The aim of this study was to analyze the relative frequency, clinical characteristics, disease onset and progression in f-IPF vs. sporadic IPF (s-IPF).Familial IPF index patients and their family members were recruited into the European IPF registry/biobank (eurIPFreg) at the Universities of Giessen and Marburg (UGMLC). Initially, we employed wide range criteria of f-IPF . After narrowing down the search to occurrence of idiopathic interstitial pneumonia (IIP) in at least one first grade relative, 28 index patients were finally identified, prospectively interviewed and examined. Their family members were phenotyped with establishment of pedigree charts.p < 0.001). On average, the f-IPF patients presented a significantly milder extent of functional impairment at the time point of inclusion vs. the s-IPF group . In contrast, the decline in FVC was found to be faster in the f-IPF vs. the s-IPF group . The average age of death in f-IPF group was 67 years vs. 71.8 years in s-IPF group (p = 0.059). The f-IIP group displayed diverse inheritance patterns, mostly autosomal-dominant with variable penetrance. In the f-IPF, the younger generations showed a tendency for earlier manifestation of IPF vs. the older generation .Within the 28 IPF families, overall 79 patients with f-IPF were identified. In the same observation period, 286 f-IIP and s-IIP patients were recruited into the eurIPFreg at our UGMLC sites, corresponding to a familial versus s-IPF of 9.8%. The both groups showed no difference in demographics , smoking history, and exposure to any environmental triggers known to cause lung fibrosis. The f-IPF group differed by an earlier age at the onset of the disease (55.4 vs. 63.2 years; The 28 f-IPF index patients presented an earlier onset and more aggressive natural course of the disease. The disease seems to affect consecutive generations at a younger age.NCT02951416http://www.www.clinicaltrials.govNr.  Idiopathic Pulmonary Fibrosis (IPF) is a specific form of chronic, progressive fibrosing idiopathic interstitial pneumonia (IIP), a subgroup of diffuse parenchymal lung diseases (DPLD). The disease is characterized by progressive scarring of the lungs, with a consecutive decline in lung function, and is associated with diverse impairments and a poor prognosis –3. AlthoThe strongest epidemiological evidence for a genetic predisposition is the observation of familial clustering, which has been reported in monozygotic twins raised apart, in consecutive generations of families, and in family members separated at an early age . As oppoIn fact, any IIP of known or unknown cases can occur in a familial setting and be related to genetic cause . In geneThe reported degree of relation for a diagnosis of f-IPF ranges from 3rd degree, to 2nd degree and to a required first degree relationship , 13. OthMany studies have been looking for genetic associations in IPF; during the last years, mutations in components of the shelterin or the telomerase complex as well as in the surfactant proteins A and C have been identified to cause IPF , 21. AboIn general, the telomere length seems to play an important role in IPF development , 24. NatDespite the clear proof of monogenetic transmission in the pathogenesis of f-IPF’s, the penetrance appears to vary substantially, resulting in a clinically apparent disease in some cases within 4 months after birth, whereas others members of the same family are over 60 years of age at first diagnosis. Due to these observations, a “two-hit” concept has been proposed, being based on a genetic predisposition resulting in epithelial injury and second environmental hits, including exposure to known fibrogenic materials (e.g. outdoor pollution and cigarette smoke) , 31. TheThe intention of this study was to analyse the relative frequency of f-IPF in our patients’ cohort in relation to the sporadic form, as well as to profoundly characterize clinical differences between the f-IPF and s-IPF. We also studied occurrence of the anticipation phenomenon (increasing severity of illness and / or earlier manifestation of the disease in younger generations), as well as the types of inheritance by analysing pedigree charts of our f-IPF cohort.The phenotyping data from IIP patients and their relatives were collected via the European IPF Registry (eurIPFreg) to evaluate the differences between f-IIP and s-IIP groups (with a focus on IPF as a subgroup). All patients were recruited at our UGMLC sites in Giessen and Greifenstein. The purpose was to identify all patients with familial background.http://www.pulmonary-fibrosis.net) [The eurIPFreg is as an Internet-based, multicenter registry interlinked with the European IPF Biobank  . The dathttp://www.pulmonary-fibrosis.net) [The clinical data were collected at the time of enrolment (baseline) and in intervals 3 to 12 month thereafter via patient and physician baseline questionnaires, which can be retrieved upon logging in to our website (sis.net) .Grade A - First line relative lives with or died from IPF / NSIPGrade B - First line relative lives with or died from DPLDGrade C - First line relative died because of a lung disease not further specifiedAll 28 index patients were diagnosed accordingly ATS/ERS/JRS/ALAT Guidelines 2011 . The defAs first line relatives, the biological parents, children and full siblings were taken into account. The f-IPF index patients as well as their families were enrolled into the eurIPFreg. When employing wide range criteria of f-IIP incidence (evidence grade C), 60 index patients were identified. However, after refining the criteria to occurrence of either nonspecific interstitial pneumonia (NSIP) or IPF in one first grade relative , the search narrowed down to 28 index IPF patients.The following exclusion criteria were applied: patients under 18 years of age, missing informed consent, death of the patient, other lung diseases apart from IIP, adoption of the patient, as well as evidence level C of f-IPF.These 28 index patients were prospectively interviewed, examined, and analyzed using the patient and physician questionnaires of the eurIPFreg, including establishment of pedigree charts. The patient questionnaire included patient’s demographics, a detailed medical history and complaints. The physician questionnaire contained data of physical examination and laboratory tests, pulmonary function, HRCT, echocardiography, 6 MWD, co-morbidities as well as other information concerning relevant patient’s diagnosis and therapy . In addiAdult relatives of the f-IPF index patients were also recruited into the eurIPFreg, and were invited to our DPLD outpatient clinic in order to undergo examination as part of this study. On site, a detailed medical history, a physical examination, a blood collection, a whole body plethysmography, a DLCO test and a blood gas analysis were performed. In total, 52 healthy relatives were included, 26 of whom have carried out all the above-mentioned examinations. One of the 52 family members was diagnosed with an early form of IPF and was re-classified as an affected relative.The pedigree charts were developed in collaboration with affected families during the clinical presentation of the patient either in our outpatient clinic or via telephone interview. All family members from all known generations were taken into account.The following data were collected by each individual subject: family relationship, date of birth, date or at least age at death, cause of death, complaints and all known illnesses. In particular, we were looking for the presence of lung diseases. Individuals with known or suspected IIP were a subject to a more extensive survey: the explorative diagnostics with evaluation of the entity and stage of pulmonary fibrosis, age at first manifestation and diagnosis, smoking status, exposure to occupational and environmental risks. In 24 DPLD patients, included in the pedigree charts, only medical history data could be collected. The pedigree charts were generated with the program GenoPro, version 2.5.4.1.n = 147), f-IPF vs. s-NSIP (n = 17), f-IPF vs. unclassifiable s-IIP (n = 69), f-IPF vs. s-COP (n = 42).The data of all s-IIP patients (incl. IPF) recruited at the same sites as the familial form were extracted from the eurIPFreg, applying inclusion and exclusion criteria mentioned above. The following comparison groups were formed: f-IPF vs. s-IPF  was performed between the f-IPF index patients and s-IPF patients. The main requirement for statistical analysis here was the presence of at least five FVC measurements. The required conditions were met by 21 index patients and 54 s-IPF patients. The last recorded FVC value was the last measurement before lost to follow-up, death of the patient, start on antifibrotics, or lung transplantation.p-value for statistical significance was set as p ≤ 0.05. For distribution tests, we performed the Shapiro-Wilk test as well as the Anderson-Darling test. For normally distributed variables, an independent two-tailed t-test or Mann-Whitney test were conducted, both with data adjustment. For nominal scaled parameters, the Chi-square test and the Fischer test were performed.The statistical analyses were carried out in cooperation with the Department of Medical Statistics of the Justus Liebig University Giessen. For the data evaluation, we used software “R Statistics” (R version 3.1.3). The individual groups were independent of each other. The Of the 28 identified index patients, all had the diagnosis IPF with following levels of diagnostic confidence: 24 definite, three probable, one possible. Together with the 51 affected family members , these patients were diagnosed to have IPF (56%), nonspecific interstitial pneumonia , unclassifiable IIP  and other DPLD (40%). The IPF diagnosis was done in accordance with an Official ATS/ERS/JRS/ALAT Clinical Practice Guideline 2011 . In thosIn total, 314 IIP patients  were recruited at our two UGMLC sites. The 28 f-IPF index patients represented 8.92% of all IIP cases. The confidence levels of IPF diagnosis were defined accordingly ATS/ERS/JRS/ALAT Guideline 2011 and are shown in Table When relating the diagnosis of all familial cases to the sporadic comparator group, percentage values from 1.4% (u-IIP) up to 23% (IPF) were encountered (see Table All IPF index patients underwent high-resolution computed tomography (HRCT) for diagnosis prior to this study as a part of diagnostic routine. In all cases, the radiological findings were consistent with a UIP, of them definite UIP in 66.7% and possible UIP in 33.3% of the cases. In total, 14 index patients additionally underwent a lung biopsy for histopathological diagnosis; a video-assisted thoracoscopic surgery (VATS) was performed in 11 patients; in all of the 14 cases UIP pattern was seen.Twenty-one index patients received a bronchoscopy with a bronchoalveolar lavage (BALF); alveolar macrophages were present in 74.8 ± 37.47%, neutrophilic granulocytes in 10 ± 31.81%, eosinophilic granulocytes in 6.54 ± 0.71% and lymphocytes in 8.2 ± 4.95% of the cases . In 25 cases autoantibodies  were tested and not found to be abnormal; in