Validation of human microRNA target pathways enables evaluation of target prediction tools.

MicroRNAs are regulators of gene expression. A wide-spread, yet not validated, assumption is that the targetome of miRNAs is non-randomly distributed across the transcriptome and that targets share functional pathways. We developed a computational and experimental strategy termed high-throughput miRNA interaction reporter assay (HiTmIR) to facilitate the validation of target pathways. First, targets and target pathways are predicted and prioritized by computational means to increase the specificity and positive predictive value. Second, the novel webtool miRTaH facilitates guided designs of reporter assay constructs at scale. Third, automated and standardized reporter assays are performed. We evaluated HiTmIR using miR-34a-5p, for which TNF- and TGFB-signaling, and Parkinson's Disease (PD)-related categories were identified and repeated the pipeline for miR-7-5p. HiTmIR validated 58.9% of the target genes for miR-34a-5p and 46.7% for miR-7-5p. We confirmed the targeting by measuring the endogenous protein levels of targets in a neuronal cell model. The standardized positive and negative targets are collected in the new miRATBase database, representing a resource for training, or benchmarking new target predictors. Applied to 88 target predictors with different confidence scores, TargetScan 7.2 and miRanda outperformed other tools. Our experiments demonstrate the efficiency of HiTmIR and provide evidence for an orchestrated miRNA-gene targeting.

A Novel nonsense variant in the CDH2 gene associated with ACOGS: A case report.

Agenesis of Corpus Callosum, Cardiac, Ocular, and Genital Syndrome (ACOGS; OMIM #618929) is a rare genetic disorder characterized by global developmental delay, agenesis or hypoplasia of corpus callosum, craniofacial dysmorphism, ocular, cardiac, and genital anomalies. ACOGS is caused by variations in the CDH2 gene. Our patient had a novel finding besides the classical findings of ACOGS. To the best of our knowledge, only 14 patients with ACOGS have been reported. Here, we reported the fifteenth patient with ACOGS, having a novel de novo nonsense variant in the CDH2 gene, and the first patient from Turkey with a novel finding. Our patient was the first female to have a renal anomaly since only genital malformations were reported in male patients (cryptorchidism, micropenis) so far.

Is there a relationship between ACTN3 R577X gene polymorphism and sarcopenia?

BACKGROUND AND AIMS: The alpha-actinin (ACTN) genes are important structural components of the sarcomere. Sarcopenia is a common geriatric syndrome characterized by morbidity and mortality. Our study aimed to examine the relationship between the ACTN3 R577X gene and sarcopenia in community-dwelling Turkish adults. METHODS: We designed a cross-sectional study among the patients aged ≥ 65 years admitted to the geriatric outpatient clinic. We recorded the general characteristics of the patients. We used the Jamar hand dynamometer to evaluate handgrip strength. Body composition was estimated using bioimpedance analysis. Sarcopenia was diagnosed according to the European Working Group on Sarcopenia in Older People2 criteria with population-specific cutoffs. We performed analyses of low muscle mass (LMM) with skeletal muscle mass index adjusted for body mass index [SMMI(BMI)]. We further categorized the SMMI(BMI) cutoffs into tenths. The analyzes were performed according to the 90th percentile SMMI(BMI) cutoffs. Peripheral blood samples were collected to determine the ACTN3 genotypes. RESULTS: 197 participants were included [mean age: 76.3 ± 6.1 years, 151 (76.6%) women]. The proportions of the ACTN3 genotypes were as follows: RX (45.1%) > RR (31%) > XX (23.9%). The significant difference between genotypes was found only for low SMMI(BMI) according to the 90th percentile (p = 0.025). In multivariate analysis, only gender (female) was independently associated with LMM. CONCLUSION: We did not find any association between ACTN3 R577X gene polymorphism and probable sarcopenia, confirmed sarcopenia and LMM. Besides, much more research is needed to reveal how ethnicity affects the muscles of older adults with ACTN3 R577X gene polymorphism.

microRNA neural networks improve diagnosis of acute coronary syndrome (ACS).

