Systematic P2Y receptor survey identifies P2Y11 as modulator of immune responses and virus replication in macrophages.

The immune system is in place to assist in ensuring tissue homeostasis, which can be easily perturbed by invading pathogens or nonpathogenic stressors causing tissue damage. Extracellular nucleotides are well known to contribute to innate immune signaling specificity and strength, but how their signaling is relayed downstream of cell surface receptors and how this translates into antiviral immunity is only partially understood. Here, we systematically investigated the responses of human macrophages to extracellular nucleotides, focusing on the nucleotide-sensing GPRC receptors of the P2Y family. Time-resolved transcriptomic analysis showed that adenine- and uridine-based nucleotides induce a specific, immediate, and transient cytokine response through the MAPK signaling pathway that regulates transcriptional activation by AP-1. Using receptor trans-complementation, we identified a subset of P2Ys (P2Y1, P2Y2, P2Y6, and P2Y11) that govern inflammatory responses via cytokine induction, while others (P2Y4, P2Y11, P2Y12, P2Y13, and P2Y14) directly induce antiviral responses. Notably, P2Y11 combined both activities, and depletion or inhibition of this receptor in macrophages impaired both inflammatory and antiviral responses. Collectively, these results highlight the underappreciated functions of P2Y receptors in innate immune processes.

Two cGAS-like receptors induce antiviral immunity in Drosophila.

In mammals, cyclic GMP-AMP (cGAMP) synthase (cGAS) produces the cyclic dinucleotide 2'3'-cGAMP in response to cytosolic DNA and this triggers an antiviral immune response. cGAS belongs to a large family of cGAS/DncV-like nucleotidyltransferases that is present in both prokaryotes1 and eukaryotes2-5. In bacteria, these enzymes synthesize a range of cyclic oligonucleotides and have recently emerged as important regulators of phage infections6-8. Here we identify two cGAS-like receptors (cGLRs) in the insect Drosophila melanogaster. We show that cGLR1 and cGLR2 activate Sting- and NF-κB-dependent antiviral immunity in response to infection with RNA or DNA viruses. cGLR1 is activated by double-stranded RNA to produce the cyclic dinucleotide 3'2'-cGAMP, whereas cGLR2 produces a combination of 2'3'-cGAMP and 3'2'-cGAMP in response to an as-yet-unidentified stimulus. Our data establish cGAS as the founding member of a family of receptors that sense different types of nucleic acids and trigger immunity through the production of cyclic dinucleotides beyond 2'3'-cGAMP.

Topical Neuropeptide Y for Ischemic Skin Wounds.

Our objective was to investigate the effects of topically applied neuropeptide Y (NPY) on ischemic wounds. Initially, the animal model for ischemic wound healing was validated using 16 male Sprague Dawley albino rats. In the intervention study, an additional 28 rats were divided into three groups: NPY (0.025%), the positive control insulin-like growth factor-I (IGF-I, 0.0025%), and the hydrogel carrier alone (control). The hydrogel was selected due to its capacity to prolong NPY release (p < 0.001), as demonstrated in a Franz diffusion cell. In the animals, an 8 mm full-thickness wound was made in a pedunculated dorsal ischemic skin flap. Wounds were then treated and assessed for 14 days and collected at the end of the experiment for in situ hybridization analysis (RNAscope®) targeting NPY receptor Y2R and for meticulous histologic examination. Wound healing rates, specifically the percentage changes in wound area, did not show an increase with NPY (p = 0.907), but there was an increase with rhIGF-I (p = 0.039) compared to the control. Y2R mRNA was not detected in the wounds or adjacent skin but was identified in the rat brain (used as a positive control). Light microscopic examination revealed trends of increased angiogenesis and enhanced inflammatory cell infiltration with NPY compared to control. An interesting secondary discovery was the presence of melanophages in the wounds. Our findings suggest the potential of NPY to enhance neovascularization under ischemic wound healing conditions, but further optimization of the carrier and dosage is necessary. The mechanism remains elusive but likely involves NPY receptor subtypes other than Y2R.

Can reintroduction of beavers improve insect biodiversity?

