Search

Le Dantec virus (LDV), assigned to the species Ledantevirus ledantec, genus Ledantevirus, family Rhabdoviridae has been associated with human disease but has gone undetected since the 1970s. We describe the detection of LDV in a human case of undifferentiated fever in Uganda by metagenomic sequencing and demonstrate a serological response using ELISA and pseudotype neutralisation. By screening 997 individuals sampled in 2016, we show frequent exposure to ledanteviruses with 76% of individuals seropositive in Western Uganda, but lower seroprevalence in other areas. Serological cross-reactivity as measured by pseudotype-based neutralisation was confined to ledanteviruses, indicating population seropositivity may represent either exposure to LDV or related ledanteviruses. We also describe the discovery of a closely related ledantevirus in blood from the synanthropic rodent Mastomys erythroleucus. Ledantevirus infection is common in Uganda but is geographically heterogenous. Further surveys of patients presenting with acute fever are required to determine the contribution of these emerging viruses to febrile illness in Uganda., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Shepherd et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Background. Gestational weight gain (GWG) constitutes an essential aspect of the gestational process. Due to factors such as pregestational body mass index (BMI), nutritional intake, level of physical activity, and psychological aspects, the recommended GWG may not be achieved, leading to adverse neonatal outcomes. Adolescents, due to their physiological and mental developmental stage, are at a higher risk of inappropriate GWG. Our aim is to highlight the importance of GWG in our population and to determine the correlation with perinatal outcomes. Methods. Pregnant adolescents who attended a tertiary care institution for prenatal care were included; maternal data such as preBMI and GWG were used to determine maternal and neonatal outcomes using the chi-square test and OR determination. Results. A total of 202 adolescent pregnant patients were included, comprising those with inadequate GWG ( n = 70), adequate GWG ( n = 85), and excessive GWG ( n = 47). A statistically significant association was found between low BMI and inadequate GWG. Patients with inadequate GWG demonstrated a correlation with IUGR and low birth weight, while patients with excessive GWG gave birth to macrosomic neonates. Conclusion. We concluded that previous habits play a significant role in determining weight gain throughout pregnancy. GWG has a direct impact on neonatal growth and development.

Background: Streptococcus pyogenes (group A streptococcus, GAS) causes a variety of diseases ranging from mild superficial infections of the throat and skin to severe invasive infections, such as necrotizing soft tissue infections (NSTIs). Tissue passage of GAS often results in mutations within the genes encoding for control of virulence (Cov)R/S two component system leading to a hyper-virulent phenotype. Dendritic cells (DCs) are innate immune sentinels specialized in antigen uptake and subsequent T cell priming. This study aimed to analyze cytokine release by DCs and other cells of monocytic origin in response to wild-type and natural covR/S mutant infections., Methods: Human primary monocyte-derived (mo)DCs were used. DC maturation and release of pro-inflammatory cytokines in response to infections with wild-type and covR/S mutants were assessed via flow cytometry. Global proteome changes were assessed via mass spectrometry. As a proof-of-principle, cytokine release by human primary monocytes and macrophages was determined., Results: In vitro infections of moDCs and other monocytic cells with natural GAS covR/S mutants resulted in reduced secretion of IL-8 and IL-18 as compared to wild-type infections. In contrast, moDC maturation remained unaffected. Inhibition of caspase-8 restored secretion of both molecules. Knock-out of streptolysin O in GAS strain with unaffected CovR/S even further elevated the IL-18 secretion by moDCs. Of 67 fully sequenced NSTI GAS isolates, 28 harbored mutations resulting in dysfunctional CovR/S. However, analyses of plasma IL-8 and IL-18 levels did not correlate with presence or absence of such mutations., Conclusions: Our data demonstrate that strains, which harbor covR/S mutations, interfere with IL-18 and IL-8 responses in monocytic cells by utilizing the caspase-8 axis. Future experiments aim to identify the underlying mechanism and consequences for NSTI patients., (© 2024. The Author(s).)

The isolation of DNA from placental tissue suspected of infection is an important tool for identifying microorganisms such as bacteria, fungi, and viruses associated with complications during and after pregnancy. While experts primarily process placental tissue, the preservation methods employed pose challenges to extracting complete DNA. Therefore, selecting the appropriate protocol is paramount to achieving greater efficiency in obtaining genetic material., (© 2024. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Mother-infant interactions during the early postnatal period are critical for infant survival and the scaffolding of infant development. Rodent models are used extensively to understand how these early social experiences influence neurobiology across the lifespan. However, methods for measuring postnatal dam-pup interactions typically involve time-consuming manual scoring, vary widely between research groups, and produce low density data that limits downstream analytical applications. To address these methodological issues, we developed the Automated Maternal Behavior during Early life in Rodents (AMBER) pipeline for quantifying home-cage maternal and mother-pup interactions using open-source machine learning tools. DeepLabCut was used to track key points on rat dams (32 points) and individual pups (9 points per pup) in postnatal day 1-10 video recordings. Pose estimation models reached key point test errors of approximately 4.1-10 mm (14.39 pixels) and 3.44-7.87 mm (11.81 pixels) depending on depth of animal in the frame averaged across all key points for dam and pups respectively. Pose estimation data and human-annotated behavior labels from 38 videos were used with Simple Behavioral Analysis (SimBA) to generate behavior classifiers for dam active nursing, passive nursing, nest attendance, licking and grooming, self-directed grooming, eating, and drinking using random forest algorithms. All classifiers had excellent performance on test frames, with F 1 scores above 0.886. Performance on hold-out videos remained high for nest attendance (F 1  = 0.990), active nursing (F 1  = 0.828), and licking and grooming (F 1  = 0.766) but was lower for eating, drinking, and self-directed grooming (F 1  = 0.534-0.554). A set of 242 videos was used with AMBER and produced behavior measures in the expected range from postnatal 1-10 home-cage videos. This pipeline is a major advancement in assessing home-cage dam-pup interactions in a way that reduces experimenter burden while increasing reproducibility, reliability, and detail of data for use in developmental studies without the need for special housing systems or proprietary software., (© 2023. Springer Nature Limited.)

