Cyanovirin-N Binding to N-Acetyl-d-glucosamine Requires Carbohydrate-Binding Sites on Two Different Protomers.

Cyanovirin-N (CV-N) binds high-mannose oligosaccharides on enveloped viruses with two carbohydrate-binding sites, one bearing high affinity and one low affinity to Manα(1-2)Man moieties. A tandem repeat of two CV-N molecules (CVN2) was tested for antiviral activity against human immunodeficiency virus type I (HIV-1) by using a domain-swapped dimer. CV-N was shown to bind N-acetylmannosamine (ManNAc) and N-acetyl-d-glucosamine (GlcNAc) when the carbohydrate-binding sites in CV-N were free to interact with these monosaccharides independently. CVN2 recognized ManNAc at a Kd of 1.4 µM and bound this sugar in solution, regardless of the lectin making amino acid side chain contacts on the targeted viral glycoproteins. An interdomain cross-contacting residue Glu41, which has been shown to be hydrogen bonding with dimannose, was substituted in the monomeric CV-N. The amide derivative of glucose, GlcNAc, achieved similar high affinity to the new variant CVN-E41T as high-mannose N-glycans, but binding to CVN2 in the nanomolar range with four binding sites involved or binding to the monomeric CVN-E41A. A stable dimer was engineered and expressed from the alanine-to-threonine-substituted monomer to confirm binding to GlcNAc. In summary, low-affinity binding was achieved by CVN2 to dimannosylated peptide or GlcNAc with two carbohydrate-binding sites of differing affinities, mimicking biological interactions with the respective N-linked glycans of interest and cross-linking of carbohydrates on human T cells for lymphocyte activation.

Heterosubtypic, cross-reactive immunity to human Cytomegalovirus glycoprotein B.

Cytomegalovirus (CMV) genome is highly variable and heterosubtypic immunity should be considered in vaccine development since it can enhance protection in a cross-reactive manner. Here, we developed a protein array to evaluate heterosubtypic immunity to CMV glycoprotein B (gB) in natural infection and vaccination. DNA sequences of four antigenic domains (AD1, AD2, AD4/5, and AD5) of gB were amplified from six reference and 12 clinical CMV strains, and the most divergent genotypes were determined by phylogenetic analysis. Assigned genotypes were in vitro translated and immobilized on protein array. Then, we tested immune response of variable serum groups (primarily infected patients, reactivated CMV infections and healthy individuals with latent CMV infection, as well gB-vaccinated rabbits) with protein in situ array (PISA). Serum antibodies of all patient cohorts and gB-vaccinated rabbits recognized many genetic variants of ADs on protein array, including but not limited to the subtype of infecting strain. High-grade cross-reactivity was observed. In several patients, we observed none or neglectable immune response to AD1 and AD2, while the same patients showed high antibody response to AD4/5 and AD5. Among the primary infected patients, AD5 was the predominant AD, in antibody response. The most successful CMV vaccine to date contains gB and demonstrates only 50% efficacy. In this study, we showed that heterosubtypic and cross-reactive immunity to CMV gB is extensive. Therefore, the failure of CMV gB vaccines cannot be explained by a highly, strain-specific immunity. Our observations suggest that other CMV antigens should be addressed in vaccine design.

Oral Abundance of Actinomyces spp. in Breast Cancer Patients.

