HIV-1 capsids enter the FG phase of nuclear pores like a transport receptor.

HIV-1 infection requires nuclear entry of the viral genome. Previous evidence suggests that this entry proceeds through nuclear pore complexes (NPCs), with the 120 × 60 nm capsid squeezing through an approximately 60-nm-wide central channel1 and crossing the permeability barrier of the NPC. This barrier can be described as an FG phase2 that is assembled from cohesively interacting phenylalanine-glycine (FG) repeats3 and is selectively permeable to cargo captured by nuclear transport receptors (NTRs). Here we show that HIV-1 capsid assemblies can target NPCs efficiently in an NTR-independent manner and bind directly to several types of FG repeats, including barrier-forming cohesive repeats. Like NTRs, the capsid readily partitions into an in vitro assembled cohesive FG phase that can serve as an NPC mimic and excludes much smaller inert probes such as mCherry. Indeed, entry of the capsid protein into such an FG phase is greatly enhanced by capsid assembly, which also allows the encapsulated clients to enter. Thus, our data indicate that the HIV-1 capsid behaves like an NTR, with its interior serving as a cargo container. Because capsid-coating with trans-acting NTRs would increase the diameter by 10 nm or more, we suggest that such a 'self-translocating' capsid undermines the size restrictions imposed by the NPC scaffold, thereby bypassing an otherwise effective barrier to viral infection.

Antibiotic resistances from slaughterhouse effluents and enhanced antimicrobial blue light technology for wastewater decontamionation.

The frequencies of 6 different facultative pathogenic bacteria of the ESKAPE group (priority list WHO) and a total of 14 antibiotic resistance genes (ARGs) with different priorities for human medicine were quantified in wastewaters of poultry and pig slaughterhouses using molecular biological approaches. Raw sewage from poultry and pig slaughterhouses was found to be contaminated not only with facultative pathogenic bacteria but also with various categories of clinically relevant ARGs, including ARGs against the reserve antibiotics group. The concentration of the different gene targets decreased after on-site conventional biological or advanced oxidative wastewater treatments, but was not eliminated. Hence, the antimicrobial BlueLight (aBL) in combination with a porphyrin photo-sensitizer was studied with ESKAPE bacteria and real slaughterhouse wastewaters. The applied broad LED-based blue light (420-480 nm) resulted in groups of sensitive, intermediate, and non-sensitive ESKAPE bacteria. The killing effect of aBL was increased in the non-sensitive bacteria Klebsiella pneumoniae and Enterococcus faecium due to the addition of porphyrins in concentrations of 10-6 M. Diluted slaughterhouse raw wastewater was treated with broad spectrum aBL and in combination with porphyrin. Here, the presence of the photo-sensitizer enhanced the aBL biocidal impact.

Free-floating extracellular DNA (exDNA) in different wastewaters: Status quo on exDNA-associated antimicrobial resistance genes.