Ecosystem engineering species, such as beavers, may help the restoration of biodiversity. Through the building of dams and lodges and altering the natural hydrology, beavers change the habitat structure and create multiple habitats that facilitate a wide variety of other organisms including terrestrial invertebrate communities. Here we study the effect of beaver reintroduction in Klosterheden in Denmark on biomass of flying invertebrates and diversity of moths. Further, aerial photos were used to assess riparian structure and productivity using the normalized difference vegetation index (NDVI). Our findings show that the presence of beavers affected flying invertebrate biomass, but that this was dependent on time of the year. Further, a strong effect of presence of beavers was found on diversity of moths. The results also show an increase in vegetation productivity and structural heterogeneity at sites with presence of beavers. Overall, our results demonstrate the importance of beavers as important ecosystem engineers that affect invertebrate species composition and abundance, as well as riparian structure and productivity.

Immune cell analyses of the tumor microenvironment in prostate cancer highlight infiltrating regulatory T cells and macrophages as adverse prognostic factors.

Improved risk stratification is needed for patients with localized prostate cancer. This study characterized and assessed the prognostic potential of distinct immune cell infiltration patterns in the prostate tumor microenvironment. Using tissue microarrays, multiplex immunohistochemistry/immunofluorescence, and automated digital pathology, we analyzed radical prostatectomy specimens from two large patient cohorts (training: n = 470; validation: n = 333) to determine infiltration levels of seven immune cell types in malignant versus benign prostate tissue: CD3+ CD8- FoxP3- T helper cells, CD3+ CD8+ FoxP3- cytotoxic T cells (CTLs), CD3+ CD8- FoxP3+ regulatory T cells (Tregs ), CD20+ B cells, CD68+ CD163- M1 macrophages, CD68+ CD163+ M2 macrophages, and tryptase+ mast cells. Results were further validated by cell type enrichment analyses of bulk tumor RNAseq data from a third independent patient cohort (n = 99). Prognostic potential was assessed by Kaplan-Meier and uni-/multi-variate Cox regression analyses. Clinical endpoint was biochemical recurrence. All seven immune cell types were enriched in prostate cancer versus benign stroma, while there was selective enrichment for B cells, Tregs , M1 and M2 macrophages, and depletion of mast cells and CTLs in prostate cancer epithelium. In all three cohorts, high levels of infiltrating Tregs , M1, and M2 macrophages in stroma and/or epithelium were associated with biochemical recurrence (p < 0.05; log-rank test). After adjustment for routine clinical variables, Tregs and M2 macrophages remained significant adverse predictors of biochemical recurrence (p < 0.05; multivariate Cox regression). Our comprehensive analyses of immune cell infiltration patterns in the prostate tumor microenvironment highlight infiltrating Tregs , M1, and M2 macrophages as adverse predictors of prostate cancer outcome. © 2021 The Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Characterization of distinct molecular interactions responsible for IRF3 and IRF7 phosphorylation and subsequent dimerization.

IRF3 and IRF7 are critical transcription factors in the innate immune response. Their activation is controlled by phosphorylation events, leading to the formation of homodimers that are transcriptionally active. Phosphorylation occurs when IRF3 is recruited to adaptor proteins via a positively charged surface within the regulatory domain of IRF3. This positively charged surface also plays a crucial role in forming the active homodimer by interacting with the phosphorylated sites stabilizing the homodimer. Here, we describe a distinct molecular interaction that is responsible for adaptor docking and hence phosphorylation as well as a separate interaction responsible for the formation of active homodimer. We then demonstrate that IRF7 can be activated by both MAVS and STING in a manner highly similar to that of IRF3 but with one key difference. Regulation of IRF7 appears more tightly controlled; while a single phosphorylation event is sufficient to activate IRF3, at least two phosphorylation events are required for IRF7 activation.

SARS-CoV-2 infection in pregnancy in Denmark-characteristics and outcomes after confirmed infection in pregnancy: A nationwide, prospective, population-based cohort study.

