Background Uridine diphosphate-glucuronosyl transferase 1A1 (UGT1A1), which is the major UGT1 gene product, is located on chromosome 2q37. The expression of UGT1A1 is relatively managed by a polymorphic dinucleotide repeat inside the promoter TATA box consisting of 5-8 copies of a TA repeat. A (TA) 6TAA is considered as the wild type. The A (TA) 7TAA allele has been identified as the most frequent allele in the Caucasian populations while A (TA) 8TAA allele remains the rarest allele worldwide in North Africa, including the Arab populations. Methods The spectrum of UGT1A1 genetic mutations in seventeen Tunisian children affected by persistent unconjugated hyperbilirubinemias is represented in addition to their relatives, notably parents, sisters, and brothers. Tunisian children, from 16 unrelated families as well as a 17(th) family without CN1 affected child, were originated from the West Center of Tunisia. The promoter region and coding exons of the UGT1A1 were PCR amplified, subsequently subjected to Sanger sequencing. Results The frequencies of genotypes in CN1 patients were as follows (TA) (7/7) (12/17: 70.6%) and (TA) (8/8) (5/17: 29.4%). All patients harbored the c.1070A>G mutation of exon 3 (UGT1A1*16) in the homozygous state. Among relatives of our patients (n = 16), who were all heterozygotes for UGT1A1*16, 13/16 (81.25%) had a heterozygous state for UGT1A1*1/UGT1A1*28 or (TA) (6/7) and, 18.75% (3/16) were heterozygous for UGT1A1*28/UGT1A1*37 or (TA) (7/8) of the promoter polymorphisms. Conclusion UGT1A1*16 accompanied with UGT1A1*28 or UGT1A1*37 had a specific geographic and ethnic distribution for CN pathogenesis in this Tunisian cohort.

Forensic molecular autopsies have emerged as a tool for medical examiners to establish the cause of death. It is particularly useful in sudden unexplained deaths where the cause of death cannot be determined with a regular medical autopsy. We provide the first study of exome data from formalin-fixed paraffin-embedded samples (FFPE) paired with data from high-quality blood samples in forensic applications. The approach allows exploration of the potential to use FFPE samples for molecular autopsies and identify variants in extensive exome data. We leverage the high uniformity of the hybridization capture approach provided by Twist Bioscience to target the complete exome and sequence the libraries on a NextSeq 550. Our findings suggest that exome sequencing is feasible for 24 out of a total of 35 included FFPE samples. When successful, the coverage across the exome is comparatively high (> 90% covered to 20X) and uniform (fold80 below 1.5). Detailed variant comparisons for matched FFPE and blood samples show high concordance with few false variants (positive predictive value of 0.98 and a sensitivity of 0.97) with no distinct FFPE artefacts. Ultimately, we apply carefully constructed forensic gene panels in a stepwise manner to find genetic variants associated with the clinical phenotype and with relevance to the sudden unexplained death.

Sudden cardiac death (SCD) is a tragic and traumatic event. SCD is often associated with hereditary genetic disease and in such cases, sequencing of stored formalin fixed paraffin embedded (FFPE) tissue is often crucial in trying to find a causal genetic variant. This study was designed to compare two massive parallel sequencing assays for differences in sensitivity and precision regarding variants related to SCD in FFPE material. From eight cases of SCD where DNA from blood had been sequenced using HaloPlex, corresponding FFPE samples were collected six years later. DNA from FFPE samples were amplified using HaloPlex HS, sequenced on MiSeq, representing the first method, as well as amplified using modified Twist and sequenced on NextSeq, representing the second method. Molecular barcodes were included to distinguish artefacts from true variants. In both approaches, read coverage, uniformity and variant detection were compared using genomic DNA isolated from blood and corresponding FFPE tissue, respectively. In terms of coverage uniformity, Twist performed better than HaloPlex HS for FFPE samples. Despite higher overall coverage, amplicon-based HaloPlex technologies, both for blood and FFPE tissue, suffered from design and/or performance issues resulting in genes lacking complete coverage. Although Twist had considerably lower overall mean coverage, high uniformity resulted in equal or higher fraction of genes covered at >= 20X. By comparing variants found in the matched samples in a pre-defined cardiodiagnostic gene panel, HaloPlex HS for FFPE material resulted in high sensitivity, 98.0% (range 96.6-100%), and high precision, 99.9% (range 99.5-100%) for moderately fragmented samples, but suffered from reduced sensitivity (range 74.2-91.1%) in more severely fragmented samples due to lack of coverage. Twist had high sensitivity, 97.8% (range 96.8-98.7%) and high precision, 99.9% (range 99.3-100%) in all analyzed samples, including the severely fragmented samples.

Carbapenem resistant Enterobacteriaceae (CRE) are a threat to public health globally, yet the role of the environment in the epidemiology of CRE remains elusive. Given that wild birds can acquire CRE, likely from foraging in anthropogenically impacted areas, and may aid in the maintenance and dissemination of CRE in the environment, a spatiotemporal comparison of isolates from different regions and timepoints may be useful for elucidating epidemiological information. Thus, we characterized the genomic diversity of CRE from fecal samples opportunistically collected from gulls (Larus spp.) inhabiting Alaska (USA), Chile, Spain, Turkey, and Ukraine and from black kites (Milvus migrans) sampled in Pakistan and assessed evidence for spatiotemporal patterns of dissemination. Within and among sampling locations, a high diversity of carbapenemases was found, including Klebsiella pneumoniae carbapenemase (KPC), New Delhi metallo-beta-lactamase (NDM), oxacillinase (OXA), and Verona integron Metallo beta-lactamase (VIM). Although the majority of genomic comparisons among samples did not provide evidence for spatial dissemination, we did find strong evidence for dissemination among Alaska, Spain, and Turkey. We also found strong evidence for temporal dissemination among samples collected in Alaska and Pakistan, though the majority of CRE clones were transitory and were not repeatedly detected among locations where samples were collected longitudinally. Carbapenemase-producing hypervirulent K. pneumoniae was isolated from gulls in Spain and Ukraine and some isolates harbored antimicrobial resistance genes conferring resistance to up to 10 different antibiotic classes, including colistin. Our results are consistent with local acquisition of CRE by wild birds with spatial dissemination influenced by intermediary transmission routes, likely involving humans. Furthermore, our results support the premise that anthropogenicallyassociated wild birds may be good sentinels for understanding the burden of clinically-relevant antimicrobial resistance in the local human population.

The human vagina harbor a rich microbiota. The optimal state is dominated by lactobacilli that help to maintain health and prevent various diseases. However, the microbiota may rapidly change to a polymicrobial state that has been linked to a number of diseases. In the present study, the temporal changes of the vaginal microbiota in patients treated for sexually transmitted diseases or bacterial vaginosis (BV) and in untreated controls were studied for 26 days. The patients included 52 women treated with azithromycin, tetracyclines or moxifloxacin for present or suspected infection withChlamydia trachomatisorMycoplasma genitalium. Women with concurrent BV were also treated with metronidazole. The controls were 10 healthy women of matching age. The microbiota was analyzed by 16S rRNA gene deep sequencing, specific qPCRs and microscopy. There was generally good correlation between Nugent score and community state type (CST) and qPCR confirmed the sequencing results. By sequencing, more than 600 different taxa were found, but only 33 constituted more than 1 parts per thousand of the sequences. In both patients and controls the microbiota could be divided into three different community state types, CST-I, CST-III and CST-IV. Without metronidazole, the microbiota remained relatively stable regarding CST although changes were seen during menstrual periods. Administration of metronidazole changed the microbiota from CST-IV to CST-III in approximately 50% of the treated patients. In contrast, the CST was generally unaffected by azithromycin or tetracyclines. In 30% of the BV patients,Gardnerella vaginaliswas not eradicated by metronidazole. The majority of women colonized withUreaplasma parvumremained positive after azithromycin whileU.urealyticumwas eradicated.

Objectives: Laser Doppler Flowmetry (LDF) is a non-invasive technique for the assessment of tissue blood flow, but increased reproducibility would facilitate longitudinal studies. The aim of the study was to assess the interday reproducibility of Laser Doppler Flowmetry (LDF) at rest, at elevated local temperatures, and with the use of the vasodilator Methyl Nicotinate (MN) in six interconnected protocols for the measurement of the blood supply to the microvascular bed of the gingiva. Methods: Ten healthy volunteers were included. Interweek LDF measurements with custom-made acrylic splints were performed. Six protocols were applied in separate regions of interest (ROI): 1; basal LDF, 2; LDF with thermoprobe 42 degrees C, 3; LDF with thermoprobe 45 degrees C, 4; LDF with thermoprobe 42 degrees C and MN, 5; LDF with thermoprobe 45 C and MN and 6; LDF with MN. Results: Intra-individual reproducibility was assessed by the within -subject coefficient of variation (wCV) and the intraclass correlation coefficient (ICC). Basal LDF measurements demonstrated high reproducibility with wCV 11.1 in 2 min and 10.3 in 5 min. ICC was 0.9 and 0.92. wCV after heat and MN was 4.9-10.3 and ICC 0.82-0.93. The topically applied MN yielded increased blood flow. Conclusion: This is the first study evaluating the reproducibility of basal LDF compared to single or multiple vasodilatory stimuli in gingiva. Multiple collector fibers probe and stabilizing acrylic splints are recommended. Vasodilatory stimulation showed a tendency toward higher reproducibility. Furthermore, MN yields vasodilation in gingiva.

