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OBJECTIVE The objective of this study was to determine bacterial flora throughout the gastrointestinal tract of a saltwater crocodile (Crocodylus porosus) using 16S rRNA gene analysis. ANIMALS A convention on international trade in endangered species (CITES) of wild fauna and flora registered crocodile farm, provided a healthy male saltwater crocodile, Crocodylus porosus for this study. PROCEDURES Three samples were taken from the oral cavity, 3 samples from the proximal region of the small intestine (jejunum), and 3 samples from the distal part of the large intestine of the gastrointestinal tract of C. porosus were obtained using sterile cotton swabs. Next, swabs were placed in 15 mL sterile centrifuge tubes, individually, and kept on ice for immediate transportation to the laboratory. This was followed by 16S rRNA gene analysis using specific primers (341F-CCTAYGGGRBGCASCAG, and 806R-GGACTACNNGGGTATCTAAT). Amplicons were sequenced on Illumina paired-end platform, and bacterial gastrointestinal communities, the relative abundance of taxa, and principal component and coordinate analysis were performed. RESULTS The findings revealed that bacterial community structures from differing regions exhibited several differences. The number of observed bacterial operational taxonomic units (OTUs) was 153 in the oral cavity, 239 in the small intestine, and 119 in the large intestine of C. porosus. The small intestine reflects the highest richness. In contrast, the large intestine exhibited the least richness of microbial communities. Relative abundance of taxa showed that Proteobacteria, Bacteroidetes, and Firmicutes were dominant in all 3 sample sites. Pseudomonas differed in the oral cavity and the large intestine, with the latter exhibiting less distribution of Pseudomonas. Stenotrophomonas and Castellaniella were higher in the oral cavity, while the relative abundance of Comamonas and Salmonella was higher in the small intestine. Conversely, the relative abundance of Salmonella and Pannonibacter was augmented in the large intestine. CLINICAL RELEVANCE For the first time, this study demonstrates the bacterial diversity along the segments of the gastrointestinal tract of C. porosus. Bacterial flora varies throughout the gastrointestinal tract. Although further studies using large cohorts are warranted; however, our findings suggest that microbiome composition may have the potential as a biomarker in determining the overall health and well-being of C. porosus. [ABSTRACT FROM AUTHOR]

Reptiles are ectothermic amniotes in a world dominated by endotherms. Reptiles originated more than 300 million years ago and they often dwell in polluted environments which may expose them to pathogenic micro‐organisms, radiation and/or heavy metals. Reptiles also possess greater longevity and may live much longer than similar‐sized land mammals, for example, turtles, tortoises, crocodiles and tuatara are long‐lived reptiles living up to 100 years or more. Many recent studies have emphasized the pivotal role of the gut microbiome on its host; thus, we postulated that reptilian gut microbiome and/or its metabolites and the interplay with their robust immune system may contribute to their longevity and overall hardiness. Herein, we discuss the composition of the reptilian gut microbiome, immune system–gut microbiome cross‐talk, antimicrobial peptides, reptilian resistance to infectious diseases and cancer, ageing, as well the current knowledge of the genome and epigenome of these remarkable species. Preliminary studies have demonstrated that microbial gut flora of reptiles such as crocodiles, tortoises, water monitor lizard and python exhibit remarkable anticancer and antibacterial properties, as well as comprise novel gut bacterial metabolites and antimicrobial peptides. The underlying mechanisms between the gut microbiome and the immune system may hold clues to developing new therapies overall for health, and possible extrapolation to exploit the ancient defence systems of reptiles for Homo sapiens benefit. [ABSTRACT FROM AUTHOR]

The ability of aerobic microorganisms to regulate internal and external concentrations of the reactive oxygen species (ROS) superoxide directly influences the health and viability of cells. Superoxide dismutases (SODs) are the primary regulatory enzymes that are used by microorganisms to degrade superoxide. SOD is not one, but three separate, non‐homologous enzymes that perform the same function. Thus, the evolutionary history of genes encoding for different SOD enzymes is one of convergent evolution, which reflects environmental selection brought about by an oxygenated atmosphere, changes in metal availability, and opportunistic horizontal gene transfer (HGT). In this study, we examine the phylogenetic history of the protein sequence encoding for the nickel‐binding metalloform of the SOD enzyme (SodN). The genomic potential to produce SodN is widespread among bacteria, including Actinobacteriota (Actinobacteria), Chloroflexota (Chloroflexi), Cyanobacteria, Proteobacteria, Patescibacteria, and others. The gene is also present in many archaea, with Thermoplasmatota and Nanoarchaeota representing the vast majority of archaeal sodN diversity. A comparison of organismal and SodN protein phylogenetic trees reveals several instances of HGT, including multiple inter‐domain transfers of the sodN gene from the bacterial domain to the archaeal domain. Nearly half of the archaeal members with sodN live in the photic zone of the marine water column. The sodN gene is widespread and characterized by apparent vertical gene transfer in some sediment‐ or soil‐associated lineages within the Actinobacteriota and Chloroflexota phyla, suggesting the ancestral sodN likely originated in one of these clades before expanding its taxonomic and biogeographic distribution to additional microbial groups in the surface ocean in response to decreasing iron availability. In addition to decreasing iron quotas, nickel‐binding SOD has the added benefit of withstanding high reactant and product ROS concentrations without damaging the enzyme, making it particularly well suited for the modern surface ocean. [ABSTRACT FROM AUTHOR]

Student problem behaviors in early elementary school are associated with fewer learning opportunities, poor relationships with teachers, and academic and behavioral problems, including increased risk of emotional/behavioral disorders (EBDs). This study examined the effect of BEST in CLASS–Elementary (BEST in CLASS-E), a Tier 2 intervention delivered by teachers, on student problem behavior, academic achievement, teacher–student relationships, and classroom quality using a randomized controlled trial design. Participants were 45 kindergarten to grade 3 students identified at risk of development of EBDs and their 26 teachers from three elementary schools located in an urban school district. BEST in CLASS-E was found to decrease teacher-reported student problem behavior and increase teacher–student closeness; no effects were noted for academic achievement or overall classroom quality. Results suggest the promise of BEST in CLASS-E as a Tier 2 intervention delivered by teachers in elementary classrooms. Implications and limitations of the study are discussed. [ABSTRACT FROM AUTHOR]