BACKGROUND: Cardiac troponins are the preferred biomarkers of acute myocardial infarction. Despite superior sensitivity, serial testing of Troponins to identify patients suffering acute coronary syndromes is still required in many cases to overcome limited specificity. Moreover, unstable angina pectoris relies on reported symptoms in the troponin-negative group. In this study, we investigated genome-wide miRNA levels in a prospective cohort of patients with clinically suspected ACS and determined their diagnostic value by applying an in silico neural network. METHODS: PAXgene blood and serum samples were drawn and hsTnT was measured in patients at initial presentation to our Chest-Pain Unit. After clinical and diagnostic workup, patients were adjudicated by senior cardiologists in duty to their final diagnosis: STEMI, NSTEMI, unstable angina pectoris and non-ACS patients. ACS patients and a cohort of healthy controls underwent deep transcriptome sequencing. Machine learning was implemented to construct diagnostic miRNA classifiers. RESULTS: We developed a neural network model which incorporates 34 validated ACS miRNAs, showing excellent classification results. By further developing additional machine learning models and selecting the best miRNAs, we achieved an accuracy of 0.96 (95% CI 0.96-0.97), sensitivity of 0.95, specificity of 0.96 and AUC of 0.99. The one-point hsTnT value reached an accuracy of 0.89, sensitivity of 0.82, specificity of 0.96, and AUC of 0.96. CONCLUSIONS: Here we show the concept of neural network based biomarkers for ACS. This approach also opens the possibility to include multi-modal data points to further increase precision and perform classification of other ACS differential diagnoses.

The sncRNA Zoo: a repository for circulating small noncoding RNAs in animals.

The repertoire of small noncoding RNAs (sncRNAs), particularly miRNAs, in animals is considered to be evolutionarily conserved. Studies on sncRNAs are often largely based on homology-based information, relying on genomic sequence similarity and excluding actual expression data. To obtain information on sncRNA expression (including miRNAs, snoRNAs, YRNAs and tRNAs), we performed low-input-volume next-generation sequencing of 500 pg of RNA from 21 animals at two German zoological gardens. Notably, none of the species under investigation were previously annotated in any miRNA reference database. Sequencing was performed on blood cells as they are amongst the most accessible, stable and abundant sources of the different sncRNA classes. We evaluated and compared the composition and nature of sncRNAs across the different species by computational approaches. While the distribution of sncRNAs in the different RNA classes varied significantly, general evolutionary patterns were maintained. In particular, miRNA sequences and expression were found to be even more conserved than previously assumed. To make the results available for other researchers, all data, including expression profiles at the species and family levels, and different tools for viewing, filtering and searching the data are freely available in the online resource ASRA (Animal sncRNA Atlas) at https://www.ccb.uni-saarland.de/asra/.

Rational drug use habits of patients with chronic diseases: A cross-sectional examination focusing on the use of technological devices.

OBJECTIVE: The aim of this study was to investigate the factors affecting rational drug use habits and the use of technological devices in patients with chronic diseases. METHODS: Adults who applied to the internal medicine outpatient clinics of a university hospital between March and December 2019, who used medications for chronic disease were included in the study. Using a questionnaire, data on demographic characteristics, technology use, smoking and alcohol use, knowledge and behaviour on rational drug use were collected. RESULTS: Of the patients, 73.3% (n = 220) had smartphones, 28.0% (n = 84) tablets, 8.7% (n = 26) smartwatches, 6.0% (n = 18) were using smart bracelets, 52.3% (n = 157) knew the e-pulse application of the Ministry of Health, 53.3% (n = 160) forgot on occasions the time to take their medications, 51.7% (n = 155) threw away some drugs because the expiration date has passed, 64.0% (n = 192) had at home never-used or unfinished medications, 21.3% (n = 64) had medications to be used 'in case', 19.0% (n = 57) recommend drugs to others and 34.3% (n = 103) were getting advice from their environment on drug use. Women were 2.35 times more likely to use technology than men (95% CI: 1.19-4.64). Decreasing age was associated with an increased likelihood of using technology. Those with an income of more than twice the minimum wage compared with those with an income of minimum wage and below had 3.41 times (95% CI: 1.06-10.94) higher possibility of using technological devices, while compared with the illiterate, those with secondary education or university education had 14.96 times (95% GA: 3.67-60.93) higher possibility of using technological devices. CONCLUSION: The patients with chronic diseases demonstrate crucial deficiencies regarding rational drug use. The widespread use of technological devices may be an opportunity for preventive and remedial projects to be developed through these devices. Smartphone-based self-management tools should be developed and introduced to chronic patients.

