Sex-specific associations between particulate matter exposure and gene expression in independent discovery and validation cohorts of middle-aged men and women

BACKGROUND: Particulate matter (PM) exposure leads to premature death, mainly due to respiratory and cardiovascular diseases. OBJECTIVES: Identification of transcriptomic biomarkers of air pollution exposure and effect in a healthy adult population. METHODS: Microarray analyses were performed in 98 healthy volunteers (48 men, 50 women). The expression of eight sex-specific candidate biomarker genes (significantly associated with PM(10) in the discovery cohort and with a reported link to air pollution-related disease) was measured with qPCR in an independent validation cohort (75 men, 94 women). Pathway analysis was performed using Gene Set Enrichment Analysis. Average daily PM(2.5) and PM(10) exposures over 2-years were estimated for each participant’s residential address using spatiotemporal interpolation in combination with a dispersion model. RESULTS: Average long-term PM(10) was 25.9 (± 5.4) and 23.7 (± 2.3) μg/m(3) in the discovery and validation cohorts, respectively. In discovery analysis, associations between PM(10) and the expression of individual genes differed by sex. In the validation cohort, long-term PM(10) was associated with the expression of DNAJB5 and EAPP in men and ARHGAP4 (p = 0.053) in women. AKAP6 and LIMK1 were significantly associated with PM(10) in women, although associations differed in direction between the discovery and validation cohorts. Expression of the eight candidate genes in the discovery cohort differentiated between validation cohort participants with high versus low PM(10) exposure (area under the receiver operating curve = 0.92; 95% CI: 0.85, 1.00; p = 0.0002 in men, 0.86; 95% CI: 0.76, 0.96; p = 0.004 in women). CONCLUSIONS: Expression of the sex-specific candidate genes identified in the discovery population predicted PM(10) exposure in an independent cohort of adults from the same area. Confirmation in other populations may further support this as a new approach for exposure assessment, and may contribute to the discovery of molecular mechanisms for PM-induced health effects. CITATION: Vrijens K, Winckelmans E, Tsamou M, Baeyens W, De Boever P, Jennen D, de Kok TM, Den Hond E, Lefebvre W, Plusquin M, Reynders H, Schoeters G, Van Larebeke N, Vanpoucke C, Kleinjans J, Nawrot TS. 2017. Sex-specific associations between particulate matter exposure and gene expression in independent discovery and validation cohorts of middle-aged men and women. Environ Health Perspect 125:660–669; http://dx.doi.org/10.1289/EHP37

Golgi function and dysfunction in the first COG4-deficient CDG type II patient

The conserved oligomeric Golgi (COG) complex is a hetero-octameric complex essential for normal glycosylation and intra-Golgi transport. An increasing number of congenital disorder of glycosylation type II (CDG-II) mutations are found in COG subunits indicating its importance in glycosylation. We report a new CDG-II patient harbouring a p.R729W missense mutation in COG4 combined with a submicroscopical deletion. The resulting downregulation of COG4 expression additionally affects expression or stability of other lobe A subunits. Despite this, full complex formation was maintained albeit to a lower extent as shown by glycerol gradient centrifugation. Moreover, our data indicate that subunits are present in a cytosolic pool and full complex formation assists tethering preceding membrane fusion. By extending this study to four other known COG-deficient patients, we now present the first comparative analysis on defects in transport, glycosylation and Golgi ultrastructure in these patients. The observed structural and biochemical abnormalities correlate with the severity of the mutation, with the COG4 mutant being the mildest. All together our results indicate that intact COG complexes are required to maintain Golgi dynamics and its associated functions. According to the current CDG nomenclature, this newly identified deficiency is designated CDG-IIj

Genetic and cell-biological analysis of defects in Golgi structure and function in novel CDG-IIx patients