The primary purpose of this paper is to review how BEST in CLASS, an evidence-based program targeting children at risk for emotional and behavior disorders, interacts with a diverse range of social ecological factors to promote successful implementation. Recent findings from two BEST in CLASS studies (BEST in CLASS-Prekindergarten and BEST in CLASS- Elementary) are reviewed in relation to barriers and facilitators of high quality implementation. The importance of taking into account factors associated with teacher delivery of interventions in classrooms, particularly for children with and at-risk for emotional and behavioral disorders, when designing and evaluating interventions is discussed. Implications for future research and practice are provided. [ABSTRACT FROM AUTHOR]

Naegleria fowleri causes an uncommon but deadly disease called primary amoebic meningoencephalitis (PAM). There has been an increase of reported PAM cases, particularly since 2000. Although water is the dominant route of transmission of PAM, infection through soil/dust is a possible alternative route. We have observed differences in epidemiology between the southern states in the USA and the Indian subcontinent (ISC). The patient age range is greater in the ISC than in the USA, and there are more infections in the ISC which are not water-associated. We show that PAM is under-reported and argue that climate change will increase the incidence of PAM, and that the geographic range of N. fowleri will spread polewards. The incidence of reported PAM cases caused by N. fowleri has seen an increase which is especially marked after around 2000. The two best documented areas, the southern states of the USA and the ISC, appear to differ in epidemiology. There are indications that climate change is likely beginning to increase the abundance and range of N. fowleri , and this trend will continue. [ABSTRACT FROM AUTHOR]

Abstract: The ability of micro‐organisms to oxidize manganese (Mn) from Mn(II) to Mn(III/IV) oxides transcends boundaries of biological clade or domain. Many bacteria and fungi oxidize Mn(II) to Mn(III/IV) oxides directly through enzymatic activity or indirectly through the production of reactive oxygen species. Here, we determine the oxygen isotope fractionation factors associated with Mn(II) oxidation via various biotic (bacteria and fungi) and abiotic Mn(II) reaction pathways. As oxygen in Mn(III/IV) oxides may be derived from precursor water and molecular oxygen, we use a twofold approach to determine the isotope fractionation with respect to each oxygen source. Using both 18O‐labeled water and closed‐system Rayleigh distillation approaches, we constrain the kinetic isotope fractionation factors associated with O atom incorporation during Mn(II) oxidation to −17.3‰ to −25.9‰ for O2 and −1.9‰ to +1.8‰ for water. Results demonstrate that stable oxygen isotopes of Mn(III/IV) oxides have potential to distinguish between two main classes of biotic Mn(II) oxidation: direct enzymatic oxidation in which O2 is the oxidant and indirect enzymatic oxidation in which superoxide is the oxidant. The fraction of Mn(III/IV) oxide‐associated oxygen derived from water varies significantly (38%–62%) among these bio‐oxides with only weak relationship to Mn oxidation state, suggesting Mn(III) disproportionation may account for differences in the fraction of mineral‐bound oxygen from water and O2. Additionally, direct incorporation of molecular O2 suggests that Mn(III/IV) oxides contain a yet untapped proxy of δ 18 O O 2 of environmental O2, a parameter reflecting the integrated influence of global respiration, photorespiration, and several other biogeochemical reactions of global significance. [ABSTRACT FROM AUTHOR]

We describe a new species of cyst-producing soil amoeba Vannella pentlandii from course pasture in the Pentland Hills, Scotland. Analysis of the 18S rDNA gene reveals that it belongs to the sub-group within the genus, presently composed of V. placida, V. epipetala and V. fimicola (the PEF group). This group share features such as longitudinal folds/ridges on the lamella (the anterior hyaline region of the trophozoite), stubby floating forms and cyst production. While each PEF species contain cyst producing strains, not all strains within these species do so. V. fimicola produces cysts on stalks leading to its former classification as a slime mould, however no such stalks were evident in the V. pentlandii , instead groups of cysts become piled on top of each other forming clumps. The encysting amoebae crawl toward each other, pushing some off the surface to form these mounds. The V. pentlandii trophozoites are of typical size for the genus but the cysts at 6.9 μm in diameter, are the smallest so far described in genus Vannella . Other cyst producing species are found in various branches within the Vannella phylogenetic tree, probably meaning that this ability was ancestral but lost in many branches (particularly in marine species), and perhaps re-gained in others. [ABSTRACT FROM AUTHOR]

We report the isolation of a new species of Allovahlkampfia, a small cyst-forming heterolobosean soil amoeba. Phylogenetic analysis of the 18S rDNA and the internal transcribed spacers indicates that Allovahlkampfia is more closely related to the acrasids than to other heterolobosean groups and indicates that the new strain (GF1) groups with Allovahlkampfia tibetiensis and A. nederlandiensis despite being significantly smaller than these and any other described Allovahlkampfia species. GF1 forms aggregated cyst masses similar to the early stages of Acrasis sorocarp development, in agreement with the view that it shares ancestry with the acrasids. Time-lapse video microscopy reveals that trophozoites are attracted to individuals that have already begun to encyst or that have formed cysts. Although some members of the genus are known to be pathogenic the strain GF1 does not grow above 28oC nor at elevated osmotic conditions, indicating that it is unlikely to be a pathogen. [ABSTRACT FROM AUTHOR]

While some amoebae reproduce sexually, many amoebae (e.g., Acanthamoeba , Naegleria ) reproduce asexually and therefore, according to popular doctrine, are likely to have been genetically disadvantaged as a consequence. In the absence of sex, mutations are proposed to accumulate by a mechanism known as Muller's ratchet. I hypothesise that amoebae can escape the ravages of accumulated mutation by virtue of their being polyploid. The polyploid state reduces spontaneous mutation accumulation by gene conversion, the freshly mutated copy being corrected by the presence of the many other wild-type copies. In this manner these amoebae reap the benefits of an asexual reproductive existence: principally, that it is rapid and convenient. Evidence for this mechanism comes from polyploid plants, bacteria, and archaea. [ABSTRACT FROM AUTHOR]

