Long-term Musculoskeletal Consequences of Chemotherapy in Pediatric Mice.

Thanks to recent progress in cancer research, most children treated for cancer survive into adulthood. Nevertheless, the long-term consequences of anticancer agents are understudied, especially in the pediatric population. We and others have shown that routinely administered chemotherapeutics drive musculoskeletal alterations, which contribute to increased treatment-related toxicity and long-term morbidity. Yet, the nature and scope of these enduring musculoskeletal defects following anticancer treatments and whether they can potentially impact growth and quality of life in young individuals remain to be elucidated. Here, we aimed at investigating the persistent musculoskeletal consequences of chemotherapy in young (pediatric) mice. Four-week-old male mice were administered a combination of 5-FU, leucovorin, irinotecan (a.k.a., Folfiri) or the vehicle for up to 5 wk. At time of sacrifice, skeletal muscle, bones, and other tissues were collected, processed, and stored for further analyses. In another set of experiments, chemotherapy-treated mice were monitored for up to 4 wk after cessation of treatment. Overall, the growth rate was significantly slower in the chemotherapy-treated animals, resulting in diminished lean and fat mass, as well as significantly smaller skeletal muscles. Interestingly, 4 wk after cessation of the treatment, the animals exposed to chemotherapy showed persistent musculoskeletal defects, including muscle innervation deficits and abnormal mitochondrial homeostasis. Altogether, our data support that anticancer treatments may lead to long-lasting musculoskeletal complications in actively growing pediatric mice and support the need for further studies to determine the mechanisms responsible for these complications, so that new therapies to prevent or diminish chemotherapy-related toxicities can be identified.

Barriers to Healthy Family Dinners and Preventing Child Obesity: Focus Group Discussions with Parents of 5-to-8-Year-Old Children.

BACKGROUND: Although numerous physical and mental health benefits for children have been linked to family dinners, many families still do not have regular family meals together. This study sought to identify the barriers that keep families from having dinners together. METHODS: We interviewed 42 parents of 5-to-8-year-old children in small focus groups to identify barriers and challenges that keep families from having healthy and consistent dinners together. RESULTS: Parents reported the main barriers were time (e.g., time strain and overscheduling, mismatched schedules, long work hours, etc.), lack of meal planning or failure to follow plans, lack of skills (e.g., cooking skills or nutritional awareness), external factors (e.g., daycare, schools, or extended family, and competing with advertising), and food-related challenges (e.g., picky eating, food allergies). Parents also suggested potential solutions to overcome these barriers. CONCLUSIONS: Overall, parents had a desire to have family dinners with their children, but they felt that there are many barriers keeping them from establishing or maintaining consistent family mealtimes. Future research, as well as child obesity prevention and intervention efforts, should consider these barriers and suggested solutions in efforts to promote healthy and consistent family meals as a means of lowering the prevalence of childhood obesity.

Myeloid cell-specific mutation of Spi1 selectively reduces M2-biased macrophage numbers in skeletal muscle, reduces age-related muscle fibrosis and prevents sarcopenia.

Intramuscular macrophages play key regulatory roles in determining the response of skeletal muscle to injury and disease. Recent investigations showed that the numbers and phenotype of intramuscular macrophages change during aging, suggesting that those changes could influence the aging process. We tested that hypothesis by generating a mouse model that harbors a myeloid cell-specific mutation of Spi1, which is a transcription factor that is essential for myeloid cell development. The mutation reduced the numbers of macrophages biased to the CD163+/CD206+ M2 phenotype in muscles of aging mice without affecting the numbers of CD68-expressing macrophages and reduced the expression of transcripts associated with the M2-biased phenotype. The mutation did not affect the colony-forming ability or the frequency of specific subpopulations of bone marrow hematopoietic cells and did not affect myeloid/lymphoid cell ratios in peripheral blood leukocyte populations. Cellularity of most myeloid lineage cells was not influenced by the mutation. The Spi1 mutation in bone marrow-derived macrophages in vitro also did not affect expression of transcripts that indicate the M2-biased phenotype. Thus, myeloid cell-targeted mutation of Spi1 influences macrophage phenotype in muscle but did not affect earlier stages of differentiation of cells in the macrophage lineage. The mutation reduced age-related muscle fibrosis, which is consistent with the reduction of M2-biased macrophages, and reduced expression of the pro-fibrotic enzyme arginase. Most importantly, the mutation prevented sarcopenia. Together, our observations indicate that intramuscular, M2-biased macrophages play significant roles in promoting detrimental, age-related changes in muscle.