general, prior to IPF diagnosis, connective tissue diseases-associated ILD were excluded.Of the 147 sporadic IPF patients in 97% of the cases function tests were performed, 98% underwent HRCT, 76% performed 6 MWD, 33% of the diagnoses were additionally confirmed by histology. Bronchoscopy was performed in 73% of the patients, echocardiography in 63% of patients.p < 0.001) and broader age range (36–72 years). With an average of 67.0 years, f-IPF index patients died earlier than sporadic cases (71.8 years), although the result was not statistically significant (p = 0.059). The results are shown in Table Both groups showed no difference in demographics  and smoking history. The f-IPF group differed by an earlier age at the onset of the disease , similar to that in the s-IPF group. Interestingly, f-IPF patients reported more frequently on persistent, dry cough without expectoration, as compared to s-IPF group . During physical examination, “velcro-like” crackles were found in 96% of f-IPF patients, nail widening in 74% and finger clubbing in 13% of all cases. Oxygen supplementation (LTOT) hat 4.76% of the f-IPF patients with mean flow of 2.5 l, as compared to 22.13% of the s-IPF group (mean flow 3.01 l).p = 0.027, FVC p = 0.011, RV/TLC p = 0.026, DLCO p = 0.006, pO2 p = 0.015). The results are shown in Table On average, f-IPF index patients showed a milder extent of functional impairment at the time point of diagnosis vs. the s-IPF group (VC p = 0.208). Lung transplantation was performed in 15% of f-IPF patients, as compared to 5% in s-IPF patients (p = 0.094). The average age of death in f-IPF group was 67 years, as compared to 71.8 years in s-IPF group (p = 0.059).The most common co-morbidity in the f-IPF group was pulmonary hypertension (26%); obstructive sleep apnea was seen in 7% of the cases, and pulmonary embolism in 7% of the patients. The percentage of patients ultimately treated with antifibrotic drugs was similar in both groups (p = 0.013).The f-IPF group displayed diverse inheritance patterns, mainly autosomal-dominant with variable penetrance. In the f-IPF, the younger generations revealed a tendency for earlier manifestation of IPF vs. the older generation, thus displaying a phenomenon of anticipation  showed an average, relative FVC decline of 0.028% per day (95% CI: 0.018–0.039%). This corresponded to an average, relative decline of 4.94% (95% CI: 3.155–6.71%) in 6 months, or 9.88% per year. The mean percentage starting point of the FVC of f-IPF was 73.63% predicted (95% CI: 65.76–82.47%).Patients with f-IPF (n = 54) lost 0.014% (95% CI: 0.009–0.019%) of the FVC on average per day (relative decline). Corresponding to this, the average decline in 6 months was 2.48% (95% CI: 1.568–3.382%), or 4.96% per year. The mean value of the starting point of the FVC in s-IPF group was 62% predicted. However, possibly due to the broad scattering of data, the difference in FVC decline between the groups did not reach statistical significance (p = 0.12). In a logarithmic analysis of FVC decline, the starting value in the f-IPF group was 4.30 (95% CI: 4.19–4.41). In contrast, the logarithmic starting point of FVC for sporadic IPF was 4.18 (95% CI: 4.11% - 4.25). The interpolated slope of the FVC for f-IPF was − 0.00028 (95% CI: − 0.00018 – − 0.00039), and for s-IPF group was − 0.00014 (95%-CI: − 0.00009 – − 0.00019). The data are shown in Fig. The s-IPF group . In one family, it was noticed that all four affected IIP patients died within a very short time after diagnosis due to disease progression. Furthermore, it was observed in five pedigree charts that the younger generation became ill over a decade earlier than the generation of the parents (phenomenon of anticipation).Within the families, altogether 79 cases of DPLD including the 28 index patients, as well as 51 further patients with DPLD were detected . In fourteen families, two members were affected; another seven families had three affected members per family, six families had four DPLD cases, and one family had six relatives with DPLD.Pedigree charts could be completed for 25 of the 28 f-IPF patients. In four pedigree charts, a father to son inheritance was observed. In two pedigree charts, the disease appeared to originate from both parental sites. In contrast, in 16 families (ten times inheritance mother to child and six times inheritance father to child), the disease only seemed to originate from one parent. Of these, in one family, children of the same mother (but different father) were affected. Only one generation was affected in seven families .In the following, four pedigree charts are presented as example. The index patient is marked with an arrow. All members suffering from DPLD are marked yellow with a blue border. Patients suffering from another lung disease are marked with a blue border. Family members without DPLD are marked with a magenta border. The relationships in pedigree charts always refer to the index patient.This family tree comprised four generations with 13 individuals. The grade of evidence of f-IPF was level A, because the index patient (III: 3) had at least one immediate relative diagnosed with IPF or NSIP. In this family, the brother (III: 5) fulfilled this condition. The disease seemed to have its origin on the maternal side. In the mother (II: 3) no diagnosed pulmonary fibrosis could be detected. However, shortly before her death she began suffering from progressive dyspnea and cyanosis without history of nicotine consumption. The aunt of the index patient suffered from pulmonary fibrosis. In the third generation, all three members were affected by an IPF. Inheritance pattern here seems to be autosomal dominant, since all children were affected, despite the fact that they have different biological fathers. The data are shown in Fig. This family included six generations with 39 members. The index patient is in position III: 4. There was an evidence level A (index patient III: 4 and sister III: 5). The cousin was also affected by diffuse parenchymal lung disease (III: 7). He was accidentally diagnosed with lung fibrosis because of auscultation of velcro-crackles in the routine checkup. His daughter (IV: 2) also did not have any complaints at the time of first diagnosis of pulmonary fibrosis. In total, ten other blood relatives of the index patient suffered from some form of lung disease. In particular, childhood pneumonia was very common . Since only one generation was affected with IPF, inheritance mode is challenging to interpret in this example. The results are shown in Fig. The pedigree chart 3 shown here comprised six generations with 42 family members and fulfilled the evidence level B (IV: six and III: 4). Four members of the family were known to be affected by pulmonary fibrosis . The index patient (IV: 6) had died of a rapidly progressive lung fibrosis with subsequent respiratory failure at the age of 71 years. More family members had died due to pulmonary fibrosis: his father (III: four at the age of 64), his uncle (III: seven at the age of 64) and a cousin (IV: 10 at the age of 71). The index patient had no siblings. The aunt (III: 5) had died at the age of 80 and had no pulmonary fibrosis during her lifetime. Other children of the DPLD-affected uncle  died early at the age of 43 and 49, respectively, without having developed (or diagnosed) pulmonary fibrosis during their lifetime. The most likely inheritance seems to be an autosomal dominant inheritance, as both father and uncle seem to pass on the gene Fig. The pedigree chart four observed five generations consisting of 53 individuals. Evidence level B was met by the index patient (III: 5) and her father (II: 6). Three generations were affected by pulmonary fibrosis . The sister (III: 4), the daughter (IV: 9) and two nieces (IV 14 and 15) of the index patient had shown no evidence of diffuse parenchymal lung disease at the time of the study. In the oldest generation (I), the grandmother (I: 4) died at the age of 86. In the following generation (II), the father and the uncle died earlier than the grandmother. The father was diagnosed with IPF at the age of 79 years and so the uncle (II: 7) was diagnosed with IPF with 67 years. At age 53, the index patient (III: 5) was diagnosed much earlier than relatives in the previous generations. Due to the rapid progression of the disease, the index patient received a lung transplant at age 64. Up to now, no cases of pulmonary fibrosis have been observed in generations IV and V. This family tree demonstrates the phenomenon of anticipation. The younger the generation, the sooner the disease was diagnosed, but also the sooner the patients died or become lung transplant Fig. p = 0.013). The affected family members of youngest generation died on average at the age of 66 years. In the parental generation, 25 persons were diagnosed with DPLD (38%), and they died on average at the age of 71 years. The grandparent generation was first diagnosed at the age of 72 and died on average at age 79 . In this generation, the age at first diagnosis was 58 ± 11 years , which was significantly lower as compared to the year of first diagnosis in the parents’ generation , which suffered or died from IPF / NSIP (evidence level A) or other DPLD (evidence level B). In previous studies, affected family members have often been diagnosed on third-party medical history or questionnaires only , 20. ThiWith regard to f-IPF, the majority of studies indicated that f-IPF does not differ clinically or histologically from its sporadic form. The only difference being observed was an earlier age at onset of familial cases , 14, 15.n = 17) for all f-IPF cases [Loyd at al commented that nine out of 47 (=19%) pulmonary fibrosis patients who underwent lung transplantation at their study center had at least one relative with pulmonary fibrosis . MarshalBy analyzing pedigree charts of f-IPF, we observed mostly autosomal dominant inheritance pathway (16 of 25 pedigrees) with variable penetrance, which has been confirmed by other authors as well , 39. HowBennett et al. retrospectively stratified 46 f-IPF patients according to HRCT pattern and BAL cellular composition and analysed functional and radiological follow-up data of patients with f-IPF, showing that it may manifests with several HRCT patterns with different rates of progression. The study found UIP pattern at HRCT in 54.3% of patients, possible UIP in 21.8% and inconsistent UIP in 23.9% . Our resOf note, there were nine families with multiple cases of bronchial carcinoma. This phenomenon has been linked by Wang et al. to a surfactant protein A2 mutation  and openp = 0.013).Within the f-IPF families, the age at onset of disease and of death was found to come down with each generation, hence reflecting the phenomenon of anticipation. The affected family members of youngest generation died on average at the age of 66 years, as compared to 71 years in the parental generation. The grandparent generation was first diagnosed on average at the age of 72 and died on average at age 79, as shown in Table In genetics, anticipation is a phenomenon whereby