Dermatitis herpetiformis has been investigated in the past; however, only a limited number of studies have reported its incidence based on validated nationwide population-based registries. To address this gap, the aims of this study are to estimate the incidence of dermatitis herpetiformis in Sweden and to validate the National Patient Register (NPR) for diagnosis of dermatitis herpetiformis. A population-based open cohort study was conducted, including all patients diagnosed with dermatitis herpetiformis (International Classification of Diseases 10th revision; ICD-10 code L13.0) in Sweden from 2005 to 2018 (n = 1,724), identified from the NPR. The diagnosis of dermatitis herpetiformis in the NPR was validated using medical records, histopathological and immunopathological data, yielding a positive predictive value (PPV) of 62.5%. The mean annual incidence of dermatitis herpetiformis was 0.93/100,000 (95% confidence interval 0.79-1.08), female to male ratio 1:1, and mean age at diagnosis 60.9 years. In conclusion, this large nationwide cohort study showed a low validity for diagnosis of dermatitis herpetiformis in the NPR, and the adjusted incidence rate of dermatitis herpetiformis in Sweden was estimated to be 0.93/100,000, which is lower than that in previous Swedish studies.

Evidence about the association of bullous pemphigoid and the risk of cancer is conflicting. Patients diagnosed with bullous pemphigoid (n = 5,739) between 2005 and 2016 were matched with a control cohort from the general population (n = 17,168) to estimate their overall and specific risk of cancer. The risk of squamous cell cancer of the skin (cSCC) was increased in patients with bullous pemphigoid (hazard ratio (HR) 1.3; 95% confidence interval (CI) 1.1-1.6). The risk of lymphoma within one year after bullous pemphigoid diagnosis was also increased (HR 3.1; 95% CI 1.3-7.6). While overall cancer risk prior to diagnosis of bullous pemphigoid was similar in cases and controls (prevalence odds ratio (POR) 1.0; 95% CI 0.9-1.0), the risk of male genital cancer within one year prior to diagnosis of bullous pemphigoid was lower in cases (POR 0.4; 95% CI 0.2-0.8). Clinicians must be aware of the increased risk of cSCC and lymphoma in patients with bullous pemphigoid.

Rising consumer demands for safer, more natural, and sustainable topical products have led to increased interest in finding alternative excipients, while retaining functionality and cosmetic appeal. Particle-stabilized Pickering creams have emerged as possible alternatives to replace traditional surfactant-stabilized creams and are thus one of the focuses in this study. The aim of this paper was to study relationships between sensorial characteristics and physical properties to understand how different excipients affect these aspects, comparing one starch particle-stabilized and three surfactant-stabilized formulations. A human panel was used to evaluate sensorial perception, while physical properties were deduced by rheology and tactile friction, together with in vivo and ex vivo skin hydration measurements. The results show that sensorial attributes related to the application phase can be predicted with rheology, while afterfeel attributes can be predicted with tactile friction studies. Differences in rheological and sensory properties among surfactant-based creams could mainly be attributed to the type of emollients used, presence of thickeners and surfactant composition. Differences between surfactant-based creams and a Pickering cream were more evident in relation to the afterfeel perception. Presence of starch particles in the residual film on skin results in high tactile friction and low perception of residual coating, stickiness, greasiness, and slipperiness in sensorial afterfeel.

African populations are underrepresented in medical genomics studies. For the Somali population, there is virtually no information on genomic markers with significance to precision medicine. Here, we analyzed nearly 900,000 genomic markers in samples collected from 95 unrelated individuals in the North Eastern Somalia. ADMIXTURE program for estimation of individual ancestries revealed a homogenous Somali population. Principal component analysis with PLINK software showed approximately 60% East African and 40% West Eurasian genes in the Somali population, with a close relation to the Cushitic and Semitic speaking Ethiopian populations. We report the unique features of human leukocyte antigens (HLA) in the Somali population, which seem to differentiate from all other neighboring regions compared. Current study identified high prevalence of the diabetes type 1 (T1D) predisposing HLA DR-DQ haplotypes in Somalia. This finding may explain the increased T1D risk observed among Somali children. In addition, ethnic Somalis were found to host the highest frequencies observed thus far for several pharmacogenetic variants, including UGT1A4*2. In conclusion, we report that the Somali population displays genetic traits of significance to health and disease. The Somali dataset is publicly available and will add more information to the few genomic datasets available for African populations.

Background Accurate predictions of tumor dissemination risks and medical treatment outcomes are critical to personalize therapy. Patient-derived xenograft (PDX) models in mice have demonstrated high accuracy in predicting therapeutic outcomes, but methods for predicting tumor invasiveness and early stages of vascular/lymphatic dissemination are still lacking. Here we show that a zebrafish tumor xenograft (ZTX) platform based on implantation of PDX tissue fragments recapitulate both treatment outcome and tumor invasiveness/dissemination in patients, within an assay time of only 3 days. Methods Using a panel of 39 non-small cell lung cancer PDX models, we developed a combined mouse-zebrafish PDX platform based on direct implantation of cryopreserved PDX tissue fragments into zebrafish embryos, without the need for pre-culturing or expansion. Clinical proof-of-principle was established by direct implantation of tumor samples from four patients. Results The resulting ZTX models responded to Erlotinib and Paclitaxel, with similar potency as in mouse-PDX models and the patients themselves, and resistant tumors similarly failed to respond to these drugs in the ZTX system. Drug response was coupled to elevated expression of EGFR, Mdm2, Ptch1 and Tsc1 (Erlotinib), or Nras and Ptch1 (Paclitaxel) and reduced expression of Egfr, Erbb2 and Foxa (Paclitaxel). Importantly, ZTX models retained the invasive phenotypes of the tumors and predicted lymph node involvement of the patients with 91% sensitivity and 62% specificity, which was superior to clinically used tests. The biopsies from all four patient tested implanted successfully, and treatment outcome and dissemination were quantified for all patients in only 3 days. Conclusions We conclude that the ZTX platform provide a fast, accurate, and clinically relevant system for evaluation of treatment outcome and invasion/dissemination of PDX models, providing an attractive platform for combined mouse-zebrafish PDX trials and personalized medicine.

SwedAD, a Swedish nationwide registry for patients with atopic dermatitis receiving systemic pharma-cotherapy, was launched on 1 September 2019. We describe here the establishment of a user-friendly re-gistry to the benefit of patients with atopic dermati-tis. By 5 November 2022, 38 clinics had recorded 931 treatment episodes in 850 patients with an approxi-mate national coverage rate of 40%. Characteristics at enrolment included median Eczema Area and Seve-rity Index (EASI) 10.2 (interquartile range 4.0, 19.4), Patient-Oriented Eczema Measure (POEM) 18.0 (10.0, 24.0), Dermatology Life Quality Index (DLQI) 11.0 (5.0, 19.0) and Peak Itch Numerical Rating Scale-11 (NRS-11) 6.0 (3.0, 8.0). At 3 months, median EASI was 3.2 (1.0, 7.3) and POEM, DLQI, and NRS-11 were im-proved. Regional coverage varied, reflecting the distri-bution of dermatologists, the ratio of public to private healthcare, and difficulties in recruiting certain clinics. This study highlights the importance of a nationwide registry when managing systemic pharmacotherapy of atopic dermatitis.

Comprehensive and scalable proteomic profiling of plasma samples can improve the screening and diagnosis of cancer patients. Here, the authors use the Olink Proximity Extension Assay technology to characterise the plasma proteomes of 1477 patients across twelve cancer types, and use machine learning to obtain a protein panel for cancer classification. A comprehensive characterization of blood proteome profiles in cancer patients can contribute to a better understanding of the disease etiology, resulting in earlier diagnosis, risk stratification and better monitoring of the different cancer subtypes. Here, we describe the use of next generation protein profiling to explore the proteome signature in blood across patients representing many of the major cancer types. Plasma profiles of 1463 proteins from more than 1400 cancer patients are measured in minute amounts of blood collected at the time of diagnosis and before treatment. An open access Disease Blood Atlas resource allows the exploration of the individual protein profiles in blood collected from the individual cancer patients. We also present studies in which classification models based on machine learning have been used for the identification of a set of proteins associated with each of the analyzed cancers. The implication for cancer precision medicine of next generation plasma profiling is discussed.

