RUSHing back: Kinetic analysis of adaptor protein complex-1 (AP-1)-mediated retrograde traffic.

Numerous biomedically important cargoes depend on adaptor protein complex-1 (AP-1) for their localization. However, controversy surrounds whether AP-1 mediates traffic from or to the Golgi. Robinson et al. (https://www.doi.org/10.1083/jcb.202310071) present compelling evidence that AP-1 mediates recycling to the Golgi.

Whole-cortex in situ sequencing reveals input-dependent area identity.

The cerebral cortex is composed of neuronal types with diverse gene expression that are organized into specialized cortical areas. These areas, each with characteristic cytoarchitecture1,2, connectivity3,4 and neuronal activity5,6, are wired into modular networks3,4,7. However, it remains unclear whether these spatial organizations are reflected in neuronal transcriptomic signatures and how such signatures are established in development. Here we used BARseq, a high-throughput in situ sequencing technique, to interrogate the expression of 104 cell-type marker genes in 10.3 million cells, including 4,194,658 cortical neurons over nine mouse forebrain hemispheres, at cellular resolution. De novo clustering of gene expression in single neurons revealed transcriptomic types consistent with previous single-cell RNA sequencing studies8,9. The composition of transcriptomic types is highly predictive of cortical area identity. Moreover, areas with similar compositions of transcriptomic types, which we defined as cortical modules, overlap with areas that are highly connected, suggesting that the same modular organization is reflected in both transcriptomic signatures and connectivity. To explore how the transcriptomic profiles of cortical neurons depend on development, we assessed cell-type distributions after neonatal binocular enucleation. Notably, binocular enucleation caused the shifting of the cell-type compositional profiles of visual areas towards neighbouring cortical areas within the same module, suggesting that peripheral inputs sharpen the distinct transcriptomic identities of areas within cortical modules. Enabled by the high throughput, low cost and reproducibility of BARseq, our study provides a proof of principle for the use of large-scale in situ sequencing to both reveal brain-wide molecular architecture and understand its development.

In-Depth Proteome Coverage of In Vitro-Cultured Treponema pallidum and Quantitative Comparison Analyses with In Vivo-Grown Treponemes.

Previous mass spectrometry (MS)-based global proteomics studies have detected a combined total of 86% of all Treponema pallidum proteins under infection conditions (in vivo-grown T. pallidum). Recently, a method was developed for the long-term culture of T. pallidum under in vitro conditions (in vitro-cultured T. pallidum). Herein, we used our previously reported optimized MS-based proteomics approach to characterize the T. pallidum global protein expression profile under in vitro culture conditions. These analyses provided a proteome coverage of 94%, which extends the combined T. pallidum proteome coverage from the previously reported 86% to a new combined total of 95%. This study provides a more complete understanding of the protein repertoire of T. pallidum. Further, comparison of the in vitro-expressed proteome with the previously determined in vivo-expressed proteome identifies only a few proteomic changes between the two growth conditions, reinforcing the suitability of in vitro-cultured T. pallidum as an alternative to rabbit-based treponemal growth. The MS proteomics data have been deposited in the MassIVE repository with the data set identifier MSV000093603 (ProteomeXchange identifier PXD047625).

Diversity of true bugs (Insecta: Hemiptera: Heteroptera) from the El Impenetrable National Park, Argentina.