Tumor-on-chips have become an effective resource in cancer research. However, their widespread use remains limited due to issues related to their practicality in fabrication and use. To address some of these limitations, we introduce a 3D-printed chip, which is large enough to host ~1 cm 3 of tissue and fosters well-mixed conditions in the liquid niche, while still enabling the formation of the concentration profiles that occur in real tissues due to diffusive transport. We compared the mass transport performance in its rhomboidal culture chamber when empty, when filled with GelMA/alginate hydrogel microbeads, or when occupied with a monolithic piece of hydrogel with a central channel, allowing communication between the inlet and outlet. We show that our chip filled with hydrogel microspheres in the culture chamber promotes adequate mixing and enhanced distribution of culture media. In proof-of-concept pharmacological assays, we biofabricated hydrogel microspheres containing embedded Caco2 cells, which developed into microtumors. Microtumors cultured in the device developed throughout the 10-day culture showing >75% of viability. Microtumors subjected to 5-fluorouracil treatment displayed <20% cell survival and lower VEGF-A and E-cadherin expression than untreated controls. Overall, our tumor-on-chip device proved suitable for studying cancer biology and performing drug response assays.

Chronic wounds have become one of the most important issues for healthcare systems and are a leading cause of death worldwide. Wound dressings are necessary to facilitate wound treatment. Engineering wound dressings may substantially reduce healing time, reduce the risk of recurrent infections, and reduce the disability and costs associated. In the path of engineering of an ideal wound dressing, hydrogels have played a leading role. Hydrogels are 3D hydrophilic polymeric structures that can provide a protective barrier, mimic the native extracellular matrix (ECM), and provide a humid environment. Due to their advantages, hydrogels (with different architectural, physical, mechanical, and biological properties) have been extensively explored as wound dressing platforms. Here we describe recent studies on hydrogels for wound healing applications with a strong focus on the interplay between the fabrication method used and the architectural, mechanical, and biological performance achieved. Moreover, we review different categories of additives which can enhance wound regeneration using 3D hydrogel dressings. Hydrogel engineering for wound healing applications promises the generation of smart solutions to solve this pressing problem, enabling key functionalities such as bacterial growth inhibition, enhanced re-epithelialization, vascularization, improved recovery of the tissue functionality, and overall, accelerated and effective wound healing., Competing Interests: The authors declare no conflict of interest., (© 2022 The Authors.)

Genomic sequencing (GS) can reveal secondary findings (SFs), findings unrelated to the reason for testing, that can be overwhelming to both patients and providers. An effective approach for communicating all clinically significant primary and secondary GS results is needed to effectively manage this large volume of results. The aim of this study was to develop a comprehensive approach to communicate all clinically significant primary and SF results. A genomic test report with accompanying patient and provider letters were developed in three phases: review of current clinical reporting practices, consulting with genetic and non-genetics experts, and iterative refinement through circulation to key stakeholders. The genomic test report and consultation letters present a myriad of clinically relevant GS results in distinct, tabulated sections, including primary (cancer) and secondary findings, with in-depth details of each finding generated from exome sequencing. They provide detailed variant and disease information, personal and familial risk assessments, clinical management details, and additional resources to help support providers and patients with implementing healthcare recommendations related to their GS results. The report and consultation letters represent a comprehensive approach to communicate all clinically significant SFs to patients and providers, facilitating clinical management of GS results., (© 2022. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Genetic variants in α-actinin-2 (ACTN2) are associated with several forms of (cardio)myopathy. We previously reported a heterozygous missense (c.740C>T) ACTN2 gene variant, associated with hypertrophic cardiomyopathy, and characterized by an electro-mechanical phenotype in human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs). Here, we created with CRISPR/Cas9 genetic tools two heterozygous functional knock-out hiPSC lines with a second wild-type (ACTN2wt) and missense ACTN2 (ACTN2mut) allele, respectively. We evaluated their impact on cardiomyocyte structure and function, using a combination of different technologies, including immunofluorescence and live cell imaging, RNA-seq, and mass spectrometry. This study showed that ACTN2mut presents a higher percentage of multinucleation, protein aggregation, hypertrophy, myofibrillar disarray, and activation of both the ubiquitin-proteasome system and the autophagy-lysosomal pathway as compared to ACTN2wt in 2D-cultured hiPSC-CMs. Furthermore, the expression of ACTN2mut was associated with a marked reduction of sarcomere-associated protein levels in 2D-cultured hiPSC-CMs and force impairment in engineered heart tissues. In conclusion, our study highlights the activation of proteolytic systems in ACTN2mut hiPSC-CMs likely to cope with ACTN2 aggregation and therefore directs towards proteopathy as an additional cellular pathology caused by this ACTN2 variant, which may contribute to human ACTN2 -associated cardiomyopathies.

Objective: It has been demonstrated in vitro that acetylsalicylic acid (ASA) treatment halves hepatitis C virus (HCV) expression in hepatocarcinoma cells. However, the signaling pathway that promotes this ASA-induced antiviral effect has not yet been identified., Aim: The aim of this work was to identify alterations in the transcriptional profile of Huh-7-HCV-subgenomic replicon cells with vs. without ASA treatment. This comparison sheds light onto the signaling pathways and molecular mechanisms involved in the antiviral effects of ASA., Methods: Human hepatocellular carcinoma (Huh-7) cells that express non-structural HCV proteins (Huh-7-HCV-replicon cells) were exposed to 4 mM ASA for 0, 24, 48, and 72 hours. Total RNA was isolated, and cDNA was synthesized. Transcripts were then tagged with biotin and purified. Thereafter, they were fragmented and hybridized on HG-U133 Plus 2 Gene Expression chips. Hybridization signals were captured using a GeneChip 3000 7G Scanner and analyzed via Expression Console and dChip Software., Results: When exposed to ASA, hepatocarcinoma cells with non-structural HCV proteins were found to differentially regulate genes with oxidative roles in the cell. The most upregulated genes were interleukin 8 (IL-8), cytochrome P450 (CYP450), and metallothioneins (MTs), while the most downregulated genes were ribonucleotide reductases (RRs)., Conclusion: These results show that ASA modulates the expression of genes with antioxidant functions. This suggests that ASA induces a remodeling of the antioxidant microenvironment, which may in turn interfere with the replication of HCV., (© 2022 by the Association of Clinical Scientists, Inc.)