Crocodiles are renowned for their resilience and capacity to withstand environmental stressors, likely influenced by their unique gut microbiome. In this study, we determined whether selected gut bacteria of Crocodylus porosus exhibit anti-inflammatory effects in response to stress, by measuring nitric oxide release, interleukin 1-beta, tumor necrosis factor-alpha, and prostaglandin E2 in cerebrovascular endothelial cells. Using the Griess assay, the findings revealed that among several C. porosus gut bacterial isolates, the conditioned media containing the metabolites of two bacterial strains (CP27 and CP36) inhibited nitric oxide production significantly, in response to the positive control, i.e., taxol-treatment. Notably, CP27 and CP36 were more potent at reducing nitric oxide production than senloytic compounds (fisetin, quercetin). Using enzyme linked immunosorbent assays, the production of pro-inflammatory cytokines (IL-1β, TNF-α, PGE2), was markedly reduced by treatment with CP27 and CP36, in response to stress. Both CP27 and CP36 contain a plethora of metabolites to exact their effects [(3,4-dihydroxyphenylglycol, 5-methoxytryptophan, nifedipine, 4-chlorotestosterone-17-acetate, 3-phenoxypropionic acid, lactic acid, f-Honaucin A, l,l-Cyclo(leucylprolyl), 3-hydroxy-decanoic acid etc.], indicative of their potential in providing protection against cellular stress. Further high-throughput bioassay-guided testing of gut microbial metabolites from crocodiles, individually as well as in combination, together with the underlying molecular mechanisms, in vitro and in vivo will elucidate their value in the rational development of innovative therapies against cellular stress/gut dysbiosis. [ABSTRACT FROM AUTHOR]

Objective: PI susceptibility results from a complex interplay between protease and Gag proteins, with Gag showing wide variation across HIV-1 subtypes.We explored the impact of pre-treatment susceptibility on the outcome of lopinavir/ritonavir monotherapy. Methods: Treatment-naive individuals who experienced lopinavir/ritonavir monotherapy failure from the MONARK studywere matched (by subtype, viral load and baseline CD4 count) with those who achieved virological response (‘successes’). Successes were defined by viral load ,400 copies/mL after week 24 and ,50 copies/mL from week 48 to week 96. Full-length Gag–protease was amplified from patient samples for in vitro phenotypic susceptibility testing, with susceptibility expressed as fold change (FC) relative to a subtype B reference strain. Results: Baseline lopinavir susceptibility was lower in viral failures compared with viral successes, but the differences were not statistically significant (median lopinavir susceptibility: 4.4 versus 8.5, respectively, P¼0.17). Among CRF02_AG/G patients, there was a significant difference in lopinavir susceptibility between the two groups (7.1 versus 10.4, P¼0.047), while in subtype B the difference was not significant (2.7 versus 3.4, P¼0.13). Subtype CRF02_AG/G viruses had a median lopinavir FC of 8.7 compared with 3.1 for subtype B (P¼0.001). Conclusions: We report an association between reduced PI susceptibility (using full-length Gag–protease sequences) at baseline and subsequent virological failure on lopinavir/ritonavir monotherapy in antiretroviralnaive patients harbouring subtype CRF02_AG/G viruses. We speculate that this may be important in the context of suboptimal adherence in determining viral failure. [ABSTRACT FROM AUTHOR]

Highlights: [•] Lac resin samples were studied using GCMS with quaternary ammonium reagents. [•] Diagnostic methyl derivatives of aliphatic and terpene hydroxyacids were identified. [•] The analyses enabled identification and discrimination of various types of lac resin. [•] The methods were applied successfully to microgram-sized samples from museum objects. [•] The results shed light on artists’ practices and inform conservation of works of art. [Copyright &y& Elsevier]

The plankton assemblage of the permanently open Sundays Estuary in South Africa was sampled seasonally to provide further information on the feeding ecology of the larval stages of fishes in temperate estuaries. Collections were made between winter 2007 and autumn 2009 at eight sites by means of boat-based plankton tows. Physico-chemical variability in the water column was typical for temperate systems. Mean density of larval fishes and zooplankton showed similar seasonal and spatial trends. Gut content analyses were conducted in five dominant larval fish species:Gilchristella aestuaria(Clupeidae),Pomadasys commersonnii(Haemulidae),Monodactylis falciformis(Monodactylidae),Myxus capensis(Mugilidae) andRhabdosargus holubi(Sparidae). The first record of selective consumption of copepod eggs byG. aestuariais reported. Insect larvae (chironimids) dominated the diet of some marine fish larvae (M. falciformisandM. capensis). The copepodPseudodiaptomus hesseiformed the largest volume of the diet ofP. commersonniiwhereasR. holubiselected for the amphipodCorophium triaenonyx, withP. hesseialso being a frequently consumed prey item. Seasonal and spatial variability with salinity was evident in the diet. Larvae also showed changes in diet at different stages of development. Pelagic prey species constituted the largest volume of the diet ofG. aestuariawhereas all other larvae consumed large volumes of bentho-pelagic prey. All larvae selectedP. hesseias a prey item.Myxus capensisandR. holubialso selected this copepod and showed the largest diversity in diet. [ABSTRACT FROM PUBLISHER]

The zooplankton assemblage in the permanently open Sundays Estuary, on the south-east coast of South Africa, was studied seasonally. Samples were collected once per season between winter 2007 and autumn 2009 by means of boat-based plankton tows. A total of 19 taxa was recorded. Numerically, the copepodPseudodiaptomus hesseidominated the zooplankton, withParacartia longipatellaandHalicyclopssp. also contributing significantly to the assemblage. Mysids were dominated byMesopodopsis wooldridgeiandRhopalopthalmus terranatalis. Among the Brachyura,Hymenosoma orbicularelarvae andParatylodiplax edwardsiilarvae were prevalent. Zooplankton density varied with season, site and salinity zone, as has been reported in previous studies of temperate estuaries worldwide. Density peaked in summer, with means of 3 331 ind. m−3in 2008 and 8 031 ind. m−3in 2009. Highest densities ofP. longipatellaoccurred in the lower estuary (Sites 1–3) andP. hesseiin the upper estuary (Sites 8–10). Highest density occurred in the euhaline zone, due largely to the high density ofP. longipatella. Site 1, closest to the mouth, typically supported the highest diversity of zooplankton. Patterns in zooplankton density and diversity resulted from the combined influence of salinity, temperature and fresh water inflow. [ABSTRACT FROM AUTHOR]