The objective of this study was to evaluate emotions of depression and anxiety in psoriatic patients that due to insufficient response to tumor necrosis factor-alpha inhibition (TNF-alpha), underwent a treatment switch from TNF-alpha to interleukin 17 inhibition using brodalumab. The Self-rated Montgomery-Asberg Depression Rating Scale and the Hospital Anxiety and Depression Scale were used to assess depression and anxiety. A total of 20 patients with psoriasis were enrolled in the study. They were monitored for a period of 3 months following the transition to brodalumab treatment. The results showed a significant improvement in both the Psoriasis Area and Severity Index as well as symptoms of depression; anxiety symptoms showed a reduction, though not statistically significant. Perhaps of more interest, the positive effects on depression and anxiety seem to be independent of the reduction in skin related psoriatic lesions. These findings highlight the importance of addressing depressive and anxiety symptoms, together with psoriasis severity and quality of life, when managing patients with psoriasis.

Background

Myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) is a chronic disease considered to be triggered by viral infections in a majority of cases. Symptoms overlap largely with those of post-acute sequelae of COVID-19/long-COVID implying common pathogenetic mechanisms. SARS-CoV-2 infection is risk factor for sustained latent virus reactivation that may account for the symptoms of post-viral fatigue syndromes. The aim of this study was first to investigate whether patients with ME/CFS and healthy donors (HDs) differed in their antibody response to mild/asymptomatic SARS-CoV-2 infection. Secondly, to analyze whether COVID-19 imposes latent virus reactivation in the cohorts.

Methods

Anti-SARS-CoV-2 antibodies were analyzed in plasma and saliva from non-vaccinated ME/CFS (n=95) and HDs (n=110) using soluble multiplex immunoassay. Reactivation of human herpesviruses 1-6 (HSV1, HSV2, VZV, EBV, CMV, HHV6), and human endogenous retrovirus K (HERV-K) was detected by anti-viral antibody fingerprints in saliva.

Results

At 3-6 months after mild/asymptomatic SARS-CoV-2 infection, virus-specific antibodies in saliva were substantially induced signifying a strong reactivation of latent viruses (EBV, HHV6 and HERV-K) in both cohorts. In patients with ME/CFS, antibody responses were significantly stronger, in particular EBV-encoded nuclear antigen-1 (EBNA1) IgG were elevated in patients with ME/CFS, but not in HDs. EBV-VCA IgG was also elevated at baseline prior to SARS-infection in patients compared to HDs.

Conclusion

Our results denote an altered and chronically aroused anti-viral profile against latent viruses in ME/CFS. SARS-CoV-2 infection even in its mild/asymptomatic form is a potent trigger for reactivation of latent herpesviruses (EBV, HHV6) and endogenous retroviruses (HERV-K), as detected by antibody fingerprints locally in the oral mucosa (saliva samples). This has not been shown before because the antibody elevation is not detected systemically in the circulation/plasma.

Myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) is a chronic disease presenting with severe fatigue, post-exertional malaise, and cognitive disturbances-among a spectrum of symptoms-that collectively render the patient housebound or bedbound. Epigenetic studies in ME/CFS collectively confirm alterations and/or malfunctions in cellular and organismal physiology associated with immune responses, cellular metabolism, cell death and proliferation, and neuronal and endothelial cell function. The sudden onset of ME/CFS follows a major stress factor that, in approximately 70% of cases, involves viral infection, and ME/CFS symptoms overlap with those of long COVID. Viruses primarily linked to ME/CFS pathology are the symbiotic herpesviruses, which follow a bivalent latent-lytic lifecycle. The complex interaction between viruses and hosts involves strategies from both sides: immune evasion and persistence by the viruses, and immune activation and viral clearance by the host. This dynamic interaction is imperative for herpesviruses that facilitate their persistence through epigenetic regulation of their own and the host genome. In the current article, we provide an overview of the epigenetic signatures demonstrated in ME/CFS and focus on the potential strategies that latent viruses-particularly Epstein-Barr virus-may employ in long-term epigenetic reprograming in ME/CFS. Epigenetic studies could aid in elucidating relevant biological pathways impacted in ME/CFS and reflect the physiological variations among the patients that stem from environmental triggers, including exogenous viruses and/or altered viral activity. image

Titanium dioxide (TiO₂) and silver (Ag) NPs are among the most used engineered inorganic nanoparticles (NPs); however, their potential effects to marine demersal fish species, are not fully understood. Therefore, this study aimed to assess the proteomic alterations induced by sub-lethal concentrations citrate-coated 25 nm ("P25") TiO₂ or polyvinylpyrrolidone (PVP) coated 15 nm Ag NPs to turbot, Scophthalmus maximus. Juvenile fish were exposed to the NPs through daily feeding for 14 days. The tested concentrations were 0, 0.75 or 1.5 mg of each NPs per kg of fish per day. The determination of NPs, Titanium and Ag levels (sp-ICP-MS/ICP-MS) and histological alterations (Transmission Electron Microscopy) supported proteomic analysis performed in the liver and kidney. Proteomic sample preparation procedure (SP3) was followed by LC-MS/MS. Label-free MS quantification methods were employed to assess differences in protein expression. Functional analysis was performed using STRING web-tool. KEGG Gene Ontology suggested terms were discussed and potential biomarkers of exposure were proposed. Overall, data shows that liver accumulated more elements than kidney, presented more histological alterations (lipid droplets counts and size) and proteomic alterations. The Differentially Expressed Proteins (DEPs) were higher in Ag NPs trial. The functional analysis revealed that both NPs caused enrichment of proteins related to generic processes (metabolic pathways). Ag NPs also affected protein synthesis and nucleic acid transcription, among other processes. Proteins related to thyroid hormone transport (Serpina7) and calcium ion binding (FAT2) were suggested as biomarkers of TiO₂ NPs in liver. For Ag NPs, in kidney (and at a lower degree in liver) proteins related with metabolic activity, metabolism of exogenous substances and oxidative stress (e.g.: NADH dehydrogenase and Cytochrome P450) were suggested as potential biomarkers. Data suggests adverse effects in turbot after medium/long-term exposures and the need for additional studies to validate specific biological applications of these NPs.

Background: Patients with type 1 diabetes have a high prevalence of upper extremity impairments (UEIs), such as frozen shoulder, carpal tunnel syndrome, and trigger finger. The UEIs are strongly associated with activity limitations and impaired quality of life. The etiology of the UEI is not clear. Vitamin D deficiency has been considered to play a role in the pathogenesis of type 1 diabetes and in the development of macro- and microvascular complications in diabetes. Aim: To characterize vitamin D status in a large population of patients with type 1 diabetes, if vitamin D deficiency is associated with metabolic factors and possible association with UEI. Material and methods: Patients who diagnosed before 35 years of age, whose diabetes duration >20 years, and who are not older than 65 years were invited to participate in this cross-sectional case-control, multicenter study. Controls matched for age and sex were obtained from the national population registry. Fasting blood samples were collected and stored at -80 degrees C until analyzed regarding 25-hydroxy-vitamin D (25(OH)D3) by a liquid chromatographic-mass spectrometric method (LC-MS/MS). Results: Vitamin D levels varied with season as expected in the northern hemisphere. The association between 25(OH)D3 and clinical variables was analyzed in a univariate general linear model, which indicated no difference in 25(OH)D3 in men with and without diabetes but higher values in women with diabetes. About 30% of both patients and controls had vitamin D deficiency (=50 nmol/L). Analyzed by binary logistic regression UEIs was not associated with 25(OH)D3 levels. In both patients and controls, 25(OH)D3 was correlated to apolipoprotein A1 (r = 0.153; 0.220, P < 0.001). Conclusion: In patients with type 1 diabetes and a duration of 20 years or more, vitamin D level is not lower than in nondiabetic controls and is not associated with UEIs.

OBJECTIVE To evaluate HbA(1c) followed from diagnosis, as a predictor of severe microvascular complications (i.e., proliferative diabetic retinopathy [PDR] and nephropathy [macroalbuminuria]). RESEARCH DESIGN AND METHODS In a population-based observational study, 447 patients diagnosed with type 1 diabetes before 35 years of age from 1983 to 1987 in southeast Sweden were followed from diagnosis until 2019. Long-term weighted mean HbA(1c) (wHbA(1c)) was calculated by integrating the area under all HbA(1c) values. Complications were analyzed in relation to wHbA(1c) categorized into five levels. RESULTS After 32 years, 9% had no retinopathy, 64% non-PDR, and 27% PDR, and 83% had no microalbuminuria, 9% microalbuminuria, and 8% macroalbuminuria. Patients with near-normal wHbA(1c) did not develop PDR or macroalbuminuria. The lowest wHbA(1c) values associated with development of PDR and nephropathy (macroalbuminuria) were 7.3% (56 mmol/mol) and 8.1% (65 mmol/mol), respectively. The prevalence of PDR and macroalbuminuria increased with increasing wHbA(1c), being 74% and 44% in the highest category, wHbA(1c) >9.5% (>80 mmol/mol). In comparison with the follow-up done after 20-24 years duration, the prevalence of PDR had increased from 14 to 27% and macroalbuminuria from 4 to 8%, and both appeared at lower wHbA(1c) values. CONCLUSIONS wHbA(1c) followed from diagnosis is a very strong biomarker for PDR and nephropathy, the prevalence of both still increasing 32 years after diagnosis. To avoid PDR and macroalbuminuria in patients with type 1 diabetes, an HbA(1c) <7.0% (53 mmol/mol) and as normal as possible should be recommended when achievable without severe hypoglycemia and with good quality of life.