Detailed characterization of transmitted HIV-1 variants in Uganda is fundamentally important to inform vaccine design, yet studies on the transmitted full-length strains of subtype D viruses are limited. Here, we amplified single genomes and characterized viruses, some of which were previously classified as subtype D by sub-genomic pol sequencing that were transmitted in Uganda between December 2006 to June 2011. Analysis of 5' and 3' half genome sequences showed 73% (19/26) of infections involved single virus transmissions, whereas 27% (7/26) of infections involved multiple variant transmissions based on predictions of a model of random virus evolution. Subtype analysis of inferred transmitted/founder viruses showed a high transmission rate of inter-subtype recombinants (69%, 20/29) involving mainly A1/D, while pure subtype D variants accounted for one-third of infections (31%, 9/29). Recombination patterns included a predominance of subtype D in the gag / pol region and a highly recombinogenic envelope gene. The signal peptide-C1 region and gp41 transmembrane domain (Tat2/Rev2 flanking region) were hotspots for A1/D recombination events. Analysis of a panel of 14 transmitted/founder molecular clones showed no difference in replication capacity between subtype D viruses ( n = 3) and inter-subtype mosaic recombinants ( n = 11). However, individuals infected with high replication capacity viruses had a faster CD4 T cell loss. The high transmission rate of unique inter-subtype recombinants is striking and emphasizes the extraordinary challenge for vaccine design and, in particular, for the highly variable and recombinogenic envelope gene, which is targeted by rational designs aimed to elicit broadly neutralizing antibodies.

Purpose: Metformin has been widely used for the treatment of Type 2 Diabetes Mellitus (T2DM), hyperglycemia and polycystic ovarian syndrome. Recent studies have suggested the potential of this substance as a cancer chemopreventive agent. We evaluated the antitumoral effect of iRNA-PFK-1 and the combined therapy iRNA-PFK-1 + metformin in RKO p53-positive cells., Methods: mRNA levels of tumor suppressor genes AMPK, APC, and c-MYC, KRAS oncogenes were measured by qRT-PCR in RKO cells treated with 25 µM metformin alone or combined with iRNA-PFK-1, to evaluate the effect of both treatments., Results: At 72 h after treatment with either 25 µM metformin, 150 nM iRNA-PFK-1, or the combined treatment, the transcriptional levels of these biomarkers were decreased by ~73% (p˂0.05), ~99.9%, (p˂0.01), and ~76% (p˂0.05), respectively., Conclusion: These in vitro results support the potential therapeutic role of metformin and PFK-1 in the treatment of colon cancer via down-modulation of the expression of several important cancer biomarkers.

Massive worldwide serological testing for SARS-CoV-2 is needed to determine the extent of virus exposure in a particular region, the ratio of symptomatic to asymptomatic infected persons, and the duration and extent of immunity after infection. To achieve this, the development and production of reliable and cost-effective SARS-CoV-2 antigens is critical. We report the bacterial production of the peptide S-RBD N318-V510 , which contains the receptor-binding domain of the SARS-CoV-2 spike protein (region of 193 amino acid residues from asparagine-318 to valine-510) of the SARS-CoV-2 spike protein. We purified this peptide using a straightforward approach involving bacterial lysis, his-tag-mediated affinity chromatography, and imidazole-assisted refolding. The antigen performances of S-RBD N318-V510 and a commercial full-length spike protein were compared in ELISAs. In direct ELISAs, where the antigen was directly bound to the ELISA surface, both antigens discriminated sera from non-exposed and exposed individuals. However, the discriminating resolution was better in ELISAs that used the full-spike antigen than the S-RBD N318-V510 . Attachment of the antigens to the ELISA surface using a layer of anti-histidine antibodies gave equivalent resolution for both S-RBD N318-V510 and the full-length spike protein. Results demonstrate that ELISA-functional SARS-CoV-2 antigens can be produced in bacterial cultures, and that S-RBD N318-V510 may represent a cost-effective alternative to the use of structurally more complex antigens in serological COVID-19 testing.

The ability to efficiently establish a new infection is a critical property for human immunodeficiency virus type 1 (HIV-1). Although the envelope protein of the virus plays an essential role in receptor binding and internalization of the infecting virus, the structural proteins, the polymerase and the assembly of new virions may also play a role in establishing and spreading viral infection in a new host. We examined Ugandan viruses from newly infected patients and focused on the contribution of the Gag-Pol genes to replication capacity. A panel of Gag-Pol sequences generated using single genome amplification from incident HIV-1 infections were cloned into a common HIV-1 NL4.3 pol/env backbone and the influence of Gag-Pol changes on replication capacity was monitored. Using a novel protein domain approach, we then documented diversity in the functional protein domains across the Gag-Pol region and identified differences in the Gag-p6 domain that were frequently associated with higher in vitro replication.

Objectives: We implemented the WHO cross-sectional survey protocol to determine rates of HIV viral load (VL) suppression (VLS), and weighted prevalence, predictors and patterns of acquired drug resistance (ADR) in individuals with virological failure (VF) defined as VL ≥1000 copies/mL., Methods: We enrolled 547 and 1064 adult participants on first-line ART for 12 (±3) months (ADR12) and ≥48 months (ADR48), respectively. Dried blood spots and plasma specimens were collected for VL testing and genotyping among the VFs., Results: VLS was 95.0% (95% CI 93.4%-96.5%) in the ADR12 group and 87.9% (95% CI 85.0%-90.9%) in the ADR48 group. The weighted prevalence of ADR was 96.1% (95% CI 72.9%-99.6%) in the ADR12 and 90.4% (95% CI 73.6-96.8%) in the ADR48 group, out of the 30 and 95 successful genotypes in the respective groups. Initiation on a zidovudine-based regimen compared with a tenofovir-based regimen was significantly associated with VF in the ADR48 group; adjusted OR (AOR) 1.96 (95% CI 1.13-3.39). Independent predictors of ADR in the ADR48 group were initiation on a zidovudine-based regimen compared with tenofovir-based regimens, AOR 3.16 (95% CI 1.34-7.46) and ART duration of ≥82 months compared with <82 months, AOR 1.92 (95% CI 1.03-3.59)., Conclusions: While good VLS was observed, the high prevalence of ADR among the VFs before they underwent the recommended three intensive adherence counselling (IAC) sessions followed by repeat VL testing implies that IAC prior to treatment switching may be of limited benefit in improving VLS., (© The Author(s) 2020. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy.)