Characterizing dark state kinetics and single molecule fluorescence of FusionRed and FusionRed-MQ at low irradiances.

The presence of dark states causes fluorescence intermittency of single molecules due to transitions between "on" and "off" states. Genetically encodable markers such as fluorescent proteins (FPs) exhibit dark states that make several super-resolved single-molecule localization microscopy (SMLM) methods possible. However, studies quantifying the timescales and nature of dark state behavior for commonly used FPs under conditions typical of widefield and total internal reflection fluorescence (TIRF) microscopy remain scarce and pre-date many new SMLM techniques. FusionRed is a relatively bright red FP exhibiting fluorescence intermittency and has thus been identified as a potential candidate for SMLM. We herein characterize the rates for dark-state conversion and the subsequent ground-state recovery of FusionRed and its 2.5-fold brighter descendent FusionRed L175M M42Q (FusionRed-MQ) at low irradiances (1-10 W cm-2), which were previously unexplored experimental conditions. We characterized the kinetics of dark state transitions in these two FPs by using single molecule blinking and ensemble photobleaching experiments bridged with a dark state kinetic model. We find that at low irradiances, the recovery process to the ground state is minimally light-driven and FusionRed-MQ has a 1.3-fold longer ground state recovery time indicating a conformationally restricted dark-state chromophore in comparison to FusionRed. Our studies indicate that the brighter FusionRed-MQ variant exhibits higher dark state conversion rates with longer ground state recovery lifetimes, thus it is potentially a better candidate for SMLM applications than its progenitor FusionRed.

Mechanism of cooperative N-glycan processing by the multi-modular endoglycosidase EndoE.

Bacteria produce a remarkably diverse range of glycoside hydrolases to metabolize glycans from the environment as a primary source of nutrients, and to promote the colonization and infection of a host. Here we focus on EndoE, a multi-modular glycoside hydrolase secreted by Enterococcus faecalis, one of the leading causes of healthcare-associated infections. We provide X-ray crystal structures of EndoE, which show an architecture composed of four domains, including GH18 and GH20 glycoside hydrolases connected by two consecutive three α-helical bundles. We determine that the GH20 domain is an exo-ß-1,2-N-acetylglucosaminidase, whereas the GH18 domain is an endo-ß-1,4-N-acetylglucosaminidase that exclusively processes the central core of complex-type or high-mannose-type N-glycans. Both glycoside hydrolase domains act in a concerted manner to process diverse N-glycans on glycoproteins, including therapeutic IgG antibodies. EndoE combines two enzyme domains with distinct functions and glycan specificities to play a dual role in glycan metabolism and immune evasion.

Context-specific emergence and growth of the SARS-CoV-2 Delta variant

The Delta variant of concern of SARS-CoV-2 has spread globally causing large outbreaks and resurgences of COVID-19 cases1-3. The emergence of Delta in the UK occurred on the background of a heterogeneous landscape of immunity and relaxation of non-pharmaceutical interventions4,5. Here we analyse 52,992 Delta genomes from England in combination with 93,649 global genomes to reconstruct the emergence of Delta, and quantify its introduction to and regional dissemination across England, in the context of changing travel and social restrictions. Through analysis of human movement, contact tracing, and virus genomic data, we find that the focus of geographic expansion of Delta shifted from India to a more global pattern in early May 2021. In England, Delta lineages were introduced >1,000 times and spread nationally as non-pharmaceutical interventions were relaxed. We find that hotel quarantine for travellers from India reduced onward transmission from importations; however the transmission chains that later dominated the Delta wave in England had been already seeded before restrictions were introduced. In England, increasing inter-regional travel drove Deltas nationwide dissemination, with some cities receiving >2,000 observable lineage introductions from other regions. Subsequently, increased levels of local population mixing, not the number of importations, was associated with faster relative growth of Delta. Among US states, we find that regions that previously experienced large waves also had faster Delta growth rates, and a model including interactions between immunity and human behaviour could accurately predict the rise of Delta there. Deltas invasion dynamics depended on fine scale spatial heterogeneity in immunity and contact patterns and our findings will inform optimal spatial interventions to reduce transmission of current and future VOCs such as Omicron.

COVID-19-related knowledge, attitudes and practices: a mixed-mode cross-sectional survey in Liberia.