a genetic disorder is passed on to the next generation, but the symptoms of the disease become apparent at an earlier age with each age group. In some cases, an increase of severity of symptoms was also noted . The prop < 0.001) [p = 0.013). Some pedigree charts displayed that IPF manifests in the younger generation over a decade earlier vs. parents’ generation. It must be taken into account that the younger generation might be more aware of the disease and could therefore be diagnosed earlier. Nevertheless, the younger generation tends to die earlier . Despite the lack of significance, this difference of 5 years on average is considerable, as the younger generation had access to novel therapies, unlike the older generations.Scholand et al. showed that f-IPF patients have a significantly increased risk of death vs. s-IPF among the first- through third-degree relatives (< 0.001) . The evaChibbar at al showed in the analysis of a large family with many IPF patients phenomenon of anticipation in the f-IIP. The oldest generation was diagnosed at the age of 50, with an average of around 39 and the youngest at age 32 . RavagliThe limitations of this research were: the study did not state the proportion of mutated patients, although genetic testing and counseling were proposed to all IPF patients with suspected familial form. The proportion of telomerase-related and surfactant mutations within the cohort of f-IPF is the subject of our upcoming studies. Furthermore, to our understanding, there can only be one index patient per family. These were the 28 patients we had been identifying during their visit at our site. The other fIPF patients were not primary patients of our site. With regard to the concerned family members, we tried our best to get as much information as possible (including CTs) in order to judge on the predominant pattern and the IIP diagnosis. However, this was not always possible. In addition, some of the information which the index patient provided during their visit at our site was not available for their concerned relatives . Therefore, we were not able to fully expand the comparison between fIPF and sIPF to all fIPF subjects in the families.In summary, anticipation of familial IPF can be assumed. In light of the progressive shortening of telomers in successive generations of affected families in those with Terc/Tert mutations, such anticipation phenomenon could be easily explained by the underlying pathomechanisms .Eighteen of the 25 affected relatives in our study had been diagnosed by high-resolution computed tomography . Another difference to previous studies is that, in addition to the patients, their healthy relatives were invited for a medical checkup, in order to actively search for an evidence for a diffuse parenchymal lung disease. In particular, young, close relatives and people with mild respiratory symptoms volunteered gladly to take part in this study. This group of family members is precisely the cohort that may have previously unrecognized IPF/IIP; and so the number of false negatives within the families could also be kept to a minimum.Such early detection may have a significant impact on the life of these patients: prevention of respiratory infections, prevention of exposure to a fumes and gases known to contribute to disease progression  and early initiation of anti-fibrotic treatment may be mentioned as immediate consequences of such approach and justify a more rigorous screening for concerned family members on clinical grounds.Whether one needs to conduct genetic screening for routine purpose is a matter of debate and will be subject of position papers in the future: here, the advantage of identifying a disease-causing mutation and the potential to identify family members who even do not show any visible disease at the time of testing contrasts with the burden of knowing that one could develop the disease in a unpredictable time span. We therefore tend to offer clinical screening, with a special emphasis on lung auscultation and screen for velcro rales, to the families of our IPF patients, and not routine genetic testing. For research purpose, however, sequencing should be done and will also be done in our cohort.The 28 f-IPF index patients summarized here shown an earlier onset and more aggressive natural course of the disease as compared to s-IPF. The most likely mode of inheritance seems to be autosomal dominant with variable penetrance. In addition, the disease seems affect the consecutive generations at a younger age. Therefore, if f-IPF suspected, a thorough clinical screening of family members should be offered. Further studies will aim to disclose the underlying and causative genetic and epigenetic changes in our cohort of f-IPF patients. This study lays the foundation for evaluating further generations of affected families. The sample size and profound clinical phenotyping and diagnostics of our cohort are the strength of this work.


A facile and rapid method to synthesize graphene/layered double hydroxide (LDH) nanohybrids by a microwave technique is demonstrated. The synthesis procedure involves hydrothermal crystallization of Zn–Al LDH at the same time in situ reduction of graphene oxide (GO) to graphene. The microstructure, composition, and morphology of the resulting graphene/LDH nanohybrids were characterized. The results confirmed the formation of nanohybrids and the reduction of graphene oxide. The growth mechanism of LDH and in situ reduction of GO were discussed. The LDH sheet growth was found to prevent the scrolling of graphene layers in resulting hybrids. The electrochemical properties exhibit superior performance for graphene/Zn–Al LDH hybrids over pristine graphene. The present approach may open a strategy in hybridizing graphene with multimetallic nano-oxides and hydroxides using microwave method. The combination of multidimensional nanomaterials leads to the formation of hierarchical composites that can take full advantages of each kind of components, which is an effective way for the preparation of multifunctional materials with exceptional properties. Recently, nanocarbon  has emerged as the most powerful material and was used in multifunctional hybrids for various applications –3. AmongRecently, decorating layered metal hydroxides like layered double hydroxides (LDHs) with different metal compositions have been studied in order to diminish the restacking interactions and to limit the aggregation in graphene nanosheets suitable for electrochemical applications , 10. LDHMicrowave irradiation is often applied for the rapid synthesis of inorganic solids and organic synthetic reactions , 19. TheIn this work, we present a one-step synthesis method to synthesize graphene/Zn–Al LDH hybrids through microwave-assisted growth of 2D LDHs with simultaneous in situ reduction of graphene oxide (GO) to graphene under hydrothermal conditions. In this facile and rapid synthetic procedure, the exfoliated GO was reduced to graphene using in situ hydrolyzed urea (ammonia). Simultaneously, the Zn–Al LDH platelets were formed in situ and hybridized with graphene. The resulting hybrids were characterized using various physicochemical characterization techniques. Furthermore, we also demonstrated the use of graphene/Zn–Al LDHs for electrochemical applications by studying their cyclic voltammetry (CV) and galvanostatic charge/discharge measurements.All the chemicals were of analytical reagent grade and without any further purification. GO was prepared from expanded graphite by a modified Hummers method according to our previous report . Exfolia−1) was sonicated for 5 min and placed in a microwave flask, and subsequently zinc nitrate (10 mmol) and aluminum nitrate (0.33 mmol) salt mixture (molar ratio of Zn/Al = 3) was added. The mixture was magnetically stirred for 30 min in order to form a colloidal dispersion. Subsequently, an excess urea  was added to adjust the pH and to reduce GO through urea hydrolysis under microwave. Lastly, the flask containing Zn salt/GO dispersion was placed into microwave system  with cooling water circulating condenser. The system was heated gradually to 150 °C under microwave irradiation at a power of 200 W for 2 h. After cooling, the resulting black precipitate was repeatedly washed with CO2-free water via centrifugation before freeze drying. Finally, the resulting product was vacuum dried at 80 °C to remove any remaining traces of water. For the sake of comparison, pure RGO and Zn–Al LDH were also synthesized by under similar microwave-assisted technique using GO dispersion and metal salts, respectively.The RGO/LDH nanohybrids were synthesized using urea-hydrolysis reaction under hydrothermal conditions using microwave technique Fig. . In deta−1 resolution. Thermogravimetric analysis (TGA) of the resulting hybrids was studied by a TA instruments Q500 TGA at a heating rate of 10°C min−1 under inert atmosphere. X-ray photoelectron spectroscopy (XPS) was used to control the elemental composition of the samples. All reported spectra were recorded at a 90° take-off angle relative to the substrate with a VG ESCALAB 220iXL spectrometer using the monochromatized Al Kα radiation (1486.6 eV). The electrochemical properties of the nanohybrids were measured in an aqueous system (electrolyte: 30 % KOH). CV curves were measured with an electrochemistry workstation (Princeton PARSTAT2273). Galvanostatic charge/discharge measurement was conducted with a charge–discharge tester . The galvanostatic charge–discharge tests were performed on a BTS-5 V/10 mA battery-testing instrument  at room temperature. A two-electrode cell was assembled for cyclic testing of cell. During the cycling process, the cells were charged at 1C for 60 min and discharged at 1C down to 1.4 V cut-off voltages.The surface morphology, structure, and composition of graphene and graphene–LDH nanohybrids were characterized by transmission electron microscopy  and X-ray diffraction . The Raman spectra were obtained by a Renishaw Raman system Model 3000 spectrometer equipped with an integral microscope (Olympus BH2-UMA). Radiation from a He–Ne laser (633 nm) was used as the excitation source. The FT-IR was recorded on a Bruker Infrared Spectrometer using 32 scans and a 4 cmFigure When evolved, ammonia increased the pH, resulting in the controlled precipitation of metal ions. Therefore, the use of microwave activation is expected to energize the metal cations, and through their inherent ionic conduction to achieve a uniform bulk during heating the materials. Moreover, the excess ammonia present in the system was also used as a reducing agent for GO reduction at higher temperature under microwave irradiation. During microwave heating, well-crystalized LDH nanoplatelets were thereby synthesized and closely interacted with the RGO layers. These nanoplatelets also prohibit the stacking of graphene sheets by van der Waals force as evidenced hereafter.