Sampling fish gut microbiota - A genome-resolved metagenomic approach.

Despite a surge in microbiota-focused studies in teleosts, few have reported functional data on whole metagenomes as it has proven difficult to extract high biomass microbial DNA from fish intestinal samples. The zebrafish is a promising model organism in functional microbiota research, yet studies on the functional landscape of the zebrafish gut microbiota through shotgun based metagenomics remain scarce. Thus, a consensus on an appropriate sampling method accurately representing the zebrafish gut microbiota, or any fish species is lacking. Addressing this, we systematically tested four methods of sampling the zebrafish gut microbiota: collection of faeces from the tank, the whole gut, intestinal content, and the application of ventral pressure to facilitate extrusion of gut material. Additionally, we included water samples as an environmental control to address the potential influence of the environmental microbiota on each sample type. To compare these sampling methods, we employed a combination of genome-resolved metagenomics and 16S metabarcoding techniques. We observed differences among sample types on all levels including sampling, bioinformatic processing, metagenome co-assemblies, generation of metagenome-assembled genomes (MAGs), functional potential, MAG coverage, and population level microdiversity. Comparison to the environmental control highlighted the potential impact of the environmental contamination on data interpretation. While all sample types tested are informative about the zebrafish gut microbiota, the results show that optimal sample type for studying fish microbiomes depends on the specific objectives of the study, and here we provide a guide on what factors to consider for designing functional metagenome-based studies on teleost microbiomes.

Telomere dynamics as mediators of gut microbiota-host interactions.

The highly proliferative gut tissue exhibits rapid telomere shortening with systemic effects on the host organism. Recent studies have demonstrated a bidirectionality in interactions between intestinal telomere length dynamics and the composition and activity of the gut microbiome thus linking processes of inflammation, dysbiosis and aging across different vertebrate species.

Genomic context determines the effect of DNA methylation on gene expression in the gut epithelium of Atlantic salmon (Salmo salar).

The canonical view of DNA methylation, a pivotal epigenetic regulation mechanism in eukaryotes, dictates its role as a suppressor of gene activity, particularly within promoter regions. However, this view is being challenged as it is becoming increasingly evident that the connection between DNA methylation and gene expression varies depending on the genomic location and is therefore more complex than initially thought. We examined DNA methylation levels in the gut epithelium of Atlantic salmon (Salmo salar) using whole-genome bisulfite sequencing, which we correlated with gene expression data from RNA sequencing of the same gut tissue sample (RNA-seq). Assuming epigenetic signals might be pronounced between distinctive phenotypes, we compared large and small fish, finding 22 significant associations between 22 differentially methylated regions and 21 genes. We did not detect significant methylation differences between large and small fish. However, we observed a consistent signal of methylation levels around the transcription start sites (TSS), being negatively correlated with the expression levels of those genes. We found both negative and positive associations of methylation levels with gene expression further upstream or downstream of the TSS, revealing a more unpredictable pattern. The 21 genes showing significant methylation-expression correlations were involved in biological processes related to salmon health, such as growth and immune responses. Deciphering how DNA methylation affects the expression of such genes holds great potential for future applications. For instance, our results suggest the importance of genomic context in targeting epigenetic modifications to improve the welfare of aquaculture species like Atlantic salmon.

Unraveling host regulation of gut microbiota through the epigenome-microbiome axis.

Recent studies of dynamic interactions between epigenetic modifications of a host organism and the composition or activity of its associated gut microbiota suggest an opportunity for the host to shape its microbiome through epigenetic alterations that lead to changes in gene expression and noncoding RNA activity. We use insights from microbiota-induced epigenetic changes to review the potential of the host to epigenetically regulate its gut microbiome, from which a bidirectional 'epigenome-microbiome axis' emerges. This axis embeds environmentally induced variation, which may influence the adaptive evolution of host-microbe interactions. We furthermore present our perspective on how the epigenome-microbiome axis can be understood and investigated within a holo-omic framework with potential applications in the applied health and food sciences.

Musculoskeletal misdiagnoses in pediatric patients with spinal tumors.

