The impact of insect herbivory on biogeochemical cycling in broadleaved forests varies with temperature.

Herbivorous insects alter biogeochemical cycling within forests, but the magnitude of these impacts, their global variation, and drivers of this variation remain poorly understood. To address this knowledge gap and help improve biogeochemical models, we established a global network of 74 plots within 40 mature, undisturbed broadleaved forests. We analyzed freshly senesced and green leaves for carbon, nitrogen, phosphorus and silica concentrations, foliar production and herbivory, and stand-level nutrient fluxes. We show more nutrient release by insect herbivores at non-outbreak levels in tropical forests than temperate and boreal forests, that these fluxes increase strongly with mean annual temperature, and that they exceed atmospheric deposition inputs in some localities. Thus, background levels of insect herbivory are sufficiently large to both alter ecosystem element cycling and influence terrestrial carbon cycling. Further, climate can affect interactions between natural populations of plants and herbivores with important consequences for global biogeochemical cycles across broadleaved forests.

Comparison of uridine and N1-methylpseudouridine mRNA platforms in development of an Andes virus vaccine.

The rodent-borne Andes virus (ANDV) causes a severe disease in humans. We developed an ANDV mRNA vaccine based on the M segment of the viral genome, either with regular uridine (U-mRNA) or N1-methylpseudouridine (m1Ψ-mRNA). Female mice immunized by m1Ψ-mRNA developed slightly greater germinal center (GC) responses than U-mRNA-immunized mice. Single cell RNA and BCR sequencing of the GC B cells revealed similar levels of activation, except an additional cluster of cells exhibiting interferon response in animals vaccinated with U-mRNA but not m1Ψ-mRNA. Similar immunoglobulin class-switching and somatic hypermutations were observed in response to the vaccines. Female Syrian hamsters were immunized via a prime-boost regimen with two doses of each vaccine. The titers of glycoprotein-binding antibodies were greater for U-mRNA construct than for m1Ψ-mRNA construct; however, the titers of ANDV-neutralizing antibodies were similar. Vaccinated animals were challenged with a lethal dose of ANDV, along with a naïve control group. All control animals and two animals vaccinated with a lower dose of m1Ψ-mRNA succumbed to infection whereas other vaccinated animals survived without evidence of virus replication. The data demonstrate the development of a protective vaccine against ANDV and the lack of a substantial effect of m1Ψ modification on immunogenicity and protection in rodents.

Epithelial responses to fungal pathogens.

Epithelial cells orchestrate immune responses against fungal pathogens. This review highlights advances in integrating epithelial cells in immune responses against inhaled molds and dimorphic fungi, and against Candida species that colonize mucosal surfaces. In the lung, epithelial cells respond to interleukin-1 (IL-1) and interferon signaling to regulate effector cell influx and fungal killing. In the alimentary and vulvovaginal tracts, epithelial cells modulate fungal commensalism, invasive growth, and local immune tone, in part by responding to damage caused by candidalysin, a C. albicans peptide toxin, and through IL-17-dependent release of antimicrobial peptides that contribute to Candida colonization resistance. Understanding fungal-epithelial interactions in mammalian models of disease is critical to predict vulnerabilities and to identify opportunities for immune-based strategies to treat fungal infections.

Foraging dives of southern right whales (Eubalaena australis) in relation to larger zooplankton size prey availability in Golfo Nuevo, Península Valdés, Argentina.

Southern right whales (SRWs, Eubalaena australis) have been observed feeding both at and below the surface (< 10 m) in Golfo Nuevo (42°42' S, 64°30' W), Península Valdés, Argentina, an area traditionally recognized as calving ground. In addition, we documented diving feeding behavior in SRWs during their stay in this gulf, which has not been previously described. We assessed this behavior using suction-cup-attached video-imaging tags (CRITTERCAMs) on individual whales. A total of eight CRITTERCAM deployments were successful, and feeding events were documented in all SRWs successfully equipped with CRITTERCAMs. The highest speeds occurred during the ascent phase, and the average diving time was 6 min 45 s ± 3 min 41 s for SRWs. Concurrently, zooplankton samples were collected from the subsurface and bottom of the water in areas where tagged whales dived to assess differences in composition, abundance, and biomass. Copepods dominated the upper layer, while euphausiids were more abundant in the deeper sample. Furthermore, zooplankton total biomass was five times higher at depth (2515.93 mg/m3) compared to the subsurface (500.35 mg/m3). Differences in zooplankton characteristics between depths, combined with CRITTERCAM videos, indicated that SRWs exploit high concentrations of organisms near the seafloor during daytime feeding dives. This study provides baseline insights into how SRWs utilize Península Valdés during their stay in the area.