Galectin-3: can it be a diagnostic tool for pneumonia in covid-19 patients?

Background/aim: Biochemical markers are needed to show lung involvement in COVID-19 disease. Galectin-3 is known to play a key role in the inflammation and fibrosis process. We aimed to evaluate the predictive role of galectin-3 levels for pneumonia in patients with COVID-19. Materials and methods: Total of 176 patients with COVID-19, confirmed with reverse transcriptase polymerase chain reaction, admitted to the Erzurum Regional Training and Research Hospital was analyzed. The study was designed as a cross sectional. The baseline data of laboratory examinations, including galectin-3 were collected at the time of diagnosis. CT images evaluated by a single radiologist according to the recommendation of the Radiological Society of North America Expert Consensus Document for pulmonary involvement. The severity of COVID-19 pneumonia was assessed using the total severity score. Results: The mean galectin-3 level in patients with typical pneumonia was found to be significantly higher than those patients with atypical (p < 0.01) and indeterminate appearance (p < 0.01) and patients without pneumonia (p < 0.01). The severity of lung involvement was significantly associated with Galectin-3 levels (p < 0.01 r: 0.76). Stepwise logistic regression model showed that the levels of ferritin (odds ratio [OR] = 0.05, p: 0.08) and galectin-3 (OR = 0.1, p < 0.01) were significantly and independently associated with typical pneumoniain COVID-19 patients. When COVID-19 patients were evaluated in terms of typical pneumonia, we determined a cut-off value of 18.9 ng/mL for galectin-3 via ROC analysis (87% sensitivity; 73% specificity; area under curve (AUC): 0.89; p < 0.001). Conclusion: Galectin-3 was found as a diagnostic tool for COVID-19 associated typical pneumonia and as an indicator of both pneumonia and its severity.

A high-resolution map of the human small non-coding transcriptome.

Motivation: Although the amount of small non-coding RNA-sequencing data is continuously increasing, it is still unclear to which extent small RNAs are represented in the human genome. Results: In this study we analyzed 303 billion sequencing reads from nearly 25 000 datasets to answer this question. We determined that 0.8% of the human genome are reliably covered by 874 123 regions with an average length of 31 nt. On the basis of these regions, we found that among the known small non-coding RNA classes, microRNAs were the most prevalent. In subsequent steps, we characterized variations of miRNAs and performed a staged validation of 11 877 candidate miRNAs. Of these, many were actually expressed and significantly dysregulated in lung cancer. Selected candidates were finally validated by northern blots. Although isolated miRNAs could still be present in the human genome, our presented set likely contains the largest fraction of human miRNAs. Contact: c.backes@mx.uni-saarland.de or andreas.keller@ccb.uni-saarland.de. Supplementary information: Supplementary data are available at Bioinformatics online.

Spring is in the air: seasonal profiles indicate vernal change of miRNA activity.

The envisioned application of miRNAs as diagnostic or prognostic biomarkers calls for an in-depth understanding of their distribution and variability in different physiological states. While effects with respect to ethnic origin, age, or gender are known, the inter-individual variability of miRNAs across the four seasons remained largely hidden. We sequentially profiled the complete repertoire of blood-borne miRNAs for 25 physiologically normal individuals in spring, summer, fall, and winter (altogether 95 samples) and validated the results on 292 individuals (919 samples collected with the Mitra home sampling device) by RT-qPCR. Principal variance component analysis suggests that the largest variability observed in miRNA expression is due to individual variability and the individuals' gender. But the results also highlight a deviation of miRNA activity in samples collected during spring time. Following adjustment for multiple testing, remarkable differences are observed between spring and fall (77 miRNAs). The two most dys-regulated miRNAs were miR-181c-5p and miR-106b-5p (adjusted p-value of 0.007). Other significant miRNAs include miR-140-3p, miR-21-3p, and let-7c-5p. The dys-regulation was validated by RT-qPCR. Systems biology analysis further provides strong evidence for the immunological origin of the signals: dys-regulated miRNAs are enriched in CD56 cells and belong to various signalling and immune-system-related pathways. Our data suggest that besides known confounding factors such as age and sex, also the season in which a test is conducted might have a considerable influence on the expression of blood-borne miRNAs and subsequently might interfere with diagnosis based on such signatures.