OBJECTIVE: Childhood spinal tumors often present with musculoskeletal symptoms, potentially causing a misdiagnosis and delays in diagnosis and treatment. This study aims to identify, characterize, and compare children with spinal tumors who had prior musculoskeletal misdiagnoses to those without, analyzing clinical presentation, diagnostic interval, and outcome. STUDY DESIGN: This retrospective cohort study evaluated all children aged 0-14 years diagnosed with a spinal tumor in Denmark from 1996 to 2018. The cohort was identified through the Danish Childhood Cancer Registry, and the registry data were supplemented with data from medical records. The survival was compared using the Kaplan-Meier method. RESULTS: Among 58 patients, 57% (33/58) received musculoskeletal misdiagnoses before the spinal tumor diagnosis. Misdiagnoses were mostly nonspecific (64%, 21/33), involving pain and accidental lesions, while 36% (12/33) were rheumatologic diagnoses. The patients with prior misdiagnosis had less aggressive tumors, fewer neurological/general symptoms, and 5.5 months median diagnostic interval versus 3 months for those without a misdiagnosis. Those with prior misdiagnoses tended to have a higher 5-year survival of 83% (95% confidence interval [CI]: 63%-92%) compared to 66% (95% CI: 44%-82%) for those without (p = .15). CONCLUSION: Less aggressive spinal tumors may manifest as gradual skeletal abnormalities and musculoskeletal symptoms without neurological/general symptoms, leading to misdiagnoses and delays.

Effect of dapagliflozin on collectins and complement activation in plasma from patients with type 2 diabetes and albuminuria: Data from the DapKid cohort.

BACKGROUND: Sodium-glucose cotransporter 2 (SGLT- 2) inhibitors exert cardiovascular and kidney-protective effects in people with diabetes. Attenuation of inflammation could be important for systemic protection. The lectin pathway of complement system activation is linked to diabetic nephropathy. We hypothesized that SGLT-2 inhibitors lower the circulating level of pattern-recognition molecules of the lectin cascade and attenuate systemic complement activation. METHODS: Analysis of paired plasma samples from the DapKid crossover intervention study where patients with type 2 diabetes mellitus (T2DM) and albuminuria were treated with dapagliflozin and placebo for 12 weeks (10 mg/day, n=36). ELISA was used to determine concentrations of collectin kidney 1 (CL-K1), collectin liver 1 (CL-L1), mannose-binding lectin (MBL), MBL-associated serine protease 2 (MASP-2), the anaphylatoxin complement factor 3a (C3a), the stable C3 split product C3dg and the membrane attack complex (sC5b-9). RESULTS: As published before, dapagliflozin treatment lowered Hba1C from 74 (14.9) mmol/mol to 66 (13.9) mmol/mol (p<0.0001), and the urine albumin/creatinine ratio from 167.8 mg/g to 122.5 mg/g (p<0.0001). Plasma concentrations of CL-K1, CL-L1, MBL, and MASP-2 did not change significantly after dapagliflozin treatment (P>0.05) compared to placebo treatment. The plasma levels of C3a (P<0.05) and C3dg (P<0.01) increased slightly but significantly, 0.6 [0.2] units/mL and 76 [52] units/mL respectively, after dapagliflozin treatment. The C9-associated neoepitope in C5b-9 did not change in plasma concentration by dapagliflozin (P>0.05). CONCLUSION: In patients with type 2 diabetes and albuminuria, SGLT-2 inhibition resulted in modest C3 activation in plasma, likely not driven by primary changes in circulating collectins and not resulting in changes in membrane attack complex. Based on systemic analyses, organ-specific local protective effects of gliflozins against complement activation cannot be excluded.

Bidirectional electrostatic MEMS-tunable VCSELs.

Microelectromechanical system (MEMS) vertical cavity surface-emitting lasers (VCSELs) are the fastest coherently tunable lasers (nm/ns) due to their unique Doppler-assisted tuning mechanism. However, in standard electrostatic actuation, the response is highly nonlinear and large (>100 V) dynamic voltages are needed for MHz sweep rates. We present a bidirectional MEMS VCSEL as a solution to these challenges where static voltages can be used to enable substantially linear and amplified wavelength tuning with respect to the fast tuning (MEMS) voltage. Using an InP/SOI MEMS bonded structure, we show a tuning range of 54.5 nm (gain limited) centered around 1586 nm at an actuation frequency of 2.73 MHz.