Human perivascular progenitor cells, including pericytes, are well-described multipotent mesenchymal cells giving rise to mesenchymal stem cells in culture. Despite the unique location of pericytes, specific antigens to distinguish human pericytes from other cell types are few. Here, we employed a human tissue microarray (Human Protein Atlas) to identify proteins that are strongly and specifically expressed in a pericytic location within human adipose tissue. Next, these results were cross-referenced with RNA sequencing data from human adipose tissue pericytes, as defined as a fluorescence activated cell sorting (FACS) purified CD146 + CD34 - CD31 - CD45 - cell population. Results showed that from 105,532 core biopsies of soft tissue, 229 proteins showed strong and specific perivascular immunoreactivity, the majority of which (155) were present in the tunica intima . Next, cross-referencing with the transcriptome of FACS-derived CD146 + pericytes yielded 25 consistently expressed genes/proteins, including 18 novel antigens. A majority of these transcripts showed maintained expression after culture propagation (56% of genes). Interestingly, many novel antigens within pericytes are regulators of osteogenic differentiation. In sum, our study demonstrates the existence of novel pericyte markers, some of which are conserved in culture that may be useful for future efforts to typify, isolate, and characterize human pericytes.

The respiratory syncytial virus (RSV) is the main pathogen associated with upper respiratory tract infections during early childhood. Vertical transmission of this virus has been suggested in humans, based on observations recorded during animal studies that revealed an association of RSV with persistent structural and functional changes in the developing lungs of the offspring. However, human placentas have not yet been evaluated for susceptibility to RSV infection. In this study, we examined the capacity of RSV to infect a human trophoblast model, the BeWo cell line. Our results suggest that BeWo cells are susceptible to RSV infection since they allow RNA viral replication, viral protein translation, leading to the production of infectious RSV particles. In this report, we demonstrate that a human placenta model system, consisting of BeWo cells, is permissive to RSV infection. Thus, the BeWo cell line may represent a useful model for studies that aim to characterize the events of a possible RSV infection at the human maternal-fetal interface.

Although fishing communities (FCs) in Uganda are disproportionately affected by HIV-1 relative to the general population (GP), the transmission dynamics are not completely understood. We earlier found most HIV-1 transmissions to occur within FCs of Lake Victoria. Here, we test the hypothesis that HIV-1 transmission in FCs is isolated from networks in the GP. We used phylogeography to reconstruct the geospatial viral migration patterns in 8 FCs and 2 GP cohorts and a Bayesian phylogenetic inference in BEAST v1.8.4 to analyse the temporal dynamics of HIV-1 transmission. Subtype A1 (pol region) was most prevalent in the FCs (115, 45.1%) and GP (177, 50.4%). More recent HIV transmission pairs from FCs were found at a genetic distance (GD) <1.5% than in the GP (Fisher's exact test, p = 0.001). The mean time depth for pairs was shorter in FCs (5 months) than in the GP (4 years). Phylogeographic analysis showed strong support for viral migration from the GP to FCs without evidence of substantial viral dissemination to the GP. This suggests that FCs are a sink for, not a source of, virus strains from the GP. Targeted interventions in FCs should be extended to include the neighbouring GP for effective epidemic control.

Recombination in HIV-1 is well documented, but its importance in the low-diversity setting of within-host diversification is less understood. Here we develop a novel computational tool (RAPR (Recombination Analysis PRogram)) to enable a detailed view of in vivo viral recombination during early infection, and we apply it to near-full-length HIV-1 genome sequences from longitudinal samples. Recombinant genomes rapidly replace transmitted/founder (T/F) lineages, with a median half-time of 27 days, increasing the genetic complexity of the viral population. We identify recombination hot and cold spots that differ from those observed in inter-subtype recombinants. Furthermore, RAPR analysis of longitudinal samples from an individual with well-characterized neutralizing antibody responses shows that recombination helps carry forward resistance-conferring mutations in the diversifying quasispecies. These findings provide insight into molecular mechanisms by which viral recombination contributes to HIV-1 persistence and immunopathogenesis and have implications for studies of HIV transmission and evolution in vivo.

Chagas disease is a parasitic infection mainly found in Latin America; it is transmitted by a triatomine, also known as assassin bug or kissing bug. In humans, the parasite causes mostly cardiac disorders. Two-thirds of the Mexican territory are regarded as risk areas for vector transmission of Trypanosoma cruzi, the causal agent. The parasite can be found as a blood-borne trypomastigote or as an intracellular amastigote. The progression and severity of lesions could be due to frequent reinfections or to infection by highly virulent strains. A total of 3,327 individuals younger than 18 years old, living in risk areas for this disease in the rural setting of the States of Queretaro, San Luis Potosi, and Veracruz, underwent a seroepidemiological study. Among them, 37 subjects were seropositive for T. cruzi, and were studied to look for signs of cardiac pathology, which has only been reported in adults. A clinical record was prepared for all included individuals, and electrocardiography (ECG) and echocardiography (ECHO) studies were performed; 25 cases showed lesions compatible with the onset of Chagas cardiomyopathy. The other 12 patients showed either normal ECG and ECHO data or showed abnormal parameters that were not regarded as significant. Lesions found in the onset of Chagas cardiomyopathy in children are herein reported, along with 14 cases of cardiac pathology compatible with Chagas disease. Our results indicate that patients younger than 18 years can show a cardiac pathology similar to that observed in adults.