Web-based NGS data analysis using miRMaster: a large-scale meta-analysis of human miRNAs.

The analysis of small RNA NGS data together with the discovery of new small RNAs is among the foremost challenges in life science. For the analysis of raw high-throughput sequencing data we implemented the fast, accurate and comprehensive web-based tool miRMaster. Our toolbox provides a wide range of modules for quantification of miRNAs and other non-coding RNAs, discovering new miRNAs, isomiRs, mutations, exogenous RNAs and motifs. Use-cases comprising hundreds of samples are processed in less than 5 h with an accuracy of 99.4%. An integrative analysis of small RNAs from 1836 data sets (20 billion reads) indicated that context-specific miRNAs (e.g. miRNAs present only in one or few different tissues / cell types) still remain to be discovered while broadly expressed miRNAs appear to be largely known. In total, our analysis of known and novel miRNAs indicated nearly 22 000 candidates of precursors with one or two mature forms. Based on these, we designed a custom microarray comprising 11 872 potential mature miRNAs to assess the quality of our prediction. MiRMaster is a convenient-to-use tool for the comprehensive and fast analysis of miRNA NGS data. In addition, our predicted miRNA candidates provided as custom array will allow researchers to perform in depth validation of candidates interesting to them.

Dedifferentiation of Primary Hepatocytes is Accompanied with Reorganization of Lipid Metabolism Indicated by Altered Molecular Lipid and miRNA Profiles.

AIM: Primary human hepatocytes (PHHs) undergo dedifferentiation upon the two-dimensional (2D) culture, which particularly hinders their utility in long-term in vitro studies. Lipids, as a major class of biomolecules, play crucial roles in cellular energy storage, structure, and signaling. Here, for the first time, we mapped the alterations in the lipid profile of the dedifferentiating PHHs and studied the possible role of lipids in the loss of the phenotype of PHHs. Simultaneously, differentially expressed miRNAs associated with changes in the lipids and fatty acids (FAs) of the dedifferentiating PHHs were investigated. METHODS: PHHs were cultured in monolayer and their phenotype was monitored morphologically, genetically, and biochemically for five days. The lipid and miRNA profile of the PHHs were analyzed by mass spectrometry and Agilent microarray, respectively. In addition, 24 key genes involved in the metabolism of lipids and FAs were investigated by qPCR. RESULTS: The typical morphology of PHHs was lost from day 3 onward. Additionally, ALB and CYP genes were downregulated in the cultured PHHs. Lipidomics revealed a clear increase in the saturated fatty acids (SFA) and monounsaturated fatty acids (MUFA) containing lipids, but a decrease in the polyunsaturated fatty acids (PUFA) containing lipids during the dedifferentiation of PHHs. In line with this, FASN, SCD, ELOVL1, ELOVL3, and ELOVL7 were upregulated but ELOVL2 was downregulated in the dedifferentiated PHHs. Furthermore, differentially expressed miRNAs were identified, and the constantly upregulated miR-27a and miR-21, and downregulated miR-30 may have regulated the synthesis, accumulation and secretion of PHH lipids during the dedifferentiation. CONCLUSION: Our results showed major alterations in the molecular lipid species profiles, lipid-metabolizing enzyme expression as wells as miRNA profiles of the PHHs during their prolonged culture, which in concert could play important roles in the PHHs' loss of phenotype. These findings promote the understanding from the dedifferentiation process and could help in developing optimal culture conditions, which better meet the needs of the PHHs and support their original phenotype.

Deregulated microRNA and mRNA expression profiles in the peripheral blood of patients with Marfan syndrome.