Host-gut microbiota interactions shape parasite infections in farmed Atlantic salmon.

Animals and their associated microbiota share long evolutionary histories. However, it is not always clear how host genotype and microbiota interact to affect phenotype. We applied a hologenomic approach to explore how host-microbiota interactions shape lifetime growth and parasite infection in farmed Atlantic salmon (Salmo salar). Multi-omics data sets were generated from the guts of 460 salmon, 82% of which were naturally infected with an intestinal cestode. A single Mycoplasma bacterial strain, MAG01, dominated the gut metagenome of large, non-parasitized fish, consistent with previous studies showing high levels of Mycoplasma in the gut microbiota of healthy salmon. While small and/or parasitized salmon also had high abundance of MAG01, we observed increased alpha diversity in these individuals, driven by increased frequency of low-abundance Vibrionaceae and other Mycoplasma species that carried known virulence genes. Colonization by one of these cestode-associated Mycoplasma strains was associated with host individual genomic variation in long non-coding RNAs. Integrating the multi-omic data sets revealed coordinated changes in the salmon gut mRNA transcriptome and metabolome that correlated with shifts in the microbiota of smaller, parasitized fish. Our results suggest that the gut microbiota of small and/or parasitized fish is in a state of dysbiosis that partly depends on the host genotype, highlighting the value of using a hologenomic approach to incorporate the microbiota into the study of host-parasite dynamics.IMPORTANCEStudying host-microbiota interactions through the perspective of the hologenome is gaining interest across all life sciences. Intestinal parasite infections are a huge burden on human and animal health; however, there are few studies investigating the role of the hologenome during parasite infections. We address this gap in the largest multi-omics fish microbiota study to date using natural cestode infection of farmed Atlantic salmon. We find a clear association between cestode infection, salmon lifetime growth, and perturbation of the salmon gut microbiota. Furthermore, we provide the first evidence that the genetic background of the host may partly determine how the gut microbiota changes during parasite-associated dysbiosis. Our study therefore highlights the value of a hologenomic approach for gaining a more in-depth understanding of parasitism.

Collaborative nowcasting of COVID-19 hospitalization incidences in Germany.

Real-time surveillance is a crucial element in the response to infectious disease outbreaks. However, the interpretation of incidence data is often hampered by delays occurring at various stages of data gathering and reporting. As a result, recent values are biased downward, which obscures current trends. Statistical nowcasting techniques can be employed to correct these biases, allowing for accurate characterization of recent developments and thus enhancing situational awareness. In this paper, we present a preregistered real-time assessment of eight nowcasting approaches, applied by independent research teams to German 7-day hospitalization incidences during the COVID-19 pandemic. This indicator played an important role in the management of the outbreak in Germany and was linked to levels of non-pharmaceutical interventions via certain thresholds. Due to its definition, in which hospitalization counts are aggregated by the date of case report rather than admission, German hospitalization incidences are particularly affected by delays and can take several weeks or months to fully stabilize. For this study, all methods were applied from 22 November 2021 to 29 April 2022, with probabilistic nowcasts produced each day for the current and 28 preceding days. Nowcasts at the national, state, and age-group levels were collected in the form of quantiles in a public repository and displayed in a dashboard. Moreover, a mean and a median ensemble nowcast were generated. We find that overall, the compared methods were able to remove a large part of the biases introduced by delays. Most participating teams underestimated the importance of very long delays, though, resulting in nowcasts with a slight downward bias. The accompanying prediction intervals were also too narrow for almost all methods. Averaged over all nowcast horizons, the best performance was achieved by a model using case incidences as a covariate and taking into account longer delays than the other approaches. For the most recent days, which are often considered the most relevant in practice, a mean ensemble of the submitted nowcasts performed best. We conclude by providing some lessons learned on the definition of nowcasting targets and practical challenges.

Efficient low-reflection fully etched vertical free-space grating couplers for suspended silicon photonics.