Studies have shown differences in the skin and gut bacterial microbiomes in patients with psoriasis, but the pharyngeal microbiome has not been studied previously. The aim of this study was to investigate differences in the bacterial microbiome of the pharynx and skin of patients with psoriasis compared with healthy controls. Swabs were taken from the pharynx and el-bow skin of 39 patients with psoriasis and 70 controls. Microbiomes were characterized by sequencing 16S rRNA genes on the Illumina MiSeq platform. Significant differences were found in alpha and beta diversity in the skin, but not in the pharynx. Significant differences were also found between several phyla and genera in both skin and pharynx. The severity of psoriasis did not correlate with any genera in the pharynx, but with Capnocytophaga, Leptotrichia, Abiotrophia and Tannerella in the skin. The composition of the pharyngeal and skin microbiome may be of importance in the pathogenesis of psoriasis.

Maturation of lymphoid cells is controlled by the action of stage and lineage-restricted transcription factors working in concert with the general transcription and chromatin remodeling machinery to regulate gene expression. To better understand this functional interplay, we used Biotin Identification in human embryonic kidney cells to identify proximity interaction partners for GATA3, TCF7 (TCF1), SPI1, HLF, IKZF1, PAX5, ID1, and ID2. The proximity interaction partners shared among the lineagerestricted transcription factors included ARID1a, a BRG1-associated factor complex component. CUT&RUN analysis revealed that ARID1a shared binding with TCF7 and GATA3 at a substantial number of putative regulatory elements in mouse T cell progenitors. In support of an important function for ARID1a in lymphocyte development, deletion of Aridla in early lymphoid progenitors in mice resulted in a pronounced developmental arrest in early T cell development with a reduction of CD4(+)CD8(+) cells and a 20-fold reduction in thymic cellularity. Exploring gene expression patterns in DN3 cells from Wt and Aridla-deficient mice suggested that the developmental block resided in the DN3a to DN3b transition, indicating a deficiency in beta-selection. Our work highlights the critical importance of functional interactions between stage and lineage-restricted factors and the basic transcription machinery during lymphocyte differentiation.

Elderly patients represent a growing proportion of individuals with glioblastoma, who however, are often excluded from clinical trials owing to poor expected prognosis. We aimed at identifying age-related molecular differences that would justify and guide distinct treatment decisions in elderly glioblastoma patients. The combined DNA methylome (450 k) of four IDH wild-type glioblastoma datasets, comprising two clinical trial cohorts, was interrogated for differences based on the patients age, DNA methylation (DNAm) age acceleration (DNAm age "Horvath-clock" minus patient age), DNA methylation-based tumor classification (Heidelberg), entropy, and functional methylation of DNA damage response (DDR) genes. Age dependent methylation included 19 CpGs (p-value <= 0.1, Bonferroni corrected), comprising a CpG located in the ELOVL2 gene that is part of a 13-gene forensic age predictor. Most of the age related CpGs (n = 16) were also associated with age acceleration that itself was associated with a large number of CpGs (n = 50,551). Over 70% age acceleration-associated CpGs (n = 36,348) overlapped with those associated with the DNA methylation based tumor classification (n = 170,759). Gene set enrichment analysis identified associated pathways, providing insights into the biology of DNAm age acceleration and respective commonalities with glioblastoma classification. Functional methylation of several DDR genes, defined as correlation of methylation with gene expression (r <= -0.3), was associated with age acceleration (n = 8), tumor classification (n = 12), or both (n = 4), the latter including MGMT. DNAm age acceleration was significantly associated with better outcome in both clinical trial cohorts, whereof one comprised only elderly patients. Multivariate analysis included treatment (RT, RT/TMZ -> TMZ; TMZ, RT), MGMT promoter methylation status, and interaction with treatment. In conclusion, DNA methylation features of age acceleration are an integrative part of the methylation-based tumor classification (RTK I, RTK II, MES), while patient age seems hardly reflected in the glioblastoma DNA methylome. We found no molecular evidence justifying other treatments in elderly patients, not owing to frailty or co-morbidities.

Quorum sensing (QS) is a mode of cell-cell communication that bacteria use to sense population density and orchestrate collective behaviors. The common opportunistic human pathogen Pseudomonas aeruginosa employs QS to regulate a large set of genes involved in virulence and host-pathogen interactions. The Las circuit positioned on the top of the QS hierarchy in P. aeruginosa makes use of N-acyl-L-homoserine lactones (AHLs) as signal molecules, like N-3-oxo-dodecanoyl-L-homoserine lactone (3O-C12-HSL). Disabling QS circuits by certain small-molecule compounds, known as quorum-sensing inhibitors (QSIs), has been proposed as a strategy to attenuate bacterial pathogenicity. In this study, four new AHL analogs were designed by incorporating a tert-butoxycarbonyl Boc group in amide and beta-keto (3-oxo) moiety. Compounds were evaluated on a molecular and phenotypic basis as a QSI using the screening strategy linked to the assignment of the Las QS system in P. aeruginosa. Using a LasR-based bioreporter, we found that the compounds decreased LasR-controlled light activity and competed efficiently with natural 3O-C12-HSL. The compounds reduced the production of the cognate 3O-C12-HSL and certain virulence traits, like total protease activity, elastase activity, pyocyanin production, and extracellular DNA release. Furthermore, a quantitative proteomic approach was used to study the effect of the compounds on QS-regulated extracellular proteins. Among the four compounds tested, one of them showed the most significant difference in the appearance of the 3O-C12-HSL-responsive reference proteins related to QS communication and virulence, i.e., a distinct activity as a QSI. Moreover, by combining experimental data with computational chemistry, we addressed the effect of LasR protein flexibility on docking precision and assessed the advantage of using a multi-conformational docking procedure for binding mode prediction of LasR modulators. Thus, the four new AHL compounds were tested for their interaction with the AHL-binding site in LasR to identify the key interferences with the activity of LasR. Our study provides further insight into molecular features that are required for small-molecule modulation of LasR-dependent QS communication in P. aeruginosa. This should facilitate rational design of the next generation of antivirulence tools to study and manipulate QS-controlled fitness in bacteria and, thereby, handle bacterial infections in a new way.

Regulatory T cells are an important component of the immune system that plays a key role in maintaining homeostasis. Identification of distinct regulatory T cell subsets is essential to understand their function. Mass cytometry or CyTOF is a technology that enables the simultaneous measurement of up to 50 markers in single cells by using antibodies tagged with heavy metals, which are then detected with time-of-flight mass spectrometry. This chapter describes a mass cytometry approach for phenotypic characterization of regulatory T cells and determination of their master transcription factor Foxp3.

The specific viscoelastic mechanical properties of Achilles tendons are highly dependent on the structural characteristics of collagen at and between all hierarchical levels. Research has been conducted on the defor-mation mechanisms of positional tendons and single fibrils, but knowledge about the coupling between the whole tendon and nanoscale deformation mechanisms of more commonly injured energy-storing tendons, such as Achilles tendons, remains sparse. By exploiting the highly periodic arrangement of tendons at the nanoscale, in situ loading of rat Achilles tendons during small-angle X-ray scattering acquisition was used to investigate the collagen structural response during load to rupture, cyclic loading and stress relaxation. The fibril strain was substantially lower than the applied tissue strain. The fibrils strained linearly in the elastic region of the tissue, but also exhibited viscoelastic properties, such as an increased stretchability and recov-ery during cyclic loading and fibril strain relaxation during tissue stress relaxation. We demonstrate that the changes in the width of the collagen reflections could be attributed to strain heterogeneity and not changes in size of the coherently diffracting domains. Fibril strain heterogeneity increased with applied loads and after the toe region, fibrils also became increasingly disordered. Additionally, a thorough evaluation of radiation damage was performed. In conclusion, this study clearly displays the simultaneous structural response and adaption of the collagen fibrils to the applied tissue loads and provide novel information about the transition of loads between length scales in the Achilles tendon. (C) 2022 The Author(s). Published by Elsevier B.V. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/)

Recovery of the collagen structure following Achilles tendon rupture is poor, resulting in a high risk for re-ruptures. The loading environment during healing affects the mechanical properties of the tendon, but the relation between loading regime and healing outcome remains unclear. This is partially due to our limited understanding regarding the effects of loading on the micro-and nanostructure of the heal-ing tissue. We addressed this through a combination of synchrotron phase-contrast X-ray microtomog-raphy and small-angle X-ray scattering tensor tomography (SASTT) to visualize the 3D organization of microscale fibers and nanoscale fibrils, respectively. The effect of in vivo loading on these structures was characterized in early healing of rat Achilles tendons by comparing full activity with immobilization. Un-loading resulted in structural changes that can explain the reported impaired mechanical performance. In particular, unloading led to slower tissue regeneration and maturation, with less and more disorganized collagen, as well as an increased presence of adipose tissue. This study provides the first application of SASTT on soft musculoskeletal tissues and clearly demonstrates its potential to investigate a variety of other collagenous tissues.