Staphylococcus aureus is a Gram-positive opportunistic pathogen that is able to cause a broad range of infectious diseases in humans. Furthermore, S. aureus is able to survive inside nonprofessional phagocytic host cell which serve as a niche for the pathogen to hide from the immune system and antibiotics therapies. Modern OMICs technologies provide valuable tools to investigate host-pathogen interactions upon internalization. However, these experiments are often hampered by limited capabilities to retrieve bacteria from such an experimental setting. Thus, the aim of this study was to develop a labeling strategy allowing fast detection and quantitation of S. aureus in cell lysates or infected cell lines by flow cytometry for subsequent proteome analyses. Therefore, S. aureus cells were labeled with the DNA stain SYTO ® 9, or Vancomycin BODIPY ® FL (VMB), a glycopeptide antibiotic binding to most Gram-positive bacteria which was conjugated to a fluorescent dye. Staining of S. aureus HG001 with SYTO 9 allowed counting of bacteria from pure cultures but not in cell lysates from infection experiments. In contrast, with VMB it was feasible to stain bacteria from pure cultures as well as from samples of infection experiments. VMB can also be applied for histocytochemistry analysis of formaldehyde fixed cell layers grown on coverslips. Proteome analyses of S. aureus labeled with VMB revealed that the labeling procedure provoked only minor changes on proteome level and allowed cell sorting and analysis of S. aureus from infection settings with sensitivity similar to continuous gfp expression. Furthermore, VMB labeling allowed precise counting of internalized bacteria and can be employed for downstream analyses, e.g., proteomics, of strains not easily amendable to genetic manipulation such as clinical isolates. © 2016 International Society for Advancement of Cytometry., (© 2016 International Society for Advancement of Cytometry.)

Saliva as major human body fluid may act as an indicator of oral disease status. Oral mucositis is a common and often treatment-limiting side effect of radiotherapy for head and neck cancer patients. In this dataset, we provide the complete proteome dataset (raw and search files) of the patients at baseline of radiotherapy treatment in patients undergoing radiotherapy analyzed by nano liquid chromatography coupled to mass spectrometry (LC-MS/MS). In the data set, 5323 tryptic peptides were identified which can be assigned to 487 distinct proteins (≥2 peptides). The MS data have been deposited to the ProteomeXchange ("ProteomeXchange provides globally coordinated proteomics data submission and dissemination" [1]) via the PRIDE partner repository with the dataset identifier PRIDE: PXD003230. The data are associated with the previously published work, "Differences in the whole saliva baseline proteome profile associated with development of oral mucositis in head and neck cancer patients undergoing radiotherapy" [2].

Prior work has demonstrated that HIV-1-specific CD8 T cells can cross-recognize variant epitopes. However, most of these studies were performed in the context of chronic infection, where the presence of viral quasispecies makes it difficult to ascertain the true nature of the original antigenic stimulus. To overcome this limitation, we evaluated the extent of CD8 T cell cross-reactivity in patients with acute HIV-1 clade B infection. In each case, we determined the transmitted founder virus sequence to identify the autologous epitopes restricted by individual HLA class I molecules. Our data show that cross-reactive CD8 T cells are infrequent during the acute phase of HIV-1 infection. Moreover, in the uncommon instances where cross-reactive responses were detected, the variant epitopes were poorly recognized in cytotoxicity assays. Molecular analysis revealed that similar antigenic structures could be cross-recognized by identical CD8 T cell clonotypes mobilized in vivo, yet even subtle differences in a single TCR-accessible peptide residue were sufficient to disrupt variant-specific reactivity. These findings demonstrate that CD8 T cells are highly specific for autologous epitopes during acute HIV-1 infection. Polyvalent vaccines may therefore be required to provide optimal immune cover against this genetically labile pathogen., (Copyright © 2016 by The American Association of Immunologists, Inc.)

Background: Identification of HIV-1 genomes responsible for establishing clinical infection in newly infected individuals is fundamental to prevention and pathogenesis research. Processing, storage, and transportation of the clinical samples required to perform these virologic assays in resource-limited settings requires challenging venipuncture and cold chain logistics. Here, we validate the use of dried-blood spots (DBS) as a simple and convenient alternative to collecting and storing frozen plasma., Methods: We performed parallel nucleic acid extraction, single genome amplification (SGA), next generation sequencing (NGS), and phylogenetic analyses on plasma and DBS., Results: We demonstrated the capacity to extract viral RNA from DBS and perform SGA to infer the complete nucleotide sequence of the transmitted/founder (TF) HIV-1 envelope gene and full-length genome in two acutely infected individuals. Using both SGA and NGS methodologies, we showed that sequences generated from DBS and plasma display comparable phylogenetic patterns in both acute and chronic infection. SGA was successful on samples with a range of plasma viremia, including samples as low as 1,700 copies/ml and an estimated ∼50 viral copies per blood spot. Further, we demonstrated reproducible efficiency in gp160 env sequencing in DBS stored at ambient temperature for up to three weeks or at -20°C for up to five months., Conclusions: These findings support the use of DBS as a practical and cost-effective alternative to frozen plasma for clinical trials and translational research conducted in resource-limited settings.