θ = 21° (002) was also observed, which reveals the reduction of GO to well-exfoliated RGO under microwave irradiation [π–π stacking interactions between the graphene sheets, leading to the formation of the agglomerates. However, the residual oxygen functionalities on the RGO surface induce electrostatic repulsion, stabilizing the graphene sheets.Figure adiation . The intθ value. The Zn–Al LDH crystals deposited on graphene can prevent them from stacking into multilayers, leading to the much lower crystalline extent of graphene, which results in the (002) peak broadening and shift toward lower angle. Moreover, the diffraction peak intensity of RGO/LDH nanohybrids nearly remains the same as of pure Zn–Al LDH. This means that under microwave irradiation, the crystallinity is improved with respect to the conventional hydrothermal method. This result suggests that the restacking of the graphene sheets is effectively prevented from a complete exfoliation state of graphene in the RGO/LDH nanohybrids, which is in well agreement with the morphological findings observed by TEM  peak is significantly increased while O 1s (533) peak intensity has been gone down. For, RGO/Zn–Al LDH hybrids, the C 1 s and O 1 s peak intensities are matching to the RGO confirming the successful GO reduction to RGO. Furthermore, the spectrum also exhibits the peaks of Zn 2p and Al 2p, suggesting the formation of the RGO/Zn–Al LDH composite. The high-resolution C 1s spectrum of GO shows three types of carbon bonds  and the carboxylate carbon (O–C=O), indicating a considerable degree of oxidation [s XPS spectrum of RGO/LDH , aromatic stretching C=C (1615 cm−1), epoxy C–O group stretching (1218 cm−1), and alkoxy C–OH group stretching vibrations (1043 cm−1) [−1 are accounted for lattice vibration modes for M–O and M–O–M , which also can be seen in FT-IR spectra of pristine Zn–Al LDH. Hence, FT-IR studies confirm the in situ reduction of GO to RGO with simultaneous growth of Zn–Al LDH under microwave irradiation.The FT-IR spectra of GO, RGO/Zn–Al LDH nanohybrids, and pristine LDHs are shown in Fig. 43 cm−1) . In comp groups 125 cm−1, ) [The TGA behaviors of the graphene/LDH nanohybrids are displayed in Fig. ) . In the −1 in 1 M KOH solution. A couple of anodic/cathodic peaks were observed for CV curves of each sample at different scan rates, including the RGO  between the anodic and cathodic peaks than RGO under the same scan rate, demonstrating a better electrochemical reversibility of the RGO/Zn–Al LDH hybrids.To evaluate the electrochemical behaviors of the as-synthesized RGO and RGO/Zn–Al LDH nanohybrids for supercapacitor application, a series of CV and galvanostatic charge/discharge measurements were carried out. Figure RGO Fig. a and RGORGO Fig. b. The re−1 are presented in Fig. −1 which significantly improved upon hybridization with LDHs. The RGO/Zn–Al LDHs exhibit prominent discharge specific capacitance 428 F g−1, which is almost 20 % higher than RGO. The improved capacitive behavior is mainly attributed to the high electronic conductivity of the RGO/Zn–Al LDH nanohybrids, which comes from two sources of intrinsic high conductivity of the graphene sheets and interaction between the RGO and the LDHs in the nanohybrids. The anchored LDH particles play an active role preventing the aggregation of the graphene sheets, which connect with each other to further establish a conductive network for facile electron transport. The high specific surface area of the RGO/Zn–Al LDH hybrids favors the electrolyte percolation to achieve high accessibility of the active LDHs materials, which also contributes to improved specific capacitance.Moreover, the area surrounded by CV curves of the nanohybrids is obviously greater than that of RGO, implying a higher electrochemical capacitance. The charge/discharge curves of RGO and RGO/Zn–Al LDHs at current density of 2 A g−1, almost 20 % higher than that of pristine graphene. Moreover, the RGO/Zn–Al LDH hybrids demonstrated good cycling stability. Hence, LDH nanosheets in conjunction with thermo-electroconductive graphene endow the resulting hybrid nanocomposites with new multifunctional properties. The effectiveness of the method described above for fabricating graphene/LDH nanohybrids could be of high importance for the preparation of other graphene-based hybrid nanocomposite electrode materials. Similarly, the use of microwave in fabrication of graphene/LDH nanohybrids provides a novel method for the development of new multifunctional nanocomposites on the basis of the existing nanomaterials.Microwave-assisted method for hybridizing graphene with LDHs is presented. This efficient and rapid process involves the in situ reduction of GO to graphene with simultaneous growth of 2D Zn-containing LDHs using urea hydrolysis under microwave conditions. The resulting hybrids show improved electrochemical-specific capacitance, which attained maximum of 428 F g


We present a low-noise photodetector based on van der Waals stacked black phosphorus (BP)/boron nitride (h-BN)/graphene tunneling junctions. h-BN acts as a tunneling barrier that significantly blocks dark current fluctuations induced by shallow trap centers in BP. The device provides a high photodetection performance at mid-infrared (mid-IR) wavelengths. While it was found that the photoresponsivity is similar to that in a BP photo-transistor, the noise equivalent power and thus the specific detectivity are nearly two orders of magnitude better. These exemplify an attractive platform for practical applications of long wavelength photodetection, as well as provide a new strategy for controlling flicker noise. Mid-infrared (mid-IR) light 3 to 15 μm) covers two infrared atmosphere windows and contains the fingerprints of most common molecular vibrations 5 μm cove. PhotodeTo circumvent this problem, we present a new strategy for compressing flicker noise using BP/boron nitride (h-BN)/graphene tunneling junctions. We propose using h-BN as a tunneling barrier to significantly block dark current fluctuations induced by shallow trap centers. To validate this new strategy, we fabricated van der Waals stacked BP/h-BN/graphene tunneling junctions. The flexibility of 2D materials allows them to be used in heterojunctions without the limitations of lattice mismatch or the thermal expansion coefficient. We measured the temperature-dependent scanning photo-current, charge transport, and low-frequency noise spectrum. While the photoresponsivity of the heterojunction at mid-IR wavelengths (up to 4 μm) is similar to that from a BP photo-transistor, the noise equivalent power and thus the specific detectivity are a factor of 100 larger. The results show that the tunneling heterojunction provides an improved sensitivity compared to that of a BP photo-transistor.2/Si substrate using an electron beam evaporator. The materials we used were obtained using a standard mechanical exfoliation process [2 film. With the aid of an optical microscope, polyvinyl alcohol (PVA) was used to pick up these two materials sequentially and transfer them to the defined source electrode to format h-BN/graphene using a deterministic transfer technique. This process was performed on an 80 °C hot plate. The top BP flake on the polydimethylsiloxane film was then dry-transferred onto the h-BN flake and the defined drain electrodes with slight pressure to form the BP/h-BN/graphene vertical heterojunction. The electrical and optoelectrical measurement results of the device were gathered by a semiconductor characterization system. Standard Atomic force microscopy (AFM) data were obtained in tapping mode using a Cypher S AFM. Raman spectra were gathered using a LABRAM-HR Raman spectrometer with an excitation wavelength of 514 nm. process . BP/h-BN process . In the −1 corresponding to the A1g, B2g, and A2g modes, respectively. These peaks are consistent with previous observations from few-layer BP flakes [2 substrate, indicating that the graphene is only a few layers thick. The additional peak at 1367 cm−1 corresponds to the interlayer h-BN film.P flakes . Moreoveb (Vb is applied to the top BP with graphene grounded) tilts the band in h-BN, forming a triangular barrier [b narrows the equivalent barrier width, allowing photo-excited carriers to tunnel from BP to graphene. As a result, tunneling photocurrent can be generated in the heterostructure device with proper bias and illumination conditions. The photo-carrier transport mechanism is similar to what occurs in a graphene/h-BN/graphene tunneling device; however, optical excitation in BP provides carrier injection here [The schematic band structure of the BP/h-BN/graphene structure is shown in  barrier . Increasion here .b increases due to the narrowed effective barrier width [b = 5 V, which is a factor 6.36 larger than the current at 80 K (22 nA). We also fabricated a BP photo-transistor as a control sample (see below for details). The current in the BP phototransistor as a function of source-drain bias at different temperatures is summarized in We first characterized the dark I-V performance of the BP/h-BN/graphene structure, as shown in er width . A sligher width . The tunb = 3.5 V while irradiated with 4 μm light from a 10 mW laser. The top BP layer, h-BN tunneling barrier, and bottom graphene electrode are outlined with a dotted line, point, and dashed lines, respectively. Clearly, the photo-current is generated in the overlap region. We will now discuss the sensitivity of the heterojunction. The photo response and dark noise were measured in order to determine the specific detectivity. b at a zero gate voltage. The results are shown in −1 at a 4 V applied voltage when the incident power changes from 10 mW to 0.01 mW (4 μm). This negative power dependence of responsivity is similar to that of a BP phototransistor. Besides, a random distribution of defects at the interfaces and vacancies, dislocations, or grain boundaries in BP can result in the formation of shallow trap states [−1, which is approximately equal to the value of a BP phototransistor control sample  at different temperatures. To further validate the appropriate method for reducing noise used here, the dark current spectral density from the BP phototransistor control sample at different temperatures is summarized in 9 Jones at a 2 V bias, 4 μm wavelength, and 300 K in our heterojunction photo. This detectivity is more than a factor of 100 larger than that of the BP phototransistor control sample.Determining the detector sensitivity requires determining the noise and responsivity. We will see that the tunneling barrier considerably compresses dark noise, resulting in giant room-temperature detectivity, even with a low responsivity. In summary, we have presented a few-layer BP/h-BN/graphene heterojunction-based tunneling junction photodetector. The device provides a high photodetection performance at mid-IR wavelengths by facilitating dark noises and the sensitivity of the device is a factor of ~100 larger than that of a BP photo-transistor. Such a great improvement can be attributed to tunneling-assisted noise management. Our novel heterojunction photodetector provides an attractive platform for practical photodetection at long wavelengths, as well as a new strategy for controlling flicker noise.