The larval fish assemblage was studied in the permanently open Sundays Estuary on the southeast coast of South Africa. Seasonal samples were collected between winter 2007 and autumn 2009 at eight sites along the estuary by means of boat-based plankton tows using two modified Working Party 2 (WP2) nets. Salinity ranged from 1.5-36.5 and temperature from 11.6-26.8°C during the study period. A total of 8174 larval and early juvenile fishes were caught, representing 12 families and 23 taxa. The Clupeidae, Gobiidae and Blenniidae were the numerically dominant fish families. Common species included Gilchristella aestuarias (Clupeidae), Omobranchus woodi (Blenniidae), Caffrogobius gilchristi, Glossogobius callidus (Gobiidae), Liza dumerilii and Myxus capensis (Mugilidae), largely reflecting the adult assemblage of the estuary. Catches varied significantly between seasons, but not between sites and salinity zones within the estuary. Highest catches were recorded in summer (mean of 464 and 928 larvae per 100 m³ in 2008 and 2009, respectively). Species diversity also varied seasonally, with highest diversity occurring during summer. Highest larval fish density occurred in the euhaline zone while the oligohaline zone supported the highest diversity. Estuarine-resident species (category I) dominated the system (91%). The preflexion stage of development dominated the catches suggesting a high degree of local production in the Sundays Estuary. Larval sizes of the estuarine-resident G. aestuarias and catadromous species M. capensis tended to increase towards the middle and upper reaches of the estuary. Important variables regulating larval fish dynamics in the Sundays Estuary included salinity, temperature and turbidity. [ABSTRACT FROM AUTHOR]

Exposure to air pollution has been linked to pulmonary diseases. Naphthalene (NA), an abundant polycyclic aromatic hydrocarbon in tobacco smoke and urban air, is a model toxicant for air pollution effects in the lung. Repeated exposures to NA in male mice result in tolerance, defined as the emergence of a resistant cell phenotype after prior exposure. Tolerance has not been studied in females. Females have sex differences in airway epithelial responses and in the prevalence of certain airway diseases. Male and female mice were exposed to a toleranceinducing regimen of NA, and lungs were examined by airway level to characterize the cellular changes associated with repeated NA exposure and to assess the expression of genes and proteins involved in NA bioactivation and detoxification. The airway epithelium in treated males resembled that of controls. Females in the tolerant state were characterized by dense populations of ciliated cells in midlevel, distal, and bifurcating airways and a lower abundance of Clara cells at all airway levels. Cytotoxicity following a secondary challenge dose was also greater in females than males. Furthermore, females had decreased gene/protein expression of CYP2F2, a P-450 that metabolizes NA to a toxic epoxide, and glutamate-cysteine ligase, the rate-limiting enzyme in glutathione synthesis, than NA-tolerant males at all airway levels examined. We conclude that, while females develop tolerance, sex differences exist in the tolerant state by airway level, and females remain more susceptible than males to repeated exposures to NA. [ABSTRACT FROM AUTHOR]

We have used fluorescence recovery after photobleaching to study the effect of muscle α-actinin on the structure of actin filaments in dilute solutions. Unexpectedly we found that α-actinin partitioned filaments into two types: those with a high mobility and those with low mobility. We have determined that the high mobility (smaller sized) population is too large to be simple monomeric actin:α-actinin complexes. Although it is known that cofilin encourages the transformation of α-actinin:actin gels into large meshworks of inter-digitating actin filament bundles (Maciver et al. ), we have found that the presence of cofilin also increases the cross-linking of actin filaments by α-actinin and hypothesize that this is due to cofilin’s ability to alter the filament twist. This effectively makes more potential α-actinin binding sites per unit of actin filament. As expected from previous work, this effect was more marked at pH 6.5 than at pH 8.0. Both effects are likely to operate in cells to deny other actin-binding proteins access to binding these particular filaments and may explain how very different actin cytoskeletal structures may co-exist in the same cell at the same time. [ABSTRACT FROM AUTHOR]

The management of pancreatic pain is a significant clinical problem so understanding of how sensory signals are generated in pancreatic tissue is fundamental. We aimed to characterize mechanosensitive and chemosensitive properties of pancreatic spinal and vagal afferents in vitro. Spinal and vagal afferent preparations from Sprague–Dawley rats were established incorporating the left splanchnic nerve or vagus nerves respectively. The common bile duct was cannulated for distension of the pancreatic duct with fluid. Nerve discharge evoked by blunt probing, duct distension or electrical stimulation was obtained from teased nerve bundles using standard extra-cellular recording. Discharge from 197 spinal afferent bundles was recorded, of which 57% displayed spontaneous activity. Blunt probing revealed 61 mechanosensitive receptive fields which were associated primarily with arteries/blood vessels (33/61) and the parenchyma (22/61). All mechanosensitive responses were slowly adapting, with 33% continuing to discharge after termination of the stimulus and 60% displaying a response threshold <10 g. Application of chemical mediators (bradykinin, histamine, 5-hydroxytryptamine, cholecystokinin octapeptide) evoked a response from 31/57 units, with 33% excitatory and 23% inhibitory. Spontaneous discharge was recorded from 72% of 135 vagal bundles. Mechanosensitive receptive fields were not identified in the pancreas but were evident in adjacent organs. No spinal or vagal afferent response to duct distension was obtained. In conclusion, pancreatic mechanosensitive spinal afferents are common, in contrast to pancreatic mechanosensitive vagal afferents indicating that pancreatic sensory innervation is predominantly spinal. Chemosensitive spinal afferent nerve endings are present in the pancreas and respond to a variety of inflammatory and physiological mediators. [ABSTRACT FROM AUTHOR]

OBJECTIVE: This study investigated the relationship between trauma survivors' goals and retrieval of self-defining memories in post-traumatic stress disorder (PTSD). METHODS: Civilian trauma survivors with PTSD, trauma survivors with no PTSD and non-trauma-exposed control participants (N = 49) provided autobiographical memories of events that they believe shaped who they are. Participants also provided details about their major personal goals. RESULTS: Participants with PTSD reported more self-defining memories that were trauma-related, negative valence and from adult years than non-PTSD and control participants. Further, retrieval of trauma-related self-defining memories was strongly associated with reporting personal goals that were related to traumatic experiences. CONCLUSIONS: These findings are discussed in terms of the proposition that trauma survivors' current concerns may direct retrieval of trauma-related memories. [ABSTRACT FROM AUTHOR]