Wastewater treatment plants (WWTPs) have been reported as major anthropogenic reservoirs for the spread of antibiotic-resistant bacteria (ARB) and antibiotic resistance genes (ARGs) into the environment, worldwide. While most studies mainly focus on the intracellular DNA (iDNA), extracellular DNA (exDNA) accounting for a significant proportion of the total DNA in wastewater, was usually neglected. Following the One Health approach, this study focuses on wastewaters of municipal, clinical, and livestock origins (n = 45) that undergo different treatment processes (i.e., conventional activated sludge, ultrafiltration, and ozonation). Water samples were analysed for 12 ARGs as indicators of the different compartments associated with iDNA and exDNA by quantitative real-time PCR (qPCR). Taxonomic profiling of exDNA-fractions, obtained using nucleic acid adsorption particles, was conducted by sequencing the V3-V4 hypervariable regions of the 16S rRNA gene. Notified exDNA concentrations varied between on-site WWTPs and treatment stages, and ranged from 314.0 ± 70.2 ng/mL in untreated livestock wastewater down to 0.7 ± 0.1 ng/mL in effluents after ultrafiltration. In general, influents exhibited higher concentrations compared to effluents, while wastewater treated by advanced treatment processes (i.e., ultrafiltration and ozonation) showed the lowest exDNA concentrations. Despite the lower concentrations, free-floating exDNA accounted for up to 80.0 ± 5.8% of the total DNA in effluents. Target ARGs were more common in the iDNA (100%, n = 45/45), compared to the exDNA-fractions (51.1%, n = 23/45), whereas exDNA-ARGs were mostly detected in clinical and slaughterhouse wastewaters as well as in the municipal influents. Compared to the iDNA-ARGs, the concentrations of exDNA-ARGs were in general lower. Nevertheless, significant higher concentrations for exDNA-associated genes were measured in clinical wastewaters for blaNDM (4.07 ± 0.15 log gene copies (GC)/L) and blaVIM-2 (6.0 ± 0.2 log GC/L). Overall, our results suggest that depending on the origin of wastewater and its treatment methods, exDNA represents an important reservoir for ARGs, particularly in clinical wastewater.

Case report: First case of Borrelia miyamotoi meningitis in an immunocompromised patient in Norway.

Background: Tick-borne disease caused by B. miyamotoi (BMD) usually manifest as a febrile illness in humans. Complications include relapsing fever and in rare occasions involvement of the central nervous system. Only a few cases of meningoencephalitis have been described, mostly in immunosuppressed patients. Case presentation: A 70-year-old female receiving immunosuppressive rituximab therapy presented with frontal headache, dizziness, nausea, vomiting and chills. Clinical laboratory blood analyses were normal. Cerebrospinal fluid (CSF) was translucent and analysis showed increased leucocyte count (187 106/L) and elevated level of protein (1056 mg/L). Empiric antibiotic treatment was initiated. The patient showed an early symptomatic relief and 24 h after admission she was discharged from the hospital and antibiotic treatment was discontinued. Two weeks after hospitalisation the B. miyamotoi specific PCR turned out positive in both CSF and serum. At the time, the patient was recovered with mild residual headache. She was treated with high dose doxycycline and her subtle symptoms disappeared. Conclusions: To our knowledge, we present the first patient with BMD-associated meningitis in Norway, one of eight cases reported worldwide. The patient had mild symptoms and received an early diagnosis. A more severe progression or relapse of disease may have been prevented by antibiotic treatment. BMD should be considered as causes of aseptic meningitis, especially in immunosuppressed patients living in endemic areas.

FHODs: Nuclear tethered formins for nuclear mechanotransduction.

In this review, we discuss FHOD formins with a focus on recent studies that reveal a new role for them as critical links for nuclear mechanotransduction. The FHOD family in vertebrates comprises two structurally related proteins, FHOD1 and FHOD3. Their similar biochemical properties suggest overlapping and redundant functions. FHOD1 is widely expressed, FHOD3 less so, with highest expression in skeletal (FHOD1) and cardiac (FHOD3) muscle where specific splice isoforms are expressed. Unlike other formins, FHODs have strong F-actin bundling activity and relatively weak actin polymerization activity. These activities are regulated by phosphorylation by ROCK and Src kinases; bundling is additionally regulated by ERK1/2 kinases. FHODs are unique among formins in their association with the nuclear envelope through direct, high affinity binding to the outer nuclear membrane proteins nesprin-1G and nesprin-2G. Recent crystallographic structures reveal an interaction between a conserved motif in one of the spectrin repeats (SRs) of nesprin-1G/2G and a site adjacent to the regulatory domain in the amino terminus of FHODs. Nesprins are components of the LINC (linker of nucleoskeleton and cytoskeleton) complex that spans both nuclear membranes and mediates bidirectional transmission of mechanical forces between the nucleus and the cytoskeleton. FHODs interact near the actin-binding calponin homology (CH) domains of nesprin-1G/2G enabling a branched connection to actin filaments that presumably strengthens the interaction. At the cellular level, the tethering of FHODs to the outer nuclear membrane mechanically couples perinuclear actin arrays to the nucleus to move and position it in fibroblasts, cardiomyocytes, and potentially other cells. FHODs also function in adhesion maturation during cell migration and in the generation of sarcomeres, activities distant from the nucleus but that are still influenced by it. Human genetic studies have identified multiple FHOD3 variants linked to dilated and hypertrophic cardiomyopathies, with many mutations mapping to "hot spots" in FHOD3 domains. We discuss how FHOD1/3's role in reinforcing the LINC complex and connecting to perinuclear actin contributes to functions of mechanically active tissues such as striated muscle.