Glycosylatie, het aanhechten van suikerketens aan eiwitten, is één van de belangrijkste post-translationele modificaties. Gezien het belang ervan in de stabiliteit, opvouwing en interactie van eiwitten is het ni et verwonderlijk dat defecten in glycosylatie aan de basis liggen van ee n steeds groter wordende groep van aangeboren glycosylatiestoornissen, m et name de Congenital Disorders of Glycosylation,. De symptomen bij deze aandoeningen zijn vaak zeer ernstig en hebben betrekking op verschillen de orgaansystemen, waarbij neurologische en leverafwijkingen het meest v oorkomend zijn. De basis voor dit project was de ontdekking van mutaties in één van de subeenheden van het Conserved Oligomeric Golgi (COG) complex, betrokken in intra-Golgi en Golgi-naar-ER transport. Deze obse rvatie toonde duidelijk aan dat normaal intracellulair transport essenti eel is voor de glycosylatie ter hoogte van het Golgi apparaat. In de grote groep van onopgeloste CDG-II patiënten, waarover we beschikk en in ons centrum, werd op twee verschillende manieren gezocht naar even tuele mutaties in eiwitten betrokken in intracellulair transport. In de eerste plaats hebben we een screening aan de hand van western blot uitge voerd op 21 patiëntencellijnen, voor 34 verschillende eiwitten betrokken in het transport, de structuur en de werking van het Golgi apparaat. Di t onderzoek heeft echter niet onmiddellijk geleid tot de identificatie v an een oorzakelijk gen. In de toekomst kunnen, na de ontdekking van een gendefect, deze data wel gebruikt worden om na te gaan welke eiwitten hi erdoor gedestabiliseerd zijn. Als tweede methode hebben we gebruik gemaa kt van de Brefeldin A (BFA) assay, waarmee we defecten in anterograad en retrograad transport tussen het endoplasmitisch reticulum (ER) en het G olgi kunnen bestuderen. In verschillende patiëntencellijnen werden vertr agingen in het retrograde Golgi-naar-ER transport opgemerkt. In het tran sport van het ER naar het Golgi apparaat hebben we echter geen defecten kunnen identificeren. Door het combineren van beide methoden zijn we eri n geslaagd om verschillende gevallen op te helderen: we hebben patiënten gevonden met defecten in een component van het v-ATPase. Ook werd een g enomische screening voor COG defecten op punt gesteld. Tenslotte hebben we een begin gemaakt met de toepassing van de nieuwste grootschalige seq uencing methoden voor de identificatie van nieuwe oorzakelijke genen bij CDG-Iix patiënten. De combinatie van een puntmutatie en een deletie in de COG4-deficiënte p atiënt wekte onze interesse, omdat de puntmutatie de eerste, relatief mi lde, mutatie was die beschreven werd in het COG complex. Dit gaf ons de mogelijkheid om subtielere effecten van mutaties op de werking en vormin g van het complex te bestuderen. Voor zowel de COG4 als de COG8 mutatie kon de pathogeniciteit bevestigd worden in de patiëntencellen. Bij overe xpressie werden ernstige dominant-negatieve effecten van het COG4 proteï ne gevonden, wat in overeenstemming is met de literatuur. Beide cellijne n van de patiënten vertoonden duidelijke instabiliteit van andere COG su beenheden, glycosyltransferases en/of eiwitten betrokken in het intracel lulaire transport. Dit duidt erop dat de effecten van de mutaties niet b eperkt blijven tot een destabilisatie van het complex zelf. Glycerol gra diënt centrifugatie van cytosolische en membraan extracten van fibroblas ten van een controle en beide patiënten bevestigde de centrale rol die C OG8 inneemt in het complex door het koppelen van beide subcomplexen. De destabilisatie van COG4 had, wellicht door zijn meer perifere localisati e, echter geen invloed op de vorming van het volledige complex. Bijkomen de analyses suggereren dat de COG subeenheden in het cytosol voornamelij k aanwezig zijn als subcomplexen, terwijl op de membraan zowel het volle dige als de subcomplexen teruggevonden kunnen worden. Op basis hiervan s tellen we een nieuw model voor waarin de subcomplexen aan de membraan va n het vesikel of het organel binden en zorgen voor aanhechting van het v esikel aan de membraan van het Golgi door vorming van het volledige comp lex. Een verdere vergelijking van de verschillende beschreven COG-deficiënte patiënten toonde aan dat er een vrij goede overeenkomst was tussen de tr ansport- en morfologische afwijkingen en de ernst van het klinisch beeld . De gevonden glycosylatiedefecten zijn ook in overeenstemming met de be schreven effecten van het COG complex op de localizatie en stabiliteit v an Golgi enzymes zoals GlcNAcT1 en ManII. Aan de andere kant is het verr assend dat, ondanks enorme structurele afwijkingen, de cellen toch in st aat zijn om de eiwitten in een belangrijke mate (normaal) te glycosylere n. Een mogelijke verklaring hiervoor zou gevonden kunnen worden in het r ecente two-phase model van de Golgi organisatie. De cellen van de COG- deficiënte patiënten zouden kunnen helpen in het testen van dit model. D oor transmissie EM werd waargenomen dat het Golgi apparaat van de versch illende patiëntencellijnen ernstig verstoord is, met een belangrijke mat e van dilatatie, fragmentatie en vesiculatie van de membraan. Op basis van het klinische beeld werden ook een aantal patiënten gegroep ereerd met een opmerkelijke cutis laxa, een grote open fontanel en N- en (in sommige gevallen) O-glycosylatiedefecten. In samenwerking met Dr. U Kornak en Prof. S. Mundlos (Max Planck Institute, Berlin) zijn we erin geslaagd om de oorzakelijke mutatie te localizeren in het ATP6V0A2 gen. Het resulterende eiwit is één van de vier isoformen van de a-subeen heid van de ATP-afhankelijke proton pomp. Deze pomp is aanwezig in de me mbraan van het Golgi apparaat en de lysosomen. Het ATP6V0A2 eiwit is voo rnamelijk gelocaliseerd in de trans-Golgi membraan, wat de specifieke si alylatiedefecten in deze patiënten verklaart. De identificatie van dit g en was interessant omdat het de eerste bevestiging was van de hypothese dat verstoringen in de micro-omgeving van het Golgi apparaat de rechtstr eekse oorzaak kunnen zijn van glycosylatiedefecten. Met behulp van de BF A assay hebben we kunnen aantonen dat al de cellen van deze patiënten oo k een belangrijke vertraging vertoonden in het Golgi-naar-ER transport. Een rol voor de a-subeenheid van deze protonpomp in dit transport was en kel eens kort geopperd in de literatuur, maar nooit bevestigd. Uiteindelijk heeft dit project geleid tot de identificatie van verschill ende nieuwe mutaties als onderliggende oorzaak van CDG. Diepgaande studi e COG4- en COG8-deficiënte patiënten heeft ons in staat gesteld om een d eel van het actiemechanisme van het COG complex bloot te leggen en een m odel voor zijn werking voor te stellen. De observatie dat defecten in de regulering van Golgi pH en de dynamiek van microtubuli ook aanleiding k unnen geven tot CDG heeft geleid tot een enorme uitbreiding van de set m ogelijke kandidaat-genen. In de toekomst zullen nieuwe defecten de betro kkenheid van verschillende cellulaire systemen voor het behouden van de Golgi structuur en werking nog verder benadrukken. Daarom zullen deze st udies van nieuwe defecten bij CDG-patiënten ook verder bijdragen tot een beter begrip van hoe het Golgi apparaat georganiseerd en gereguleerd wo rdt.status: publishe