The study of the Heteroptera (Hemiptera) fauna of the El Impenetrable National Park, resulted in an inventory composed of: Alydidae (2 spp.), Aradidae (1 sp.), Belostomatidae (5 spp.), Berytidae (1 sp.), Blissidae (1 sp.), Coreidae (11 spp.), Corixidae (2 spp.), Geocoridae (1 sp.), Gerridae (1 sp.), Hebridae (1 sp.), Largidae (4 spp.), Lygaeidae (5 spp.), Miridae (17 spp.), Nabidae (1 sp.), Notonectidae (1 sp.), Oxycarenidae (1 sp.), Pachygronthidae (1 sp.), Pachynomidae (2 spp.), Pentatomidae (16 spp.), Pleidae (1 sp.), Pyrrhocoridae (1 sp.), Reduviidae (30 spp.), Rhopalidae (5 spp.), Rhyparochromidae (12 spp.), Saldidae (1 sp.), Scutelleridae (2 spp.), Tingidae (1 sp.), and Veliidae (1 sp.). These findings include six new records for the Argentinean fauna: Prytanes foedus (Stl), Saldula pallipes (Fabricius), Camirus brevilinea (Walker), Atopozelus opsimus Elkins, Doldina bicarinata Stl, Rocconota sextuberculata St and 39 new records for Chaco Province.

Adherence and dropout in exercise-based interventions in childhood obesity: A systematic review of randomized trials.

Our objective was to systematically examine the characteristics of exercise interventions on adherence and dropout in children and adolescents with obesity. PubMed, Embase, PsycINFO, Lilacs, Scielo, and The Cochrane Central Register of Controlled Trials and reference lists of relevant articles were searched. We included randomized controlled trials with exercise interventions for pediatric patients with obesity presenting data on dropout and/or adherence. Two reviewers screened the records independently for eligibility with disagreements being resolved by a third reviewer. Twenty-seven studies with 1268 participants were included. Because of high heterogeneity and poor reporting of adherence, it was not possible to perform a meta-analysis. Dropout prevalence was calculated, and subgroup analyses comparing different types of exercise and a meta-regression with potential moderators were performed. We found a dropout rate of 13%. Subgroup analyses did not identify significant differences. The duration of the exercise presented a moderating effect on dropout, suggesting that longer exercise sessions may lead to higher dropout in children and adolescents with obesity. Because of the poor adherence data, it is not clear which exercise characteristics may moderate adherence. To improve the quality of childhood obesity care, it is mandatory that future studies present adherence data. Systematic review registration: PROSPERO CRD42021290700.

Ecological modeling, biogeography, and phenotypic analyses setting the tiger cats' hyperdimensional niches reveal a new species.

Recently, the tiger-cat species complex was split into Leopardus tigrinus and Leopardus guttulus, along with other proposed schemes. We performed a detailed analysis integrating ecological modeling, biogeography, and phenotype of the four originally recognized subspecies-tigrinus, oncilla, pardinoides, guttulus-and presented a new multidimensional niche depiction of the species. Species distribution models used > 1400 records from museums and photographs, all checked for species accuracy. Morphological data were obtained from institutional/personal archives. Spotting patterns were established by integrating museum and photographic/camera-trap records. Principal component analysis showed three clearly distinct groups, with the Central American specimens (oncilla) clustering entirely within those of the Andes, namely the pardinoides group of the cloud forests of the southern Central-American and Andean mountain chains (clouded tiger-cat); the tigrinus group of the savannas of the Guiana Shield and central/northeastern Brazil (savanna tiger-cat); and the guttulus group in the lowland forests of the Atlantic Forest domain (Atlantic Forest tiger-cat). This scheme is supported by recent genetic analyses. All species displayed different spotting patterns, with some significant differences in body measurements/proportions. The new distribution presented alarming reductions from the historic range of - 50.4% to - 68.2%. This multidimensional approach revealed a new species of the elusive and threatened tiger-cat complex.

Fast-evolving cofactors regulate the role of HEATR5 complexes in intra-Golgi trafficking.