Simple Summary The most prevalent and lethal primary brain tumor, glioblastoma multiforme (GBM), exhibits fast growth and widespread invasion and has a poor prognosis. The recurrence and mortality rates of GBM patients are still significant due to the intricacy of their molecular process. Therefore, screening GBM biomarkers is urgently required to demonstrate the therapy impact and enhance the prognosis. The findings of this study revealed 11 genes (UBC, HDAC1, CTNNB1, TRIM28, CSNK2A1, RBBP4, TP53, APP, DAB1, PINK1, and RELN), five miRNAs (has-mir-221-3p, hsa-mir-30a-5p, hsa-mir-15a-5p, has-mir-130a-3p, and hsa-let-7b-5p), six metabolites (HDL, N6-acetyl-L-lysine, cholesterol, formate, N, N-dimethylglycine/xylose, and X2. piperidinone), and 15 distinct signaling pathways that are essential for the development of GBM disease. The top genes, miRNAs, and metabolite signatures identified in this study may be used to develop early diagnosis procedures and construct individualized therapeutic approaches to GBM. The most aggressive primary malignant brain tumor in adults is glioblastoma (GBM), which has poor overall survival (OS). There is a high relapse rate among patients with GBM despite maximally safe surgery, radiation therapy, temozolomide (TMZ), and aggressive treatment. Hence, there is an urgent and unmet clinical need for new approaches to managing GBM. The current study identified modules (MYC, EGFR, PIK3CA, SUZ12, and SPRK2) involved in GBM disease through the NeDRex plugin. Furthermore, hub genes were identified in a comprehensive interaction network containing 7560 proteins related to GBM disease and 3860 proteins associated with signaling pathways involved in GBM. By integrating the results of the analyses mentioned above and again performing centrality analysis, eleven key genes involved in GBM disease were identified. ProteomicsDB and Gliovis databases were used for determining the gene expression in normal and tumor brain tissue. The NetworkAnalyst and the mGWAS-Explorer tools identified miRNAs, SNPs, and metabolites associated with these 11 genes. Moreover, a literature review of recent studies revealed other lists of metabolites related to GBM disease. The enrichment analysis of identified genes, miRNAs, and metabolites associated with GBM disease was performed using ExpressAnalyst, miEAA, and MetaboAnalyst tools. Further investigation of metabolite roles in GBM was performed using pathway, joint pathway, and network analyses. The results of this study allowed us to identify 11 genes (UBC, HDAC1, CTNNB1, TRIM28, CSNK2A1, RBBP4, TP53, APP, DAB1, PINK1, and RELN), five miRNAs (hsa-mir-221-3p, hsa-mir-30a-5p, hsa-mir-15a-5p, hsa-mir-130a-3p, and hsa-let-7b-5p), six metabolites (HDL, N6-acetyl-L-lysine, cholesterol, formate, N, N-dimethylglycine/xylose, and X2. piperidinone) and 15 distinct signaling pathways that play an indispensable role in GBM disease development. The identified top genes, miRNAs, and metabolite signatures can be targeted to establish early diagnostic methods and plan personalized GBM treatment strategies.

Current approaches based on electrophoretic, chromatographic or immunochemical principles have allowed characterizing multiple allergens, mapping their epitopes, studying their mechanisms of action, developing detection and diagnostic methods and therapeutic strategies for the food and pharmaceutical industry. However, some of the common structural features related to the allergenic potential of food proteins remain unknown, or the pathological mechanism of food allergy is not yet fully understood. In addition, it is also necessary to evaluate new allergens from novel protein sources that may pose a new risk for consumers. Technological development has allowed the expansion of advanced technologies for which their whole potential has not been entirely exploited and could provide novel contributions to still unexplored molecular traits underlying both the structure of food allergens and the mechanisms through which they sensitize or elicit adverse responses in human subjects, as well as improving analytical techniques for their detection. This review presents cutting-edge instrumental techniques recently applied when studying structural and functional aspects of proteins, mechanism of action and interaction between biomolecules. We also exemplify their role in the food allergy research and discuss their new possible applications in several areas of the food allergy field.

Alzheimers disease (AD) is the most common form of dementia, affecting millions of people and currently lacking available disease-modifying treatments. Appropriate disease models are necessary to investigate disease mechanisms and potential treatments. Drosophila melanogaster models of AD include the A beta fly model and the A beta PP-BACE1 fly model. In the A beta fly model, the A beta peptide is fused to a secretion sequence and directly overexpressed. In the A beta PP-BACE1 model, human A beta PP and human BACE1 are expressed in the fly, resulting in in vivo production of A beta peptides and other A beta PP cleavage products. Although these two models have been used for almost two decades, the underlying mechanisms resulting in neurodegeneration are not yet clearly understood. In this study, we have characterized toxic mechanisms in these two AD fly models. We detected neuronal cell death and increased protein carbonylation (indicative of oxidative stress) in both AD fly models. In the A beta fly model, this correlates with high A beta(1-42) levels and down-regulation of the levels of mRNA encoding lysosomal-associated membrane protein 1, lamp1 (a lysosomal marker), while in the A beta PP-BACE1 fly model, neuronal cell death correlates with low A beta(1-42) levels, up-regulation of lamp1 mRNA levels and increased levels of C-terminal fragments. In addition, a significant amount of A beta PP/A beta antibody (4G8)-positive species, located close to the endosomal marker rab5, was detected in the A beta PP-BACE1 model. Taken together, this study highlights the similarities and differences in the toxic mechanisms which result in neuronal death in two different AD fly models. Such information is important to consider when utilizing these models to study AD pathogenesis or screening for potential treatments.

The membrane proteins are essential targets for understanding cellular function. The unbiased identification of membrane protein targets is still the bottleneck for a system-level understanding of cellular response to stimuli or perturbations. It has been suggested to enrich the soluble proteome with membrane proteins by introducing nonionic surfactants in the solubilization solution. This strategy aimed to simultaneously identify the globular and membrane protein targets by thermal proteome profiling principles. However, the thermal shift assay would surpass the cloud point temperature from the nonionic surfactants frequently utilized for membrane protein solubilization. It is expected that around the cloud point temperature, the surfactant micelles would suffer structural modifications altering protein solubility. Here, we show that the presence of nonionic surfactants can alter protein thermal stability from a mixed, globular, and membrane proteome. In the presence of surfactant micelles, the changes in protein solubility analyzed after the thermal shift assay was affected by the thermally dependent modification of the micellar size and its interaction with proteins. We demonstrate that the introduction of nonionic surfactants for the solubilization of membrane proteins is not compatible with the principles of target identification by thermal proteome profiling methodologies. Our results lead to exploring thermally independent strategies for membrane protein solubilization to assure confident membrane protein target identification. The proteome-wide thermal shift methods have already shown their capability to elucidate mechanisms of action from pharma, biomedicine, analytical chemistry, or toxicology, and finding strategies, free from surfactants, to identify membrane protein targets would be the next challenge.

In today's post-genomic era, it is crucial to rethink the concept of model organisms. While a few historically well-established organisms, e.g. laboratory rodents, have enabled significant scientific breakthroughs, there is now a pressing need for broader inclusion. Indeed, new organisms and models, from complex microbial communities to holobionts, are essential to fully grasp the complexity of biological principles across the breadth of biodiversity. By fostering collaboration between biology, advanced molecular science and omics communities, we can collectively adopt new models, unraveling their molecular functioning, and uncovering fundamental mechanisms. This concerted effort will undoubtedly enhance human health, environmental quality, and biodiversity conservation. The concept of model organisms in biological studies needs to be re-evaluated to reflect novel technological advances and help further scientific discovery.

HIV transmission via genital and colorectal mucosa are the most common routes of dissemination. Here, we explored the effects of free and complement-opsonized HIV on colorectal tissue. Initially, there was higher antiviral responses in the free HIV compared to complementopsonized virus. The mucosal transcriptional response at 24 hr revealed the involvement of activated T cells, which was mirrored in cellular responses observed at 96 hr in isolated mucosal T cells. Further, HIV exposure led to skewing of T cell phenotypes predominantly to inflammatory CD4+ T cells, that is Th17 and Th1Th17 subsets. Of note, HIV exposure created an environment that altered the CD8+ T cell phenotype, for example expression of regulatory factors, especially when the virions were opsonized with complement factors. Our findings suggest that HIV-opsonization alters the activation and signaling pathways in the colorectal mucosa, which promotes viral establishment by creating an environment that stimulates mucosal T cell activation and inflammatory Th cells.