We design and fabricate a grating coupler for interfacing suspended silicon photonic membranes with free-space optics while being compatible with single-step lithography and etching in 220 nm silicon device layers. The grating coupler design simultaneously and explicitly targets both high transmission into a silicon waveguide and low reflection back into the waveguide by means of a combination of a two-dimensional shape-optimization step followed by a three-dimensional parameterized extrusion. The designed coupler has a transmission of -6.6 dB (21.8 %), a 3 dB bandwidth of 75 nm, and a reflection of -27 dB (0.2 %). We experimentally validate the design by fabricating and optically characterizing a set of devices that allow the subtraction of all other sources of transmission losses as well as the inference of back-reflections from Fabry-Pérot fringes, and we measure a transmission of 19 % ± 2 %, a bandwidth of 65 nm and a reflection of 1.0 % ± 0.8 %.

Intestinal epigenotype of Atlantic salmon (Salmo salar) associates with tenacibaculosis and gut microbiota composition.

It remains a challenge to obtain the desired phenotypic traits in aquacultural production of Atlantic salmon, and part of the challenge might come from the effect that host-associated microorganisms have on the fish phenotype. To manipulate the microbiota towards the desired host traits, it is critical to understand the factors that shape it. The bacterial gut microbiota composition can vary greatly among fish, even when reared in the same closed system. While such microbiota differences can be linked to diseases, the molecular effect of disease on host-microbiota interactions and the potential involvement of epigenetic factors remain largely unknown. The aim of this study was to investigate the DNA methylation differences associated with a tenacibaculosis outbreak and microbiota displacement in the gut of Atlantic salmon. Using Whole Genome Bisulfite Sequencing (WGBS) of distal gut tissue from 20 salmon, we compared the genome-wide DNA methylation levels between uninfected individuals and sick fish suffering from tenacibaculosis and microbiota displacement. We discovered >19,000 differentially methylated cytosine sites, often located in differentially methylated regions, and aggregated around genes. The 68 genes connected to the most significant regions had functions related to the ulcerous disease such as epor and slc48a1a but also included prkcda and LOC106590732 whose orthologs are linked to microbiota changes in other species. Although the expression level was not analysed, our epigenetic analysis suggests specific genes potentially involved in host-microbiota interactions and more broadly it highlights the value of considering epigenetic factors in efforts to manipulate the microbiota of farmed fish.

Nanometer-scale photon confinement in topology-optimized dielectric cavities.

Nanotechnology enables in principle a precise mapping from design to device but relied so far on human intuition and simple optimizations. In nanophotonics, a central question is how to make devices in which the light-matter interaction strength is limited only by materials and nanofabrication. Here, we integrate measured fabrication constraints into topology optimization, aiming for the strongest possible light-matter interaction in a compact silicon membrane, demonstrating an unprecedented photonic nanocavity with a mode volume of V ~ 3 × 10-4 λ3, quality factor Q ~ 1100, and footprint 4 λ2 for telecom photons with a λ ~ 1550 nm wavelength. We fabricate the cavity, which confines photons inside 8 nm silicon bridges with ultra-high aspect ratios of 30 and use near-field optical measurements to perform the first experimental demonstration of photon confinement to a single hotspot well below the diffraction limit in dielectrics. Our framework intertwines topology optimization with fabrication and thereby initiates a new paradigm of high-performance additive and subtractive manufacturing.

Targeting two distinct epitopes on human CD73 with a bispecific antibody improves anticancer activity.

BACKGROUND: Immunosuppressive extracellular adenosine is generated by the enzymatic activity of CD73. In preclinical models, antibodies (Abs) targeting different epitopes on CD73 exert anticancer activity through distinct mechanisms such as inhibition of enzymatic activity, engagement of Fc receptors, and spatial redistribution of CD73. METHODS: Using controlled Fab arm exchange, we generated biparatopic bispecific antibodies (bsAbs) from parental anti-CD73 Abs with distinct anticancer activities. The resulting anticancer activity was evaluated using in vitro and in vivo models. RESULTS: We demonstrate that different anticancer activities can be combined in a biparatopic bsAb. Remarkably, the bsAb significantly improved the enzyme inhibitory activity compared with the parental Abs, which led to neutralization of adenosine-mediated T-cell suppression as demonstrated by proliferation and interferon gamma (IFN-γ) production and prolonged survival of tumor-bearing mice. Additionally, the bsAb caused more efficient internalization of cell surface CD73 and stimulated potent Fc-mediated engagement of human immune effector cells in vitro and in vivo. CONCLUSIONS: Our data collectively demonstrate that complementary anticancer mechanisms of action of distinct anti-CD73 Abs can be combined and enhanced in a biparatopic bsAb. The multiple mechanisms of action and superior activity compared with the monospecific parental Abs make the bsAb a promising candidate for therapeutic targeting of CD73 in cancer. This concept may greatly improve future Ab design.