BACKGROUND: MicroRNAs (miRNAs) are small RNAs regulating gene expression post-transcriptionally. While acquired changes of miRNA and mRNA profiles in cancer have been extensively studied, little is known about expression changes of circulating miRNAs and messenger RNAs (mRNA) in monogenic constitutional anomalies affecting several organ systems, like Marfan syndrome (MFS). We performed integrated miRNA and mRNA expression profiling in blood samples of Marfan patients in order to investigate deregulated miRNA and mRNA networks in these patients which could serve as potential diagnostic and prognostic tools for MFS therapy. METHODS: MiRNA and mRNA expression profiles were determined in blood samples from MFS patients (n = 7) and from healthy volunteer controls (n = 7) by microarray analysis. Enrichment analyses of altered mRNA expression were identified using bioinformatic tools. RESULTS: A total of 28 miRNAs and 32 mRNAs were found to be significantly altered in MFS patients compared to controls (> 2.0-fold change, adjusted P < 0.05). The expression of 11 miRNA and 6 mRNA candidates was validated by RT-qPCR in an independent cohort of 26 MFS patients and 26 matched HV controls. Significant inverse correlations were evident between 8 miRNAs and 5 mRNAs involved in vascular pathology, inflammation and telomerase regulation. Significant positive correlations were present for 7 miRNAs with age, for 2 miRNAs with the MFS aortic root status (Z-score) and for 7 miRNAs with left ventricular end-diastolic diameter in MFS patients. In addition, miR-331-3p was significantly up-regulated in MFS patients without mitral valve prolapse (MVP) as compared with patients with MVP. CONCLUSIONS: Our data show deregulated gene and miRNA expression profiles in the peripheral blood of MFS patients, demonstrating several candidates for prognostic biomarkers for cardiovascular manifestations in MFS as well as targets for novel therapeutic approaches. A deregulation of miRNA expression seems to play an important role in MFS, highlighting the plethora of effects on post-transcriptional regulation of miRNAs and mRNAs initiated by constitutional mutations in single genes. Trial registration Nr: EA2/131/10 . Registered 28 December, 2010.

Prioritizing and selecting likely novel miRNAs from NGS data.

Small non-coding RNAs play a key role in many physiological and pathological processes. Since 2004, miRNA sequences have been catalogued in miRBase, which is currently in its 21st version. We investigated sequence and structural features of miRNAs annotated in the miRBase and compared them between different versions of this reference database. We have identified that the two most recent releases (v20 and v21) are influenced by next-generation sequencing based miRNA predictions and show significant deviation from miRNAs discovered prior to the high-throughput profiling period. From the analysis of miRBase, we derived a set of key characteristics to predict new miRNAs and applied the implemented algorithm to evaluate novel blood-borne miRNA candidates. We carried out 705 individual whole miRNA sequencings of blood cells and collected a total of 9.7 billion reads. Using miRDeep2 we initially predicted 1452 potentially novel miRNAs. After excluding false positives, 518 candidates remained. These novel candidates were ranked according to their distance to the features in the early miRBase versions allowing for an easier selection of a subset of putative miRNAs for validation. Selected candidates were successfully validated by qRT-PCR and northern blotting. In addition, we implemented a web-server for ranking potential miRNA candidates, which is available at:www.ccb.uni-saarland.de/novomirank.

Technical Stability and Biological Variability in MicroRNAs from Dried Blood Spots: A Lung Cancer Therapy-Monitoring Showcase.

BACKGROUND: Different work flows have been proposed to use miRNAs as blood-borne biomarkers. In particular, the method used for collecting blood from patients can considerably influence the diagnostic results. METHODS: We explored whether dried blood spots (DBSs) facilitate stable miRNA measurements and compared its technical stability with biological variability. First, we tested the stability of DBS samples by generating from 1 person 18 whole-genome-wide miRNA profiles of DBS samples that were exposed to different temperature and humidity conditions. Second, we investigated technical reproducibility by performing 7 replicates of DBS again from 1 person. Third, we investigated DBS samples from 53 patients with lung cancer undergoing different therapies. Across these 3 stages, 108 genome-wide miRNA profiles from DBS were generated and evaluated biostatistically. RESULTS: In the stability analysis, we observed that temperature and humidity had an overall limited influence on the miRNomes (average correlation between the different conditions of 0.993). Usage of a silica gel slightly diminished DBS' technical reproducibility. The 7 technical replicates had an average correlation of 0.996. The correlation with whole-blood PAXGene miRNomes of the same individual was remarkable (correlation of 0.88). Finally, evaluation of the samples from the 53 patients with lung cancer exposed to different therapies showed that the biological variations exceeded the technical variability significantly (P < 0.0001), yielding 51 dysregulated miRNAs. CONCLUSIONS: We present a stable work flow for profiling of whole miRNomes on the basis of samples collected from DBS. Biological variations exceeded technical variations significantly. DBS-based miRNA profiles will potentially further the translational character of miRNA biomarker studies.