HPV integration and cervical cancer: a failed evolutionary viral trait.

Countless efforts have been made to eradicate cervical cancer worldwide, including improving disease screening and human papillomavirus (HPV) vaccination programs. Nevertheless, cervical cancer still claims the lives of more than 300 000 women every year. Persistent infections with high-risk HPV genotypes 16 and 18 are the main cause of cancer and may result in HPV integration into the host genome. The central dogma is that HPV integration is an important step in oncogenesis, but in fact, it impedes the virus from replicating and spreading. HPV causing cervical cancer can therefore be perceived as a failed evolutionary viral trait. Here we outline the occurrence and mechanisms of HPV integration and how this process results in oncogenic transformation.

Temporal composition of the cervicovaginal microbiome associates with hrHPV infection outcomes in a longitudinal study.

BACKGROUND: Persistent infections with high-risk human papillomavirus (hrHPV) can cause cervical squamous intraepithelial lesions (SIL) that may progress to cancer. The cervicovaginal microbiome (CVM) correlates with SIL, but the temporal composition of the CVM after hrHPV infections has not been fully clarified. METHODS: To determine the association between the CVM composition and infection outcome, we applied high-resolution microbiome profiling using the circular probe-based RNA sequencing technology on a longitudinal cohort of cervical smears obtained from 141 hrHPV DNA-positive women with normal cytology at first visit, of whom 51 were diagnosed by cytology with SIL six months later. RESULTS: Here we show that women with a microbial community characterized by low diversity and high Lactobacillus crispatus abundance at both visits exhibit low risk to SIL development, while women with a microbial community characterized by high diversity and Lactobacillus depletion at first visit have a higher risk of developing SIL. At the level of individual species, we observed that a high abundance for Gardnerella vaginalis and Atopobium vaginae at both visits associate with SIL outcomes. These species together with Dialister micraerophilus showed a moderate discriminatory power for hrHPV infection progression. CONCLUSIONS: Our results suggest that the CVM can potentially be used as a biomarker for cervical disease and SIL development after hrHPV infection diagnosis with implications on cervical cancer prevention strategies and treatment of SIL.

Alpha hemolysin of Escherichia coli induces a necrotic-like procoagulant state in platelets.

Uropathogenic strains of E. coli (UPEC) is a leading cause of sepsis, deploying multiple virulence factors to evade host immune responses. Notably, alpha-hemolysin (HlyA) produced by UPEC is implicated in septic symptoms associated with bacteremia, correlating with thrombocytopenia, a critical indicator of organ dysfunction and a predictor of poorer patient prognosis. This study meticulously explores the impact of sublytic concentrations of HlyA on platelets. Findings reveal that HlyA triggers an increase in intracellular calcium, activating calpain and exposing phosphatidylserine to the cell surface, as validated by flow cytometric experiments. Electron microscopy reveals a distinctive balloon-like shape in HlyA-treated platelets, indicative of a procoagulant state. The toxin induces the release of procoagulant extracellular vesicles and the secretion of alpha and dense granules. Overall, the results point to HlyA inducing a necrotic-like procoagulant state in platelets. The effects of sublytic concentrations of HlyA on both erythrocytes and platelets could have a potential impact on capillary microcirculation. Targeting HlyA emerges as a viable therapeutic strategy to mitigate the adverse effects of UPEC infections, especially in South American countries where these infections are endemic, underscoring its significance as a potential therapeutic target.

Targeting Megasphaera species to promote cervicovaginal health.