Gelsolin is an abundant calcium dependent actin filament severing and capping protein. In the absence of calcium the molecule is compact but in the presence of calcium, as its six similar domains alter their relative position, a generally more open configuration is adopted to reveal the three actin binding sites. It is generally held that a ‘helical-latch’ at the C-terminus of gelsolin's domain 6 (G6), binds domain 2 (G2) to keep gelsolin in the calcium-free compact state, and that the crutial calcium binding site(s) reside in the C-terminal half of gelsolin perhaps involving the C-terminal helix itself has to be bound to release this latch. Here we provide evidence for a calcium dependent conformational change within G2 (K d = ≈15 µm). We also report a calcium dependent binding site for the C-terminus (G4–6) within G2 and delimit this further to a specific region formed by residues 203–225 and 159–193. It is known that the activation of gelsolin involves multiple calcium binding events (around 6) the first of which (in G6) may release the latch. We propose that the calcium-dependent conformational change in G2 may be a subsequent step that is necessary for the dissociation of G2 from G4–6, and that this movement occurs in sympathy with calcium induced conformational changes within G6 by the physical coupling of the two calcium binding sites within G2 and G6. Additional calcium binding in other domains then result in the complete opening and activation of the gelsolin molecule. [ABSTRACT FROM AUTHOR]

Studies the amino acid sequence of the actin monomer binding/actin filament severing protein actophorin from acanthamoeba castellani. Automated degradation of peptide fragments; Sequencing of full-length DNA; Single copy gene; Two distinct mRNA species; Blocking of NH2 terminus; More.

Actin depolymerizing factor (ADF) from vertebrates and actophorin from Acanthamoeba castellanii are members of a protein family that bind monomeric and polymeric actin and have been shown by microscopy to sever filaments. Here, we compare the properties of recombinant human ADF and actophorin using rabbit muscle actin. ADF binds tenfold more strongly than actophorin to monomeric actin (G-actin)-ATP, and both bind co-operatively to F-actin. ADF decorates filaments below pH 7.3 and induces substantial depolymerization at higher pH values [Hawkins, M., Pope, B., Maciver, S. K. & Weeds, A. G. (1993) Human actin depolymerizing factor mediates a pH-sensitive destruction of actin filaments, Biochemistry 32, 9985-9993], but, at all pH values tested, actophorin binds to filaments in a similar manner to ADF at pH 6.5. Both proteins increase the depolymerization rate at the pointed ends of gelsolin-capped filaments, but the effect of ADF is more marked at pH 8.0. Both proteins accelerate the nucleating activity when mixed with filamentous actin (F-actin), but not with gelsolin-capped filaments, and they rapidly decrease the lengths of filaments as evidenced by electron microscopy. Both of these effects are best explained by a weak severing activity. Our results are discussed in relation to earlier models and to the structural changes observed when ADF binds F-actin [McGough, A., Pope, B., Chiu, W. & Weeds, A. (1997) Cofilin changes the twist of F-actin : implications for actin filament dynamics and cellular function, J. Cell Biol. 138, 771-781]. We also discuss the relevance of these observations to their possible roles in facilitating actin turnover in cells, thereby regulating filament dynamics in cell motility. [ABSTRACT FROM AUTHOR]

Intron retention (IR) refers to the mechanism of alternative splicing in which an intron is not excised from the mature transcript. IR in the cosmopolitan free-living amoeba Acanthamoeba castellanii has not been studied. We performed an analysis of RNA sequencing data during encystment to identify genes that presented differentially retained introns during this process. We show that IR increases during cyst formation, indicating a potential mechanism of gene regulation that could help downregulate metabolism. We identify 69 introns from 67 genes that are differentially retained comparing the trophozoite stage and encystment after 24 and 48 h. These genes include several hypothetical proteins. We show different patterns of IR during encystment taking as examples a lipase, a peroxin-3 protein, an Fbox domain containing protein, a proteasome subunit, a polynucleotide adenylyltransferase, and a tetratricopeptide domain containing protein. A better understanding of IR in Acanthamoeba, and even other protists, could help elucidate changes in life cycle and combat disease such as Acanthamoeba keratitis in which the cyst is key for its persistence. [ABSTRACT FROM AUTHOR]

The control of African trypanosomiasis (AT) in Eastern and Southern Africa, including Zambia, faces huge challenges due to the involvement of wild and domestic animal reservoirs. Free-roaming dogs in wildlife-populated and tsetse-infested villages of Zambia's Mambwe district are exposed to infectious tsetse bites. Consuming fresh raw game meat and bones further exacerbates their risk of contracting AT. We focus on the reservoir role of such dogs in maintaining and transmitting diverse species of trypanosomes that are infective to humans and livestock in Zambia's Mambwe district. A cohort of 162 dogs was enrolled for follow-up at 3 different time points from June to December 2018 in selected villages of Malama, Mnkhanya, and Nsefu chiefdoms of Mambwe district, eastern Zambia. Blood and serum were screened for AT by microscopy, GM6 ELISA, PCR (ITS1 and SRA), and Sanger sequencing. Out of the 162 dogs in the cohort, 40 were lost to follow-up and only 122 remained traceable at the end of the study. GM6 ELISA detected Trypanosoma antibodies in 121 dogs (74.7%) and ITS1-PCR detected DNA involving single and mixed infections of T. congolense, T. brucei, and suspected T. simiae or T. godfreyi in 115 dogs (70.9%). The human-infective T. b. rhodesiense was detected by SRA PCR in 67 dogs (41.4%), and some sequence data that support the findings of this study have been deposited in the GenBank under accession numbers OL961811, OL961812, and OL961813. Our study demonstrates that the Trypanosoma reservoir community in Zambia is wider than was thought and includes domesticated dogs. As dogs are active carriers of human and livestock-infective trypanosomes, they pose a risk of transmitting AT in endemic villages of Mambwe district as they are neglected and left untreated. To fully bring AT under control, countries such as Zambia where the role of animal reservoirs is important, should not limit their prevention and treatment efforts to livestock (especially cattle) but also include dogs that play an integral part in most rural communities. [ABSTRACT FROM AUTHOR]