OBJECTIVES: Pathophysiology of medication-related osteonecrosis of the jaw (MRONJ) is still unclear, and disease development is associated with adverse reaction of bisphosphonates and denosumab, and Actinomyces spp. as well. In this study, we evaluated the abundance of Actinomyces spp. in breast cancer patients undergoing chemotherapy compared to healthy controls. METHODS: Oropharyngeal samples were collected from treatment-naive early-stage breast cancer patients, who were scheduled for standard of care therapy (eight samples throughout chemotherapy, one prior to radiotherapy and one after a year of start), as well as from healthy controls at matched timepoints. We quantified Actinomyces spp. in the samples with a highly sensitive and specific quantitative polymerase chain reaction. RESULTS: Twenty-one patients and 16 healthy subjects were enrolled. Forty-eight percent of patients suffered from estrogen receptor-positive/progesterone receptor-positive or -negative/human epidermal growth factor receptor 2 (HER2)-negative disease, 38% were HER2-positive, and 14% were triple-negative. Comparison of Actinomyces spp. loads in cancer patients and healthy controls did not reveal significant difference. Fluctuations on bacterial quantity were observed in both groups over time. Tumor receptor status or different chemotherapy schemes of patients were not correlated with a particular pattern on abundance of Actinomyces spp. CONCLUSIONS: We suggest that Actinomyces spp. are not the initiative factors in MRONJ development. These bacteria are not altered in abundance during chemotherapy, but they behave opportunistic when there is a bone disruption in the oropharynx in the first place caused by antiresorptive drugs or dental trauma and proliferate in their new niche. Thus, Actinomyces spp. plays a latter role in MRONJ development, rather than a primary causative one.

Letermovir for the compassionate therapeutic use of cytomegalovirus infection.

PURPOSE: Data on the efficacy, dosing and safety of letermovir for the compassionate therapeutic use of CMV infections are limited. METHODS: Clinical and virological efficacy of letermovir was assessed in a retrospective single-centre study of patients who received letermovir for the compassionate therapeutic use of CMV infections. RESULTS: Letermovir initiation yielded prompt treatment response in 7 out of 9 patients (77.7%). CONCLUSION: Letermovir may be an effective and well tolerated option in the compassionate treatment of CMV infections, although recurrence of CMV and emergence of resistance may be issues.

Bone turnover markers can predict healing time in medication-related osteonecrosis of the jaw.

OBJECTIVES: Medication-related osteonecrosis of the jaw (MRONJ) is a severe and difficult-to-treat adverse event of bone-modifying agents. Therefore predictive strategies determining patients at risk for a prolonged healing duration are needed to optimize treatment. Thus, the present study evaluates whether or not bone turnover markers can be used to predict the healing duration in MRONJ patients. MATERIALS AND METHODS: The present study is a retrospective data analysis of patients suffering from MRONJ and positive histology for Actinomyces spp., who were identified at the General Hospital Vienna from 2014 to 2018. During the first visit, the patients' demographics and levels of bone formation parameters were compiled. Healing times were analysed by Cox regression in dependence on these factors. RESULTS: A total of 52 patients were identified who fulfilled the inclusion criteria. The indication for bone-modifying agents was breast cancer (n = 21), prostate cancer (n = 14), multiple myeloma (n = 6) and other malignant diseases (n = 11). In 43 (82.7%) of our patients, we were able to document complete mucosal healing. Furthermore, patients who responded faster to therapy showed higher levels of C-telopeptide (P < 0.05), osteocalcin (P < 0.05) and bone-specific alkaline phosphatase (P < 0.05), but lower levels of 1.25-dihydroxyvitamin D (P < 0.05) than slower responding patients. No correlation was found regarding parathyroid hormone or calcitonin levels. Interestingly, patients who had a slower response were less likely to report dental procedures, but more likely to report a history of chemotherapy. CONCLUSION: CTX and osteocalcin levels may be used for predicting healing duration for MRONJ.

Correction to: Real­world experience with dalbavancin therapy in gram­positive skin and soft tissue infection, bone and joint infection.

The original version of this article unfortunately contained a mistake. The presentation of Fig. 1 was incorrect. The corrected figure is given below.

Minimal Reconstitution of Membranous Web Induced by a Vesicle-Peptide Sol-Gel Transition.

Positive strand RNA viruses replicate in specialized niches called membranous web within the cytoplasm of host cells. These virus replication organelles sequester viral proteins, RNA, and a variety of host factors within a fluid, amorphous matrix of clusters of endoplasmic reticulum (ER) derived vesicles. They are thought to form by the actions of a nonstructural viral protein NS4B, which remodels the ER and produces dense lipid-protein condensates. Here, we used in vitro reconstitution to identify the minimal components and elucidate physical mechanisms driving the web formation. We found that the N-terminal amphipathic domain of NS4B (peptide 4BAH2) and phospholipid vesicles (∼100-200 nm in diameter) were sufficient to produce a gel-like, viscoelastic condensate. This condensate coexists with the surrounding aqueous phase and affords rapid exchange of molecules. Together, it recapitulates the essential properties of the virus-induced membranous web. Our data support a novel phase separation mechanism in which phospholipid vesicles provide a supramolecular template spatially organizing multiple self-associating peptides thereby generating programmable multivalency de novo and inducing macroscopic phase separation.