Maintaining a healthy cervicovaginal microbiome (CVM) is vital for women's wellbeing; it is dependent primarily on Lactobacillus dominance. Microbiome imbalances, driven by Megasphaera species, contribute to infections and disease. Comprehensive research into Megasphaera biology and interventions is crucial for personalized women's healthcare, and additional efforts are required to mitigate the risks posed by cervicovaginal dysbiosis.

BTK drives neutrophil activation for sterilizing antifungal immunity.

We describe a previously-unappreciated role for Bruton's tyrosine kinase (BTK) in fungal immune surveillance against aspergillosis, an unforeseen complication of BTK inhibitors (BTKi) used for treating B-cell lymphoid malignancies. We studied BTK-dependent fungal responses in neutrophils from diverse populations, including healthy donors, BTKi-treated patients, and X-linked agammaglobulinemia patients. Upon fungal exposure, BTK was activated in human neutrophils in a TLR2-, Dectin-1-, and FcγR-dependent manner, triggering the oxidative burst. BTK inhibition selectively impeded neutrophil-mediated damage to Aspergillus hyphae, primary granule release, and the fungus-induced oxidative burst by abrogating NADPH oxidase subunit p40phox and GTPase RAC2 activation. Moreover, neutrophil-specific Btk deletion in mice enhanced aspergillosis susceptibility by impairing neutrophil function, not recruitment or lifespan. Conversely, GM-CSF partially mitigated these deficits by enhancing p47phox activation. Our findings underline the crucial role of BTK signaling in neutrophils for antifungal immunity and provide a rationale for GM-CSF use to offset these deficits in susceptible patients.

Parsing the roles of DExD-box proteins DDX39A and DDX39B in alternative RNA splicing.

DExD-box RNA proteins DDX39A and DDX39B are highly homologous paralogs that are conserved in vertebrates. They are required for energy-driven reactions involved in RNA processing. Although we have some understanding of how their functions overlap in RNA nuclear export, our knowledge of whether or not these proteins have specific or redundant functions in RNA splicing is limited. Our previous work has shown that DDX39B is responsible for regulating the splicing of important immune transcripts IL7R and FOXP3. In this study, we aimed to investigate whether DDX39A, a highly homologous paralog of DDX39B, plays a similar role in regulating alternative RNA splicing. We find that DDX39A and DDX39B have significant redundancy in their gene targets, but there are targets that uniquely require one or the other paralog. For instance, DDX39A is incapable of complementing defective splicing of IL7R exon 6 when DDX39B is depleted. This exon and other cassette exons that specifically depend on DDX39B have U-poor/C-rich polypyrimidine tracts in the upstream intron and this variant polypyrimidine tract is required for DDX39B dependency. This study provides evidence that despite a high degree of functional redundancy, DDX39A and DDX39B are selectively required for the splicing of specific pre-mRNAs.

Inefficient recruitment of DDX39B impedes pre-spliceosome assembly on FOXP3 introns.

Forkhead box P3 (FOXP3) is the master fate-determining transcription factor in regulatory T (Treg) cells and is essential for their development, function, and homeostasis. Mutations in FOXP3 cause immunodysregulation polyendocrinopathy enteropathy X-linked (IPEX) syndrome, and aberrant expression of FOXP3 has been implicated in other diseases such as multiple sclerosis and cancer. We previously demonstrated that pre-mRNA splicing of FOXP3 RNAs is highly sensitive to levels of DExD-box polypeptide 39B (DDX39B), and here we investigate the mechanism of this sensitivity. FOXP3 introns have cytidine (C)-rich/uridine (U)-poor polypyrimidine (py) tracts that are responsible for their inefficient splicing and confer sensitivity to DDX39B. We show that there is a deficiency in the assembly of commitment complexes (CCs) on FOXP3 introns, which is consistent with the lower affinity of U2AF2 for C-rich/U-poor py tracts. Our data indicate an even stronger effect on the conversion of CCs to pre-spliceosomes. We propose that this is due to an altered conformation that U2AF2 adopts when it binds to C-rich/U-poor py tracts and that this conformation has a lower affinity for DDX39B. As a consequence, CCs assembled on FOXP3 introns are defective in recruiting DDX39B, and this leads to the inefficient assembly of pre-spliceosome complexes.

Complete genome sequence of the Microbacterium foliorum bacteriophage Garey24.