Dose-Dependent Anti-Inflammatory Effect of Ketamine in Liver Ischemia-Reperfusion Injury.

Introduction: Hepatic ischemia-reperfusion (I/R) injury is commonly observed in severe sepsis, hemorrhagic shock, liver transplantation, hepatic resection, and major trauma. Ketamine suppresses the production of cytokines, such as IL-6 and TNF-α, via NF-κB inhibition. We investigated the anti-inflammatory effects of ketamine in liver I/R injury. Materials and Methods: Female Wistar-Albino rats (n = 18), weighing 150-200g, were divided into three groups (n = 6 each). Group I underwent reperfusion for 4h following 30 min of ischemia. Group II received 2.5 mg/kg ketamine IM following 30 min of ischemia and 4h of reperfusion and Group III received 10 mg/kg ketamine IM following 30 min of ischemia and 4h of reperfusion. Blood samples were obtained before and after ischemia and reperfusion. MDA, AST, ALT, TNF-α, IL-1ß, IL-6, and NO levels were determined. Liver tissue samples were evaluated histologically. Results: Increased TNF-α, IL-1ß, and IL-6 levels were observed in all groups post-ischemia versus pre-ischemia (p <0.05). The TNF-α, IL-1ß, and IL-6 levels in Group III increased less than they did in Groups I and II (p <0.05). Higher MDA, NO, AST, and ALT levels were found during the ischemia and reperfusion periods compared with during the pre-ischemia period in all groups (p <0.05). The MDA, NO, AST, and ALT levels of rats that received ketamine increased less than did those of Group I (p <0.05). Significantly less injury was observed in the histopathological analysis of livers of rats administered ketamine (p <0.05). Conclusions: Ketamine showed a dose-dependent anti-inflammatory effect in I/R injury in the liver when administered after ischemia.

[Diffusion magnetic resonance imaging of thorax]. / Toraks difüzyon manyetik rezonans görüntüleme.

Although magnetic resonance imaging (MRI) of Thorax is only useful for morphological evaluation, currently we can use it for functional evaluation such as diffusion weighted imaging. Currently, we can obtain higher quality images because of new technologies such as software that decrease motion artefact, use of multi-channel, parallel imaging and fast sequences. Many promising results of thorax MRI have been published. It may also be an alternative to thorax computed tomography in some indications without radiation exposure risk. In this review, we evaluated current literature on use of DWI MRI examination on the pathologies of lungs and mediastinum and aimed to present what kind of information is provided on recognition and characterization of thoracic pathologies.

miR-Blood - a small RNA atlas of human blood components.

miR-Blood is a high-quality, small RNA expression atlas for the major components of human peripheral blood (plasma, erythrocytes, thrombocytes, monocytes, neutrophils, eosinophils, basophils, natural killer cells, CD4+ T cells, CD8+ T cells, and B cells). Based on the purified blood components from 52 individuals, the dataset provides a comprehensive repository for the expression of 4971 small RNAs from eight non-coding RNA classes.

Quantitative analysis of lung parenchymal involvement in Behçet's disease by the novel automatic program.