Cardiac dysfunction in mixed connective tissue disease: a nationwide observational study.

We aimed to identify cardiac function in patients with established mixed connective tissue disease (MCTD). This was a cross-sectional case-control study of well-characterised MCTD patients who had previously been included in a nationwide cohort. Assessments comprised protocol transthoracic echocardiography, electrocardiogram and blood samples. In patients only, we evaluated the findings of high-resolution pulmonary computed tomography and disease activity. We assessed 77 MCTD patients (mean age 50.5 ± 12.3 years) with a mean disease duration of 16.4 years, and 59 age- and sex-matched healthy controls (49.9 ± 11.7 years). By echocardiography, measures of left ventricular function, i.e. fractional shortening (38.1 ± 6.4% vs. 42.3 ± 6.6%, p < 0.001), mitral annulus plane systolic excursion (MAPSE) (13.7 ± 2.1 mm vs. 15.3 ± 2.3 mm, p < 0.001) and early diastolic velocity of the mitral annulus (e') (0.09 ± 0.02 m/s vs. 0.11 ± 0.03 m/s, p = 0.002) were subclinical and lower in patients than controls. Right ventricular dysfunction was found in patients assessed by tricuspid annular plane systolic excursion (TAPSE) (22.7 ± 4.0 mm vs. 25.5 ± 4.0 mm, p < 0.001). While cardiac dysfunction was not associated with pulmonary disease, e' and TAPSE were found to correlate with disease activity at baseline. In this cohort of MCTD patients, echocardiographic examinations demonstrated a higher frequency of cardiac dysfunction than in matched controls. Cardiac dysfunction was associated with disease activity at baseline, but was independent of cardiovascular risk factors and pulmonary disease. Our study indicates that cardiac dysfunction is part of the multi-organ affliction seen in MCTD.

Adipokine profile in long-term juvenile dermatomyositis, and associations with adipose tissue distribution and cardiac function: a cross-sectional study.

OBJECTIVES: In long-term juvenile dermatomyositis (JDM), altered adipose tissue distribution and subclinical cardiac dysfunction have been described. Our aims were to compare adipokine levels in patients with JDM after long-term disease with controls, and explore associations between adipokines and (1) adipose tissue distribution and (2) cardiac function. METHODS: The study cohort included 59 patients with JDM (60% female, mean age 25.2 years, mean disease duration 16.9 years), and 59 age/sex-matched controls. Updated Pediatric Rheumatology International Trials Organization criteria for clinically inactive JDM were used to stratify patients into active (JDM-active) or inactive (JDM-inactive) disease groups. Lipodystrophy was clinically assessed in all patients. In all study participants, we measured adipose tissue distribution by dual-energy X-ray absorptiometry and cardiac function by echocardiography. Serum adipokines (adiponectin, apelin-12, lipocalin-2, leptin, visfatin and resistin) were analysed using ELISA. RESULTS: Patients with JDM had higher leptin levels compared with controls (p≤0.01). In JDM-active, apelin-12 and visfatin were higher compared with JDM-inactive (p≤0.05). In JDM-total and JDM-active, lower adiponectin correlated with lipodystrophy and total fat mass. Also, systolic dysfunction correlated with: lower adiponectin in JDM-total, JDM-inactive and JDM-active, and with lower apelin-12 in JDM-total and JDM-active and resistin in JDM-active (all p≤0.05). Lower adiponectin correlated with diastolic dysfunction in JDM-total and JDM-active. CONCLUSION: After long-term disease, leptin levels were unfavourably regulated in patients with JDM compared with controls, and apelin-12 and visfatin in JDM-active versus JDM-inactive. We found associations between adipokines and both adipose tissue distribution and cardiac systolic function in all patients with JDM, which was most prominent in patients with active disease.