INTRODUCTION: Assessing the risk factors for and consequences of infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) during pregnancy is essential to guide clinical care. Previous studies on SARS-CoV-2 infection in pregnancy have been among hospitalized patients, which may have exaggerated risk estimates of severe outcomes because all cases of SARS-CoV-2 infection in the pregnant population were not included. The objectives of this study were to identify risk factors for and outcomes after SARS-CoV-2 infection in pregnancy independent of severity of infection in a universally tested population, and to identify risk factors for and outcomes after severe infection requiring hospital admission. MATERIAL AND METHODS: This was a prospective population-based cohort study in Denmark using data from the Danish National Patient Register and Danish Microbiology Database and prospectively registered data from medical records. We included all pregnancies between March 1 and October 31, 2020 and compared women with a positive SARS-CoV-2 test during pregnancy to non-infected pregnant women. Cases of SARS-CoV-2 infection in pregnancy were both identified prospectively and through register linkage to ensure that all cases were identified and that cases were pregnant during infection. Main outcome measures were pregnancy, delivery, maternal, and neonatal outcomes. Severe infection was defined as hospital admission due to coronavirus disease 2019 (COVID-19) symptoms. RESULTS: Among 82 682 pregnancies, 418 women had SARS-CoV-2 infection during pregnancy, corresponding to an incidence of 5.1 per 1000 pregnancies, 23 (5.5%) of which required hospital admission due to COVID-19. Risk factors for infection were asthma (odds ratio [OR] 2.19, 95% CI 1.41-3.41) and being foreign born (OR 2.12, 95% CI 1.70-2.64). Risk factors for hospital admission due to COVID-19 included obesity (OR 2.74, 95% CI 1.00-7.51), smoking (OR 4.69, 95% CI 1.58-13.90), infection after gestational age (GA) 22 weeks (GA 22-27 weeks: OR 3.77, 95% CI 1.16-12.29; GA 28-36 weeks: OR 4.76, 95% CI 1.60-14.12), and having asthma (OR 4.53, 95% CI 1.39-14.79). We found no difference in any obstetrical or neonatal outcomes. CONCLUSIONS: Only 1 in 20 women with SARS-CoV-2 infection during pregnancy required admission to hospital due to COVID-19. Risk factors for admission comprised obesity, smoking, asthma, and infection after GA 22 weeks. Severe adverse outcomes of SARS-CoV-2 infection in pregnancy were rare.

Decellularised Human Umbilical Artery as a Vascular Graft Elicits Minimal Pro-Inflammatory Host Response Ex Vivo and In Vivo.

Small diameter (<6 mm) vessel grafts still pose a challenge for scientists worldwide. Decellularised umbilical artery (dUA) remains promising as small diameter tissue engineered vascular graft (TEVG), yet their immunogenicity remains unknown. Herein, we evaluated the host immune responses, with a focus on the innate part, towards human dUA implantation in mice, and confirmed our findings in an ex vivo allogeneic human setup. Overall, we did not observe any differences in the number of circulating white blood cells nor the number of monocytes among three groups of mice (1) dUA patch; (2) Sham; and (3) Mock throughout the study (day -7 to 28). Likewise, we found no difference in systemic inflammatory and anti-inflammatory cytokine levels between groups. However, a massive local remodelling response with M2 macrophages were observed in the dUA at day 28, whereas M1 macrophages were less frequent. Moreover, human monocytes from allogeneic individuals were differentiated into macrophages and exposed to lyophilised dUA to maximize an eventual M1 response. Yet, dUA did not elicit any immediate M1 response as determined by the absence of CCR7 and CXCL10. Together this suggests that human dUA elicits a minimal pro-inflammatory response further supporting its use as a TEVG in an allogeneic setup.

Can reed harvest be used as a management strategy for improving invertebrate biomass and diversity?

The succession-driven reed bed habitat hosts a unique flora and fauna including several endangered invertebrate species. Reed beds can be managed through commercial winter harvest, with implications for reed bed conservation. However, the effects of winter harvest on the invertebrate community are not well understood and vary across studies and taxonomic levels. The aim of this study was to investigate the effects of reed harvest on invertebrate communities. Ground-dwelling and aerial invertebrates were continuously sampled for 10 weeks in the largest coherent reed bed of Scandinavia in order to assess how time since last reed harvest (0, 3, and 25-years) influences invertebrate biomass, biodiversity and community structure across taxonomic levels. Biomass was measured and all specimens were sorted to order level, and Coleoptera was even sorted to species level. The invertebrate community showed distinct compositional differences across the three reed bed ages. Furthermore, biomass of both aerial and ground-dwelling invertebrates was highest in the age-0 reed bed and lowest in the age-25 reed bed. Generally, biodiversity showed an opposite trend with the highest richness and diversity in the age-25 reed bed. We conclude that it is possible to ensure high insect biomass and diversity by creating a mosaic of reed bed of different ages through small-scale harvest in the largest coherent reed bed in Scandinavia. The youngest red beds support a high invertebrate biomass whereas the oldest reed beds support a high biodiversity. Collectively, this elevate our understanding of reed harvest and the effects it has on the invertebrate communities, and might aid in future reed bed management and restoration.

Frequently used bioinformatics tools overestimate the damaging effect of allelic variants.