How Golgi glycosylation meets and needs trafficking: the case of the COG complex

Protein glycosylation is one of the major biosynthetic functions occurring in the endoplasmic reticulum (ER) and Golgi compartments. It requires an amazing number of enzymes, chaperones, lectins and transporters whose actions delicately secure the fidelity of glycan structures. Over the past 30 years, glycobiologists hammered that glycan structures are not mere decorative elements but serve crucial cellular functions. This becomes dramatically illustrated by a group of mostly severe, inherited human disorders named Congenital Disorders of Glycosylation (CDG). To date many types of CDG have been defined genetically and most of the time the defects impair the biosynthesis, transfer and remodeling of N-glycans. Recently, the identification of several types of CDG caused by deficiencies in the Conserved Oligomeric Golgi (COG) complex, a complex involved in vesicular Golgi trafficking, expanded the field of CDG but also brought novel insights in glycosylation. The molecular mechanisms underlying the complex pathway of N-glycosylation in the Golgi are far from understood. The availability of COG deficient CDG patients and patients' cells offered a new way to study how COG, and its different subunits, could influence the Golgi N-glycosylation machinery and localization. This review summarizes the recent findings on the implication of COG in Golgi glycosylation. It highlights the need for a dynamic, finely tuned balance between anterograde and retrograde trafficking for the correct localization of Golgi enzymes to assure the stepwise maturation of N-glycan chains.status: publishe

Serial Detection of Circulating Mucorales DNA in Invasive Mucormycosis: A Retrospective Multicenter Evaluation