Drug-induced aseptic meningitis. / Legemiddelindusert aseptisk meningitt.

Drug-induced aseptic meningitis is a rare but serious condition that should be suspected in patients with meningitis who test negative for a microbiological agent. The medical history is presented here of a woman with recurrent urinary tract infections where meningitis symptoms arose after repeated exposure to a frequently prescribed drug.

Adipose tissue distribution is associated with cardio-metabolic alterations in adult patients with juvenile-onset dermatomyositis.

OBJECTIVES: Primary aims were to compare adipose tissue distribution in adult patients with juvenile-onset DM (JDM), with matched controls. Secondary aims were to explore how adipose tissue distribution is associated with cardio-metabolic status (cardiac dysfunction and metabolic syndrome) in patients. METHODS: Thirty-nine JDM patients (all aged ≥18 y, mean age 31.7 y and 51% female) were examined mean 22.7 y (s.d. 8.9 y) after disease onset and compared with 39 age/sex-matched controls. In patients, disease activity and lipodystrophy were assessed by validated tools and use of prednisolone noted. In all participants, dual-energy X-ray absorptiometry (DXA) and echocardiography were used to measure visceral adipose tissue (VAT)(g) and cardiac function, respectively. Risk factors for metabolic syndrome were measured and associations with adipose tissue distribution explored. For primary and secondary aims, respectively, P-values ≤0.05 and ≤0.01 were considered significant. RESULTS: Patients exhibited a 2.4-fold increase in VAT, and reduced HDL-cholesterol values compared with controls (P-values ≤ 0.05). Metabolic syndrome was found in 25.7% of the patients and none of the controls. Cardiac dysfunction (systolic and/or diastolic) was found in 23.7% of patients and 8.1% of controls (P = 0.07). In patients, VAT levels were correlated with age, disease duration and occurrence of metabolic syndrome and cardiac dysfunction. Occurrence of lipodystrophy (P = 0.02) and male sex (P = 0.04) tended to be independently associated with cardiac dysfunction. CONCLUSION: Adults with JDM showed more central adiposity and cardio-metabolic alterations than controls. Further, VAT was found increased with disease duration, which was associated with development of cardio-metabolic syndrome.

Antibacterial Inorganic Coating of Calcium Silicate Hydrate Substrates by Copper Incorporation.

Under environmental conditions, biofilms can oftentimes be found on different surfaces, accompanied by the structural degradation of the substrate. Since high-copper-content paints were banned in the EU, a solution for the protection of these surfaces has to be found. In addition to hydrophobation, making the surfaces inherently biofilm-repellent is a valid strategy. We want to accomplish this via the metal exchange in calcium silicate hydrate (CSH) substrates with transition metals. As has been shown with Europium, even small amounts of metal can have a great influence on the material properties. To effectively model CSH surfaces, ultrathin CSH films were grown on silicon wafers using Ca(OH)2 solutions. Subsequently, copper was incorporated as an active component via ion exchange. Biofilm development is quantified using a multiple-resistant Pseudomonas aeruginosa strain described as a strong biofilm former cultivated in the culture medium for 24 h. Comprehensive structural and chemical analyses of the substrates are done by environmental scanning electron microscopy (ESEM), transmission Fourier transform infrared spectroscopy (FT-IR), X-ray photoelectron spectroscopy (XPS), and time-of-flight secondary ion mass spectrometry (ToF-SIMS). Results do not show any structural deformation of the substrates by the incorporation of the Cu combined with three-dimensional (3D) homogeneous distribution. While the copper-free CSH phase shows a completely random distribution of the bacteria in biofilms, the samples with copper incorporation reveal lower bacterial colonization of the modified surfaces with an enhanced cluster formation.