The highly conserved HEATR5 proteins are best known for their roles in membrane traffic mediated by the adaptor protein complex-1 (AP1). HEATR5 proteins rely on fast-evolving cofactors to bind to AP1. However, how HEATR5 proteins interact with these cofactors is unknown. Here, we report that the budding yeast HEATR5 protein, Laa1, functions in two biochemically distinct complexes. These complexes are defined by a pair of mutually exclusive Laa1-binding proteins, Laa2 and the previously uncharacterized Lft1/Yml037c. Despite limited sequence similarity, biochemical analysis and structure predictions indicate that Lft1 and Laa2 bind Laa1 via structurally similar mechanisms. Both Laa1 complexes function in intra-Golgi recycling. However, only the Laa2-Laa1 complex binds to AP1 and contributes to its localization. Finally, structure predictions indicate that human HEATR5 proteins bind to a pair of fast-evolving interacting partners via a mechanism similar to that observed in yeast. These results reveal mechanistic insight into how HEATR5 proteins bind their cofactors and indicate that Laa1 performs functions besides recruiting AP1.

Minimal adverse events occur when inducing emesis with apomorphine in brachycephalic, mesocephalic, and dolichocephalic dogs.

OBJECTIVE: To determine risks of complications with emesis induction and whether facial conformation is associated with the frequency of complications. ANIMALS: 1,788 client-owned dogs that presented immediately or by referral from a primary care veterinarian following ingestion of toxic or foreign materials. METHODS: Patients with emesis induced with apomorphine for removal of toxic or foreign materials were retrospectively identified. Collected data included patient factors, routes of apomorphine administration, other therapies, adverse events, and patient outcomes. RESULTS: 2 types of complications were identified in a very small number of patients (11 [0.6%]), with 3 (0.17%) having regurgitation postemesis and 8 (0.44%) having prolonged vomiting. No significant difference was found in the rates of repeated vomiting or regurgitation between brachycephalic dogs and nonbrachycephalic dogs (P = .375 and P = 1.00, respectively). Brachycephalic dogs had 1.6 times greater odds of having emesis induction due to toxin ingestion compared to foreign material ingestion. The presence of clinical signs of toxicity at the time of emesis induction was associated with regurgitation (P < .001), and the development of regurgitation was associated with admission to hospital (P = .001). CLINICAL RELEVANCE: This study found no increased risk of complications when emesis was induced using apomorphine in brachycephalic breeds compared to nonbrachycephalic breeds, regardless of indication for emesis induction. Facial conformation is not a reason to withhold emesis induction.

Two functionally distinct HEATR5 protein complexes are defined by fast-evolving co-factors in yeast.

The highly conserved HEATR5 proteins are best known for their roles in membrane traffic mediated by the adaptor protein complex-1 (AP1). HEATR5 proteins rely on fast-evolving co-factors to bind to AP1. However, how HEATR5 proteins interact with these co-factors is unknown. Here, we report that the budding yeast HEATR5 protein, Laa1, functions in two biochemically distinct complexes. These complexes are defined by a pair of mutually exclusive Laa1-binding proteins, Laa2 and the previously uncharacterized Lft1/Yml037c. Despite limited sequence similarity, biochemical analysis and structure predictions indicate that Lft1 and Laa2 bind Laa1 via structurally similar mechanisms. Both Laa1 complexes function in intra-Golgi recycling. However, only the Laa2-Laa1 complex binds to AP1 and contributes to its localization. Finally, structure predictions indicate that human HEATR5 proteins bind to a pair of fast-evolving interacting partners via a mechanism similar to that observed in yeast. These results reveal mechanistic insight into how HEATR5 proteins bind their co-factors and indicate that Laa1 performs functions besides recruiting AP1.

Quantitative proteomics reveals unique responses to antimicrobial treatments in clinical Pseudomonas aeruginosa isolates.