Review: Epidemiology of Helicobacter pylori infection.

This review summarizes recent publications on the epidemiology of Helicobacter pylori. Two major systemic analyses, from Malaysia and Ethiopia, were published. The Brazilian Consensus Conference has stated that H pylori infection is an infectious disease with an indication for antimicrobial therapy. A continuous decrease in H pylori prevalence was reported from many regions worldwide, including Korea, China, Iran, and Austria. A cross-sectional H pylori prevalence study conducted in the United Arab Emirates found 41% prevalence in a group of healthy children and adults. Several studies from Asia addressed H pylori prevalence in adults undergoing regular checkup. The largest of such studies, performed in Korea, involved 24 471 subjects and reported 41.5% seroprevalence. A relatively smaller study from East China on 3252 subjects reported 27.5% prevalence. In contrast, a study from Spain reported 87.2% seroprevalence. A report on the association between smoking and H pylori seropositivity was published on behalf of the Stomach Cancer Pooling (StoP) Project-a consortium of epidemiological studies of gastric cancer. Also, other potential risk factors, including occupational risk factors, water supply, and food were analyzed. Gastroesophageal reflux and sexual partners has been associated with a higher risk for H pylori acquisition, and gut microbiota was suggested to play a role in intrafamilial transmission of H pylori. Finally, in a few studies (from Mexico and Japan), the catalytic model for predicting the potential risk of acquiring H pylori infection in the future was used. As anticipated, a further decline in H pylori-related disease was demonstrated by applying the modeling.

Real-world experience with dalbavancin therapy in gram-positive skin and soft tissue infection, bone and joint infection.

PURPOSE: Dalbavancin is a novel lipoglycopeptide with potent activity against several gram-positive pathogens, an excellent safety profile and a long elimination half-life. METHODS: In this case series observed at the University Hospital of Vienna between 2015 and 2017, all adult patients with gram-positive infections who received at least one dosage of dalbavancin were screened (n = 118). A total of 72 patients were included in the final analysis. The number of included patients stratified by the source of infection was: skin and soft tissue infection (SSTI) (n = 26), osteomyelitis (n = 20), spondylodiscitis (n = 14), acute septic arthritis (n = 4) and prosthetic joint infection (n = 8). RESULTS: In 46 patients (64%), clinical cure was detected at the end of dalbavancin therapy without additional antibiotic therapy. Of the 26 patients who received additional antibiotic therapy other than dalbavancin, 15 patients (21%) showed no clinical improvement under dalbavancin therapy, four patients (5%) had side effects (nausea n = 1, exanthema n = 2, hyperglycemia n = 1), and in seven patients (10%) clinical improvement under dalbavancin therapy was detected but antibiotic therapy was de-escalated to an oral drug. CONCLUSION: We demonstrated high clinical effectiveness of dalbavancin for acute gram-positive infections primarily acute SSTI, acute septic arthritis, acute osteomyelitis and spondylodiscitis. In patients with biofilm-associated infection (chronic infection or joint prosthesis), source control was absolutely necessary for treatment success.

Dalbavancin as Primary and Sequential Treatment for Gram-Positive Infective Endocarditis: 2-Year Experience at the General Hospital of Vienna.

The clinical outcomes and safety of dalbavancin as primary and sequential treatment of gram-positive bacteremia with infective endocarditis were evaluated retrospectively. The clinical success rate was high (92.6%), but in 24 of 27 patients dalbavancin was used only after clearance of bacteria from the bloodstream.

Viruses comprise an extensive pool of mobile genetic elements in eukaryote cell cultures and human clinical samples.