The notion that eukaryotes are ancestrally sexual has been gaining attention. This idea comes in part from the discovery of sets of "meiosis‐specific genes" in the genomes of protists. The existence of these genes has persuaded many that these organisms may be engaging in sex, even though this has gone undetected. The involvement of sex in protists is supported by the view that asexual reproduction results in the accumulation of mutations that would inevitably result in the decline and extinction of such lineages. It is argued that this phenomenon can be obviated by polyploidy and that the "meiosis‐specific genes" are used in other processes, including polyploidy control and homologous recombination, independent of meiosis. These phenomena account for the finding that these genes are expressed in cultures devoid of apparent cell fusion events. Hence, it is also proposed that asexual, and not sexual, reproduction is the ancestral condition. [ABSTRACT FROM AUTHOR]

Of the more than 150 ctenophore species, the oceanic ctenophore Eurhamphaea vexilligera is notable for its bright orange-yellow ink, secreted from numerous small vesicles that line its substomodeal comb rows. To date, in situ observations by scuba divers have proved the most fruitful method of observing these animals' natural behavior. We present the results of one such contemporary scuba-based observation of E. vexilligera , conducted in the Gulf Stream waters off the coast of Florida, using high-resolution photography and video. Utilizing underwater camera systems purpose built for filming gelatinous zooplankton, we observed E. vexilligera ink release and swimming behavior in situ. From these data, we describe the timeline and mechanics of E. vexilligera ink release in detail, as well as the animal's different swimming behaviors and resulting ink dispersal patterns. We also describe a rolling swimming behavior, accompanied and possibly facilitated by a characteristic change in overall body shape. These observations provide further insight into the behavioral ecology of this distinctive ctenophore and may serve as the foundation for future kinematic studies. [ABSTRACT FROM AUTHOR]

Acanthamoeba castellanii, a ubiquitous protozoan, is responsible for significant diseases such as Acanthamoeba keratitis and granulomatous amoebic encephalitis. A crucial survival strategy of A. castellanii involves the formation of highly resistant cysts during adverse conditions. This study delves into the cellular processes underpinning encystment, focusing on gene expression changes related to reactive oxygen species (ROS) balance, with a particular emphasis on mitochondrial processes. Our findings reveal a dynamic response within the mitochondria during encystment, with the downregulation of key enzymes involved in oxidative phosphorylation (COX, AOX, and NADHalt) during the initial 48 h, followed by their overexpression at 72 h. This orchestrated response likely creates a pro-oxidative environment, facilitating encystment. Analysis of other ROS processing enzymes across the cell reveals differential expression patterns. Notably, antioxidant enzymes, such as catalases, glutaredoxins, glutathione S-transferases, peroxiredoxins, and thioredoxins, mirror the mitochondrial trend of downregulation followed by upregulation. Additionally, glycolysis and gluconeogenesis are downregulated during the early stages in order to potentially balance the metabolic requirement of the cyst. Our study underscores the importance of ROS regulation in Acanthamoeba encystment. Understanding these mechanisms offers insights into infection control and identifies potential therapeutic targets. This work contributes to unraveling the complex biology of A. castellanii and may aid in combatting Acanthamoeba-related infections. Further research into ROS and oxidase enzymes is warranted, given the organism’s remarkable respiratory versatility. [ABSTRACT FROM AUTHOR]

Acanthamoeba is a widely distributed opportunistic parasite which causes a vision-threatening keratitis and a life-threatening encephalitis. The cyst stage of this amoeba is especially resistant to currently used therapeutics and so alternative agents are urgently required. Growing evidence supports the existence of a programmed cell death system (PCD) in Acanthamoeba and while some features are shared by higher eukaryote cells, others differ. It is hoped that by understanding these differences we can exploit them as targets for novel drug intervention to activate PCD pathways in the amoebae but not the invaded human tissue. Here, we use the aminoglycoside G418 to activate PCD in Acanthamoeba. This drug caused a shape change in the treated amoebae. Cells rounded up and contracted, and after 6 h fragments of cells resembling the 'apoptotic bodies' of vertebrate cells were observed. G418 causes an increase in intracellular calcium from a resting level of 24 nM to 60 nM after 6 h of treatment. Mitochondrial function as assayed by the ΔΨm reporting dye JC-1 and CTC a redox dye becomes inhibited during treatment and we have found that cytochrome c is released from the mitochondria. Cells stained with Hoechst showed first an alteration in chromatin structure and then a vesiculation of the nucleus with G418 treatment, although we found no obvious breakdown in genomic DNA in the early stages of PCD. [ABSTRACT FROM AUTHOR]

Eastern boundary systems support major fisheries of species whose early stages depend on upwelling production. However, upwelling can be highly variable at the regional scale, leading to complex patterns of feeding, growth, and survival for taxa that are broadly distributed in space and time. The northern California Current (NCC) is characterized by latitudinal variability in the seasonality and intensity of coastal upwelling. We examined the diet and larval growth of a dominant myctophid (Stenobrachius leucopsarus) in the context of their prey and predators in distinct NCC upwelling regimes. Larvae exhibited significant differences in diet and growth, with greater seasonal than latitudinal variability. In winter, during reduced upwelling, growth was substantially slower, guts less full, and diets dominated by copepod nauplii. During summer upwelling, faster-growing larvae had guts that were more full from feeding on calanoid copepods and relying less heavily on lower trophic level prey. Yet, our findings revealed a dome-shaped relationship with the fastest growth occurring at moderate upwelling intensity. High zooplanktivorous predation pressure led to above average growth, which may indicate the selective loss of slower-growing larvae. Our results suggest that species whose spatio-temporal distributions encompass multiple regional upwelling regimes experience unique feeding and predation environments throughout their range with implications for larval survivorship. [ABSTRACT FROM AUTHOR]