We selected two sets of naturally occurring human missense allelic variants within innate immune genes. The first set represented eleven non-synonymous variants in six different genes involved in interferon (IFN) induction, present in a cohort of patients suffering from herpes simplex encephalitis (HSE) and the second set represented sixteen allelic variants of the IFNLR1 gene. We recreated the variants in vitro and tested their effect on protein function in a HEK293T cell based assay. We then used an array of 14 available bioinformatics tools to predict the effect of these variants upon protein function. To our surprise two of the most commonly used tools, CADD and SIFT, produced a high rate of false positives, whereas SNPs&GO exhibited the lowest rate of false positives in our test. As the problem in our test in general was false positive variants, inclusion of mutation significance cutoff (MSC) did not improve accuracy.

Spawning by the European eel across 2000 km of the Sargasso Sea.

It has been known for about a century that European eels have a unique life history that includes offshore spawning in the Sargasso Sea about 5000-7000 km away from their juvenile and adult habitats in Europe and northern Africa. Recently hatched eel larvae were historically collected during Danish, German and American surveys in specific areas in the southern Sargasso Sea. During a 31 day period of March and April 2014, Danish and German research ships sampled for European eel larvae along 15 alternating transects of stations across the Sargasso Sea. The collection of recently hatched eel larvae (≤12 mm) from 70° W and eastward to 50° W showed that the European eel had been spawning across a 2000 km wide region of the North Atlantic Ocean. Historical collections made from 1921 to 2007 showed that small larvae had also previously been collected in this wide longitudinal zone, showing that the spatial extent of spawning has not diminished in recent decades, irrespective of the dramatic decline in recruitment. The use of such a wide spawning area may be related to variations in the onset of the silver eel spawning migration, individual differences in their long-term swimming ability, or aspects of larval drift.

The effect of gender on early colonic anastomotic wound healing.

PURPOSE: Clinically, male patients subjected to colorectal surgery are more prone to develop anastomotic leakage than female patients by unknown mechanisms. Our aim was to investigate the impact of gender on anastomotic wound healing using an experimental model. METHODS: One-layer colonic anastomosis was constructed in 8-week-old 28 male and 32 female Sprague-Dawley rats. Animals of one group (n = 30) were sacrificed immediately after surgery day 0 and the other group (n = 30) on postoperative day 3. Anastomotic breaking strength, total collagen (hydroxyproline), soluble collagen (Sircol), matrix metalloproteinase (MMP)-9, and transforming growth factor (TGF)-ß1 were measured. RESULTS: The anastomotic breaking strength decreased from day 0 to day 3 with no significant gender differences either in the extent of decline (P = 0.122) or absolute day 3 strengths (P = 0.425). Analogously, total collagen concentration in the anastomotic wounds decreased postoperatively and were lower (P = 0.043) in the male compared with the female rats on day 3. MMP-9 levels increased in the anastomoses postoperatively, but they did not differ (P = 0.391) between male and female animals. Soluble collagen levels were lower in the day-3 anastomoses of male versus female rats (P = 0.015) and correlated positively with total TGF-ß1 levels (rS = 0.540, P = 0.006). Although TGF-ß1 tended to be lower in male compared with the female rats, the differences did not reach statistical significance. CONCLUSION: Our findings point towards a less favorable collagen metabolism in colonic anastomoses of male compared with female rats during early wound healing.

Structural and functional analysis reveals that human OASL binds dsRNA to enhance RIG-I signaling.

The oligoadenylate synthetase (OAS) enzymes are cytoplasmic dsRNA sensors belonging to the antiviral innate immune system. Upon binding to viral dsRNA, the OAS enzymes synthesize 2'-5' linked oligoadenylates (2-5As) that initiate an RNA decay pathway to impair viral replication. The human OAS-like (OASL) protein, however, does not harbor the catalytic activity required for synthesizing 2-5As and differs from the other human OAS family members by having two C-terminal ubiquitin-like domains. In spite of its lack of enzymatic activity, human OASL possesses antiviral activity. It was recently demonstrated that the ubiquitin-like domains of OASL could substitute for K63-linked poly-ubiquitin and interact with the CARDs of RIG-I and thereby enhance RIG-I signaling. However, the role of the OAS-like domain of OASL remains unclear. Here we present the crystal structure of the OAS-like domain, which shows a striking similarity with activated OAS1. Furthermore, the structure of the OAS-like domain shows that OASL has a dsRNA binding groove. We demonstrate that the OAS-like domain can bind dsRNA and that mutating key residues in the dsRNA binding site is detrimental to the RIG-I signaling enhancement. Hence, binding to dsRNA is an important feature of OASL that is required for enhancing RIG-I signaling.