Chromatin accessibility is crucial for gene expression regulation in specific cells and in multiple biological processes. Assay for Transposase Accessible Chromatin with high-throughput sequencing (ATAC-seq) is an effective way to reveal chromatin accessibility at a genome-wide level. Through ATAC-seq, produced reads from a small number of cells reflect accessible regions that correspond to nucleosome positioning and transcription factor binding sites, due to probing hyperactive Tn5 transposase to DNA sequence.In this review, we summarize both principle and features of ATAC-seq, highlight its applications in basic and clinical research. ATAC-seq has generated comprehensive chromatin accessible maps, and is becoming a powerful tool to understand dynamic gene expression regulation in stem cells, early embryos and tumors. In eukaryotic cells, chromatin is a basic hereditary unit, which consists of DNA, histone proteins and other genetic materials, and regulates cell type-specific gene expression , 2. ChroIn general, the regulatory elements selectively localize in the accessible chromatin, which is crucial to transcriptional regulation . AlthougChanges of the chromatin structure occur at specific ribozyme accessibility sites that are associated with transcriptional initiation or some specific DNA structures such as specific hypersensitive sites . These sHere we summarize the principle of the ATAC-seq method, highlight its usage in understanding basic transcription programs in specific cell types of humans and mice, and in revealing genetic reasoning of human diseases.ATAC-seq is an innovative epigenetic technology, which is a method for mapping chromatin accessibility by probing hyperactive Tn5 transposase to DNA sequence at a genome-wide level  Fig. 1bb1b. DNA The construction of ATAC-seq library consists of three steps: nuclei preparation, transposition and amplification  Fig. 2)2). FirstQuality control of the ATAC-seq library should be performed prior to sequencing to guarantee that the library concentration reaches the sequencing criteria. After library sequencing, raw reads are collected through sequencing the qualified library. After filtering data through sequencing data quality assessment, clean reads are further obtained by evaluating sequencing quality and summarizing data production , 28, 29.The ATAC-seq method was first developed as an alternative or supplement to sequencing of Micrococcal Nuclease sensitive sites (MNase-seq), Formaldehyde-Assisted Isolation of Regulatory Elements (FAIRE-seq), and Deoxyribonuclease I hypersensitive sites sequencing (DNase-seq) –38 Tabl. For theNoteworthy, ATAC-seq has several advantages: first, the transposase method can reduce experimental time to 2–3 h to achieve DNA fragmentation using a simple enzymatic reaction, which avoids the tedious conventional DNA fragmentation, terminal repairing and adapter connection reaction . On the There are also some limitations of the ATAC-seq technology. First, the Tn5 transposase simultaneously fragments and tags unprotected regions of DNA with sequencing adapters through the “cut and paste” mechanism. The adapter joints at both ends of each DNA fragment are random, which leads to a 50% probability of that the adapters at both ends of one fragment are the same, generating half unusable fragments for enrichment, amplification and sequencing . Second,To obtain pure nuclear genome reads and to reduce mitochondrial contamination, two methods can be used: using the cell lysis buffer without detergent , and usiSince the ATAC-seq method was first developed, it has been improved in order to adapt broader usage in research. Single-cell ATAC-seq (scATAC-seq) provides the first insightful examination of cell-to-cell variability in chromatin organization, which can be achieved by a programmable microfluidic device or combinatorial cellular indexing scheme. The scATAC-seq can be used as a genome-wide vehicle to map chromatin accessibility in all specific cell types of an organism , 63–65. Moreover, Omni-ATAC-seq is another improved ATAC-seq protocol to detect chromatin accessibility . Based oATAC-seq obtains the information of accessible chromatin by breaking up cells, so it cannot describe the three-dimensional structure of these accessible genomic regions. Assay for Transposase Accessible Chromatin with Visualization (ATAC-see) uses the same enzymatic methods as ATAC-seq, and adds fluorescent clusters together with DNA markers, which allows visualization of three-dimensional immobilized nuclei .Applying ATAC-seq has advanced our understanding of the machinery of gene expression regulation, such as chromatin accessibility between different samples, nucleosome positions, and genome-wide binding sites of transcription factors , 69, 70.Mapping the accessible chromatin landscape can obtain information of spatial changes in chromatin structures and transcription factors associated with gene expression . This inFGFR2 and EOMES as important regulatory players in cortical neurogenesis [In developmental biology, the lineage-specific open chromatin regions and changes have been mapped using ATAC-seq in epidermal differentiation, and in trophoblast stem cell differentiation in placenta , 78. In ogenesis . Moreoveogenesis , 82–86.In summary, applying ATAC-seq has generated comprehensive accessible chromatin landscapes of various cell types in different tissues and organs, which has provided valuable insights into the complexity of gene transcription.cis-regulatory factors, which confirms that cis-regulatory elements play a significant role in early development [Chromatin reprogramming actively occurs during early embryonic development . Studieselopment , 91. ATAelopment . Chromatelopment .DUX4 are activated by endogenous genes KDM4E and ZSCAN4 that are expressed only in cleavage-stage of human embryos, which subsequently initiates transition of embryonic stem cells to 2-cells stage with totipotency [Moreover, to study embryonic genome activation, ATAC-seq has been used to detect transcriptome sequences . These sipotency –97.RAD21 and STAG2 genes, which encode the chromosome cohesion complex, have been shown to be key elements in malignancy formation in acute myeloid leukemia (AML) [ATAC-seq is highly applicable to capture the tissue-specific chromatin activity of regulatory regions in tumors , 98–100.ia (AML) –104. Stuia (AML) –108.ARID1A mutations usually occur in many kinds of tumors, such as melanoma, glioblastoma and other human malignancies [ARID1B mutations are usually found in neuroblastoma, hepatocellular carcinoma and breast invasive ductal carcinoma [ARID1A and ARID1B complex are frequently associated with tumorigenesis via altering promoter and enhancer activities to modulate downstream gene expression [ARID1A leads to H3K27ac reduction at enhancer regions of downstream genes for ARID1A [ARID1A plays an important role in maintaining chromatin accessibility at enhancers. In particular, the expression of MET gene has been changed in ARID1A mutant ovarian cancer cells, while ARID1B deletion displays the same effect only in the context of ARID1A mutation, indicating an important role of ARID1A in ovarian cancer cells [Moreover, gnancies –113, andarcinoma –117. Stupression . During r ARID1A , 120. ARp53 is a well-studied cancer suppressor gene. The protein encoded by p53 has a role of suppressing cancer under normal circumstances, and promoting cancer development when mutations occur [p53 has a prior binding to the enhancer in healthy fibroblasts detected using ATAC-seq [p53 gene is activated to maintain genome stability [Furthermore, ns occur , 122. Whns occur . Studiesns occur , 125. ItATAC-seq . When DNtability .ATAC-seq uses high-throughput sequencing approach to identify all active regulatory sequences in the genome using a small amount of cells. ATAC-seq has been widely used in the acquisition of open chromatin regions and transcription factor binding sites to reveal a real time profile of chromatin accessibility. It has been rapidly applied and accepted to investigate gene expression dynamics in stem cells, early embryos, and various tumors, and even to detect potential biomarkers. Taking advantage of optimization of ATAC-seq methodology to simplify the experimental procedure and to reduce the cost, ATAC-seq should soon have a broader usage in basic research and clinical diagnostics.


Sarcoidosis is a rare inflammatory disease characterized by the development of granulomas in various organs, especially in the lungs and lymph nodes. Clinics of the disease largely depends on the organ involved and may range from mild symptoms to life threatening manifestations. Over the last two decades, significant advances in the diagnosis, clinical assessment and treatment of sarcoidosis have been achieved, however, the precise etiology of this disease remains unknown. Current evidence suggests that, in genetically predisposed individuals, an excessive immune response to unknown antigen/s is crucial for the development of sarcoidosis. Epidemiological and microbiological studies suggest that, at least in a fraction of patients, microbes or their products may trigger the immune response leading to sarcoid granuloma formation. In this article, we discuss the scientific evidence on the interaction of microbes with immune cells that may be implicated in the immunopathogenesis of sarcoidosis, and highlight recent studies exploring potential implications of human microbiota in the pathogenesis and the clinical course of sarcoidosis. Sarcoidosis is a unique inflammatory disease characterized by the formation of non-caseating granulomas that can affect any body organ but most often affects the lungs and lymph nodes and less commonly the skin, eyes, liver, heart, and brain , 44. ExtCutibacterium acne (C. acne) (previously known as propionibacterium acne) and mycobacterium tuberculosis (mTB) may be implicated in the development of sarcoidosis, and thus various authors have proposed the possibility of including antibiotics as part of the standard treatment of this disease [In addition, the existence of cases of familial sarcoidosis along with the observations that certain HLA loci and single nucleotide polymorphisms (SNPs) in non-HLA genes are associated with increased risk of sarcoidosis suggest that a genetic predisposition also plays a role in disease pathogenesis , 60. In  disease , 12, 19. disease .With the development of advanced tools for microbe research, such as new generation sequencing (NGS) and the incorporation of more sophisticated methods form microbiome research, it will be possible to determine whether or not, there is a link between specific alterations in the composition of certain microbiota niches (namely dysbiosis of the gut or dysbiosis of the lung microbiota) and the development or progression of sarcoidosis.In this article, we revise the scientific evidence on the potential role that host/microbe interplay may have in the pathogenesis of sarcoidosis. The clinical relevance of previous reports and ongoing clinical trials testing the therapeutic utility of antimicrobials for the management of this disease is also highlighted.The incidence rate of sarcoidosis varies depending on the geographic region. For example, high incidence rates are reported in Northern European countries, such as Sweden and Iceland, with nearly 60 cases per 100,000 population . In AsiaSarcoidosis more commonly occurs in people younger than 40, with a peak in the age-group from 25 to 40 years, but the disease has been diagnosed in people of all ages . IntriguDisease severity and clinical manifestations also show certain racial patterns. For instance, in African Americans the disease is more likely to be a chronic and severe disorder involving several organs that can lead to death . ErythemSince sarcoidosis is a multisystem inflammatory disease, symptoms largely depend on the organs affected  Table 11. StrikiPulmonary involvement, which is the most frequent form of the disease, may present with cough and shortness of breath and less commonly hemoptysis , 84. TheExcept for Japanese individuals, in whom cardiac sarcoidosis is a common manifestation of this disease, heart affectation has been considered a rare phenomenon . HoweverFurthermore, sarcoidosis can cause a wide variety of musculoskeletal complaints affecting the bones, joints, or muscles with joint involvement manifested as acute or chronic arthritis , 137. CuFinally, Sarcoidosis affecting the nervous system (neuro-sarcoidosis) is a rare condition that can manifest as a space-occupying lesion in the central