Structure and specificity of an anti-chloramphenicol single domain antibody for detection of amphenicol residues.

Antibiotics in aquaculture prevent bacterial infection of fish, but their misuse is a public health risk and contributes to the unintentional creation of multiresistant pathogens. Regulatory agencies cannot do the rigorous, expensive testing required to keep up with the volume of seafood shipments. Current rapid test kits for these drugs enable the increase in testing needed for adequate monitoring of food supply chains, but they lack a high degree of accuracy. To combat this, we set out to discover and engineer single-domain antibodies (VHHs) that bind to small molecule antibiotics, and that can be used in rapid test kits. The small size, solubility, and stability of VHHs are useful properties that can improve the reliability and shelf-life of test kits for these adulterants. Here, we report a novel anti-chloramphenicol VHH (Chl-VHH) with a disassociation constant of 57 nM. This was achieved by immunizing a llama against a chloramphenicol-keyhole limpet hemocyanin (KLH) conjugate and screening for high affinity binders through phage display. The crystal structure of the bound-VHH to chloramphenicol was key to identifying a mutation in the binding pocket that resulted in a 16-fold improvement in binding affinity. In addition, the structure provides new insights into VHH-hapten interactions that can guide future engineering of VHHs against additional targets.

Facultative pathogenic bacteria and antibiotic resistance genes in swine livestock manure and clinical wastewater: A molecular biology comparison.

Manure contains vast amounts of biological contaminants of veterinary origin. Only few studies analyse clinically critical resistance genes against reserve antibiotics in manure. In general, resistances against these high priority antibiotics involve a high potential health risk. Therefore, their spread in the soil as well as the aquatic environment has to be prevented. Manures of 29 different swine livestock were analysed. Abundances of facultative pathogenic bacteria including representatives of the clinically critical ESKAPE-pathogens (P. aeruginosa, K. pneumoniae, A. baumannii, E. faecium) and E. coli were investigated via qPCR. Antibiotic resistance genes against commonly used veterinary antibiotics (ermB, tetM, sul1) as well as various resistance genes against important (mecA, vanA) and reserve antibiotics (blaNDM, blaKPC3, mcr-1), which are identified by the WHO, were also obtained by qPCR analysis. The manures of all swine livestock contained facultative pathogenic bacteria and commonly known resistance genes against antibiotics used in veterinary therapies, but more important also a significant amount of clinically critical resistance genes against reserve antibiotics for human medicine. To illustrate the impact the occurrence of these clinically critical resistance genes, comparative measurements were taken of the total wastewater of a large tertiary care hospital (n = 8). Both manure as well as raw hospital wastewaters were contaminated with significant abundances of gene markers for facultative pathogens and with critical resistance genes of reserve antibiotics associated with genetic mobile elements for horizontal gene transfer. Hence, both compartments bear an exceptional potential risk for the dissemination of facultative pathogens and critical antibiotic resistance genes.

Membrane bioreactor followed by solar photo-Fenton oxidation: Bacterial community structure changes and bacterial reduction.