OBJECTIVES: To examine the knowledge, attitudes and practices (KAP) of COVID-19 of rural and urban residents in Liberia to inform the development of local social and behaviour change communication strategies. DESIGN: Cross-sectional, mixed-mode (online and telephone) survey using non-probability sampling. SETTING: All 15 counties in Liberia with a focus on Maryland County. PARTICIPANTS: From 28 May to 28 June 2020, data were collected from a total of 431 adults aged 18 years and older (telephone 288 (66.8%); online 143 (33.2%)) out of a total of 741 contacts. MAIN OUTCOME MEASURES: KAP scores. Frequencies and proportions were calculated, followed by univariate and multivariable analyses to examine the association between KAP scores and the sociodemographic variables. RESULTS: Around 69% of the online survey respondents were younger than 35 years of age, compared with 56% in the telephone interviews. The majority (87%) of online respondents had completed tertiary education, compared with 77% of the telephone respondents. Male participants, on average, achieved higher knowledge (52%) and attitude scores (72%), in contrast to females (49% and 67%, respectively). Radio (71%) was the most cited source for COVID-19 information, followed by social media (63%). After controlling for sociodemographic variables, adaptive regression modelling revealed that survey mode achieved 100% importance for predicting knowledge and practice levels with regard to COVID-19. CONCLUSIONS: The survey population demonstrated moderate COVID-19 knowledge, with significant differences between survey mode and educational level. Correct knowledge of COVID-19 was associated with appropriate practices in Maryland County. Generalisation of survey findings must be drawn carefully owing to the limitations of the sampling methods. Yet, given the differences in knowledge gaps between survey modes, sex, education, occupation and place of residence, it is recommended that information is tailored to different audiences.

Engineering of a Brighter Variant of the FusionRed Fluorescent Protein Using Lifetime Flow Cytometry and Structure-Guided Mutations.

The development of fluorescent proteins (FPs) has revolutionized biological imaging. FusionRed, a monomeric red FP (RFP), is known for its low cytotoxicity and correct localization of target fusion proteins in mammalian cells but is limited in application by low fluorescence brightness. We report a brighter variant of FusionRed, "FR-MQV," which exhibits an extended fluorescence lifetime (2.8 ns), enhanced quantum yield (0.53), higher extinction coefficient (∼140 000 M-1 cm-1), increased radiative rate constant, and reduced nonradiative rate constant with respect to its precursor. The properties of FR-MQV derive from three mutations-M42Q, C159V, and the previously identified L175M. A structure-guided approach was used to identify and mutate candidate residues around the para-hydroxyphenyl and the acylimine sites of the chromophore. The C159V mutation was identified via lifetime-based flow cytometry screening of a library in which multiple residues adjacent to the para-hydroxyphenyl site of the chromophore were mutated. The M42Q mutation is located near the acylimine moiety of the chromophore and was discovered using site-directed mutagenesis guided by X-ray crystal structures. FR-MQV exhibits a 3.4-fold higher molecular brightness and a 5-fold increase in the cellular brightness in HeLa cells [based on fluorescence-activated cell sorting (FACS)] compared to FusionRed. It also retains the low cytotoxicity and high-fidelity localization of FusionRed, as demonstrated through assays in mammalian cells. These properties make FR-MQV a promising template for further engineering into a new family of RFPs.

Colpoda secrete viable Listeria monocytogenes within faecal pellets.

Transmission electron microscopy was used to demonstrate that co-cultures of the ciliate Colpoda RR (an environmental isolate) and Colpoda MLS-5 (a food processing environment isolate) with the pathogenic Listeria monocytogenes DRDC8 resulted in secretion of faecal pellets containing intact DRDC8 cells. A green fluorescent protein expressing variant of DRDC8 was used in co-cultures to confirm that the pellet-associated bacterial cells were L. monocytogenes. Viability was confirmed by plate counts, and assay of microbial respiratory activity-proved DRDC8 cells present within faecal pellets was metabolically active. Following treatment of faecal pellets secreted by Colpoda RR and MLS-5 with gentamycin and sodium hypochlorite (NaOCl), no loss of viability of the pellet-located DRDC8 cells was observed, indicating that faecal pellet encapsulated DRDC8 cells are resistant to biocidal agents. This work suggests that Colpoda-derived faecal pellets may provide a mechanism for transmission of L. monocytogenes and other pathogenic bacteria. Furthermore, bacteria encapsulated by faecal pellets may be resistant to disinfectants and cleaning agents used in food manufacturing and preparation facilities.

Structural analysis of the essential resuscitation promoting factor YeaZ suggests a mechanism of nucleotide regulation through dimer reorganization.