nervous system or as peripheral neuropathies , 162.Findings in conventional Chest X-rays may be the first clue that suggest the diagnosis of sarcoidosis, which is complemented with more advanced imaging techniques such as high resolution CT scan (HRCT) and magnetic resonance (MRI) imaging.In addition, 18F-fluorodeoxyglucose positron emission tomography–computed tomography (FDG-PET/CT) scan has become an extremely useful technique not only for the diagnosis of sarcoidosis but also for evaluating treatment response , 84, 100In patients with lung involvement, pulmonary function tests are useful to assess lung functioning and bronchoscopy for bronchial inspection and biopsy extraction are frequently utilized. The development of ultrasound imaging coupled with bronchoscopy has improved the diagnosis of pulmonary sarcoidosis as it increases the yield of tissue aspiration of hilar and/or mediastinal lymph nodes .According to the inclusion criteria from the American Thoracic Society/European Respiratory Society/World Association of Sarcoidosis and other Granulomatous Disorders (ATS/ERS/WASOG), there are three criteria for diagnosis sarcoidosis: (1) a compatible clinical and radiologic presentation, (2) pathologic evidence of typical lesions in more than one organ, and (3) exclusion of other diseases known to cause granulomatous disease. However, since the clinical presentation of sarcoidosis is quite variable and there is not a single diagnostic test or procedure to definitely diagnosis sarcoidosis, in many cases the diagnosis of this disease is challenging . RecentlA recently study that included 2163 Caucasian patients with sarcoidosis who were evaluated at 31 study centers in Europe, revealed that patients with acute onset were mainly female, young and presented with scadding type I or II. High frequency of eye and skin involvement along with fatigue were also frequently observed in female patients. More importantly, according to the predominant organs affected, patients could be consistently stratified into five distinct subgroups: 1) abdominal organ involvement, 2) ocular–cardiac–cutaneous–central nervous system disease involvement, 3) musculoskeletal–cutaneous involvement, 4) pulmonary and intrathoracic lymph node involvement, and 5) extrapulmonary involvement . In addiTreatment recommendations for patients with sarcoidosis will not be reviewed in this article, however, it is important to mention that since spontaneous resolution frequently occurs, many patients with pulmonary disease do not require any specific treatment and can be monitored over a period of time . In thosIn patients who do not respond to corticosteroids, more potent immunosuppressant drugs such as methotrexate, azathioprine and mycophenolate may be required, although due to the considerable secondary effects associated with the long-term use of these drugs, patients need to be carefully monitored , 126.In addition, biological agents, including anti-TNF-α monoclonal antibodies (infliximab) and anti-CD20 monoclonal antibodies (rituximab) have shown promising therapeutic potential in select group of patients, especially in those with severe or refractory sarcoidosis, , 83, 126Recently the Innate Repair Receptor (IRR) activator ARA 290, has been designed an orphan drug for the treatment of sarcoidosis by the U.S. Food and Drug Administration (FDA) and the European Union and was granted as Fast Track designation by the FDA for the treatment of painful small fiber neuropathy in patients with sarcoidosis. ARA290 has been evaluated in patients with small fiber neuropathy associated with sarcoidosis or type 2 diabetes mellitus. In the sarcoidosis group, ARA290 improved quality of life and significantly ameliorated neuropathic and autonomic symptoms and thus ARA290 will be useful for ameliorating pain associated with this form of sarcoidosis , 155.As mentioned above, the key pathogenic component of sarcoidosis is the development of noncaseating granulomas that can arise in different organs. Current evidence suggests that a combination of genetic predisposition and environmental conditions play a central role in the excessive immune reaction leading to the development of sarcoidosis , 125. GrHistologically, sarcoidal granulomas are characterized by the presence of centrally organized collections of epithelioid histiocytes and macrophages surrounded by giant cells and lymphocytes, mostly Th1 lymphocytes, and a rim of fibrosis , 91. UnlThe immune signature of sarcoidosis is the excessive immune response mediated by CD4+ type 1 helper-like cells (Th1-cells), including hyperactivation of Th1-cells and increased levels of Th1 cytokines  and transforming growth factor β (TGF-β) or through mechanisms dependent on cell contact via the CD25 molecules constitutively expressed on their surfaces [Regulatory cells (Treg cells), which are defined as CD4surfaces . High nusurfaces  likely dsurfaces . Contrarsurfaces . Notablysurfaces , which isurfaces . Interessurfaces  indicatiThe appearance of familiarly clustered cases of sarcoidosis and the racial differences in disease incidence and clinical progression support a possible link of genetics with the development of this disease , 154. ThHLA-DRB1∗01/∗04  region, comprising of HLA class I  and class II  genes, being HLA class II variants, particularly HLA-DRB1 and DQB1 the alleles most prevalently reported in association with sarcoidosis , 158. For, 2015; , 130). I disease , 61, 123 disease , 158.CCR2, CCR5, IL1A, IL23R, TNF-α and NOD2, FCGR, have been also reported in association with the disease [On the other hand, SNPs in non-HLA genes, including  disease , 139. It disease ) and som disease , 138.Despite that numerous genetic risk factors in association with sarcoidosis have been identified, in the majority of cases, the biologic functions of specific variants and their precise interaction with environmental exposures remain unknown. The use of new generation sequencing based HLA typing will be helpful to identify potential new associations and to elucidate the specific contribution of particular gene variants, especially within the HLA region, to disease susceptibility and may provide new insights on the pathogenesis of this disease.Sarcoidosis has been also linked with certain environments factors such as exposure to inorganic particles, insecticides, and moldy environments. Occupational studies have also found an increased risk of sarcoidosis among metalworkers and fire fighters . In a reFurthermore, tattoos-induced sarcoidosis has been described in the literature, and although the etiology of this phenomenon is unknown, it has been attributed to an excessive immune reaction against pigment particles, in genetically predisposed individuals. Notably, the sarcoidal granulomas, contain pigment particles, pigment laden macrophages and polarizing material and some patients may develop symptoms of pulmonary sarcoidosis .mycobacterium tuberculosis hsp (mTB-hsp) and HSP, this may lead to an excessive immune response that ultimately may cause sarcoidosis. On the other hand, in non-infectious causes of sarcoidosis, the persistent exposure to an array of damaging factors such as phagocyted metal fumes, pigments and other chemicals may result in the release of damage-associated molecular patterns (DAMPs), also known as danger-associated molecular patterns, which are host biomolecules capable of initiating and perpetuating a noninfectious inflammatory response [In line with these observations, it has been proposed that sarcoid formation may occur in the context of infectious and non-infectious causes, depending on the genetic background of the host. In this model, the exposure to certain microorganisms expressing microbial hsps or pathogen-associated molecular patterns (PAMPs), which are sensed by immune cells via pattern recognition receptors (PRR), may generate an increased and persistent release of both human HSPs and microbial hsp. Given the high homology of mycobacterial hsp, particularly response .Intriguingly, patients with sarcoidosis have increased risk of developing lymphomas, a condition that has been called sarcoidosis-lymphoma syndrome, with sarcoidosis preceding lymphoma in most cases, though sarcoidosis following lymphoma has been also reported  and sarcThe potential involvement of microbes in the pathogenesis of sarcoidosis was proposed many years ago , 49. ThiC. acnes, resulted in a considerable attenuation of the granulomatous pulmonary inflammation compared to wild-type C57BL/6 animals [+ lymphocytes in BAL of patients with sarcoidosis. Increased TLR9 expression in alveolar macrophages derived from patients with sarcoidosis was also reported and these cells secreted higher levels of cytokines in response to in vitro stimulation [In addition, a putative involvement of microbes in the pathogenesis of sarcoidosis is indirectly supported by studies reporting considerable activation PRRs, which are essential components of the innate immune system involved in the control of infective microorganisms. Among the altered PRRs, enhanced expression toll-like receptors (TLR) and their activated downstream signals have been documented in various cell subsets of patients with sarcoidosis. For example, a study that assessed global transcriptomic changes of miRNAs of circulating Tregs and CD4+/CD25 + Tregs from BAL, identified alterations in TLR-2 signaling pathway. Notably, induction of TLR-2 expression was found not only in Tregs, but also in the heterogeneous population of peripheral blood mononuclear cells of patients with pulmonary sarcoidosis . In addi animals . Anothermulation . TLR9 ismulation .C. acnes and sarcoidosis . Strong association was also found with mycobacterium tuberculosis (mTB), as this bacterium was detected within the sarcoidal lesions of nearly 25% of sarcoidosis patients (OR 6.8). Other microbes, including Borrelia, HHV-8, as well as Rickettsia helvetica, Chlamydia pneumoniae, Epstein-Barr virus and Retrovirus were not associated with sarcoidosis [C. acnes and mTB with sarcoidosis.So far, the most comprehensive study aimed to clarify the role of microbes in sarcoidosis is a meta-analysis that included 58 case–control studies  reporting the presence of microorganisms in samples of patients with sarcoidosis using culture methods or molecular biology techniques concluded that an etiological link exists between coidosis . In the C. acnes, is a slow-growing anaerobic Gram-positive bacterium rod that is etiologically linked to the skin disorder acne. In addition, C. acnes can cause chronic blepharitis, and endophthalmitis and is an opportunistic agent that can cause endocarditis, corneal ulcer, septic arthritis and is a contaminating agent of areas subcutaneous such as after craniotomy. This bacterium is largely commensal and part of the skin microbiota detectable on healthiest adult humans’ skin and may be also found throughout the gastrointestinal tract.C. acnes can be found in BAL of the majority of patients with sarcoidosis and its presence has been associated with disease activity, however the microbe can be also found in around 20% of controls [C. acnes has been identified in granulomas and inflammatory cells of myocardial tissues [C. acnes constitutes the only microorganisms that has been isolated from sarcoidal specimens by bacterial culture [controls . In addi tissues , lymph n tissues  neurolog tissues , the gra tissues  and many culture .C. acnes, Propionibacterium granulosum, mTB, Mycobacterium avium subsp. paratuberculosis, and Escherichia coli (as the control). The authors concluded that Propionibacterium spp. are more likely than Mycobacteria spp. to be involved in the etiology of sarcoidosis and the association occurred not only in Japanese but also in European patients with sarcoidosis [C. acnes genomes were also found in FFPE derived from Chinese patients with sarcoidosis but not in tuberculosis or lung cancer [One of the first attempts to systematically study a potential link between sarcoidosis and pathogens was an international study included formalin-fixed and paraffin-embedded (FFPE) sections of lymph nodes of patients from Japan, Italy, Germany, and England. The specimens  were subjected to quantitative real-time PCR to identify genomes of coidosis . High frg cancer .C. acnes-specific monoclonal antibodies that react with cell-membrane-bound lipoteichoic acid (PAB antibody), the authors detected small round bodies within sarcoid granulomas in 20/27 (74%) video-assisted thoracic surgery lung samples, 24/50 (48%) transbronchial lung biopsy samples, 71/81 (88%) Japanese lymph