The removal of antibiotic resistance genes (ARGs) and taxon-specific markers, the bacterial community structure changes and the permanent inactivation of total bacteria including their antibiotic-resistant counterparts (ARB) in actual wastewater during a Membrane BioReactor (MBR) application followed by solar photo-Fenton oxidation at bench- and then pilot-scale under solar irradiation, were investigated. The presence of enterococci- and pseudomonad-specific taxon markers and of sul1 and ampC ARGs in the MBR effluent was confirmed, indicating the challenge of such processes, for the removal of biological molecules. On the other hand, >99 % reduction of all types of cultivable bacteria examined was observed after MBR treatment, with a 5-log reduction of E. coli and 6-log reduction of P. aeruginosa and Klebsiella spp. There was a shift in the bacterial community structure in the MBR effluent after the bench- and pilot-scale solar photo-Fenton oxidation. Notably, thermotolerant bacterial genera like Ignavibacterium and Thermomonas were prevalent during the pilot-scale process operated at a high ambient temperature, while the most prevalent genera were Mycobacterium, Nocardioides and Mesorhizobium, which are primarily not pathogenic and plant-related. In agreement, a different bacterial community structure according to the G-C content after DGGE analysis was noted between the MBR and solar photo-Fenton oxidation-treated effluents, but interestingly also between the bench- and pilot-scale oxidation-treated effluents. There was complete absence of ARGs after the bench-scale solar photo-Fenton oxidation application but not after the pilot-scale treatment (1.56 and 1.53 log10 CE 100 ng-1 DNA, of sul and ermB, respectively). Taxon-specific markers were found in both oxidation setups. Inactivation of cultivable Escherichia coli, Pseudomonas aeruginosa and Klebsiella spp. (including ARB) was achieved during both oxidation setups, with no further re-activation observed.

Droplet Microarray as a Powerful Platform for Seeking New Antibiotics Against Multidrug-Resistant Bacteria.

Multidrug-resistant (MDR) bacteria is a severe threat to public health. Therefore, it is urgent to establish effective screening systems for identifying novel antibacterial compounds. In this study, a highly miniaturized droplet microarray (DMA) based high-throughput screening system is established to screen over 2000 compounds for their antimicrobial properties against carbapenem-resistant Klebsiella pneumoniae and methicillin resistant Staphylococcus aureus (MRSA). The DMA consists of an array of hydrophilic spots divided by superhydrophobic borders. Due to the differences in the surface wettability between the spots and the borders, arrays of hundreds of nanoliter-sized droplets containing bacteria and different drugs can be generated for screening applications. A simple colorimetric viability readout utilizing a conventional photo scanner is developed for fast single-step detection of the inhibitory effect of the compounds on bacterial growth on the whole array. Six hit compounds, including coumarins and structurally simplified estrogen analogs are identified in the primary screening and validated with minimum inhibition concentration assay for their antibacterial effect. This study demonstrates that the DMA-based high-throughput screening system enables the identification of potential antibiotics from novel synthetic compound libraries, offering opportunities for development of new treatments against multidrug-resistant bacteria.

The effect of ultrafiltration process on the fate of antibiotic-related microcontaminants, pathogenic microbes, and toxicity in urban wastewater.

Ultrafiltration (UF) was assessed at chemical, microbiological, genetical and toxicological level and in terms of removing specific antibiotic-related microcontaminants from urban wastewater. The UF capacity to remove various antibiotics (clarithromycin, erythromycin, ampicillin, ofloxacin, sulfamethoxazole, trimethoprim, and tetracycline; [A0] = 100 µg L-1) was optimised with respect to the feed recirculation rate (25-50%) and feed/transmembrane pressure (1.5-3/1.5-2.4 bar, respectively). Here, we tested the UF capacity to reduce the cultivable bacteria (faecal coliforms, total heterotrophs, Enterococci, Pseudomonas aeruginosa), enteric opportunistic pathogens, including antibiotic-resistant bacteria (ARB) and antibiotic-resistance genes (ARGs) load. Moreover, the toxicity towards Daphnia magna and three plant species was investigated. Upon optimisation of UF, the removal of antibiotics ranged from 19% for trimethoprim to 95% for clarithromycin. The concentration of cultivable faecal coliforms in the permeate was significantly reduced compared to the feed (P < 0.001), whereas all the bacterial species decreased by more than 3 logs. A similar pattern of reduction was observed for the ARGs (P < 0.001) and enteric opportunistic pathogens (~3-4 logs reduction). A nearly complete removal of the antibiotics was obtained by UF followed by granular activated carbon adsorption (contact time: 90 min), demonstrating the positive contribution of such combination to the abatement of chemical microcontaminants.