Viruses shape a diversity of ecosystems by modulating their microbial, eukaryotic, or plant host metabolism. The complexity of virus-host interaction networks is progressively fathomed by novel metagenomic approaches. By using a novel metagenomic method, we explored the virome in mammalian cell cultures and clinical samples to identify an extensive pool of mobile genetic elements in all of these ecosystems. Despite aseptic treatment, cell cultures harbored extensive and diverse phage populations with a high abundance of as yet unknown and uncharacterized viruses (viral dark matter). Unknown phages also predominated in the oropharynx and urine of healthy individuals and patients infected with cytomegalovirus despite demonstration of active cytomegalovirus replication. The novelty of viral sequences correlated primarily with the individual evaluated, whereas relative abundance of encoded protein functions was associated with the ecologic niches probed. Together, these observations demonstrate the extensive presence of viral dark matter in human and artificial ecosystems.-Thannesberger, J., Hellinger, H.-J., Klymiuk, I., Kastner, M.-T., Rieder, F. J. J., Schneider, M., Fister, S., Lion, T., Kosulin, K., Laengle, J., Bergmann, M., Rattei, T., Steininger, C. Viruses comprise an extensive pool of mobile genetic elements in eukaryote cell cultures and human clinical samples.

Human cytomegalovirus phosphoproteins are hypophosphorylated and intrinsically disordered.

Protein phosphorylation has important regulatory functions in cell homeostasis and is tightly regulated by kinases and phosphatases. The tegument of human cytomegalovirus (CMV) contains not only several proteins reported to be extensively phosphorylated but also cellular protein phosphatases (PP1 and PP2A). To investigate this apparent inconsistency, we evaluated the phosphorylation status of the tegument proteins pUL32 and pp65 by enzymatic dephosphorylation and MS. Enzymatic dephosphorylation with bacterial λ phosphatase, but not with PP1, shifted the pUL32-specific signal on reducing SDS-PAGE from ~150 to ~148 kDa, a mass still much larger than the ~118 kDa obtained from our diffusion studies and from the calculated protein mass of ~113 kDa. Remarkably, inhibition of phosphatases through treatment with the phosphatase inhibitors calyculin A and okadaic acid resulted in a shift to ~190 or ~180 kDa, respectively, indicating that a considerable number of potential phosphorylated residues on pUL32 are not phosphorylated under normal conditions. MS revealed a general state of hypophosphorylation of CMV phosphoproteins with only 17 phosphorylated residues detected on pUL32 and 19 on pp65, respectively. Moreover, bioinformatics analysis shows that the C-terminal two-thirds of pUL32 are intrinsically disordered and that most phosphorylations map to this region. In conclusion, we show that important CMV tegument proteins are indeed phosphorylated, though to a lesser extent than previously reported, and the difference in mobility on SDS-PAGE and calculated mass of pUL32 may not be attributed to phosphorylation but more likely due to the partially intrinsically disordered nature of pUL32.

Prevalence of clarithromycin-resistant Helicobacter pylori strains in gastric mucosa-associated lymphoid tissue lymphoma patients.

Gastric MALT lymphoma is closely associated with Helicobacter pylori infection. Bacterial eradication therapy comprising clarithromycin is the first-line treatment in gastric MALT lymphoma patients. However, antimicrobial resistance to clarithromycin has been increasing in Europe, and thus far, it has not been examined in gastric MALT lymphoma patients. Based upon histopathological investigation, 17 adult gastric MALT lymphoma patients were identified to be related with H. pylori infection between 1997 and 2014. Detection of H. pylori infection in these patients and clarithromycin susceptibility testing were performed by 23S rRNA gene real-time PCR. Twelve of the patients were confirmed with H. pylori infection by real-time PCR. Among these patients, only two were found to be infected with clarithromycin-resistant H. pylori strain. In one of them, both the clarithromycin-resistant and sensitive genotype were detected. The rate of clarithromycin resistance was 15.4 %. Clarithromycin resistance pattern in gastric MALT lymphoma patients is under the predictions since a previous study performed in Central Europe revealed a rate of 36.6 % in Austria. Considering the low antimicrobial resistance rate, clarithromycin is still an option in gastric MALT lymphoma management.

Acne inversa complicated by Actinomyces neuii.