node samples, and 34/38 (89%) German lymph node samples. Notably, PAB antibody did not react with non-sarcoid granulomas in any of the 45 tuberculosis samples or the 34 samples with sarcoid reaction, suggesting that this indigenous bacterium might be the cause of granuloma formation in many sarcoid patients [Negi and colleagues examined formalin-fixed and paraffin-embedded samples of lungs and lymph nodes from 196 patients with sarcoidosis, as well as control samples derived from 275 patients with non-sarcoidosis diseases. Using immunohistochemistry with patients .C. acnes secretes propionic acid and other short-chain fatty acids metabolites (SCFAs) such as formic acid (C1), acetic acid (C2), butyric acid (C4), and valeric acid (C5). Interesting SCFAs secreted by gut microbiota exert modulatory effects on the immune system, including the differentiation and proliferation of Treg cells [ic acid C, and valeg cells , and regeg cells , 150.C. acnes may promote the proliferation of Treg cells and thus it could theoretically prevent the development of sarcoidosis rather that promoting granuloma formation, however, in individuals with new onset pulmonary sarcoidosis, a large infiltration of Treg cells, along with Treg cytokines such as IL-10 and TGF-β are increased but are not functional [One may expect that propionic acid released by nctional . TherefoC. acnes, however is worthy to notice that the vast majority of the studies reported so far have been conducted by Japanese groups testing Japanese patients, while only a few testing specimens obtained from patients from African American or Caucasian patients having been published. More studies are needed to demonstrate a direct causal link between this pathogen and sarcoidosis.In summary, there is abundant laboratory and clinical evidence supporting an association between sarcoidosis and The link between mTB and sarcoidosis was proposed many years ago  given thPathological studies have documented the presence of mTB-hsps, including mTB-hsp70, mTB-hsp65, and mTB-hsp16 in lymph node tissues of patients with sarcoidosis, being mTB-hsp16 predominantly detected in the early stage of the disease and mTB-hsp70 in stage II sarcoidosis . As expeThere is a relative high prevalence of sarcoidosis in certain countries of northern Europe and for many years it was assumed that whereas sarcoidosis was a disease of the developed world, tuberculosis was a disease that mainly affect people in the developing world . HoweverEarly observations published during the mid-twentieth reported the presence of mTB bacilli-like structures identified by bacteriological methods in inflamed tissues of some patients with suspected sarcoidosis , 133, alA meta-analysis assessing the molecular evidence of the role of mycobacteria in sarcoidosis analyzed 31 studies in which PCR for nucleic acid amplification followed by identification of nucleic acid sequences specific for different types of mycobacteria concluded that 231 out of 874 patients were positive for mycobacteria with a positive signal rate of 26.4 (23.6–29.5%), and the odds of finding mycobacteria in samples of patients with sarcoidosis versus controls were 9.67 (4.56–20.5%). It must be noted however that methodological and statistical heterogeneity and publication bias was evident in most of the studies assessed . However+ monocytes have the potential to differentiate into two functionally distinct subpopulations M1 and M2 macrophages. Notably, M2 macrophages, also known as “product of Th2 activation”, are the result of macrophage polarization driven by immunoregulatory cytokines such IL-4, IL-10 and TGF-β and tumor-infiltrating macrophages are typically classified as M2 and are well-known to promote malignant cells growing and immune evasion [A comprehensive assessment of immune infiltration of sarcoidosis or tuberculosis granulomas using immunohistochemistry revealed that, although there was no significance difference in single CD4+, CD8+, CD22+, CD14+ and CD68+ staining between sarcoidosis and tuberculosis sections, CD163 expression (a marker of M2 macrophages) was significantly increased in sarcoidosis sections compared with those from tuberculosis . Althoug evasion . Interes evasion .Mycobacterial or their products can exert differential effects on immune from patients with sarcoidosis. For example, higher percentages of CD8(+) αβ (+) T-cells, with a concomitant increase in the release of pro-inflammatory cytokines was observed when peripheral blood mononuclear cells (PBMCs) from patients with sarcoidosis were stimulated in vitro with Mtb-hsp, in comparison with PBMCs from controls or from patients with tuberculosis . NotablyTaken together, the above discussed evidence appear to link mTB, other mycobacteria or their products with the excessive immune responses leading to sarcoidosis in a proportion of patients with sarcoidosis. Although this association in unlikely to occur in all patients and may be more significant in countries where tuberculosis is endemic.Human microbiota composition is functionally implicated in the regulation of several physiological processes, including metabolic functions and immune homeostasis. Although gut microbiota is the most studied human microbiota niche, well-defined microbiota communities have been identified in virtually any body niche, including the skin and the respiratory tract , 53, 111Atopobium spp. was detectable in 68% of sarcoidosis specimens but in none of healthy controls. Similarly, Fusobacterium spp. were also identified as sarcoidosis-associated bacteria. In the same study, gene association analysis identified an association of the variant rs2076530 in BTNL2 associated with a decrease in bacterial burden [Microbiome analysis in healthy individuals reveal that airway and lung microbiome composition is similar to oropharyngeal microbiome with no evidence of site specific enrichment of bacterial communities indicating and the presence of certain microorganisms, which reach the lungs mainly via passive microaspiration, may directly modulates local immune responses . Not surl burden . Of notel burden , 153.n = 93) and control subjects without sarcoidosis (n = 72). Sarcoidosis specimens analyzed included two independent sets of formalin-fixed, paraffin-embedded (FFPE) granulomatous tissue biopsies from patients with newly identified sarcoidosis and control patients, and BAL from patients with newly diagnosed sarcoidosis and healthy control subjects. A set of 20 specimens (19 Kveim reagent and one fresh spleen) that had been prepared under sterile conditions were also analyzed. Specimens were interrogated using diverse sequencing strategies including 16S ribosomal RNA gene sequencing , shotgun metagenomics  and internal transcribed spacer ribosomal RNA gene sequencing (for fungi). In one tissue set, fungi in the Cladosporiaceae family were enriched in sarcoidosis compared with nonsarcoidosis tissues; in the other tissue set, several bacterial lineages were enrichment in sarcoidosis but not Cladosporiaceae. In BAL fluid samples, Corynebacteria was enriched along with a limited enrichment of Aspergillus fungi. Interestingly, in the 20 sterile-collected samples available (Kveim and spleen tissue), whole-genome shotgun sequencing analysis revealed the enrichment of several microbial lineages, especially Cladosporium [Clarke and colleagues reported performed metagenomics sequencing analysis of specimens from subjects with sarcoidosis (osporium . DespitePropionibacteria (which have been linked with sarcoidosis in previous studies) may be present in both environmental controls and patient tissues, along with the fact that mycobacteria DNA extraction is difficult, likely contributed to the false negative results reported in the study. Finally, spleen and Kveim specimens were not matched with negative control human specimens. A recent study that included 16sRNA gene sequencing of bronchoscopy obtained specimens from 31 patients with sarcoidosis and 19 with interstitial lung disease found no significant differences in the microbiome composition between the two groups, being Firmicutes, Proteobacteria, Acinetobacter, Bacteroidetes, Fusobacteriales, and Spirochaetales the most dominant microorganisms identified. The absence of a healthy control group and the heterogeneity of the interstitial lung disease group were important limitations in this study [However, it must be noted that there were various technical limitations associated with this study. First, most samples utilized in this study were FFPE and formalin degrades DNA and also it is well-known that the de-crosslinking step necessary to undo the formalin fixation results in DNA damage which may have impaired the sensitivity for microbe detection in these samples. In addition, although in this study various negative control specimens, representing a variety of potential sources of contamination, were analyzed to exclude false positive findings, the fact that mycobacteria and is study .Furthermore, as previous studies have shown, alterations in the gut microbiome may result in the development of systemic inflammatory conditions, such as rheumatoid arthritis and atherosclerosis , 143, whC. acnes or Mtb could modify the clinical course of the disease. Indeed, a few studies testing the clinical efficacy of antibiotics for sarcoidosis have been reported, although most studies have been conducted in small series of cases and essentially in patients with cutaneous sarcoidosis. Measurable clinical response was observed in 10 of the 12 patients with cutaneous sarcoidosis treated with either minocycline or doxycycline, although no control group was included in this small study [One may speculate that using antibiotics capable of eradicating microbes potentially linked with sarcoidosis such as ll study . In a rall study . The samll study .C. acnes within the granulomas from some patients with sarcoidosis [Although some studies utilizing electron microscopic images and various molecular biology techniques have reported the presence of infective coidosis , 108, thcoidosis .C. acnes) or standard corticosteroid therapy alone [Currently, a multicenter open-label clinical trial (J-ACNES) of sarcoidosis is being conducted in Japan. This study, which is the largest so far, will assess the clinical response of patients with cardiac sarcoidosis in response to antibiotic therapy for which patients will be randomized to receive either standard corticosteroid therapy plus antibiotic therapy  tools may be very useful for this purpose, since AI has tremendous potential in the development of algorithms for the diagnosis, treatment and prognosis of complex human diseases. AI can integrate large number of data encompassing laboratory data, imaging studies, and clinical data to better characterize disease and developing predictive models , 114.For more than one century, utilizing a variety of approaches and testing numerous specimens, researchers have tried to identify the cause of sarcoidosis to ultimately design a cure for this disease. Some of those studies have provided important insights for understanding the etiology of the sarcoidosis. For example, immunohistochemistry and molecular biology studies have delineated the cellular components of sarcoidal granulomas and the unique features that distinguish sarcoidosis from other granulomatous disorders including tuberculosis. Gene association studies have identified genetic risk factors of sarcoidosis and epidemiological studies have contributed to identify specific environmental and occupational factors strongly associated with the disease.C. acnes appear to be the strongest candidates. Although current evidence does not support a direct causal role of these pathogens in sarcoidosis, some studies have reported encouraging results on the potential utility of antibiotics for the management of patients with sarcoidosis and there is a great expectation about the results that will generate ongoing randomized clinical trials of antibiotics in sarcoidosis. Further studies are needed to better understand the specific role of the above mentioned agents in the development of sarcoidosis. For example, although articles published so far have failed to identify a lung microbiome signature in patients with sarcoidosis, data generated from those studies could not definitely rule out that an alteration in the microbiome composition of the respiratory tract or in other body niche, such as dysbiosis of the gut has some role in the development or progression of the disease. Given the complexity of the disease, future studies will require the combination of various approaches using microbiological, immunological, epidemiological and molecular biology techniques to unravel the unknown aspects of this disease. In this regard, the utilization of machine learning and deep learning algorithms will be extremely useful for future studies as artificial intelligence tools can identify causal associations that are undetectable using standard statistical analysis methods [Several studies have tested the hypothesis that microbes may be the cause of sarcoidosis and among the numerous microorganisms investigated, mTB and  methods . Finally methods , 77, app