IMPORTANCE: Pseudomonas aeruginosa is an important pathogen often associated with hospital-acquired infections and chronic lung infections in people with cystic fibrosis. P. aeruginosa possesses a wide array of intrinsic and adaptive mechanisms of antibiotic resistance, and the regulation of these mechanisms is complex. Label-free quantitative proteomics is a powerful tool to compare susceptible and resistant strains of bacteria and their responses to antibiotic treatments. Here we compare the proteomes of three isolates of P. aeruginosa with different antibiotic resistance profiles in response to five challenge conditions. We uncover unique and shared proteome changes for the widely used laboratory strain PAO1 and two isolates of the Liverpool epidemic strain of P. aeruginosa, LESlike1 and LESB58. Our data set provides insight into antibiotic resistance in clinically relevant Pseudomonas isolates and highlights proteins, including those with uncharacterized functions, which can be further investigated for their role in adaptive responses to antibiotic treatments.

Gating of homeostatic regulation of intrinsic excitability produces cryptic long-term storage of prior perturbations.

Neurons and neuronal circuits must maintain their function throughout the life of the organism despite changing environments. Previous theoretical and experimental work suggests that neurons monitor their activity using intracellular calcium concentrations to regulate their intrinsic excitability. Models with multiple sensors can distinguish among different patterns of activity, but previous work using models with multiple sensors produced instabilities that lead the models' conductances to oscillate and then to grow without bound and diverge. We now introduce a nonlinear degradation term that explicitly prevents the maximal conductances to grow beyond a bound. We combine the sensors' signals into a master feedback signal that can be used to modulate the timescale of conductance evolution. Effectively, this means that the negative feedback can be gated on and off according to how far the neuron is from its target. The modified model recovers from multiple perturbations. Interestingly, depolarizing the models to the same membrane potential with current injection or with simulated high extracellular K+ produces different changes in conductances, arguing that caution must be used in interpreting manipulations that serve as a proxy for increased neuronal activity. Finally, these models accrue traces of prior perturbations that are not visible in their control activity after perturbation but that shape their responses to subsequent perturbations. These cryptic or hidden changes may provide insight into disorders such as posttraumatic stress disorder that only become visible in response to specific perturbations.

Giant Sex Chromosomes in Omophoita Species (Coleoptera, Chrysomelidae): Structural and Evolutionary Relationships Revealed by Zoo-FISH and Comparative Genomic Hybridization (CGH).

The beetles of the subtribe Oedionychina (Chrysomelidae, Alticinae) are the only ones that have the atypical giant and achiasmatic sex chromosomes, which are substantially larger than the autosomes. Previous cytogenetic analyses suggest a large accumulation of repetitive DNA in the sex chromosomes. In this study, we examined the similarity of X and Y chromosomes in four Omophoita species and compared genomic differentiation to better understand the evolutionary process and the giant sex chromosomes origin. Intraspecific genomic comparation using male and female genomes of O. octoguttata and interespecific analyses using genomic DNA of O. octoguttata, O. sexnotata, O. magniguttis, and O. personata were performed. In addition, whole chromosome painting (WCP) experiments were performed with X and Y chromosome probes of O. octogutatta. CGH analysis revealed great genomic similarity between the sexes and a sex-specific region on the Y chromosome, and interspecific analysis revealed a genomic divergence between species. In contrast, WCP results revealed that the sex chromosomes of O. octoguttata have high intra- and interspecific similarity with the studied species. Our data support a common origin under the canonical evolution of the sex chromosomes in this group, as they have high genomic similarity between them.

Circulating Small RNA Profiling of Patients with Alveolar and Cystic Echinococcosis.

Alveolar (AE) and cystic (CE) echinococcosis are two parasitic diseases caused by the tapeworms Echinococcus multilocularis and E. granulosus sensu lato (s. l.), respectively. Currently, AE and CE are mainly diagnosed by means of imaging techniques, serology, and clinical and epidemiological data. However, no viability markers that indicate parasite state during infection are available. Extracellular small RNAs (sRNAs) are short non-coding RNAs that can be secreted by cells through association with extracellular vesicles, proteins, or lipoproteins. Circulating sRNAs can show altered expression in pathological states; hence, they are intensively studied as biomarkers for several diseases. Here, we profiled the sRNA transcriptomes of AE and CE patients to identify novel biomarkers to aid in medical decisions when current diagnostic procedures are inconclusive. For this, endogenous and parasitic sRNAs were analyzed by sRNA sequencing in serum from disease negative, positive, and treated patients and patients harboring a non-parasitic lesion. Consequently, 20 differentially expressed sRNAs associated with AE, CE, and/or non-parasitic lesion were identified. Our results represent an in-depth characterization of the effect E. multilocularis and E. granulosus s. l. exert on the extracellular sRNA landscape in human infections and provide a set of novel candidate biomarkers for both AE and CE detection.