Invasive mucormycosis is a fungal infection with high mortality. Early diagnosis and initiation of appropriate treatment is essential to improve survival. However, current diagnostic tools suffer from low sensitivity, leading to delayed or missed diagnoses. Recently, several PCR assays for the detection of Mucorales DNA have been developed. We retrospectively assessed the diagnostic and kinetic properties of a commercial Mucorales PCR assay (MucorGenius, PathoNostics) on serial blood samples from patients with culture-positive invasive mucormycosis and found an overall sensitivity of 75%. Importantly, a positive test preceded a positive culture result by up to 81 days (median eight days, inter-quartile range 1.75-16.25). After initiation of appropriate therapy, the average levels of circulating DNA decreased after one week and stabilized after two weeks. In conclusion, detection of circulating Mucorales DNA appears to be a good, fast diagnostic test that often precedes the final diagnosis by several days to weeks. This test could be especially useful in cases in which sampling for culture or histopathology is not feasible.status: publishe

Lifestyle factors in multiple sclerosis disability progression and silent brain damage : a cross-sectional study

Objective: To determine the association between lifestyle risk factors with 1/ the Multiple Sclerosis Severity Score (MSSS) and 2/ ongoing subclinical brain damage in non-active MS patients on high-efficacy treatment. Methods: Cross-sectional study in persons with Multiple Sclerosis (PwMS) investigating lifestyle factors including cognitive reserve (CR), physical activity (PA), smoking status, alcohol use, dietary habits, body mass index (BMI), blood pressure (BP) and cholesterol ratio. Data were collected through validated questionnaires, clinical and laboratory examination. Serum Neurofilament light chain (sNfL) levels were used as a proxy for ongoing brain damage in a subgroup of persons with non-active MS on high-efficacy treatment. Multiple regression analysis (MRA) models explored the relationship between lifestyle factors with the MSSS score and sNfL. Results: 351 PwMS were included (43.04 +/- 11.77 years, 69.8% female). Higher CR and PA were associated with a lower MSSS; overweight or obesity and higher systolic BP with a higher MSSS. The MRA model explained 22.2% of the variance for MSSS (R-2.255, adjusted R-2.222). Higher BMI and BP were related to lower sNfL. Twenty-3% (R-2.279, adjusted R-2.230) of the variance was explained in the MRA model for sNfL. Conclusion: Our study suggests an association between a 'brain healthy lifestyle' with disability progression in MS. A cognitive and physical active lifestyle alongside a normal body weight and blood pressure may help to prevent future disability in MS. Longitudinal and interventional research is necessary to gain insight in the causal pathway of these risk factors in preventing disability progression in MS

Immune senescence markers predict the cellular immune response to BNT162b2 vaccination in hemodialysis patients

Background Chronic kidney disease is associated with increased risk of frailty and accelerated immune senescence, potentially affecting the immune response to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccination. Methods Humoral and cellular responses against the spike protein of SARS-CoV-2 were determined in 189 COVID-naive hemodialysis patients at week 4 and 8 after vaccination with 2 doses of BNT162b2. Frailty indicators and immune senescence markers were determined at baseline to identify predictors of the immune response. Results Controlling for age, activities of daily living (ADLs), instrumental ADLs, walking pace, and the clinical frailty score correlated negatively and hand grip strength positively with the humoral response. Controlling for age, the proportions of memory CD4(+) T cells, memory CD8(+) T cells, CD28(null) T cells, and CD57(+)CD8(+) T cells correlated negatively with the humoral response, whereas the proportions of memory CD4(+) T cells and CD28(null) T cells correlated negatively and the CD4/CD8 ratio positively with the cellular response. In a multivariate model, only the proportions of memory CD4(+) T cells and CD28(null) T cells independently predicted the cellular response. Conclusions Markers of immune senescence, but not frailty indicators, independently predict the cellular immune response after vaccination in hemodialysis patients, overruling the effect of chronological age

Diagnosing Invasive Pulmonary Aspergillosis in Hematology Patients: a Retrospective Multicenter Evaluation of a Novel Lateral Flow Device