It is assumed that case-based questions require higher-order cognitive processing, whereas questions that are not case-based require lower-order cognitive processing. In this study, we investigated to what extent case-based and non-case-based questions followed this assumption based on Bloom’s taxonomy.In this article, 4,800 questions from the Interuniversity Progress Test of Medicine were classified based on whether they were case-based and on the level of Bloom’s taxonomy that they involved. Lower-order questions require students to remember or/and have a basic understanding of knowledge. Higher-order questions require students to apply, analyze, or/and evaluate. The phi coefficient was calculated to investigate the relationship between whether questions were case-based and the required level of cognitive processing.Our results demonstrated that 98.1% of case-based questions required higher-level cognitive processing. Of the non-case-based questions, 33.7% required higher-level cognitive processing. The phi coefficient demonstrated a significant, but moderate correlation between the presence of a patient case in a question and its required level of cognitive processing .Medical instructors should be aware of the association between item format (case-based versus non-case-based) and the cognitive processes they elicit in order to meet the desired balance in a test, taking the learning objectives and the test difficulty into account. It is increasingly accepted that the purpose of assessments is not only to evaluate what has been learned but also to drive students’ learning ,2. HowevThe Dutch progress test is a systematic longitudinal assessment that measures students’ knowledge at the level that they are expected to achieve by the end of their program ,5. On thA well-established framework to categorise questions is Bloom’s taxonomy, which provides a hierarchical model of knowledge acquisition that allows questions to be classified by the level of cognitive processing that they require. In theory, students achieve higher levels of cognitive processing only after mastering the lower levels of cognitive processing. The latter levels, in Bloom’s taxonomy, comprise knowledge reproduction and a minimal understanding of pieces of knowledge. The next level is application of information, followed by synthesising, evaluating, and creating information ,8. An ilQuestions that require higher-order cognitive processing facilitate further knowledge consolidation, since students must recall their previous knowledge and then reason about that knowledge to make a decision. Questions that require higher-order cognitive processing have a greater impact on students’ knowledge retention. Studying with higher-order questions has show to improve students’ performance on lower- and higher-order questions when compared studying with lower-order questions .Case-based questions thus require students to apply, synthesise, and evaluate their knowledge, assessing students’ ability to engage in higher-order cognitive processing. Non-case-based questions are expected to require students to remember and recall their knowledge, assessing students’ lower-order cognitive processing.Instructors are often asked to produce case-based or non-case-based questions, with no specification of the appropriate cognitive level. One of the underlying assumptions of case-based questions is that they require higher-order cognitive processing, whereas questions that are not case-based usually require lower-order cognitive processing. In the current study, we investigated to what extent case-based and non-case-based questions followed this assumption based on Bloom’s taxonomy, since this may have implications for how instructors should formulate questions. More importantly, this may reflect the alignment between the objectives and the quality of the items of the Dutch progress test.For this study, we followed the Declaration of Helsinki and the privacy policy of the University of Groningen, and all data were anonymised and handled with confidentiality. Under Dutch law, no ethical approval was needed, because reanalysis of historical data is exempt.This is a retrospective study.In this study, we analysed questions from the Dutch Interuniversity Progress Test of Medicine from 2007 to 2013. The progress test is a systematic, longitudinal assessment that aims to measure students’ knowledge at the level that they are expected to achieve by the end of their program, and the questions are based on the Dutch National Blueprint for the Medical Curriculum. The progress test is simultaneously administered 4 times a year to all undergraduate medical students, from the first to the sixth year. At the time of the study, 4 of the 8 medical schools in the Netherlands participated in the progress test. Each progress test consisted of 200 questions, which resulted in a total of 4,800 questions. This progress test is particularly suitable because each test contains 200 unique questions developed by a large and diverse group of instructors at different medical schools, it is independent of any curriculum, and it contains both case-based questions and non-case-based questions [Two of the authors (WA and RT) independently established for 50 questions whether a patient case was presented and what the required level of cognitive processing was according to Bloom’s taxonomy. Items were coded as case-based questions when a patient and illness were present. Items were coded as lower-order questions when items only required students to remember and/or show a basic understanding of knowledge, and when it was not necessary to use patient information to answer the question. Items were coded as higher-order questions when they required students to apply, analyse, or/and evaluate existing knowledge, and when students had to reason about the case to answer the question. The authors agreed on all questions about whether they were case-based, and there was only 1 disagreement about Bloom’s taxonomy. After discussing this question, the authors came to a consensus. Because of this high interrater agreement, the rest of the items were labelled by a single author (WA).To study the relationship between the presence of a patient case and the required level of cognitive processing, the phi coefficient was calculated. The phi coefficient indicates the correlation between 2 binary variables . The chiOf the 4,800 questions, 1,138 were case-based (23.7%) and 3,662 were non-case-based. Regarding the level of cognitive processing that was required, 2,350 questions required higher-order cognitive processing and 2,450 required lower-order cognitive processing. There was a significant, but moderate association between the presence of a patient case in the question and its required level of cognitive processing .2(1)= 1,239.7, P< 0.001). The raw data are available in Of the 1,138 case-based questions, 1,116 required higher-level cognitive processing, while only 22 required lower-level cognitive processing. Of the 3,662 non-case-based questions, 1,234 required higher-level cognitive processing and 2,428 questions required lower-level cognitive processing . This diThe current study investigated the relationship between whether a question was case-based and the level of cognitive processing, according to Bloom’s taxonomy, required to answer it. Case-based questions usually required higher-level cognitive processing, as expected. Interestingly, one-third of non-case-based questions also required higherlevel cognitive processing.Our findings suggest that writing a case-based question causes instructors to produce questions that require a higher order of cognitive processing without explicitly asking them to do so. This may be because these questions are more closely related to real-life situations that are familiar to medical doctors in clinical practice. One might even argue that if a question is only aimed to measure recall or basic understanding, it may be redundant to include a patient case, since the reality of clinical practice usually involves dealing with a complex set of variables. If instructors find it challenging to produce higher-order questions, one might suggest that they use a relevant patient case as a basis for such questions.Our findings also suggest that even without an explicit specification of the questions to be developed, more than one-third of the questions without a patient case required higher-order cognitive processing. Non-case-based questions can still require students to do more than just reproduce and show basic understanding. It is important to realise that the type of questions on a test should reflect the learning objectives and students’ level of development. Asking instructors to produce non-case-based questions and assuming that those questions will measure lower-order cognitive processing is unwarranted. Thus, it is imperative to provide guidance to instructors to write at the appropriate level of cognitive processing. If instructors write non-case-based questions, they must be instructed to write items requiring lower-level cognitive processing. Novice students will answer more lower-order than higher-order questions correctly, since they do not possess the necessary knowledge to handle cases that require knowledge application .Because of the formative function and the repetitive character of the progress test ,10, it iStudies have investigated the content of questions on large-scale exams, such as the use of questions involving race/ethnicity  and the


Methane-producing microbial communities are of ecological and biotechnological interest. Syntrophic interactions among sulfate reducers and aceto/hydrogenotrophic and obligate hydrogenotrophic methanogens form a key component of these communities, yet, the impact of these different syntrophic routes on methane production and their stability against sulfate availability are not well understood. Here, we construct model synthetic communities using a sulfate reducer and two types of methanogens representing different methanogenesis routes. We find that tri-cultures with both routes increase methane production by almost twofold compared to co-cultures and are stable in the absence of sulfate. With increasing sulfate, system stability and productivity decreases and does so faster in communities with aceto/hydrogenotrophic methanogens despite the continued presence of acetate. We show that this is due to a shift in the metabolism of these methanogens towards co-utilization of hydrogen with acetate. These findings indicate the important role of hydrogen dynamics in the stability and productivity of syntrophic communities. InteracTowards deciphering ecological and evolutionary drivers of function and functional stability in microbial communities, methanogenic anaerobic digestion (AD) offers an ideal model system, where the production of methane from complex organic substrates can be taken as a proxy for a community function. AD communities are found in many environments including ocean and lake sediments, soil and animal guts and are used in biotechnological re-valuation of organic waste . It is w2 and acetate, which can be used by aceto- and hydrogenotrophic methanogens prod. need then be combined with the observed residual levels of these compounds in the system, to estimate the levels that were available to Mb ([Acetate]Mb and [H2]Mb):The estimated [Acetate]4]calc) by Mb, through reactions 1, 2 and 6. The actual amounts of acetate used in reactions 2 and 6, as well as the actual amounts of H2 used in reactions 1 and 6, are unknown. If we assume a full conversion through the three reactions, we would have the following stoichiometric balances:xi and yi denote the amounts of acetate and H2 used in reaction i, respectively. These three equalities can then be re-arranged to yield the overall theoretical methane production.We can now use these values to calculate the estimated stoichiometric, theoretical methane production ([CH