A practical guide to expert learner skills in the research environment.

The ability to complete an experiment successfully without mistakes is at the core of the scientific enterprise. Some scientists seem more able to avoid mistakes and missteps than others. In the field of cell biology, their success is often attributed to having naturally good hands. Here I propose that rather than possessing "good hands," an innate and unlearnable aptitude for science, such scientists possess expert learning skills that can be effectively mastered. I share a straightforward approach to gaining and teaching expert learning skills in the research lab environment. I also discuss ongoing efforts by others to develop curricula that teach expert learning skills in laboratory courses.

Antibiofilm Effect of Biogenic Silver Nanoparticles Combined with Oregano Derivatives against Carbapenem-Resistant Klebsiella pneumoniae.

Resistant bacteria may kill more people than COVID-19, so the development of new antibacterials is essential, especially against microbial biofilms that are reservoirs of resistant cells. Silver nanoparticles (bioAgNP), biogenically synthesized using Fusarium oxysporum, combined with oregano derivatives, present a strategic antibacterial mechanism and prevent the emergence of resistance against planktonic microorganisms. Antibiofilm activity of four binary combinations was tested against enteroaggregative Escherichia coli (EAEC) and Klebsiella pneumoniae carbapenemase-producing K. pneumoniae (KPC): oregano essential oil (OEO) plus bioAgNP, carvacrol (Car) plus bioAgNP, thymol (Thy) plus bioAgNP, and Car plus Thy. The antibiofilm effect was accessed using crystal violet, MTT, scanning electron microscopy, and Chromobacterium violaceum anti-quorum-sensing assays. All binary combinations acted against preformed biofilm and prevented its formation; they showed improved antibiofilm activity compared to antimicrobials individually by reducing sessile minimal inhibitory concentration up to 87.5% or further decreasing biofilm metabolic activity and total biomass. Thy plus bioAgNP extensively inhibited the growth of biofilm in polystyrene and glass surfaces, disrupted three-dimensional biofilm structure, and quorum-sensing inhibition may be involved in its antibiofilm activity. For the first time, it is shown that bioAgNP combined with oregano has antibiofilm effect against bacteria for which antimicrobials are urgently needed, such as KPC.

Characterization of KPC-2-producing Klebsiella pneumoniae and affected patients of a pediatric hospital in Brazil.

Carbapenemase-producing Klebsiella pneumoniae (CPKp) infections are important threats to pediatric populations. Thus, a retrospective study was conducted in a Brazilian reference pediatric hospital, and 26 CPKp isolates obtained from 23 patients were characterized. The affected population had important underlying diseases, reflecting previous hospitalization and antibiotic use. Most CPKp isolates were resistant to all antibiotic classes, and blaKPC-2 was the only carbapenemase-encoding gene. blaCTX-M-15 was common among the isolates, and modification or absence of the mgrB gene was the cause of polymyxin B resistance. Ten different sequence types were identified, and clonal complex 258 was prevalent. Alleles wzi50 and wzi64 were the most recurrent ones regarding K-locus type, with a remarkable contribution of the epidemic ST11/KL64 lineage as a colonizer. Our findings show that lineages associated with the pediatric population are similar to those found in adults, reinforcing the need for epidemiological surveillance to effectively implement prevention and control measures.

MicroRNA-Transcription factor regulatory networks in the early strobilar development of Echinococcus granulosus protoscoleces.