Inflammatory diseases, including psoriasis, are characterized by changes in redox regulation. The MTH1 prevents the incorporation of oxidized nucleotides during DNA replication. Using MTH1 small-molecule inhibitors, we found induced apoptosis through 8-oxodeoxyguanosine triphosphate accumulation and DNA double-strand breaks after oxidative stress in normal and malignant keratinocytes. In psoriasis, we detected increased MTH1 expression in lesional skin and PBMCs compared with that in the controls. Using the imiquimod psoriasis mouse model, we found that MTH1 inhibition diminished psoriatic histological characteristics and normalized the levels of neutrophils and T cells in the skin and skin-draining lymph nodes. The inhibition abolished the expression of T helper type 17-associated cytokines in the skin, which was in line with decreased levels of IL-17-producing gd T cells in lymph nodes. In human keratinocytes, MTH1 inhibition prevented the upregulation of IL-17-downstream genes, which was independent of ROS-induced apoptosis. In conclusion, our data support MTH1 inhibition using small molecules suitable for topical application as a promising therapeutic approach to psoriasis.

Introduction 

Higher education institutions (HEIs) face challenges assessing the relevance of educational programmes. Upon graduation, the student should have acquired knowledge and understanding, competence and skills as well as good judgement and approaches to operate in a changing labour market. Ideas on new programmes and courses mainly emanate from research findings identified at the HEIs. Needs and expectations from external stakeholders have the potential to further contribute if room for collaboration is created. Extensive rapid societal changes increase this need for collaboration. 

The Standards and Guidelines for Quality Assurance in the European Higher Education Area (ESG) state that higher education aims to fulfil multiple purposes, including preparing students for active citizenship, future careers, personal development and create a broad, advanced knowledge base and stimulate research and innovation (ENQA, 2015). However, different stakeholders may have other priorities. Therefore, quality assurance needs to take these different perspectives into account. 

Relevance is considered an aspect of quality in higher education, but many HEIs in Sweden lack structures, processes, and methods for assessing relevance and involving stakeholders in these processes.

This project aimed to increase the knowledge and provide methods to systematically assess relevance and use university-industry collaborations as tools for educational development. 

Methods/Approach 

This paper is a summary of the project MERUT focused on methods for assessment of relevance in higher education. The project was carried out during 2017-2020 with financial support from Vinnova (The Swedish Innovation Agency) and involved seven Swedish HEIs. The connection to future career paths is often stated as the primary factor to describe the relevance of educational programmes and was selected as the focus of MERUT. Data have been collected using workshops, meetings, literature reviews, interview studies, and surveys. Important parts of the work have been interviews with external stakeholders in different labour-market areas who, in various ways, are involved in higher education, most often in advisory boards. Also, interviews with quality coordinators at university programmes as well as at faculty and university management levels at each HEI have been carried out.

The seven participating Swedish universities in MERUT have been Karolinska Institutet, Kristianstad University, KTH Royal Institute of Technology, Mälardalen University, Linköping University, Stockholm University, and Umeå University.

Results The project resulted in knowledge, tools, and methods to work systematically with relevance in higher education. The results can be summarized as follows:

·         Interviews with HEIs showed that they collaborate with external stakeholders in many ways, primarily around teaching and learning and to a lesser extent around programme management and quality assurance. 

·         Further, the involvement of stakeholders varied both between and within the universities (faculties, subject areas, levels of education). Therefore, it is difficult to evaluate, in a systematic way, how collaboration is included in the quality systems of the HEIs. 

·         A toolbox with methods for how to involve external stakeholders in the process of assessing and developing the relevance of a study programme. These methods can be used in continuous quality work, in major curriculum revisions as well as in the establishment of new programmes.

·         A checklist for external stakeholders' involvement in educational development to facilitate and clarify roles, structures, and tasks in connection with external stakeholders in both the development and operational phases of a study programme. 

The results show that there are many similarities between the HEIs in the study in terms of relevance assessment and dimensioning decisions. However, the potential of a systematic collaboration with stakeholders and society for relevance assessment and dimensioning of education is not yet fully being realised. 

Conclusion 

HEIs interact with external stakeholders in many ways within education. However, it is rare to find examples where external stakeholders are involved in the quality assurance process, at least not in a systematic way. The MERUT project has developed recommendations for collaboration perspectives and stakeholder participation in the governing of educational programmes. A systematic dialogue and interaction with stakeholders contribute to a mutual understanding of different stakeholder groups’ needs and expectations, and their view of quality of higher education. Furthermore, to consider relevance as a quality aspect creates a basis for a more methodical assessment process where external stakeholders can contribute in a clear role. MERUT has developed a toolbox and a checklist to facilitate such systematic interaction and collaboration with stakeholder groups. A conclusion of  this project is that a reciprocal, transparent, and systematic approach leads to a sustainable educational collaboration with improved quality and relevance of higher education.

It would constitute a large gain for society if the HEIs are able to systematically and by efficient processes take external stakeholders’ and societal needs and expectations into account when building comprehensive and systematic relevance assessment processes and in dimensioning of education.  

References

European Association for Quality Assurance in Higher Education (ENQA). (2015). Standards and Guidelines for Quality Assurance in the European Higher Education Area (ESG). Brussels, EURASHE.

Epigenetic reprogramming of innate immune cells by beta-glucan in a process called trained immunity, leads to an enhanced host response to a secondary infection. beta-glucans are structural components of plants, algae, fungi and bacteria and thus recognized as non-self by human macrophages. We selected the beta-glucans curdlan from Alcaligenes faecalis, WGP dispersible from Saccharomyces cerevisiae, and beta-glucan-rich culture supernatant of Alternaria and investigated whether they could produce trained immunity effects leading to an increased control of virulent Mycobacterium tuberculosis. We observed a significant M. tuberculosis growth-reduction in macrophages trained with curdlan and Alternaria, which also correlated with increased IL-6 and IL-1 beta release. WGP dispersible-trained macrophages were stratified into non responders and responders, according to their ability to control M. tuberculosis, with responders producing higher IL-6 levels. The addition of neutrophils to infected macrophage cultures further enhanced macrophage control of virulent M. tuberculosis, but not in a stimuli-dependent manner. Pathway enrichment analysis of DNA methylome data also highlighted hypomethylation of genes in pathways associated with signaling and cellular reorganization and motility, and responders to WGP-training were enriched in the interferon-gamma signaling pathway. This study adds evidence that certain beta-glucans show promise as immune training agents.

Supraphysiological loading induced by unstable orthopedic implants initiates osteoclast formation, which results in bone degradation. We aimed to investigate which mechanosensitive cells in the peri-implant environment produce osteoclast-stimulating factors and how the production of these factors is stimulated by supraphysiological loading. The release of osteoclast-stimulating factors by different types of isolated bone marrow-derived hematopoietic and mesenchymal stem cells from six osteoarthritic patients was analyzed after one hour of supraphysiological loading (3.0 ± 0.2 Pa, 1 Hz) by adding their conditioned medium to osteoclast precursors. Monocytes produced factors that enhanced osteoclastogenesis by 1.6 ± 0.07-fold and mesenchymal stem cells by 1.4 ± 0.07-fold. Medium from osteoprogenitors and pre-osteoblasts enhanced osteoclastogenesis by 1.3 ± 0.09-fold and 1.4 ± 0.03-fold, respectively, where medium from four patients elicited a response and two did not. Next generation sequencing analysis of osteoprogenitors revealed that genes encoding for inflammation-related pathways and cytoskeletal rearrangements were regulated differently between responders and non-responders. Our data suggest that released osteoclast-stimulating soluble factors by progenitor cells in the bone marrow after supraphysiological loading may be related to cytoskeletal arrangement in an inflammatory environment. This connection could be relevant to better understand the aseptic loosening process of orthopedic implants.