Mannan-binding lectin serine protease-2 (MASP-2) in human kidney and its relevance for proteolytic activation of the epithelial sodium channel.

Proteolytic activation of the renal epithelial sodium channel (ENaC) is increased by aldosterone. The aldosterone-sensitive protease remains unidentified. In humans, elevated circulating aldosterone is associated with increased urinary extracellular vesicle (uEVs) excretion of mannan-binding lectin associated serine protease-2 (MASP-2). We hypothesized that MASP-2 is a physiologically relevant ENaC-activating protease. It was confirmed that MASP2 mRNA is abundantly present in liver but not in human and mouse kidneys. Aldosterone-stimulation of murine cortical colleting duct (mCCD) cells did not induce MASP-2 mRNA. In human kidney collecting duct, MASP-2 protein was detected in AQP2-negative/ATP6VB1-positive intercalated cells suggestive of MASP2 protein uptake. Plasma concentration of full-length MASP-2 and the short splice variant MAp19 were not changed in a cross-over intervention study in healthy humans with low (70 mmol/day) versus high (250 mmol/day) Na+ intake despite changes in aldosterone. The ratio of MAp19/MASP-2 in plasma was significantly increased with a high Na+ diet and the ratio correlated with changes in aldosterone and fractional Na+ excretion. MASP-2 was not detected in crude urine or in uEVs. MASP2 activated an amiloride-sensitive current when co-expressed with ENaC in Xenopus oocytes, but not when added to the bath solution. In monolayers of collecting duct M1 cells, MASP2 expression did not increase amiloride-sensitive current and in HEK293 cells, MASP-2 did not affect γENaC cleavage. MASP-2 is neither expressed nor co-localized and co-regulated with ENaC in the human kidney or in urine after low Na+ intake. MASP-2 does not mediate physiological ENaC cleavage in low salt/high aldosterone settings.

Hemodynamic changes during aortic valve surgery among patients with aortic stenosis.

Introduction. Patients with severe aortic stenosis (AS) undergoing surgery are at increased risk of hypotension and hypoperfusion. Although treatable with inotropic agents or fluid, little is known about how these therapies affect central hemodynamics in AS patients under general anesthesia. We measured changes in central hemodynamics after dobutamine infusion and fluid bolus among patients with severe AS and associated these changes with preoperative echocardiography. Methods. We included 33 patients with severe AS undergoing surgical AVR. After induction of general anesthesia, hemodynamic measurements were obtained with a pulmonary artery catheter, including Cardiac index (CI), stroke volume index (SVi) and pulmonary capillary wedge pressure (PCWP). Measurements were repeated during dobutamine infusion, after fluid bolus and lastly after sternotomy. Results. General anesthesia resulted in a decrease in CI and SVi compared to preoperative values. During dobutamine infusion CI increased but mean SVi did not (38 ± 12 vs 37 ± 13 ml/m2, p = .90). Higher EF and SVi before surgery and a larger decrease in SVi after induction of general anesthesia were associated with an increase in SVi during dobutamine infusion. After fluid bolus both CI, SVi (48 ± 12 vs 37 ± 13 ml/min/m2, p < .0001) and PCWP increased. PCWP increased mostly among patients with a larger LA volume index. Conclusion. In patients with AS, CI can be increased with both dobutamine and fluid during surgery. Dobutamine's effect on SVI was highly variable and associated with baseline LVEF, and an increase in CI was mostly driven by an increase in heart rate. Fluid increased SVi at the cost of an increase in PCWP.

Quantification of the pro-form of human complement component factor D (adipsin).