Leptomyxa valladaresi was isolated from soil in a pine forest on the southern flank of Mt Teide in Tenerife, Spain. It feeds on bacteria and on a range of other amoebae, and it was possible to establish bi-axenic cultures with L. valladaresi and Acanthamoeba . It is easily propagated on a E. coli also. 18S rDNA gene sequence analysis suggests that it is most closely related to Leptomyxa variabilis , however this amoeba differs in important detail. L. valladaresi is primarily mononucleate whereas L. variabilis is multinucleate. L. valladaresi is a larger amoeba and although the cysts are similar in size, there is no sign of the pore-like structures described in L. variabilis cysts. L. valladaresi can adopt a rapid monopodal and tubular morphology similar to that described for L. neglecta and Rhizamoeba matisi , and is never reticulated as larger L. variabilis individuals tend to be. The mean generation time was found to be 18 h, in line with amoebae of this size. Like other members of the genus, L. valladaresi is reported to harbour intracellular, presumably endosymbiotic bacteria, and a Delftia sp has been identified by 16S PCR a bacterium which is also known to grow within Acanthamoeba . The availability of this easily cultured species will help to characterize of this little studied genus and family and their relationship with bacteria, both prey and symbionts. [ABSTRACT FROM AUTHOR]

Summary: Aerosol‐generating procedures are medical interventions considered high risk for transmission of airborne pathogens. Tracheal intubation of anaesthetised patients is not high risk for aerosol generation; however, patients often perform respiratory manoeuvres during awake tracheal intubation which may generate aerosol. To assess the risk, we undertook aerosol monitoring during a series of awake tracheal intubations and nasendoscopies in healthy participants. Sampling was undertaken within an ultraclean operating theatre. Procedures were performed and received by 12 anaesthetic trainees. The upper airway was topically anaesthetised with lidocaine and participants were not sedated. An optical particle sizer continuously sampled aerosol. Passage of the bronchoscope through the vocal cords generated similar peak median (IQR [range]) aerosol concentrations to coughing, 1020 (645–1245 [120–48,948]) vs. 1460 (390–2506 [40–12,280]) particles.l-1 respectively, p = 0.266. Coughs evoked when lidocaine was sprayed on the vocal cords generated 91,700 (41,907–166,774 [390–557,817]) particles.l-1 which was significantly greater than volitional coughs (p < 0.001). For 38 nasendoscopies in 12 participants, the aerosol concentrations were relatively low, 180 (120–525 [0–9552]) particles.l-1, however, five nasendoscopies generated peak aerosol concentrations greater than a volitional cough. Awake tracheal intubation and nasendoscopy can generate high concentrations of respiratory aerosol. Specific risks are associated with lidocaine spray of the larynx, instrumentation of the vocal cords, procedural coughing and deep breaths. Given the proximity of practitioners to patient‐generated aerosol, airborne infection control precautions are appropriate when undertaking awake upper airway endoscopy (including awake tracheal intubation, nasendoscopy and bronchoscopy) if respirable pathogens cannot be confidently excluded. [ABSTRACT FROM AUTHOR]

• Almost two thirds (63%) of the sample reported using their mobile phone while driving at some time point. • Exposure to social media content encouraging phone use while driving predicted increasing engagement in the behaviour. • Peer engagement in phone use while driving predicted increasing engagement in the behaviour. • Exposure to mass media content encouraging phone use while driving did not influence the behaviour. While the ever-expanding proliferation of social media is well documented, the relationship such exposure has upon road rule compliance remains completely unknown. Further, it is not clear as to whether social media independently affects such behaviour, when accounting for the influence of mass media and peers. As a result, this study examined the relative influence of exposure to content that encourages phone use while driving via these three mediums in order to identify the most salient stimuli. A total of 509 participants (aged 17–88 years, M = 33, SD = 17) from the state of Queensland, Australia, were included in this study. Participants completed an online survey, which examined self-reported levels of exposure to material encouraging hand-held mobile phone use while driving from three sources: mass media (e.g., television, movies and gaming), social media, and peer behaviour (descriptive norms). Results indicated that participants on average engaged in illegal mobile phone use while driving 16% of the time they drive. Exposure to material encouraging phone use while driving was significantly higher from mass media (M = 32% of the time) and via peers (M = 30%) compared to social media (M = 27%). However, only social media and peer behaviour (the frequency with which participants believed that their peers engage in the behaviour) significantly predicted engagement in self-reported illegal mobile phone use, such that increasing levels of exposure were associated with more frequent use while driving. These findings: (a) support an emerging body of evidence indicating that both peer behaviours and social media messages are highly salient and influential and (b) are the first to suggest that such exposure might directly affect road safety. Nonetheless, further research is warranted to elucidate the nature and direction of the relationship between social media and driving behaviour, and the mechanisms through which such messages influence behaviour. Such lines of enquiry would have important implications for road safety (e.g., by informing the development of countermeasures/interventions delivered via social media). [ABSTRACT FROM AUTHOR]

Abstract: Ethnopharmacological relevance: The island of New Britain in Papua New Guinea is an area of great floristic and cultural diversity that has received little attention from ethnobotanists. Here we present the results of a comparative medicinal ethnobotanical survey of the Bulu and inland Kaulong; two distinct people groups inhabiting lowland rainforest on different sides of the island. A high proportion of species are used in the treatment of bacterial infections and plants with antibacterial activity were identified in the field using a specially developed antibacterial assay kit. Follow up testing with human pathogens was used to evaluate active plant material in more detail. Materials and methods: Rapid appraisal techniques were used to survey both people groups with all data corroborated by three or more separate sources. Plants from both groups were tested in-the-field with a portable antibacterial test kit based on the agar diffusion assay, using a pressure cooker to sterilise glassware and media. Follow up laboratory based tests were carried out using standardised agar dilution protocols for drug resistant and drug sensitive strains of Staphylococcus aureus and Streptococcus pneumoniae. Results: We find surprisingly little overlap in the plant species used by the two people groups with only 1 out of 70 species used for the same purpose. There is also a difference in emphasis in the conditions treated with 53% of Kaulong medicinal plants dedicated to treating tropical ulcers compared with only 8% of in the Bulu group. In-the-field testing identified Garcinia dulcis bark (a Kaulong tropical ulcer treatment) to have antibacterial activity and follow up tests against a drug resistant strain of Staphylococcus aureus (a pathogen implicated in tropical ulcer pathogenesis) revealed the crude bark extract to be potently active with an MIC of just 1mg/ml. Conclusions: The results demonstrate extreme differences in medicinal plant use between two people groups living a mere 100 km apart and suggests the two medicinal plant systems have developed in isolation from one another. In-the-field antibacterial testing of plant extracts was found to be a valuable technique that enabled early identification of active plant material. [Copyright &y& Elsevier]