In this work, we report the discovery and characterization of Garey24, a bacteriophage that forms medium-size plaques with halo rings isolated from a soil sample in Funes, Argentina. Its 41,522 bp circularly permuted genome contains 63 putative protein-coding genes. Based on gene content similarity, Garey24 was assigned to subcluster EA1.

GM-CSF-mediated epithelial-immune cell crosstalk orchestrates pulmonary immunity to Aspergillus fumigatus.

Aspergillus fumigatus causes life-threatening mold pneumonia in immune compromised patients, particularly in those with quantitative or qualitative defects in neutrophils. While innate immune cell crosstalk licenses neutrophil antifungal activity in the lung, the role of epithelial cells in this process is unknown. Here, we find that that surfactant protein C (SPC)-expressing lung epithelial cells integrate infection-induced IL-1 and type III interferon signaling to produce granulocyte-macrophage colony-stimulating factor (GM-CSF) preferentially at local sites of fungal infection and neutrophil influx. Using in vivo models that distinguish the role of GM-CSF during acute infection from its homeostatic function in alveolar macrophage survival and surfactant catabolism, we demonstrate that epithelial-derived GM-CSF increases the accumulation and fungicidal activity of GM-CSF-responsive neutrophils, with the latter being essential for host survival. Our findings establish SPC + epithelial cells as a central player in regulating the quality and strength of neutrophil-dependent immunity against inhaled mold pathogens. HIGHLIGHTS: GM-CSF is essential for host defense against A. fumigatus in the lung IL-1 and IFN-λ promote GM-CSF production by lung epithelial cells in parallelEpithelial cell-derived GM-CSF increases neutrophil accumulation and fungal killing capacityEpithelial cells preferentially upregulate GM-CSF in local sites of inflammation.

Novel betulin derivatives as multidrug reversal agents targeting P-glycoprotein.

Chemotherapy is a powerful means of cancer treatment but its efficacy is compromised by the emergence of multidrug resistance (MDR), mainly linked to the efflux transporter ABCB1/P-glycoprotein (P-gp). Based on the chemical structure of betulin, identified in our previous work as an effective modulator of the P-gp function, a series of analogs were designed, synthesized and evaluated as a source of novel inhibitors. Compounds 6g and 6i inhibited rhodamine 123 efflux in the P-gp overexpressed leukemia cells, K562/Dox, at concentrations of 0.19 µM and 0.39 µM, respectively, and increased the intracellular accumulation of doxorubicin at the submicromolar concentration of 0.098 µM. Compounds 6g and 6i were able to restore the sensitivity of K562/Dox to Dox at 0.024 µM and 0.19 µM, respectively. Structure-activity relationship analysis and molecular modeling revealed important information about the structural features conferring activity. All the active compounds fitted in a specific region involving mainly transmembrane helices (TMH) 4-6 from one homologous half and TMH 7 and 12 from the other, also showing close contacts with TMH 6 and 12. Compounds that bound preferentially to another region were inactive, regardless of their free energy of binding. It should be noted that compounds 6g and 6i were devoid of toxic effects against peripheral blood mononuclear normal cells and erythrocytes. The data obtained indicates that both compounds might be proposed as scaffolds for obtaining promising P-gp inhibitors for overcoming MDR.

Inverse priority effects: A role for historical contingency during species losses.

Communities worldwide are losing multiple species at an unprecedented rate, but how communities reassemble after these losses is often an open question. It is well established that the order and timing of species arrival during community assembly shapes forthcoming community composition and function. Yet, whether the order and timing of species losses can lead to divergent community trajectories remains largely unexplored. Here, we propose a novel framework that sets testable hypotheses on the effects of the order and timing of species losses-inverse priority effects-and suggests its integration into the study of community assembly. We propose that the order and timing of species losses within a community can generate alternative reassembly trajectories, and suggest mechanisms that may underlie these inverse priority effects. To formalize these concepts quantitatively, we used a three-species Lotka-Volterra competition model, enabling to investigate conditions in which the order of species losses can lead to divergent reassembly trajectories. The inverse priority effects framework proposed here promotes the systematic study of the dynamics of species losses from ecological communities, ultimately aimed to better understand community reassembly and guide management decisions in light of rapid global change.