Oral mucositis (OM) is a common, painful and often treatment-limiting side effect of radiotherapy (RT) for head and neck cancer (HNC) patients. Unstimulated saliva was collected before the first radiotherapy application in 50 HNC patients. 41 out of 50 patients developed OM (grade III) during radiotherapy, of which 14 patients even displayed an early OM (grade III) at a low radiation dose of 30Gy. Nine patients did not develop OM (grade III). Using an LC-MS/MS approach 5323 tryptic peptides were assigned to 487 distinct proteins (≥2 peptides) in the data set. The levels of 48 proteins differed significantly (p<0.05) between patients developing OM or not. 17 proteins displayed increased levels (≥1.3-fold) and 31 proteins decreased in level in OM, respectively. Furthermore, using partial least square analysis protein patterns could be used to distinguish subjects which did not develop grade III OM even after 70Gy total dose (n=9) and those displaying early OM (grade III at <30Gy total dose, n=14). Using leave one out cross validation 37 of 41 patients (90%) developing OM could be correctly assigned indicating that prognostic proteome signatures may help identify patients that should be specifically monitored to increase overall effectiveness of RT treatment., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Staphylococcus aureus is one of the major causative agents of severe infections, and is responsible for a high burden of morbidity and mortality. Strains of increased virulence have emerged (e.g. USA300) that can infect healthy individuals in the community and are difficult to treat. To add to the knowledge about the pathophysiology of S. aureus, the adaption to iron restriction, an important in vivo stressor, was studied and the corresponding immune response of the human host characterized. Using a combination of 1D and 2D immune proteomics, the human antibody response to the exoproteomes of S. aureus USA300Δspa grown under iron restriction or with excess iron was compared. Human antibody binding to the altered exoproteome under iron restriction showed a 2.7- to 6.2-fold increase in overall signal intensity, and new antibody specificities appeared. Quantification of the secreted bacterial proteins by gel-free proteomics showed the expected strong increase in level of proteins involved in iron acquisition during iron-restricted growth compared to iron access. This was accompanied by decreased levels of superantigens and hemolysins. The latter was corroborated by functional peripheral blood mononuclear cell proliferation assays. The present data provide a comprehensive view of S. aureus exoproteome adaptation to iron restriction. Adults have high concentrations of serum antibodies specific for some of the newly induced proteins. We conclude that iron restriction is a common feature of the microenvironment, where S. aureus interacts with the immune system of its human host., (© 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Staphylococcus aureus is a human pathogen that can cause a wide range of diseases. Although formerly regarded as extracellular pathogen, it has been shown that S. aureus can also be internalized by host cells and persist within these cells. In the present study, we comparatively analyzed survival and physiological adaptation of S. aureus HG001 after internalization by two human lung epithelial cell lines (S9 and A549), and human embryonic kidney cells (HEK 293). Combining enrichment of bacteria from host-pathogen assays by cell sorting and quantitation of the pathogen's proteome by mass spectrometry we characterized S. aureus adaptation during the initial phase between 2.5 h and 6.5 h post-infection. Starting with about 2 × 10(6) bacteria, roughly 1450 S. aureus proteins, including virulence factors and metabolic enzymes were identified by spectral comparison and classical database searches. Most of the bacterial adaptation reactions, such as decreased levels of ribosomal proteins and metabolic enzymes or increased amounts of proteins involved in arginine and lysine biosynthesis, enzymes coding for terminal oxidases and stress responsive proteins or activation of the sigma factor SigB were observed after internalization into any of the three cell lines studied. However, differences were noted in central carbon metabolism including regulation of fermentation and threonine degradation. Since these differences coincided with different intracellular growth behavior, complementary profiling of the metabolome of the different non-infected host cell types was performed. This revealed similar levels of intracellular glucose but host cell specific differences in the amounts of amino acids such as glycine, threonine or glutamate. With this comparative study we provide an impression of the common and specific features of the adaptation of S. aureus HG001 to specific host cell environments as a starting point for follow-up studies with different strain isolates and regulatory mutants.

Macrophages are essential components of the innate immune system and crucial for pathogen elimination in early stages of infection. We previously observed that bone marrow-derived macrophages (BMMs) from C57BL/6 mice exhibited increased killing activity against Burkholderia pseudomallei compared to BMMs from BALB/c mice. This effect was particularly pronounced when cells were treated with IFN-γ. To unravel mechanisms that could explain these distinct bactericidal effects, a comparative combined proteome and transcriptome analysis of untreated and IFN-γ treated BALB/c and C57BL/6 BMMs under standardized serum-free conditions was carried out. We found differences in gene expression/protein abundance belonging to cellular oxidative and antioxidative stress systems. Genes/proteins involved in the generation of oxidant molecules and the function of phagosomes (respiratory chain ATPase, lysosomal enzymes, cathepsins) were predominantly higher expressed/more abundant in C57BL/6 BMMs. Components involved in alleviation of oxidative stress (peroxiredoxin, mitochondrial superoxide dismutase) were more abundant in C57BL/6 BMMs as well. Thus, C57BL/6 BMMs seemed to be better equipped with cellular systems that may be advantageous in combating engulfed pathogens. Simultaneously, C57BL/6 BMMs were well protected from oxidative burst. We assume that these variations co-determine differences in resistance between BALB/c and C57BL/6 mice observed in many infection models., Biological Significance: In this study we performed combined transcriptome and proteome analyses on BMMs derived from two inbred mouse strains that are frequently used for studies in the field of host-pathogen interaction research. Strain differences between BALB/c and C57BL/6 BMMs were found to originate mainly from different protein abundance levels rather than from different gene expression. Differences in abundance of respiratory chain complexes and lysosomal proteins as well as differential regulation of components belonging to various antioxidant stress systems help to explain long-known differences between the mouse strains concerning their different susceptibility in several infection models., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Background: Individualized Medicine aims at providing optimal treatment for an individual patient at a given time based on his specific genetic and molecular characteristics. This requires excellent clinical stratification of patients as well as the availability of genomic data and biomarkers as prerequisites for the development of novel diagnostic tools and therapeutic strategies. The University Medicine Greifswald, Germany, has launched the "Greifswald Approach to Individualized Medicine" (GANI&#95;MED) project to address major challenges of Individualized Medicine. Herein, we describe the implementation of the scientific and clinical infrastructure that allows future translation of findings relevant to Individualized Medicine into clinical practice., Methods/design: Clinical patient cohorts (N > 5,000) with an emphasis on metabolic and cardiovascular diseases are being established following a standardized protocol for the assessment of medical history, laboratory biomarkers, and the collection of various biosamples for bio-banking purposes. A multi-omics based biomarker assessment including genome-wide genotyping, transcriptome, metabolome, and proteome analyses complements the multi-level approach of GANI&#95;MED. Comparisons with the general background population as characterized by our Study of Health in Pomerania (SHIP) are performed. A central data management structure has been implemented to capture and integrate all relevant clinical data for research purposes. Ethical research projects on informed consent procedures, reporting of incidental findings, and economic evaluations were launched in parallel.

Helicobacter pylori induces less gastric inflammation in children than adults. Here we investigated whether this reduced inflammation involves dysregulated T helper type 17 (Th17) responses. H. pylori-infected children and adults in Santiago, Chile had similar levels of H. pylori colonization, proportions of bacteria containing cagA and s1/s2 vacA markers of virulence, and strain genotypes (predominantly hpEurope), but the children had significantly reduced levels of gastric inflammation and neutrophil infiltration. The reduced neutrophil accumulation in the infected children was accompanied by significantly fewer gastric Th17 cells and significantly lower levels of interleukin (IL)-17-specific mRNA and protein compared with the infected adults. The gastric mucosa of H. pylori-infected children also contained higher numbers of IL-10+ cells and increased levels of both IL-10 and Foxp3 mRNA compared with that of the infected adults. Thus, reduced gastric inflammation, including diminished neutrophil accumulation, in H. pylori-infected children compared with infected adults is likely due to downregulated gastric Th17/IL-17 responses as a consequence of enhanced mucosal regulatory T-cell activity in the children.