Factor D (also known as adipsin) is a serine protease and part of the complement system, involved in innate immune responses and effector functions of antibodies. Factor D cleaves factor B complexed with C3b, leading to the C3 convertase C3bBb. This C3 convertase is central in the alternative activation pathway and the amplification loop, which amplifies the two other complement activation pathways: the classical pathway and the lectin pathway. Adipocytes synthesize factor D as a pro-form comprising 6 additional residues that must be cleaved off to generate a mature form. The MBL-associated serine protease 3 (MASP-3), found in complex with the pattern recognition molecules of lectin activation pathway, converts the pro-form to mature factor D, which reportedly is the most abundant form found in the circulation at concentrations of 1-2 µg/ml among healthy individuals. The mature factor D is rate-limiting for complement activation, but little is known about the distribution of pro vs. mature factor D in the circulation, the regulation hereof and the potential activation stimuli of the lectin pathway, responsible for activation of MASP-3 and subsequent conversion of pro-form of factor D. In this light we established and validated an ELISA specific for measuring the pro-form of complement factor D. With a working range of 0.82-25 ng/ml, acceptable intra and inter assay CVs, and a relative recovery rate above 90%, we found that the median plasma concentration in Danish blood donors was 134 ng/ml; corresponding to that 8-15% factor D circulates as pro-form. We also found that blood sampling procedures affect conversion and hence the levels measured in serum and plasma.

Potency measurements of the complement system facilitated by antibodies targeting the zymogen form of complement factor D (Adipsin).

The serine protease complement factor D is fundamental in the activation of the complement system. In addition, it was the first adipokine described (named Adipsin) and shown to improve beta cell function in diabetes. As part of an amplification loop of complement activation, factor D is a rate-limiting enzyme, and its accessibility contributes to the potency of complement activation. The dogma has been that conversion of the zymogen form, profactor D, to mature factor D occurred during secretion by adipocytes by uncharacterized proteases. However, recent findings demonstrated that the serine protease MASP-3 of the lectin pathway of the complement system mediated this conversion, suggesting that pattern recognition of pathogen/danger-associated molecular patterns could be a prior requirement for all complement activation. To facilitate studies addressing this hypothesis, we have developed monoclonal antibodies specific for human profactor D without binding to mature factor D. We demonstrate their applications in accessing the conversion of profactor D into mature factor D and in measuring levels of profactor D.

Advancement in Production of Radiotracers.

After introduction of the first commercial combined PET and/or CT technology in 2001, this diagnostic tool quickly became a clinical success and was considered the fastest growing diagnostic imaging technology ever. However, this technique is very dependent on the availability of positron emitting isotopes and radiochemistry to incorporate the radioactive isotopes into larger molecules of physiological interest. Within this review article a historical overview starting with the first applications of positron emitting isotopes in the 1930's is presented. Afterwards a more detailed presentation summarizing the physical basis and advancements in cyclotron technology is given. Radiochemical and/or pharmaceutical advancements are presented systematically for the most significant isotopes like 15O, 13N, 11C, 18F and 68Ga Besides these major PET isotopes, advancements of other radio-metals and future perspectives regarding application of new radionuclides will be discussed. Finally, very interesting new and compact accelerator technology and microfluidic chemical reaction approaches will be discussed. Especially, new compact accelerator technology might be new quantum leap within this radiodiagnostic technology and might result in even further prevalence, ultimately envisioned by the dose-on-demand concept that will be briefly discussed.

Enzymatic extraction improves intracellular protein recovery from the industrial carrageenan seaweed Eucheuma denticulatum revealed by quantitative, subcellular protein profiling: A high potential source of functional food ingredients.

Seaweeds are regarded as a sustainable source of food protein, but protein extraction is severely impaired by the complex extracellular matrix. In this work, we investigated the protein-level effects of enzymatic extraction upstream of carrageenan extraction for the industrial red seaweed Eucheuma denticulatum. Combination of quantitative proteomics and bioinformatic prediction of subcellular localization was shown to have immense potential for process evaluation; even in the case of poorly annotated species such as E. denticulatum. Applying cell wall degrading enzymes markedly improved the relative recovery of intracellular proteins compared to treatment with proteolytic enzymes or no enzymatic treatment. Moreover, results suggest that proteomics data may prove useful for characterizing amino acid composition and that length-normalization is a viable approach for relative protein quantification in non-specific analysis. Importantly, the extracts were abundant in proteins, which contained both previously verified and novel, potential bioactive peptides, highlighting their potential for application as functional food ingredients.