Mode of delivery shapes gut colonization pattern and modulates regulatory immunity in mice.

Delivery mode has been associated with long-term changes in gut microbiota composition and more recently also with changes in the immune system. This has further been suggested to link Cesarean section (C-section) with an increased risk for development of immune-mediated diseases such as type 1 diabetes. In this study, we demonstrate that both C-section and cross-fostering with a genetically distinct strain influence the gut microbiota composition and immune key markers in mice. Gut microbiota profiling by denaturing gradient gel electrophoresis and 454/FLX-based 16S rRNA gene amplicon sequencing revealed that mice born by C-section had a distinct bacterial profile at weaning characterized by higher abundance of Bacteroides and Lachnospiraceae, and less Rikenellaceae and Ruminococcus. No clustering according to delivery method as determined by principal component analysis of denaturing gradient gel electrophoresis profiles was evident in adult mice. However, the adult C-section-born mice had lower proportions of Foxp3(+) regulatory T cells, tolerogenic CD103(+) dendritic cells, and less Il10 gene expression in mesenteric lymph nodes and spleens. This demonstrates long-term systemic effect on the regulatory immune system that was also evident in NOD mice, a model of type 1 diabetes, born by C-section. However, no effect of delivery mode was seen on diabetes incidence or insulitis development. In conclusion, the first exposure to microorganisms seems to be crucial for the early life gut microbiota and priming of regulatory immune system in mice, and mode of delivery strongly influences this.

Effects of melatonin on physical fatigue and other symptoms in patients with advanced cancer receiving palliative care: A double-blind placebo-controlled crossover trial.

BACKGROUND: Patients with advanced cancer often experience fatigue and other symptoms that negatively impact their quality of life. The current trial investigated the effect of melatonin on fatigue and other symptoms in patients with advanced cancer. METHODS: Patients who were aged ≥18 years, had a histologically confirmed stage IV cancer (TNM Classification), and who reported feeling significantly tired were recruited from the palliative care unit at the study institution. The study was a double-blind, randomized, placebo-controlled crossover trial. Patients received 1 week of melatonin at a dose of 20 mg or a placebo orally each night, before crossing over and receiving the opposite treatment for 1 week. Between the 2 periods, a washout period of 2 days was implemented. Outcomes were measured using the Multidimensional Fatigue Inventory (MFI-20) and The European Organization for Research and Treatment of Cancer Quality of Life Questionnaire. Physical fatigue from the MFI-20 was the primary outcome. The primary analysis was a complete complier analysis (ie, it included only those patients who had consumed at least 5 capsules per week and who had answered the MFI-20 on days 1, 7, 10, and 17). Sensitivity analysis using multiple imputations including all randomized patients and all patients completing the intervention were conducted. RESULTS: A total of 72 patients were randomized. Fifty patients completed the intervention and 44 patients were complete compliers. No significant differences between the placebo and melatonin periods were found for physical fatigue, secondary outcomes, or explorative outcomes. CONCLUSIONS: In the current study, oral melatonin at a dose of 20 mg was not found to improve fatigue or other symptoms in patients with advanced cancer.

Easy peak tracking in CE-UV and CE-UV-ESI-MS by incorporating temperature-correlated mobility scaling.

A simple data reconstruction technique in CE-UV-ESI-MS (where UV stands for ultraviolet) is presented to overcome the drift in mobilities caused by various factors compromising the reproducibility of such data, for example Joule heating effects and the variation in thermostatic control along the capillary, drift in EOF and the suction effect caused by the nebulizing gas in coaxial CE-MS interfaces. We present here a method to transform the traditional time-based electropherogram into the corresponding temperature-correlated mobility scale allowing tracking of analytes independent from capillary dimensions, electric field strengths, temperature control, and distance between the detectors. The main principle of this alignment is based on including the current in the mobility calculations and relating this to the initial electrical resistance of the buffer-filled capillary. The temperature-correlated mobility calculation eliminates the peak shifts due to the viscosity changes, improves the precision of peak identification using the observed temperature-correlated mobilities, and allows a direct comparison of signals from different detection combinations. The method allows peaks from normal CE-UV separations to be correlated with the corresponding peak obtained by MS detection in CE-MS even for differences in capillary dimensions and thermostatic control.

Performance of matrix-assisted laser desorption-time of flight mass spectrometry for identification of clinical yeast isolates.