Solving the nuclear pore puzzle.

Using a battery of tools, the architecture of the nuclear pore complex is revealed.

Effect of chemical and biological preservatives and ensiling stage on the dry matter loss, nutritional value, microbial counts, and ruminal in vitro gas production kinetics of wet brewer's grain silage.

This study evaluated the effects of chemical and biological preservatives and ensiling stage on spoilage, ruminal in vitro fermentation, and methane production of wet brewer's grain (WBG) silage. Treatments (TRT) were sodium lignosulfonate at 10 g/kg fresh WBG (NaL1) and 20 g/kg (NaL2), propionic acid at 5 g/kg fresh WBG (PRP, 99%), a combination inoculant (INO; Lactococcus lactis and Lactobacillus buchneri each at 4.9 log cfu per fresh WBG g), and untreated WBG (CON). Fresh WBG was treated and then ensiled for 60 d, after which mini silos were opened and aerobically exposed (AES) for 10 d. Data were analyzed as an RCBD (five blocks) with a 5 TRT × 3 stages (STG; fresh, ensiled, and AES) factorial arrangement. Results showed that ensiled PRP-treated WBG markedly preserved more water-soluble carbohydrates and starch than all other ensiled TRT (P < 0.001). Dry matter losses of ensiled PRP-treated WBG were 48% lower than all other ensiled TRT (P = 0.009) but were not different than CON in AES (P = 0.350). Due to its greater concentration of digestible nutrients, PRP-treated AES was less aerobically stable than CON (P = 0.03). Preservation was not improved by INO, NaL1, or NaL2 but the latter prevented the increase of neutral detergent fiber across STG (P = 0.392). Apparent in vitro DM digestibility (IVDMD) decreased only in ensiled CON, INO, and NaL1 relative to fresh WBG and AES NaL2 had greater IVDMD than all other AES TRT (P ≤ 0.032). In vitro ruminal fermentation of fresh WBG resulted in a greater methane concentration and yield than the other STG (P < 0.033). In conclusion, PRP was the most effective at preserving WBG during ensiling but failed to improve aerobic stability under the conditions tested.

Wet brewer's grain (WBG) is the most abundant byproduct in the manufacture of beer and its rich nutritional composition makes it a valuable feed for cattle. However, WBG is highly susceptible to spoilage so the application of cost-effective preservatives may be a viable approach to prevent nutrient losses during ensiling and feed out. The present study evaluated the effects of chemical and biological preservatives on the nutritional composition and in vitro fermentation and gas production of WBG across three silage production stages: fresh, ensiled, and aerobically exposed silage (AES). Preservatives tested were propionic acid, a bacterial inoculant, and sodium lignosulfonate (NaL) applied at 1% and 2%. Propionic acid successfully reduced the loss of nutrients and preserved more sugars and starch than all other treatments during ensiling, which resulted in higher digestibility in vitro. However, due to its greater concentration of digestible nutrients, ensiled WBG treated with propionic acid also suffered extensive spoilage in the AES. All other treatments failed to improve the preservation of ensiled or AES WBG, but NaL at 2% prevented the decrease of digestibility for AES.

Nanobodies as allosteric modulators of Parkinson's disease-associated LRRK2.