Aim: Interest in human saliva proteomics for disease-specific biomarker screening increased in the last decade. We used whole saliva samples from periodontally healthy and diseased subjects with chronic periodontitis to screen for disease-associated differences in the protein pattern., Material and Methods: We selected 20 periodontally healthy and 20 periodontally diseased subjects from the population-based cross-sectional Study of Health in Pomerania (SHIP-2 and SHIP-Trend). Saliva collection was performed with commercially available Salivette(®) (Sarstedt, Nümbrecht, Germany). Whole saliva proteins were analysed after trichloroacetic acid (TCA) precipitation and proteolytic digestion with trypsin by LC-MS/MS. MS-data were analysed and quantified using the Rosetta Elucidator software package., Results: In whole saliva we identified 344 human protein groups across all samples. For label free quantitation we only considered 152 proteins identified with more than one unique peptide. In total, 20 proteins showed 1.5-fold difference in abundance between controls and patients (p < 0.05); the majority of these proteins showed higher abundance in the periodontally diseased subjects. Functional annotation of proteins linked the periodontally diseased status with acute phase response and inflammatory processes., Conclusion: Label free proteomic analysis of whole saliva is a powerful tool to characterize the periodontal disease status and differentiate between healthy and periodontally diseased subjects., (© 2013 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Background: Intrapartum administration of single-dose nevirapine (sdNVP) reduces perinatal HIV-1 transmission in resource-limiting settings by half. Yet this strategy has limited effect on subsequent breast milk transmission, making the case for new treatment approaches to extend maternal/infant antiretroviral prophylaxis through the period of lactation. Maternal and transmitted infant HIV-1 variants frequently develop NVP resistance mutations following sdNVP, complicating subsequent treatment/prophylaxis regimens. However, it is not clear whether NVP-resistant viruses are transmitted via breastfeeding or arise de novo in the infant., Findings: We performed a detailed HIV genetic analysis using single genome sequencing to identify the origin of drug-resistant variants in an sdNVP-treated postnatally-transmitting mother-infant pair. Phylogenetic analysis of HIV sequences from the child revealed low-diversity variants indicating infection by a subtype C single transmitted/founder virus that shared full-length sequence identity with a clonally-amplified maternal breast milk virus variant harboring the K103N NVP resistance mutation., Conclusion: In this mother/child pair, clonal amplification of maternal NVP-resistant HIV variants present in systemic and mammary gland compartments following intrapartum sdNVP represents one source of transmitted NVP-resistant variants that is responsible for the acquisition of drug resistant virus by the breastfeeding infant. This finding emphasizes the need for combination antiretroviral prophylaxis to prevent mother-to-child HIV transmission.

Understanding human immunodeficiency virus type 1 (HIV-1) transmission is central to developing effective prevention strategies, including a vaccine. We compared phenotypic and genetic variation in HIV-1 env genes from subjects in acute/early infection and subjects with chronic infections in the context of subtype C heterosexual transmission. We found that the transmitted viruses all used CCR5 and required high levels of CD4 to infect target cells, suggesting selection for replication in T cells and not macrophages after transmission. In addition, the transmitted viruses were more likely to use a maraviroc-sensitive conformation of CCR5, perhaps identifying a feature of the target T cell. We confirmed an earlier observation that the transmitted viruses were, on average, modestly underglycosylated relative to the viruses from chronically infected subjects. This difference was most pronounced in comparing the viruses in acutely infected men to those in chronically infected women. These features of the transmitted virus point to selective pressures during the transmission event. We did not observe a consistent difference either in heterologous neutralization sensitivity or in sensitivity to soluble CD4 between the two groups, suggesting similar conformations between viruses from acute and chronic infection. However, the presence or absence of glycosylation sites had differential effects on neutralization sensitivity for different antibodies. We suggest that the occasional absence of glycosylation sites encoded in the conserved regions of env, further reduced in transmitted viruses, could expose specific surface structures on the protein as antibody targets.

Background: A.BY/SnJ mice are used to study pathological alterations in the heart due to enteroviral infections. Since age is a well-known factor influencing the susceptibility of mice to infection, response to stress and manifestation of cardiovascular diseases, the myocardial proteome of A.BY/SnJ mice aged 1 and 4 months was comparatively studied using two dimensional-differential in-gel electrophoresis (2D-DIGE) and liquid chromatography tandem mass spectrometry (LC-MS/MS)., Results: Complementary analyses by 2D-DIGE and gel-free LC-MS/MS revealed 96 distinct proteins displaying age associated alterations in their levels. Proteins related to protein transport, and transport chain, lipid metabolism and fatty acid transport showed significant changes in 4 months old mouse hearts compared to juvenile hearts. Proteins involved in lipid metabolism and transport were identified at significantly higher levels in older mice and dysregulation of proteins of the respiratory transport chain were observed., Conclusion: The current proteomics study discloses age dependent changes occurring in the hearts already in young mice of the strain A.BY/SnJ. Besides alterations in protein transport, we provide evidence that a decrease of ATP synthase in murine hearts starts already in the first months of life, leading to well-known low expression levels manifested in old mice thereby raising the possibility of reduced energy supply. In the first few months of murine life this seems to be compensated by an increased lipid metabolism. The functional alterations described should be considered during experimental setups in disease related studies.