Accurate and fast yeast identification is important when treating patients with invasive fungal disease as susceptibility to antifungal agents is highly species related. Matrix-assisted laser desorption-time of flight mass spectrometry (MALDI-TOF-MS) provides a powerful tool with a clear potential to improve current diagnostic practice. Two MALDI-TOF-MS-systems (BioTyper/Bruker and Saramis/AXIMA) were evaluated using: (i) A collection of 102 archived, well characterised yeast isolates representing 14 different species and (ii) Prospectively collected isolates obtained from clinical samples at two participating laboratories. Of the 102 archived isolates, 81 (79%) and 92 (90%) were correctly identified by Saramis/AXIMA and BioTyper/Bruker respectively. Saramis/AXIMA was unable to separate Candida albicans, C. africana and C. dubliniensis in 13 of 32 isolates. After manual interpretation of the mass spectra output, all 13 isolates were correctly identified, resulting in an overall identification performance of 92%. No misidentifications occurred with the two systems. Of the routine isolates one laboratory identified 99/99 (100%) and 90/99 (91%) to species level by Saramis/Axima and conventional identification, respectively, whereas the other laboratory identified 83/98 (85%) to species level by both BioTyper/Bruker and conventional identification. Both MALDI-TOF-MS systems are fast, have built-in databases that cover the majority of clinically relevant Candida species, and have an accuracy that outperforms our conventional identification systems.

eDNA Metabarcoding- and Microscopic Analysis for Diet Determination in Waterfowl, a Comparative Study in Vejlerne, Denmark.

Understanding diets and structural food webs are keys to the apprehension of ecological communities, upon which conservation and management biology are based. The understanding of grazing and habitat choice for waterfowl is one of the most important topics for avian ecologists today and can, to some degree, be answered by dietary analysis. Droppings collected from four waterfowl, the Eurasian wigeon (Anas penelope), Greylag goose (Anser anser), pink-footed goose (Anser brachyrhynchus) and Barnacle goose (Branta leucopsis) in Vejlerne (Denmark), were analysed microscopically and through eDNA metabarcoding with the use of next generation sequencing (NGS) to accumulate knowledge about the diet of these waterfowl. In total, 120 dropping samples were microscopically analysed, of which the eDNA metabarcoding analysis was done on 79 samples. The prey items were identified according to the taxonomic level of species, and a qualitative method, frequency of occurrence (FO) and FO calculated as a percentage, was used in order to compare the results from the two methods. As neither of the methods was able to encompass all species discovered when combining the two methods, it was concluded in this study that the two methods can support each other in a dietary analysis of waterfowl, but not replace one another.

The Clinical Impact of Methylated Homeobox A9 ctDNA in Patients with Non-Resectable Biliary Tract Cancer Treated with Erlotinib and Bevacizumab.

Methylated homeobox A9 (meth-HOXA9) is tumor specific and has been suggested as a prognostic biomarker in several types of cancer. ctDNA measured as meth-HOXA9 may be a valuable biomarker in the decision-making process about last-line treatment of biliary tract cancer (BTC). The aim of the study was to investigate the clinical impact of meth-HOXA9 in plasma from patients receiving erlotinib and bevacizumab for late-stage BTC and to investigate the treatment effect and adverse events. Droplet digital PCR was applied to detect meth-HOXA9 in 39 patients. Response rates were registered according to RECIST (1.1) and adverse events according to Common Terminology Criteria for Adverse Events Version 4.0 (CTCAE (4.0)). Endpoints were progression-free survival (PFS), overall survival (OS), response rate, and toxicity. A significant difference in PFS and OS between patients with increasing and non-increasing meth-HOXA9 was detected after one treatment cycle, hazard ratio (HR) 12.4 (p < 0.0001) and HR 2.75 (p = 0.04), respectively. The most common adverse events of erlotinib were fatigue, pain, and rash, and those of bevacizumab were bleeding and wounds. This study found meth-HOXA9 to be negatively associated with survival in patients with late-stage BTC. Hence, meth-HOXA9 may guide early discontinuation of ineffective treatment.

Species identification of clinical isolates of anaerobic bacteria: a comparison of two matrix-assisted laser desorption ionization-time of flight mass spectrometry systems.

We compared two matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) systems (Shimadzu/SARAMIS and Bruker) on a collection of consecutive clinically important anaerobic bacteria (n = 290). The Bruker system had more correct identifications to the species level (67.2% versus 49.0%), but also more incorrect identifications (7.9% versus 1.4%). The system databases need to be optimized to increase identification levels. However, MALDI-TOF MS in its present version seems to be a fast and inexpensive method for identification of most clinically important anaerobic bacteria.