variety of fungi, including Saccharomyces yeasts. Although many Saccharomyces killer toxins have been previously identified, their evolutionary origins remain uncertain given that many of these genes have been mobilized by double-stranded RNA (dsRNA) viruses. A survey of yeasts from the Saccharomyces genus has identified a novel killer toxin with a unique spectrum of activity produced by Saccharomyces paradoxus. The expression of this killer toxin is associated with the presence of a dsRNA totivirus and a satellite dsRNA. Genetic sequencing of the satellite dsRNA confirmed that it encodes a killer toxin with homology to the canonical ionophoric K1 toxin from Saccharomyces cerevisiae and has been named K1-like (K1L). Genomic homologs of K1L were identified in six non-Saccharomyces yeast species of the Saccharomycotina subphylum, predominantly in subtelomeric regions of the genome. When ectopically expressed in S. cerevisiae from cloned cDNAs, both K1L and its homologs can inhibit the growth of competing yeast species, confirming the discovery of a family of biologically active K1-like killer toxins. The sporadic distribution of these genes supports their acquisition by horizontal gene transfer followed by diversification. The phylogenetic relationship between K1L and its genomic homologs suggests a common ancestry and gene flow via dsRNAs and DNAs across taxonomic divisions. This appears to enable the acquisition of a diverse arsenal of killer toxins by different yeast species for potential use in niche competition.

Amyloid and Tau PET Imaging of Alzheimer Disease and Other Neurodegenerative Conditions.

Although diagnosing the syndrome of dementia is largely a clinical endeavor, neuroimaging plays an increasingly important role in accurately determining the underlying etiology, which extends beyond its traditional role in excluding other causes of altered cognition. New neuroimaging methods not only facilitate the diagnosis of the most common neurodegenerative conditions (particularly Alzheimer Disease [AD]) after symptom onset, but also show diagnostic promise even in the very early or presymptomatic phases of disease. Positron emission tomography (PET) is increasingly recognized as a key clinical tool for differentiating normal age-related changes in brain metabolism (using 18F-fluorodeoxyglucose [FDG]) from those seen in the earliest stages of specific forms of dementia. However, FDG PET only demonstrates nonspecific changes in altered parenchymal glucose uptake and not the specific etiologic proteinopathy causing the abnormal glucose uptake. A growing class of radiotracers targeting specific protein aggregates for amyloid-ß (Aß) and tau are changing the way AD is diagnosed, as these radiotracers directly label the underlying disease pathology. As these pathology-specific radiotracers are currently making their way to the clinic, it is important for the clinical neuroradiologist to understand the underlying patterns of Aß and tau deposition in the context of AD (across its clinical continuum) and in other causes of dementia, as well as understand the implications of current research.

Correction: Preclinical antivenom-efficacy testing reveals potentially disturbing deficiencies of snakebite treatment capability in East Africa.

[This corrects the article DOI: 10.1371/journal.pntd.0005969.].

The design and implementation of restraint devices for the injection of pathogenic microorganisms into Galleria mellonella.

The injection of laboratory animals with pathogenic microorganisms poses a significant safety risk because of the potential for injury by accidental needlestick. This is especially true for researchers using invertebrate models of disease due to the required precision and accuracy of the injection. The restraint of the greater wax moth larvae (Galleria mellonella) is often achieved by grasping a larva firmly between finger and thumb. Needle resistant gloves or forceps can be used to reduce the risk of a needlestick but can result in animal injury, a loss of throughput, and inconsistencies in experimental data. Restraint devices are commonly used for the manipulation of small mammals, and in this manuscript, we describe the construction of two devices that can be used to entrap and restrain G. mellonella larvae prior to injection with pathogenic microbes. These devices reduce the manual handling of larvae and provide an engineering control to protect against accidental needlestick injury while maintaining a high rate of injection.