Low-intensity loading maintains or increases bone mass, whereas lack of mechanical loading and high-intensity loading decreases bone mass, possibly via the release of extracellular vesicles by mechanosensitive bone cells. How different loading intensities alter the biological effect of these vesicles is not fully understood. Dynamic fluid shear stress at low intensity (0.7 +/- 0.3 Pa, 5 Hz) or high intensity (2.9 +/- 0.2 Pa, 1 Hz) was used on mouse hematopoietic progenitor cells for 2 min in the presence or absence of chemical compounds that inhibit release or biogenesis of extracellular vesicles. We used a Receptor activator of nuclear factor kappa-Beta ligand-induced osteoclastogenesis assay to evaluate the biological effect of different fractions of extracellular vesicles obtained through centrifugation of medium from hematopoietic stem cells. Osteoclast formation was reduced by microvesicles (10 000x g) obtained after low-intensity loading and induced by exosomes (100 000x g) obtained after high-intensity loading. These osteoclast-modulating effects could be diminished or eliminated by depletion of extracellular vesicles from the conditioned medium, inhibition of general extracellular vesicle release, inhibition of microvesicle biogenesis (low intensity), inhibition of ESCRT-independent exosome biogenesis (high intensity), as well as by inhibition of dynamin-dependent vesicle uptake in osteoclast progenitor cells. Taken together, the intensity of mechanical loading affects the release of extracellular vesicles and change their osteoclast-modulating effect. The intensity of mechanical loading strongly affects bone remodeling by either formation of bone or resorption of bone. Low-intensity loading on bone cells releases microvesicles that reduce formation of bone-resorbing osteoclasts, while high-intensity loading on bone cells releases exosomes that induce formation of bone-resorbing osteoclasts. The graphical abstract was created with image

To date, it is unclear how fluid dynamics stimulate mechanosensory cells to induce an osteoprotective or osteodestructive response. We investigated how murine hematopoietic progenitor cells respond to 2 minutes of dynamic fluid flow stimulation with a precisely controlled sequence of fluid shear stresses. The response was quantified by measuring extracellular adenosine triphosphate (ATP), immunocytochemistry of Piezo1, and sarcoplasmic/endoplasmic Ca2+ reticulum ATPase 2 (SERCA2), and by the ability of soluble factors produced by mechanically stimulated cells to modulate osteoclast differentiation. We rejected our initial hypothesis that peak wall shear stress rate determines the response of hematopoietic progenitor cells to dynamic fluid shear stress, as it had only a minor correlation with the abovementioned parameters. Low stimulus amplitudes corresponded to activation of Piezo1, SERCA2, low concentrations of extracellular ATP, and inhibition of osteoclastogenesis and resorption area, while high amplitudes generally corresponded to osteodestructive responses. At a given amplitude (3 Pa) and waveform (square), the duration of individual stimuli (duty cycle) showed a strong correlation with the release of ATP and osteoclast number and resorption area. Collectively, our data suggest that hematopoietic progenitor cells respond in a viscoelastic manner to loading, since a combination of high shear stress amplitude and prolonged duty cycle is needed to trigger an osteodestructive response. Plain Language Summary In case of painful joints or missing teeth, the current intervention is to replace them with an implant to keep a high-quality lifestyle. When exercising or chewing, the cells in the bone around the implant experience mechanical loading. This loading generally supports bone formation to strengthen the bone and prevent breaking, but can also stimulate bone loss when the mechanical loading becomes too high around orthopedic and dental implants. We still do not fully understand how cells in the bone can distinguish between mechanical loading that strengthens or weakens the bone. We cultured cells derived from the bone marrow in the laboratory to test whether the bone loss response depends on (i) how fast a mechanical load is applied (rate), (ii) how intense the mechanical load is (amplitude), or (iii) how long each individual loading stimulus is applied (duration). We mimicked mechanical loading as it occurs in the body, by applying very precisely controlled flow of fluid over the cells. We found that a mechanosensitive receptor Piezo1 was activated by a low amplitude stimulus, which usually strengthens the bone. The potential inhibitor of Piezo1, namely SERCA2, was only activated by a low amplitude stimulus. This happened regardless of the rate of application. At a constant high amplitude, a longer duration of the stimulus enhanced the bone-weakening response. Based on these results we deduce that a high loading amplitude tends to be bone weakening, and the longer this high amplitude persists, the worse it is for the bone.

Lipopolysaccharide (LPS) induces fever through cytokines like receptor-activator of nuclear factor kappa B ligand (RANKL), triggering mediators like prostaglandins (PG), endothelin-1 (ET-1), corticotrophin-releasing factor (CRF), substance P (SP) and endogenous opioids. LPS-induced fever is reduced in females compared with males except in ovariectomized (OVX) females which show increased fever mediated by PG. The present study aimed to identify the mediators involved in fever in intact and OVX female rats. Fever was induced with LPS (50 mu g/kg) intraperitoneally or CRF (2.5 mu g), ET-1 (1 pg), morphine (10 mu g) and SP (500 ng) intracerebroventricularly in sham-operated and OVX rats. The role of RANKL was evaluated with osteoprotegerin (OPG, 1 mu g, intracerebroventricularly). Expression of RANK, CRFI/II, ETB, mu-opioid (MOR) and NK1 receptors was evaluated by confocal microscopy. Besides LPS, only morphine induced fever in OVX rats while all mediators induced fever in sham-operated animals. OPG abolished LPS-induced fever in OVX but not sham-operated animals. Overall, fever involves similar central mediators in cycling females and males but only morphine induced fever in OVX females. Importantly, RANK/RANKL participates in LPS-induced fever in OVX females, as in males but not in cycling females.

Objectives Seizures as presenting symptom of glioblastoma (GBM) are known to predict prolonged survival, whereas the clinical impact of other initial symptoms is less known. Our main objective was to evaluate the influence of different presenting symptoms on survival in a clinical setting. We also assessed lead times, tumour size and localization. Methods Medical records of 189 GBM patients were reviewed regarding the first medical appointment, presenting symptom/s, date of diagnostic radiology and survival. Tumour size, localization and treatment data were retrieved. Overall survival was calculated using Kaplan-Meier and Mann-Whitney U test. Cox regression was used for risk estimation. Results Cognitive impairment as the initial symptom was often misinterpreted in primary health care leading to a delayed diagnosis. Initial global symptoms (66% of all patients) were associated with reduced survival compared to no global symptoms (median 8.4 months vs. 12.6 months). Those with the most common cognitive dysfunctions: change of behaviour, memory impairment and/or disorientation had a reduced median survival to 6.4 months. In contrast, seizures (32%) were associated with longer survival (median 11.2 months vs. 8.3 months). Global symptoms were associated with larger tumours than seizures, but tumour size had no linear association with survival. The setting of the first medical appointment was evenly distributed between primary health care and emergency units. Conclusion Patients with GBM presenting with cognitive symptoms are challenging to identify, have larger tumours and reduced survival. In contrast, epileptic seizures as the first symptom are associated with longer survival and smaller tumours.

Obesity is a worldwide epidemic and natural products may hold promise in its treatment. The chlorophyll derivative 13-2-hydroxypheophytine (hpa) was isolated in a screen with zebrafish larvae to identify lipid reducing molecules from cyanobacteria. However, the mechanisms underlying the lipid-reducing effects of hpa in zebrafish larvae remain poorly understood. Thus, investigating the mechanism of action of hpa and validation in other model organisms such as mice represents important initial steps.In this study, we identified 14 protein targets of hpa in zebrafish larvae by thermal proteome profiling, and selected two targets (malate dehydrogenase and pyruvate kinase) involved in cellular metabolism for further validation by enzymatic measurements. Our findings revealed a dose-dependent inhibition of pyruvate kinase by hpa exposure using protein extracts of zebrafish larvae in vitro, and in exposure experiments from 3 to 5 days post fertilization in vivo. Analysis of untargeted metabolomics of zebrafish larvae detected 940 mass peaks (66 increased, 129 decreased) and revealed that hpa induced the formation of various phospholipid species (phosphoinositol, phosphoethanolamine, phosphatidic acid). Inter-species validation showed that brown adipocytes exposed to hpa significantly reduced the size of lipid droplets, increased maximal mitochondrial respiratory capacity, and the expression of PPARy during adipocyte differentiation.In line with our data, previous work described that reduced pyruvate kinase activity lowered hepatic lipid content via reduced pyruvate and citrate, and improved mitochondrial function via phospholipids. Thus, our data provide new insights into the molecular mechanism underlying the lipid reducing activities of hpa in zebrafish larvae, and species overlapping functions in reduction of lipids.

Background Autosomal recessive congenital hereditary corneal dystrophy (CHED) is a rare isolated developmental anomaly of the eye characterised by diffuse bilateral corneal clouding that may lead to visual impairment requiring corneal transplantation. CHED is known to be caused by mutations in the solute carrier family 4 member 11 (SLC4A11) gene which encodes a membrane transporter protein (sodium bicarbonate transporter-like solute carrier family 4 member 11). Methods To identify SLC4A11 gene mutations associated with CHED (OMIM: #217700), genomic DNA was extracted from whole blood and sequenced for all exons and intron-exon boundaries in two large Tunisian families. Results A novel deletion SLC4A11 mutation (p. Leu479del; c.1434_1436del) is responsible for CHED in both analysed families. This non-frameshift mutation was found in a homozygous state in affected members and heterozygous in non-affected members. In silico analysis largely support the pathogenicity of this alteration that may leads to stromal oedema by disrupting the osmolarity balance. Being localised to a region of alpha-helical secondary structure, Leu479 deletion may induce protein-compromising structural rearrangements. Conclusion To the best of our knowledge, this is the first clinical and genetic study exploring CHED in Tunisia. The present work also expands the list of pathogenic genotypes in SLC4A11 gene and its associated clinical diagnosis giving more insights into genotype-phenotype correlations.