Alpha hemolysin of E. coli induces hemolysis of human erythrocytes independently of toxin interaction with membrane proteins.

Alpha hemolysin (HlyA) is a hemolytic and cytotoxic protein secreted by uropathogenic strains of E. coli. The role of glycophorins (GPs) as putative receptors for HlyA binding to red blood cells (RBCs) has been debated. Experiments using anti-GPA/GPB antibodies and a GPA-specific epitope nanobody to block HlyA-GP binding on hRBCs, showed no effect on hemolytic activity. Similarly, the hemolysis induced by HlyA remained unaffected when hRBCs from a GPAnull/GPBnull variant were used. Surface Plasmon Resonance experiments revealed similar values of the dissociation constant between GPA and either HlyA, ProHlyA (inactive protoxin), HlyAΔ914-936 (mutant of HlyA lacking the binding domain to GPA) or human serum albumin, indicating that the binding between the proteins and GPA is not specific. Although far Western blot followed by mass spectroscopy analyses suggested that HlyA interacts with Band 3 and spectrins, hemolytic experiments on spectrin-depleted hRBCs and spherocytes, indicated these proteins do not mediate the hemolytic process. Our results unequivocally demonstrate that neither glycophorins, nor Band 3 and spectrins mediate the cytotoxic activity of HlyA on hRBCs, thereby challenging the HlyA-receptor hypothesis. This finding holds significant relevance for the design of anti-toxin therapeutic strategies, particularly in light of the growing antibiotic resistance exhibited by bacteria.

AAV vectors displaying bispecific DARPins enable dual-control targeted gene delivery.

Precise delivery of genes to therapy-relevant cells is crucial for in vivo gene therapy. Receptor-targeting as prime strategy for this purpose is limited to cell types defined by a single cell-surface marker. Many target cells are characterized by combinations of more than one marker, such as the HIV reservoir cells. Here, we explored the tropism of adeno-associated viral vectors (AAV2) displaying designed ankyrin repeat proteins (DARPins) mono- and bispecific for CD4 and CD32a. Cryo-electron tomography revealed an unaltered capsid structure in the presence of DARPins. Surprisingly, bispecific AAVs transduced CD4/CD32a double-positive cells at much higher efficiencies than single-positive cells, even if present in low amounts in cell mixtures or human blood. This preference was confirmed when vector particles were systemically administered into mice. Cell trafficking studies revealed an increased cell entry rate for bispecific over monospecific AAVs. When equipped with an HIV genome-targeting CRISPR/Cas cassette, the vectors prevented HIV replication in T cell cultures. The data provide proof-of-concept for high-precision gene delivery through tandem-binding regions on AAV. Reminiscent of biological products following Boolean logic AND gating, the data suggest a new option for receptor-targeted vectors to improve the specificity and safety of in vivo gene therapy.

In-house ELISA protocols for capsid p24 detection of diverse HIV isolates.

BACKGROUND: The capsid p24 (CA-p24) antigen is a component of the viral capsid of human immunodeficiency virus (HIV) that has been commonly used for clinical diagnosis and monitoring of HIV infections in Enzyme-linked Immunosorbent Assays (ELISAs). Commercial CA-p24 ELISAs are widely used in research settings, but these kits are costly and have limited breadth for detecting diverse HIV isolates. METHODS: Commercial CA-p24 antibodies were used as capture and detection antibodies. Specific CA-p24 ELISAs were established with these antibodies and tested for the detection of HIV-1 isolates with the aim of developing in-house protocols to recognize HIV-1 infections in vitro for research purposes. RESULTS: Here we present four protocols for in-house ELISAs to detect HIV CA-p24 using commercial antibodies. The assays were able to detect the CA-p24 antigen of different HIV-1 isolates tested. Comparison between the protocols showed that these in-house ELISAs exhibit high specificity, sensitivity, and reproducibility for CA-p24 quantitation but their reactivity varied per HIV-1 isolate and subtype. CONCLUSIONS: These optimized ELISA protocols represent valuable tools to investigate HIV-1 infections in research facilities at a lower price than commercial CA-p24 kits.