Mutations in the gene coding for leucine-rich repeat kinase 2 (LRRK2) are a leading cause of the inherited form of Parkinson's disease (PD), while LRRK2 overactivation is also associated with the more common idiopathic form of PD. LRRK2 is a large multidomain protein, including a GTPase as well as a Ser/Thr protein kinase domain. Common, disease-causing mutations increase LRRK2 kinase activity, presenting LRRK2 as an attractive target for drug discovery. Currently, drug development has mainly focused on ATP-competitive kinase inhibitors. Here, we report the identification and characterization of a variety of nanobodies that bind to different LRRK2 domains and inhibit or activate LRRK2 in cells and in in vitro. Importantly, nanobodies were identified that inhibit LRRK2 kinase activity while binding to a site that is topographically distinct from the active site and thus act through an allosteric inhibitory mechanism that does not involve binding to the ATP pocket or even to the kinase domain. Moreover, while certain nanobodies completely inhibit the LRRK2 kinase activity, we also identified nanobodies that specifically inhibit the phosphorylation of Rab protein substrates. Finally, in contrast to current type I kinase inhibitors, the studied kinase-inhibitory nanobodies did not induce LRRK2 microtubule association. These comprehensively characterized nanobodies represent versatile tools to study the LRRK2 function and mechanism and can pave the way toward novel diagnostic and therapeutic strategies for PD.

Identification of critical control points for antibiotic resistance discharge in sewers.

Disrupting the spread of clinically relevant antibiotic resistance genes (ARGs) is one of the key components for the success of the One Health strategy. While waste water treatment plants (WWTPs) represent a final control point for daily discharges of antibiotic resistance genes (ARGs) to the aquatic environment, a decentralized upstream monitoring of wastewater feeds of selected urban drainage areas for blaCTX-M32, blaCTX-M15, blaOXA48, blaCMY-2, mecA, blaNDM-1, blaKPC3, vanA, and mcr-1 representing clinically relevant ARGs has been performed. Besides hospitals, also retirement homes were found to be responsible for high levels of ARG discharges compared to housing area sewer systems. The monitoring combines qPCR-based quantifications, flow volume-based analyses, and multiple antibiotic resistance analyses of isolates. As result of the study, local actions at identified critical control points could help to prevent contaminations of larger volumes of wastewaters. This strategy will support a more cost-effective treatment compared to central actions at WWTPs, only. A polluter-pays principle should be applied by this monitoring strategy.

Associations between cardiac and pulmonary involvement in patients with juvenile dermatomyositis-a cross-sectional study.

This study aimed at exploring the association between detectable cardiac and pulmonary involvement in long-term juvenile dermatomyositis (JDM) and to assess if patients with cardiac and pulmonary involvement differ with regard to clinical characteristics. 57 JDM patients were examined mean 17.3 (10.5) years after disease onset; this included clinical examination, myositis specific/associated autoantibodies (immunoblot), echocardiography, pulmonary function tests and high-resolution computed tomography. Cardiac involvement was defined as diastolic and/or systolic left ventricular dysfunction and pulmonary involvement as low diffusing capacity for carbon monoxide, low total lung capacity and/or high-resolution computed tomography abnormalities. Patients were stratified into the following four groups: (i) no organ involvement, (ii) pulmonary only, (iii) cardiac only, and (iv) co-existing pulmonary and cardiac involvement. Mean age was 25.7 (12.4) years and 37% were males. One patient had coronary artery disease, seven had a history of pericarditis, seven had hypertension and three had known interstitial lung disease prior to follow-up. There was no association between cardiac (10/57;18%) and pulmonary (41/57;72%) involvement (p = 0.83). After stratifying by organ involvement, 21% of patients had no organ involvement; 61% had pulmonary involvement only; 7% had cardiac involvement only and 11% had co-existing pulmonary or cardiac involvement. Patients with co-existing pulmonary or cardiac involvement had higher disease burden than the remaining patients. Patients with either cardiac or pulmonary involvement only, differed in clinical and autoantibody characteristics. We found no increased risk of developing concomitant cardiac/pulmonary involvement in JDM. Our results shed light upon possible different underlying mechanisms behind pulmonary and cardiac involvement in JDM.