Methylglyoxal (MG) is a highly reactive dicarbonyl and the main precursor of advanced glycation end-products (AGEs). After myocardial infarction (MI), MG-derived AGEs accumulate in the heart and contribute to adverse remodeling and loss of cardiac function. In this study, the flavonoid fisetin, a dicarbonyl scavenger, is used to reduce the negative effects of MG in the post-MI heart. A fisetin-loaded collagen type I hydrogel (fisetin-HG) is injected intramyocardially in mice at 3 h post-MI, and compared to fisetin-alone, hydrogel-alone, or saline treatment. Fisetin-HG treatment increases the level of glyoxalase-1 (the main MG-metabolizing enzyme), reduces MG-AGE accumulation, and decreases oxidative stress in the MI heart, which is associated with smaller scar size and improved cardiac function. Treatment with fisetin-HG also promotes neovascularization and increases the number of pro-healing macrophages in the infarct area, while reducing the number of pro-inflammatory macrophages. Taken together, the results demonstrate that the fisetin-collagen hydrogel therapy can reduce the accumulation and negative effects of MG post-MI. This therapy may be a promising approach to limit adverse cardiac remodeling, prevent damage, and preserve function of the infarcted heart.

Two methods combining survival and health-related quality of life (HRQoL) data in glioma trials to calculate the "net clinical benefit" were evaluated: Quality-adjusted effect sizes (QASES) and joint modeling (JM).

Background. Different analytical methods may lead to different conclusions about the impact of treatment on health-related quality of life (HRQoL). This study aimed to examine 3 different methods to evaluate change in HRQoL and to study whether these methods result in different conclusions. Methods. HRQoL data from 15 randomized clinical trials were combined (CODAGLIO project). Change in HRQoL scores, measured with the European Organization for Research and Treatment of Cancer Quality of Life Questionnaire Core 30 and BN20 questionnaires, was analyzed in 3 ways: (1) at the group level, comparing mean changes in scale/item scores between treatment arms, (2) at the patient level per scale/item, calculating the percentage of patients that deteriorated, improved, or remained stable per scale/item, and (3) at the individual patient level, combining all scales/items. Results. Baseline and first follow-up HRQoL data were available for 3727 patients. At the group scale/item level, only the item "hair loss" showed a significant and clinically relevant change (ie, >= 10 points) over time, whereas change scores on the other scales/items were statistically significant only (all P <.001; range in change score, 0.1-6.2). Although a large proportion of patients had stable HRQoL over time (range, 27%-84%) on the patient level per scale/item, many patients deteriorated (range, 6%-43%) or improved (range, 8%-32%) on a specific scale/item. At the individual patient level, the majority of patients (86%) showed both deterioration and improvement, whereas only 1% remained stable on all scales. Conclusions. Different analytical methods of changes in HRQoL result in distinct conclusions of treatment effects, all of which may be relevant for informing clinical decision making.

Background Maintenance of functioning and well-being during the progression-free survival (PFS) period is important for glioma patients. This study aimed to determine whether health-related quality of life (HRQoL) can be maintained during progression-free time, and factors associated with HRQoL deterioration in this period. Methods We included longitudinal HRQoL data from previously published clinical trials in glioma. The percentage of patients with stable HRQoL until progression was determined per scale and at the individual patient level (i.e. considering all scales simultaneously). We assessed time to a clinically relevant deterioration in HRQoL, expressed in deterioration-free survival and time-to-deterioration (the first including progression as an event). We also determined the association between sociodemographic and clinical factors and HRQoL deterioration in the progression-free period. Results Five thousand five hundred and thirty-nine patients with at least baseline HRQoL scores had a median time from randomization to progression of 7.6 months. Between 9-29% of the patients deteriorated before disease progression on the evaluated HRQoL scales. When considering all scales simultaneously, 47% of patients deteriorated on >= 1 scale. Median deterioration-free survival period ranged between 3.8-5.4 months, and median time-to-deterioration between 8.2-11.9 months. For most scales, only poor performance status was independently associated with clinically relevant HRQoL deterioration in the progression-free period. Conclusions HRQoL was maintained in only 53% of patients in their progression-free period, and treatment was not independently associated with this deterioration in HRQoL. Routine monitoring of the patients functioning and well-being during the entire disease course is therefore important, so that interventions can be initiated when problems are signaled.

Simple Summary In the pig industry, new market trends and consumer demands have emerged over the past decades, which includes increased concerns about how animals are raised on farms. As a consequence of consumers concerns, technologies capable of predicting animal welfare on farms have been explored. One of the technologies that are permeating the frontier of knowledge in this area are epigenetic biomarkers. Epigenetic biomarkers are biochemical markers surrounding the genome, which may be able to predict the exposures that individuals had during their lifetime. These markers represent an advance in the molecular level accuracy to support the current welfare indicators. In this literature review focused on pigs, we show some studies already carried out, we performed an integrative analysis of the already reported genes surrounding epi-biomarkers, and we highlight the benefits of investing efforts in this research field to enhance animal welfare and consumers trust. Epigenetics works as an interface between the individual and its environment to provide phenotypic plasticity to increase individual adaptation capabilities. Recently, a wide variety of epi-genetic findings have indicated evidence for its application in the development of putative epi-biomarkers of stress in farm animals. The purpose of this study was to evaluate previously reported stress epi-biomarkers in swine and encourage researchers to investigate potential paths for the development of a robust molecular tool for animal welfare certification. In this literature review, we report on the scientific concerns in the swine production chain, the management carried out on the farms, and the potential implications of these practices for the animals welfare and their epigenome. To assess reported epi-biomarkers, we identified, from previous studies, potentially stress-related genes surrounding epi-biomarkers. With those genes, we carried out a functional enrichment analysis of differentially methylated regions (DMRs) of the DNA of swine subjected to different stress-related conditions (e.g., heat stress, intrauterine insult, and sanitary challenges). We identified potential epi-biomarkers for target analysis, which could be added to the current guidelines and certification schemes to guarantee and certify animal welfare on farms. We believe that this technology may have the power to increase consumers trust in animal welfare.

Objective To identify and characterize genetic loci associated with the risk of developing ANCA-associated vasculitides (AAV).

Methods Genetic association analyses were performed after Illumina sequencing of 1853 genes and subsequent replication with genotyping of selected single nucleotide polymorphisms in a total cohort of 1110 Scandinavian cases with granulomatosis with polyangiitis or microscopic polyangiitis, and 1589 controls. A novel AAV-associated single nucleotide polymorphism was analysed for allele-specific effects on gene expression using luciferase reporter assay.

Results PR3-ANCA⁺ AAV was significantly associated with two independent loci in the HLA-DPB1/HLA-DPA1 region [rs1042335, P = 6.3 x 10^-61, odds ratio (OR) 0.10; rs9277341, P = 1.5 x 10^-44, OR 0.22] and with rs28929474 in the SERPINA1 gene (P = 2.7 x 10^-10, OR 2.9). MPO-ANCA⁺ AAV was significantly associated with the HLA-DQB1/HLA-DQA2 locus (rs9274619, P = 5.4 x 10^-25, OR 3.7) and with a rare variant in the BACH2 gene (rs78275221, P = 7.9 x 10^-7, OR 3.0), the latter a novel susceptibility locus for MPO-ANCA⁺ granulomatosis with polyangiitis/microscopic polyangiitis. The rs78275221-A risk allele reduced luciferase gene expression in endothelial cells, specifically, as compared with the non-risk allele.

Conclusion We identified a novel susceptibility locus for MPO-ANCA⁺ AAV and propose that the associated variant is of mechanistic importance, exerting a regulatory function on gene expression in specific cell types.

Polymyositis and inclusion body myositis are idiopathic inflammatory myopathies, with a pathology characterized by partial invasion of non-necrotic muscle fibres by CD8(+)cytotoxic T-cells, leading to fibre degeneration. Although the main effector pathway of CD8(+)T-cells is to induce apoptosis of target cells, it has remained unclear if apoptosis occurs in these diseases, and if so, if it is mediated by CD8(+)T-cells. In consecutive biopsy sections from 10 patients with partial invasion, muscle fibres and inflammatory cells were assessed by immunohistochemistry and apoptotic nuclei by the TUNEL assay. Analysis of muscle fibre morphology, staining pattern and quantification were performed on digital images, and they were compared with biopsies from 10 dermatomyositis patients and 10 controls without muscle disease. Apoptotic myonuclei were found in muscle with partial invasion, but not in the invaded fibres. Fibres with TUNEL positive nuclei were surrounded by CD8(+)T-cells, granzyme B(+)cells and macrophages, but lacked FAS receptor expression. In contrast, apoptotic myonuclei were rare in dermatomyositis and absent in controls. The findings confirm that apoptosis occurs in idiopathic inflammatory myopathies and support that it is mediated by CD8(+)cytotoxic T- cells, acting in parallel to the process of partial invasion.