BACKGROUND: Echinococcus granulosus sensu lato has a complex developmental biology with a variety of factors relating to both intermediate and final hosts. To achieve maximum parasite adaptability, the development of the cestode is dependent on essential changes in transcript regulation. Transcription factors (TFs) and miRNAs are known as master regulators that affect the expression of downstream genes through a wide range of metabolic and signaling pathways. In this study, we aimed to develop a regulatory miRNA-Transcription factor (miRNA-TF) network across early developmental stages of E. granulosus protoscoleces by performing in silico analysis, and to experimentally validate TFs expression in protoscoleces obtained from in vitro culture, and from in vivo experiments. RESULTS: We obtained list of 394 unique E. granulosus TFs and matched them with 818 differentially expressed genes which identified 41 predicted TFs with differential expression. These TFs were used to predict the potential targets of 31 differentially expressed miRNAs. As a result, eight miRNAs and eight TFs were found, and the predicted network was constructed using Cytoscape. At least four miRNAs (egr-miR-124a, egr-miR-124b-3p, egr-miR-745-3p, and egr-miR-87-3p) and their corresponding differentially expressed TFs (Zinc finger protein 45, Early growth response protein 3, Ecdysone induced protein 78c and ETS transcription factor elf 2) were highlighted in this investigation. The expression of predicted differentially expressed TFs obtained from in vitro and in vivo experiments, were experimentally validated by quantitative polymerase chain reaction. This confirmed findings of RNA-seq data. CONCLUSION: miRNA-TF networks presented in this study control some of the most important metabolic and signaling pathways in the development and life cycle of E. granulosus, providing a potential approach for disrupting the early hours of dog infection and preventing the development of the helminth in the final host.

HPV is a cargo for the COPI sorting complex during virus entry.

During entry, human papillomavirus (HPV) traffics from the cell surface to the endosome and then to the trans-Golgi network (TGN) and Golgi apparatus. HPV must transit across the TGN/Golgi and exit these compartments to reach the nucleus to cause infection, although how these steps are accomplished is unclear. Combining cellular fractionation, unbiased proteomics, and gene knockdown strategies, we identified the coat protein complex I (COPI), a highly conserved protein complex that facilitates retrograde trafficking of cellular cargos, as a host factor required for HPV infection. Upon TGN/Golgi arrival, the cytoplasmic segment of HPV L2 binds directly to COPI. COPI depletion causes the accumulation of HPV in the TGN/Golgi, resembling the fate of a COPI binding-defective L2 mutant. We propose that the L2-COPI interaction drives HPV trafficking through the TGN and Golgi stacks during virus entry. This shows that an incoming virus is a cargo of the COPI complex.

Monitoring Effects of Membrane Traffic Via Changes in Cell Polarity and Morphogenesis in Three-Dimensional Human Pluripotent Stem Cell Cysts.

Membrane traffic at the Golgi and endosomes plays many critical roles in the polarization and the morphogenesis of epithelial tissues. Studies into the roles of traffic in morphogenesis in mammals are often complicated by early embryonic lethality of mutations in membrane traffic as well as the inherent difficulty in imaging developing embryos posed by their size and location. Increasingly, human pluripotent stem cell (hPSC)-derived embryo- and organ-like systems (e.g., embryoids, organoids) provide a useful platform to illuminate the requirements of traffic in human embryonic tissue morphogenesis because these in vitro models are highly amenable to fluorescence microscopy and provide the ability to examine the role of essential genes not possible with animal studies. Here, we present a method to generate hPSC-cysts, a 3-D hPSC-based model of human epiblast lumen formation. This system provides unique opportunities to examine the role of membrane traffic during epithelial morphogenesis. We also present methods to process hPSC-cysts for immunofluorescence and staining with commonly used fluorescence labels useful for detecting defects in polarization and morphogenesis caused by defects in membrane traffic.