OBJECTIVES: We aimed to quantitatively analyze lung parenchymal changes in Behçet's patients and to detect early parenchymal quantitative changes that occur in the absence of positive visual radiological findings. METHODS: In our study, a total of 31 patients with Behçet's disease, 17 with positive radiological findings and 14 patients without positive radiological findings, and a control group of 33 individuals were evaluated. The automatic program determined lung volumes, lung densities, and opacity volume percentages by evaluating the contrast-enhanced lung computed tomography scans. RESULTS: The total lung volume was 3632.98 ± 1100.53 mL in the Behçet's disease patient group and 4925.70 ± 1098.88 mL in the control group, and there was a significant decrease in the total lung volume in Behçet's disease patients (P < 0.0001). The mean lung density was -723.73 ± 65.16 in the Behçet's disease patient group and -767.35 ± 41.17 in the control group, and there was a significant increase in the mean density in the Behçet's patients (P = 0.0023). A significantly higher correlation was observed between lung density and lung volume in Behçet's patients (P < 0.0001, r = -0.795). The lung volume of Behçet's disease patients with negative radiological findings was significantly lower than that of the control group (P = 0.0342). CONCLUSIONS: Lung volumetric changes in Behçet's disease patients are the quantitative data most affected by the disease. In addition, lung volume decreases in Behçet's patients who do not have visual parenchymal involvement.

Early Detection of Lung Cancer Using Small RNAs.

INTRODUCTION: Lung cancer remains the deadliest cancer in the world, and lung cancer survival is heavily dependent on tumor stage at the time of detection. Low-dose computed tomography screening can reduce mortality; however, annual screening is limited by low adherence in the United States of America and still not broadly implemented in Europe. As a result, less than 10% of lung cancers are detected through existing programs. Thus, there is a great need for additional screening tests, such as a blood test, that could be deployed in the primary care setting. METHODS: We prospectively recruited 1384 individuals meeting the National Lung Screening Trial demographic eligibility criteria for lung cancer and collected stabilized whole blood to enable the pipetting-free collection of material, thus minimizing preanalytical noise. Ultra-deep small RNA sequencing (20 million reads per sample) was performed with the addition of a method to remove highly abundant erythroid RNAs, and thus open bandwidth for the detection of less abundant species originating from the plasma or the immune cellular compartment. We used 100 random data splits to train and evaluate an ensemble of logistic regression classifiers using small RNA expression of 943 individuals, discovered an 18-small RNA feature consensus signature (miLung), and validated this signature in an independent cohort (441 individuals). Blood cell sorting and tumor tissue sequencing were performed to deconvolve small RNAs into their source of origin. RESULTS: We generated diagnostic models and report a median receiver-operating characteristic area under the curve of 0.86 (95% confidence interval [CI]: 0.84-0.86) in the discovery cohort and generalized performance of 0.83 in the validation cohort. Diagnostic performance increased in a stage-dependent manner ranging from 0.73 (95% CI: 0.71-0.76) for stage I to 0.90 (95% CI: 0.89-0.90) for stage IV in the discovery cohort and from 0.76 to 0.86 in the validation cohort. We identified a tumor-shed, plasma-bound ribosomal RNA fragment of the L1 stalk as a dominant predictor of lung cancer. The fragment is decreased after surgery with curative intent. In additional experiments, results of dried blood spot collection and sequencing revealed that small RNA analysis could potentially be conducted through home sampling. CONCLUSIONS: These data suggest the potential of a small RNA-based blood test as a viable alternative to low-dose computed tomography screening for early detection of smoking-associated lung cancer.

Improved Candida methylica formate dehydrogenase fermentation through statistical optimization of low-cost culture media.

NAD⁺-dependent formate dehydrogenase (FDH, EC 1.2.1.2) is of use in the regeneration of NAD(P)H coenzymes, and therefore has strong potential for practical application in chemical and medical industries. A low-cost production of recombinant Escherichia coli (E. coli) containing FDH from Candida methylica (cmFDH) was optimized in molasses-based medium by using response surface methodology (RSM) based on central composite design (CCD). The beet molasses as a sole carbon source, (NH4)2HPO4 as a nitrogen and phosphorus source, KH2PO4 as a buffer agent, and Mg2SO4 · 7H2O as a magnesium and sulfur source were used as variables in the medium. The optimum medium composition was found to be 34.694 g L⁻¹ of reducing sugar (equivalent to molasses solution), 8.536 g L⁻¹ of (NH4)2HPO4, 3.073 g L⁻¹ of KH2PO4, and 1.707 g L⁻¹ of Mg2SO4 · 7H2O. Molasses-based culture medium increased the yield of cmFDH about three times compared to LB medium. The currently developed media has the potential to be used in industrial bioprocesses with low-cost production.