INTRODUCTION: Acne inversa (AI) is a chronic and recurrent inflammatory skin disease. It occurs in intertriginous areas of the skin and causes pain, drainage, malodor and scar formation. While supposedly caused by an autoimmune reaction, bacterial superinfection is a secondary event in the disease process. METHODS: A unique case of a 43-year-old male patient suffering from a recurring AI lesion in the left axilla was retrospectively analysed. RESULTS: A swab revealed Actinomyces neuii as the only agent growing in the lesion. The patient was then treated with Amoxicillin/Clavulanic Acid 3 × 1 g until he was cleared for surgical excision. The intraoperative swab was negative for A. neuii. Antibiotics were prescribed for another 4 weeks and the patient has remained relapse free for more than 12 months now. CONCLUSION: Primary cutaneous Actinomycosis is a rare entity and the combination of AI and Actinomycosis has never been reported before. Failure to detect superinfections of AI lesions with slow-growing pathogens like Actinomyces spp. might contribute to high recurrence rates after immunosuppressive therapy of AI. The present case underlines the potentially multifactorial pathogenesis of the disease and the importance of considering and treating potential infections before initiating immunosuppressive regimens for AI patients.

Biomimetic Membranes for Multi-Redox Center Proteins.

His-tag technology was applied for biosensing purposes involving multi-redox center proteins (MRPs). An overview is presented on various surfaces ranging from flat to spherical and modified with linker molecules with nitrile-tri-acetic acid (NTA) terminal groups to bind his-tagged proteins in a strict orientation. The bound proteins are submitted to in situ dialysis in the presence of lipid micelles to form a so-called protein-tethered bilayer lipid membrane (ptBLM). MRPs, such as the cytochrome c oxidase (CcO) from R. sphaeroides and P. denitrificans, as well as photosynthetic reactions centers (RCs) from R. sphaeroides, were thus investigated. Electrochemical and surface-sensitive optical techniques, such as surface plasmon resonance, surface plasmon-enhanced fluorescence, surface-enhanced infrared absorption spectroscopy (SEIRAS) and surface-enhanced resonance Raman spectroscopy (SERRS), were employed in the case of the ptBLM structure on flat surfaces. Spherical particles ranging from µm size agarose gel beads to nm size nanoparticles modified in a similar fashion were called proteo-lipobeads (PLBs). The particles were investigated by laser-scanning confocal fluorescence microscopy (LSM) and UV/Vis spectroscopy. Electron and proton transfer through the proteins were demonstrated to take place, which was strongly affected by the membrane potential. MRPs can thus be used for biosensing purposes under quasi-physiological conditions.

Electronic Olfactory Sensor Based on A.†mellifera Odorant-Binding Protein†14 on a Reduced Graphene Oxide Field-Effect Transistor.

An olfactory biosensor based on a reduced graphene oxide (rGO) field-effect transistor (FET), functionalized by the odorant-binding protein 14 (OBP14) from the honey bee (Apis mellifera) has been designed for the in situ and real-time monitoring of a broad spectrum of odorants in aqueous solutions known to be attractants for bees. The electrical measurements of the binding of all tested odorants are shown to follow the Langmuir model for ligand-receptor interactions. The results demonstrate that OBP14 is able to bind odorants even after immobilization on rGO and can discriminate between ligands binding within a range of dissociation constants from K(d)=4 µM to K(d)=3.3 mM. The strongest ligands, such as homovanillic acid, eugenol, and methyl vanillate all contain a hydroxy group which is apparently important for the strong interaction with the protein.

A physicians' wish list for the clinical application of intestinal metagenomics.

Christoph Steininger and colleagues explore how multiple infectious, autoimmune, metabolic, and neoplastic diseases have been associated with changes in the intestinal microbiome, although a cause-effect relationship is often difficult to establish. Integration of metagenomics into clinical medicine is a challenge, and the authors highlight clinical approaches that are of high priority for the useful medical application of metagenomics. Please see later in the article for the Editors' Summary.

Recombinant antibodies encoded by IGHV1-69 react with pUL32, a phosphoprotein of cytomegalovirus and B-cell superantigen.