Background: Saliva collection devices are widely used for large-scale screening approaches. This study was designed to compare the suitability of three different whole-saliva collection approaches for subsequent proteome analyses., Methods: From 9 young healthy volunteers (4 women and 5 men) saliva samples were collected either unstimulated by passive drooling or stimulated using a paraffin gum or Salivette® (cotton swab). Saliva volume, protein concentration and salivary protein patterns were analyzed comparatively., Results: Samples collected using paraffin gum showed the highest saliva volume (4.1±1.5 ml) followed by Salivette® collection (1.8±0.4 ml) and drooling (1.0±0.4 ml). Saliva protein concentrations (average 1145 μg/ml) showed no significant differences between the three sampling schemes. Each collection approach facilitated the identification of about 160 proteins (≥2 distinct peptides) per subject, but collection-method dependent variations in protein composition were observed., Conclusion: Passive drooling, paraffin gum and Salivette® each allows similar coverage of the whole saliva proteome, but the specific proteins observed depended on the collection approach. Thus, only one type of collection device should be used for quantitative proteome analysis in one experiment, especially when performing large-scale cross-sectional or multi-centric studies., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Background: Breastfeeding is a leading cause of infant HIV-1 infection in the developing world, yet only a minority of infants exposed to HIV-1 via breastfeeding become infected. As a genetic bottleneck severely restricts the number of postnatally-transmitted variants, genetic or phenotypic properties of the virus Envelope (Env) could be important for the establishment of infant infection. We examined the efficiency of virologic functions required for initiation of infection in the gastrointestinal tract and the neutralization sensitivity of HIV-1 Env variants isolated from milk of three postnatally-transmitting mothers (n = 13 viruses), five clinically-matched nontransmitting mothers (n = 16 viruses), and seven postnatally-infected infants (n = 7 postnatally-transmitted/founder (T/F) viruses)., Results: There was no difference in the efficiency of epithelial cell interactions between Env virus variants from the breast milk of transmitting and nontransmitting mothers. Moreover, there was similar efficiency of DC-mediated trans-infection, CCR5-usage, target cell fusion, and infectivity between HIV-1 Env-pseudoviruses from nontransmitting mothers and postnatal T/F viruses. Milk Env-pseudoviruses were generally sensitive to neutralization by autologous maternal plasma and resistant to breast milk neutralization. Infant T/F Env-pseudoviruses were equally sensitive to neutralization by broadly-neutralizing monoclonal and polyclonal antibodies as compared to nontransmitted breast milk Env variants., Conclusion: Postnatally-T/F Env variants do not appear to possess a superior ability to interact with and cross a mucosal barrier or an exceptional resistance to neutralization that define their capability to initiate infection across the infant gastrointestinal tract in the setting of preexisting maternal antibodies.

Genome sequences of transmitted/founder (T/F) HIV-1 have been inferred by analyzing single genome amplicons of acute infection plasma viral RNA in the context of a mathematical model of random virus evolution; however, few of these T/F sequences have been molecularly cloned and biologically characterized. Here, we describe the derivation and biological analysis of ten infectious molecular clones, each representing a T/F genome responsible for productive HIV-1 clade B clinical infection. Each of the T/F viruses primarily utilized the CCR5 coreceptor for entry and replicated efficiently in primary human CD4(+) T lymphocytes. This result supports the conclusion that single genome amplification-derived sequences from acute infection allow for the inference of T/F viral genomes that are consistently replication competent. Studies with monocyte-derived macrophages (MDM) demonstrated various levels of replication among the T/F viruses. Although all T/F viruses replicated in MDM, the overall replication efficiency was significantly lower compared to prototypic "highly macrophage-tropic" virus strains. This phenotype was transferable by expressing the env genes in an isogenic proviral DNA backbone, indicating that T/F virus macrophage tropism mapped to Env. Furthermore, significantly higher concentrations of soluble CD4 were required to inhibit T/F virus infection compared to prototypic macrophage-tropic virus strains. Our findings suggest that the acquisition of clinical HIV-1 subtype B infection occurs by mucosal exposure to virus that is not highly macrophage tropic and that the generation and initial biological characterization of 10 clade B T/F infectious molecular clones provides new opportunities to probe virus-host interactions involved in HIV-1 transmission.

Sexual transmission of human immunodeficiency virus type 1 (HIV-1) most often results from productive infection by a single transmitted/founder (T/F) virus, indicating a stringent mucosal bottleneck. Understanding the viral traits that overcome this bottleneck could have important implications for HIV-1 vaccine design and other prevention strategies. Most T/F viruses use CCR5 to infect target cells and some encode envelope glycoproteins (Envs) that contain fewer potential N-linked glycosylation sites and shorter V1/V2 variable loops than Envs from chronic viruses. Moreover, it has been reported that the gp120 subunits of certain transmitted Envs bind to the gut-homing integrin α4β7, possibly enhancing virus entry and cell-to-cell spread. Here we sought to determine whether subtype C T/F viruses, which are responsible for the majority of new HIV-1 infections worldwide, share biological properties that increase their transmission fitness, including preferential α4β7 engagement. Using single genome amplification, we generated panels of both T/F (n = 20) and chronic (n = 20) Env constructs as well as full-length T/F (n = 6) and chronic (n = 4) infectious molecular clones (IMCs). We found that T/F and chronic control Envs were indistinguishable in the efficiency with which they used CD4 and CCR5. Both groups of Envs also exhibited the same CD4+ T cell subset tropism and showed similar sensitivity to neutralization by CD4 binding site (CD4bs) antibodies. Finally, saturating concentrations of anti-α4β7 antibodies failed to inhibit infection and replication of T/F as well as chronic control viruses, although the growth of the tissue culture-adapted strain SF162 was modestly impaired. These results indicate that the population bottleneck associated with mucosal HIV-1 acquisition is not due to the selection of T/F viruses that use α4β7, CD4 or CCR5 more efficiently.

The most relevant clinical phenotype resulting from chronic enteroviral myocarditis is dilated cardiomyopathy (DCM). Mice of the susceptible mouse strain A.BY/SnJ mimick well human DCM since they develop as a consequence of persistent infection and chronic inflammation a dilation of the heart ventricle several weeks after coxsackievirus B3 (CVB3) infection. Therefore, this model is well suited for the analysis of changes in the heart proteome associated with DCM. Here, we present a proteomic survey of the dilated hearts based on differential fluorescence gel electrophoresis and liquid chromatography-mass spectrometric centered methods in comparison to age-matched non-infected hearts. In total, 101 distinct proteins, which belong to categories immunity and defense, cell structure and associated proteins, energy metabolism and protein metabolism/modification differed in their levels in both groups. Levels of proteins involved in fatty acid metabolism and electron transport chain were found to be significantly reduced in infected mice suggesting a decrease in energy production in CVB3-induced DCM. Furthermore, proteins associated with muscle contraction (MLRV, MLRc2, MYH6, MyBPC3), were present in significantly altered amounts in infected mice. A significant increase in the level of extracellular matrix proteins in the dilated hearts indicates cardiac remodeling due to fibrosis., (Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)