BACKGROUND: The yeaZ gene product forms part of the conserved network YjeE/YeaZ/YgjD essential for the survival of many gram-negative eubacteria. Among other as yet unidentified roles, YeaZ functions as a resuscitation promoting factor required for survival and resuscitation of cells in a viable but non-culturable (VBNC) state. METHODOLOGY/PRINCIPAL FINDINGS: In order to investigate in detail the structure/function relationship of this family of proteins we have performed X-ray crystallographic studies of Vibrio parahaemolyticus YeaZ. The YeaZ structure showed that it has a classic actin-like nucleotide-binding fold. Comparisons of this crystal structure to that of available homologues from E. coli, T. maritima and S. typhimurium revealed two distinctly different modes of dimer formation. In one form, prevalent in the absence of nucleotide, the putative nucleotide-binding site is incomplete, lacking a binding pocket for a nucleotide base. In the second form, residues from the second subunit complete the nucleotide-binding site. This suggests that the two dimer architectures observed in the crystal structures correspond to a free and a nucleotide-bound form of YeaZ. A multiple sequence alignment of YeaZ proteins from different bacteria allowed us to identify a large conserved hydrophobic patch on the protein surface that becomes exposed upon nucleotide-driven dimer re-arrangement. We hypothesize that the transition between two dimer architectures represents the transition between the 'on' and 'off' states of YeaZ. The effect of this transition is to alternately expose and bury a docking site for the partner protein YgjD. CONCLUSIONS/SIGNIFICANCE: This paper provides the first structural insight into the putative mechanism of nucleotide regulation of YeaZ through dimer reorganization. Our analysis suggests that nucleotide binding to YeaZ may act as a regulator or switch that changes YeaZ shape, allowing it to switch partners between YjeE and YgjD.

Purification, crystallization and preliminary X-ray crystallographic analysis of the putative Vibrio parahaemolyticus resuscitation-promoting factor YeaZ.

Vibrio parahaemolyticus is a human pathogen associated with gastroenteritis caused by the ingestion of contaminated raw seafood. V. parahaemolyticus is able to survive exposure to low temperatures typical of those used for the refrigeration of foods by entering a viable but nonculturable (VBNC) state. The VBNC cells can regain culturability and renewed ability to cause infection upon temperature upshift. The resuscitation-promoting factor protein (Rpf, YeaZ) plays a key role in reactivation of growth. Crystals of V. parahaemolyticus YeaZ have been grown using the hanging-drop vapour-diffusion method with polyethylene glycol as a precipitating agent. The crystals belonged to the primitive monoclinic space group P2(1), with unit-cell parameters a = 81.7, b = 63.8, c = 82.3 Å, ß = 105° and four subunits in the asymmetric unit. A complete X-ray diffraction data set was collected from a single crystal to 3.1 Å resolution.

Listeria monocytogenes does not survive ingestion by Acanthamoeba polyphaga.

Listeria monocytogenes is a ubiquitous bacterium capable of infecting humans, particularly pregnant women and immunocompromised individuals. Although the intracellular invasion and pathogenesis of listeriosis in mammalian tissues has been well studied, little is known about the ecology of L. monocytogenes , and in particular the environmental reservoir for this bacterium has not been identified. This study used short-term co-culture at 15, 22 and 37 degrees C to examine the interaction of L. monocytogenes strains with Acanthamoeba polyphaga ACO12. Survival of L. monocytogenes cells phagocytosed by monolayers of trophozoites was assessed by culture techniques and microscopy. A. polyphaga trophozoites eliminated bacterial cells within a few hours post-phagocytosis, irrespective of the incubation temperature used. Wild-type L. monocytogenes and a phenotypic listeriolysin O mutant were unable to either multiply or survive within trophozoites. By contrast, Salmonella enterica serovar Typhimurium C5 cells used as controls were able to survive and multiply within A. polyphaga trophozoites. The data presented indicate that A. polyphaga ACO12 is unlikely to harbour L. monocytogenes, or act as an environmental reservoir for this bacterium.

Viability of Listeria monocytogenes in co-culture with Acanthamoeba spp.

Listeria monocytogenes is a human pathogen, ubiquitous in the environment, and can grow and survive under a wide range of environmental conditions. It contaminates foods via raw materials or food-processing environments. However, the current knowledge of its ecology and, in particular, the mode of environmental survival and transmission of this intracellular pathogen remains limited. Research has shown that several intracellular pathogens are able to survive or replicate within free-living amoebae. To examine the viability of L. monocytogenes in interaction with Acanthamoeba spp., bacteria were co-cultured with three freshly isolated amoebae, namely Acanthamoeba polyphaga, Acanthamoeba castellanii and Acanthamoeba lenticulata. The survival of bacteria and amoebae was determined using culture techniques and microscopy. Under the experimental conditions used, all amoebae were able to eliminate bacteria irrespective of the hly gene. Bacteria did not survive or replicate within amoeba cells. However, extra-amoebic bacteria grew saprophytically on materials released from amoebae, which may play an important role in the survival of bacteria under extreme environmental conditions.