Abstract: Gelsolin and calponin are well characterized actin-binding proteins that form a tight gelsolin:calponin complex (GCC). We show here that the GCC is formed through two distinct interfaces. One of these is formed between 144–182 of calponin and 25–150 of gelsolin (G1). The second is a calcium-sensitive site centred on calponin''s CH domain, and the C-terminal half of gelsolin (G4–6). The behaviour of this second interface is dependent on the presence of calcium and so it is possible that potential GCC-binding partners may be selected by calcium availability. Actin is one such GCC-binding partner and we show that a larger complex is formed with monomeric actin in calcium. The stoichiometry of this complex is determined to be 1 gelsolin/1 calponin/2 G-actins (GCA2). Both actin monomers bind the GCC through gelsolin. Both calponin and gelsolin are reported to play signaling roles in addition to their better-characterized actin-binding properties and it is possible that the GCC regulates both of these functions. [Copyright &y& Elsevier]

Abstract: Gelsolin is an actin-binding protein that is regulated by the occupancy of multiple calcium-binding sites. We have studied calcium induced conformational changes in the G1–2 and G1–3 sub-domains, and report the binding affinities for the three type II sites. A new probe for G3 has been produced and a K d of 5μM has been measured for calcium in the context of G1–3. The two halves of gelsolin, G1–3 and G4–6 bind weakly with or without calcium, suggesting that once separated by apoptotic proteolysis, G1–3 and G4–6 remain apart allowing G1–3 to sever actin in a calcium free manner. [Copyright &y& Elsevier]

Abstract: Calponins are actin-binding proteins that are implicated in the regulation of actomyosin. Calponin binds filamentous actin (F-actin) through two distinct sites ABS1 and ABS2, with an affinity in the low micromolar range. We report that smooth muscle calponin binds monomeric actin with a similar affinity (K d of 0.15μM). We show that the arrangement of binding is similar to that of F-actin by a number of criteria, most notably that the distance between Cys273 on calponin and Cys374 of actin is 29Å when measured by fluorescent resonance energy transfer, the same distance as previously reported for F-actin. [Copyright &y& Elsevier]

Gelsolin is a multidomain and multifunction protein that nucleates the assembly of filaments and severs them. The activation of gelsolin by calcium is a multistep process involving many calcium binding sites that act to unfold the molecule from a tight structure to a more loose form in which three actin-binding sites become exposed. Low pH is also known to activate gelsolin, in the absence of calcium and this too results in an unfolding of the molecule. Less is known how pH-activation occurs but we show that there are significant differences in the mechanisms that lead to activation. Crucially, while it is known that the bonds between G2 and G6 are broken by co-operative occupancy of calcium binding sites in both domains [Lagarrique, E., Maciver, S. K., Fattoum, A., Benyamin, Y. & Roustan, C. (2003) Eur. J. Biochem. 270, 2236–2243.], pH values that activate gelsolin do not result in a weakening of the G2-G6 bonds. We report the existence of pH-dependent conformational changes within G2 and in G4–6 that differ from those induced by calcium, and that low pH overrides the requirement for calcium for actin-binding within G4–6 to a modest extent so that a Kd of 1 µ m is measured, compared to 30–40 n m in the presence of calcium. Whereas the pH-dependent conformational change in G2 is possibly different from the change induced by calcium, the changes measured in G4–6 appear to be similar in both calcium and low pH. [ABSTRACT FROM AUTHOR]

A marked pH-dependent interaction with F-actin is an important property of typical members of the actin depolymerizing factor (ADF)/cofilin family of abundant actin-binding proteins. ADF/cofilins tend to bind to F-actin with a ratio of 1 : 1 at pH values around 6.5, and to G-actin at pH 8.0. We have investigated the mechanism for the pH-sensitivity. We found no evidence for pH-dependent changes in the structure of cofilin itself, nor for the interaction of cofilin with G-actin. None of the actin-derived, cofilin-binding peptides that we had previously identified [Renoult, C., Ternent, D., Maciver, S.K., Fattoum, A., Astier, C., Benyamin, Y. & Roustan, C. (1999) J. Biol. Chem. 274 , 28893–28899] bound cofilin in a pH-sensitive manner. However, we have detected a conformational change in region 75–105 in the actin subdomain 1 by the use of a peptide-directed antibody. A pH-dependent conformational change has also been detected spectroscopically in a similar peptide (84–103) on binding to cofilin. These results are consistent with a model in which pH-dependent motion of subdomain 1 relative to subdomain 2 (through region 75–105) of actin reveals a second cofilin binding site on actin (centered around region 112–125) that allows ADF/cofilin association with the actin filament. This motion requires salt in addition to low pH. [ABSTRACT FROM AUTHOR]

ADF/cofilins are actin binding proteins that bind actin close to both the N- and C-termini (site 1), and we have found a second cofilin binding site (site 2) centered around helix 112–125 [Renoult, C., Ternent, D., Maciver, S.K., Fattoum, A., Astier, C., Benyamin, Y. & Roustan, C. (1999) J. Biol. Chem. 274, 28893–28899]. We proposed a model in which ADF/cofilin intercalated between subdomains 1 and 2 of two longitudinally associated actin monomers within the actin:cofilin cofilament, explaining the change in twist that ADF/cofilins induce in the filament [McGough, A. Pope, B., Chiu, W. & Weeds, A. (1998) J. Cell Biol. 138, 771–781]. Here, we have determined the fuller extent of the cofilin footprint on site 1 of actin. Site 1 is primarily the G-actin binding site. Experiments with both peptide mimetics and fluorescently labeled cofilin suggest that site 2 only becomes available for cofilin binding within the filament, possibly due to motion between subdomains 1 and 2 within an actin monomer. We have detected motion between subdomains 1 and 2 of G-actin by FRET induced by cofilin, to reveal the second cofilin-binding site. This motion may also explain how cofilins inhibit the nucleotide exchange of actin, and why the actin:cofilin complex is polymerizable without dissociation. [ABSTRACT FROM AUTHOR]