Leukemia cells from patients with chronic lymphocytic leukemia (CLL) express a highly restricted immunoglobulin heavy variable chain (IGHV) repertoire, suggesting that a limited set of antigens reacts with leukemic cells. Here, we evaluated the reactivity of a panel of different CLL recombinant antibodies (rAbs) encoded by the most commonly expressed IGHV genes with a panel of selected viral and bacterial pathogens. Six different CLL rAbs encoded by IGHV1-69 or IGHV3-21, but not a CLL rAb encoded by IGHV4-39 genes, reacted with a single protein of human cytomegalovirus (CMV). The CMV protein was identified as the large structural phosphoprotein pUL32. In contrast, none of the CLL rAbs bound to any other structure of CMV, adenovirus serotype 2, Salmonella enterica serovar Typhimurium, or of cells used for propagation of these microorganisms. Monoclonal antibodies or humanized rAbs of irrelevant specificity to pUL32 did not react with any of the proteins present in the different lysates. Still, rAbs encoded by a germ line IGHV1-69 51p1 allele from CMV-seropositive and -negative adults also reacted with pUL32. The observed reactivity of multiple different CLL rAbs and natural antibodies from CMV-seronegative adults with pUL32 is consistent with the properties of a superantigen.

The Distinct Regulation of the Vitamin D and Aryl Hydrocarbon Receptors in COVID-19.

(1) Background: SARS-CoV-2 affects several immune pathways, including the vitamin D (VDR) and the aryl hydrocarbon receptor pathways (AhR). The aim of the study was the evaluation of the VDR and AhR pathways in the blood of COVID-19 patients with regard to the severity of disease. (2) Methods: Observational, single-center, case-control design. A total of 240 samples were selected for exploration. Patients who tested negative for SARS-CoV-2 but suffered from other respiratory infections (ORIs) served as a control group. (3) Results: VDR-specific mRNA in the blood of patients with mild symptoms (131.2 ± 198.6) was significantly upregulated relative to the VDR expression of the ORI group (23.24 ± 42.60; p < 0.0001); however, VDR expression of critically ill patients showed an impaired upregulation (54.73 ± 68.34; p < 0.001). CYP27B1 expression was not significantly regulated during SARS-CoV-2 infection. There was a downregulation of VDR and CYP27B1 compared to survivors. There was no significant difference in 25(OH)-vitamin D3 levels between critically ill patients with regard to survival (24.3 ± 9.4 vs. 27.1 ± 11.3; p = 0.433). (4) Conclusion: The VDR and AhR pathways are distinctively regulated in patients suffering from COVID-19 depending on the severity of disease. A combination treatment of antiviral drugs and vitamin D substitution should be evaluated for potentially improved prognosis in COVID-19.

Unveiling Inter- and Intra-Patient Sequence Variability with a Multi-Sample Coronavirus Target Enrichment Approach.

Amid the global challenges posed by the COVID-19 pandemic, unraveling the genomic intricacies of SARS-CoV-2 became crucial. This study explores viral evolution using an innovative high-throughput next-generation sequencing (NGS) approach. By taking advantage of nasal swab and mouthwash samples from patients who tested positive for COVID-19 across different geographical regions during sequential infection waves, our study applied a targeted enrichment protocol and pooling strategy to increase detection sensitivity. The approach was extremely efficient, yielding a large number of reads and mutations distributed across 10 distinct viral gene regions. Notably, the genes Envelope, Nucleocapsid, and Open Reading Frame 8 had the highest number of unique mutations per 1000 nucleotides, with both spike and Nucleocapsid genes showing evidence for positive selection. Focusing on the spike protein gene, crucial in virus replication and immunogenicity, our findings show a dynamic SARS-CoV-2 evolution, emphasizing the virus-host interplay. Moreover, the pooling strategy facilitated subtle sequence variability detection. Our findings painted a dynamic portrait of SARS-CoV-2 evolution, emphasizing the intricate interplay between the virus and its host populations and accentuating the importance of continuous genomic surveillance to understand viral dynamics. As SARS-CoV-2 continues to evolve, this approach proves to be a powerful, versatile, fast, and cost-efficient screening tool for unraveling emerging variants, fostering understanding of the virus's genetic landscape.