Mechanism involved in phagocytosis and killing of Listeria monocytogenes by Acanthamoeba polyphaga.

Intra-cellular pathogen, Listeria monocytogenes, is capable of invasion and survival within mammalian cells. However, Acanthamoeba polyphaga trophozoites phagocytose and rapidly degrade Listeria cells. In order to provide more information on amoeba phagocytosis and killing mechanisms, this study used several inhibitor agents known to affect the phagocytosis and killing of bacteria by eukaryotes. Amoebae were pre-treated with mannose, cytochalasin D, wortmannin, suramin, ammonium chloride, bafilomycin A and monensin followed by co-culture with bacteria. Phagocytosis and killing of bacterial cells by amoeba trophozoites was assessed using plate counting methods and microscopy. The data presented indicates that actin polymerisation and cytoskeletal rearrangement are involved in phagocytosis of L. monocytogenes cells by A. polyphaga trophozoites. Further, both phagosomal acidification and phagosome-lysosome fusion are involved in killing and degradation of L. monocytogenes cells by A. polyphaga. However, the mannose-binding protein receptor does not play an important role in uptake of bacteria by amoeba trophozoites. In conclusion, this data reveals the similar principles of molecular mechanisms used by different types of eukaryotes in uptake and killing of bacteria.

When nursing meets English: using a pathography to develop nursing students' culturally competent selves.

This article describes a collaboration between nursing and English faculty to pilot and study the use of a pathography to develop nursing students' cultural competence. The setting is a nursing program in an urban community college serving many foreign-born students. Interpreting a pathography was found to develop students' compassion for the patient and family. Exercises and assignments were used to challenge students to critically read complex transcultural interactions and apply relevant nursing concepts to analysis of these situations in health care delivery. The essay assignment presented challenges to students because of their writing skills.

Temperate phages in Salmonella enterica serovar Typhimurium: implications for epidemiology.

Salmonella enterica serovar Typhimurium is the most common Salmonella serovar isolated from humans in Australia. The most common definitive phage types (DT) include 9, 64 and 135. Induction of lysogenic phages from DT 64 with mitomycin C followed by cesium chloride gradient purification, resulted in separation of two populations of phage particles. DNA extracted from these particles that was digested with SmaI showed two distinct patterns of banding. Transmission electron microscopy showed that both phage particles belong to the podovirus family of the C1 morphotype. One of the phages, ST64T is capable of mediating both generalized transduction and bacteriophage type conversion. Crude phage lysate induced from S. Typhimurium DT 64 was capable of phage type conversion. S. Typhimurium DT 9 was converted to DT 64 and DT 135 was converted to DT 16. S. Typhimurium DT 41 was also converted to DT 29. Amplified-fragment length polymorphism revealed differences between the original isolates and the convertants. Phage type conversion raises the question of the stability of the bacterial phage types in natural settings and the possibility of its occurrence during an outbreak scenario.

Xenorhabdus bovienii T228 phase variation and virulence are independent of RecA function.

Colony pleomorphism, or phase variation, expressed by entomopathogenic bacteria belonging to the genus Xenorhabdus, is an important factor which determines the association of the bacteria with their nematode symbiont and the outcome of infection of susceptible insect larvae by the bacterium- nematode parasitic complex. The mechanism underlying phase variation is unknown. To determine whether RecA-mediated processes are linked to phase variation, the recA gene of Xenorhabdus bovienii was cloned and sequenced. When expressed in a recA-deleted strain of Escherichia coli, the X. bovienii recA clone was able to complement the loss of RecA function. X. bovienii chromosomal recA insertion mutants showed increased sensitivity to UV. Phase 1 forms did not show altered ability to convert to phase 2 and no significant differences in expression of other phase-dependent characteristics, including phospholipase C, haemolysin, protease, antibiotic activity and Congo Red binding, were noted. Furthermore, the LD(50) of the X. bovienii recA insertion mutant for Galleria mellonella larvae was not significantly different from that of wild-type strains. From these data the authors conclude that recA is unlikely to be involved in phase variation, the expression of phase-dependent characteristics, or virulence factors involved in killing of susceptible larvae.