Invasive pulmonary aspergillosis (IPA) is a potentially lethal infection in patients with hematological diseases or following allogeneic stem cell transplantation. Early diagnosis is essential, as delayed treatment results in increased mortality. Recently, a lateral flow device (LFD) for the diagnosis of IPA was CE marked and made commercially available by OLM Diagnostics. We retrospectively analyzed bronchoalveolar lavage fluid (BALf) collected from adult hematology patients from 4 centers in The Netherlands and Belgium. Galactomannan was retested in all samples. All samples were applied to an LFD and read out visually by two independent researchers blinded to the diagnosis of the patient. All samples were also read out using a digital reader. We included 11 patients with proven IPA, 68 patients with probable IPA, 44 patients with possible IPA, and 124 patients with no signs of IPA (controls). In cases of proven IPA versus controls, sensitivity and specificity were 0.82 and 0.86 for visual readout and 0.82 and 0.96 for digital readout, respectively. When comparing patients with proven and probable IPA as cases versus controls, sensitivity and specificity were found to be 0.71 and 0.86, respectively. When excluding serum and BALf galactomannan as mycological criteria from the 2008 European Organization for Research and Treatment of Cancer Invasive Fungal Infections Cooperative Group (EORTC)/Mycoses Study Group of the National Institute of Allergy and Infectious Diseases (MSG) consensus definitions, the LFD was less specific than galactomannan when comparing subjects with proven and probable IPA to controls (0.86 versus 0.96; P = 0.005) but had similar sensitivity (0.76 versus 0.85; P = 0.18). In conclusions, in this large study of the CE-marked LFD in BALf from hematology patients, the LFD had a good performance for the diagnosis of IPA.status: publishe

Lateral flow assays for diagnosing invasive pulmonary aspergillosis in adult hematology patients: A comparative multicenter study

Fast diagnosis of invasive pulmonary aspergillosis (IPA) is essential as early adequate therapy improves survival. However, current microbiological methods suffer from a low sensitivity or a long turnaround time, often as a result of batching. Recently, two lateral flow assays for diagnosing IPA have been CE (Conformité Européenne)-marked and commercialized. These assays can be used for fast single sample testing. However, clinical validation and comparative studies are lacking. We therefore sought to evaluate and compare these assays in adult hematology patients. We retrospectively tested 235 bronchoalveolar lavage fluid (BALf) samples of adult hematology patients from four centers using the AspLFD (OLM Diagnostics) and the sōna Aspergillus galactomannan LFA (IMMY). Both tests were read out independently by two researchers and by a digital reader. We included 11 patients with proven IPA, 64 with probable IPA, 43 with possible fungal disease, and 117 controls with no signs of IPA. In cases of proven IPA, the performance of both assays was similar. In cases of proven and probable IPA, we found an identical specificity for both assays, but a higher sensitivity (0.83 vs 0.69, P = .008) and a better negative predictive value (0.89 vs 0.82, P = .009) for the LFA. Digital readout improved the diagnostic performance of both tests. In conclusion, both assays showed a good performance for the diagnosis of IPA in BALf from adult hematology patients. Results were further improved by using a digital reader, especially for weakly positive results.status: publishe

Lateral flow assays for diagnosing invasive pulmonary aspergillosis in adult hematology patients : a comparative multicenter study

Fast diagnosis of invasive pulmonary aspergillosis (IPA) is essential as early adequate therapy improves survival. However, current microbiological methods suffer from a low sensitivity or a long turnaround time, often as a result of batching. Recently, two lateral flow assays for diagnosing IPA have been CE (Conformite Europeenne)-marked and commercialized. These assays can be used for fast single sample testing. However, clinical validation and comparative studies are lacking. We therefore sought to evaluate and compare these assays in adult hematology patients. We retrospectively tested 235 bronchoalveolar lavage fluid (BALf) samples of adult hematology patients from four centers using the AspLFD (OLM Diagnostics) and the sona Aspergillus galactomannan LFA (IMMY). Both tests were read out independently by two researchers and by a digital reader. We included 11 patients with proven IPA, 64 with probable IPA, 43 with possible fungal disease, and 117 controls with no signs of IPA. In cases of proven IPA, the performance of both assays was similar. In cases of proven and probable IPA, we found an identical specificity for both assays, but a higher sensitivity (0.83 vs 0.69, P = .008) and a better negative predictive value (0.89 vs 0.82, P = .009) for the LFA. Digital readout improved the diagnostic performance of both tests. In conclusion, both assays showed a good performance for the diagnosis of IPA in BALf from adult hematology patients. Results were further improved by using a digital reader, especially for weakly positive results