High blood pressure is the most significant risk factor for the development and progression of chronic kidney disease (CKD). Lowering blood pressure is a goal to prevent CKD progression. This study of adults with CKD who have hypertension aimed to determine blood pressure control rates and the treatment patterns of hypertension and to explore factors associated with control of hypertension.This cross-sectional study included all non-dialysis people with CKD stages 3A to 5 under nephrology care in three public renal clinics in Queensland, who joined the CKD.QLD registry from May 2011 to Dec 2015 and had a history of hypertension. Demographic information, other health conditions, laboratory markers and anti-hypertensive medications in use at consent were extracted from the registry.p < 0.001) more likely to have uncontrolled BP compared to those without significant albuminuria or proteinuria. Participants who had cardiovascular disease (CVD) were less likely to have uncontrolled BP compared to those without CVD . Factors associated with use of more classes of antihypertensive medicines among participants with uncontrolled BP (> 140/90 mmHg) were older age, diabetes, CVD, obesity and severe albuminuria/proteinuria (p < 0.05). Renin Angiotensin Aldosterone System inhibitors were the most frequently used medicines, regardless of the number of medicine classes an individual was prescribed.Among 1814 CKD people in these three sites in the registry who were age ≥ 18 years and had CKD stage 3A to 5, 1750 or 96% had a history of hypertension. Of these, the proportion with BP control to < 140/90 mmHg was 61.7% and to < 130/80 mmHg was 36.3%. With target BP < 140/90 mmHg or < 130/80 mmHg, participants aged ≥65 years were 1.23 (95% CI 1.06–1.42) or 1.12 (1.03–1.22) times more likely to have uncontrolled BP compared to those < 65 years old. Participants with severe albuminuria or proteinuria were 1.58 (1.32–1.87) or 1.28 (1.16–1.42, Blood pressure control rates in these hypertensive people with CKD was still far from optimal. People with CKD and hypertension aged 65 or older or with severe albuminuria or proteinuria, a group at risk of progression of kidney disease, have higher rates of uncontrolled BP. Hypertension, also known as high blood pressure, is responsible for more deaths and disease worldwide than any other single health risk factor . High blThe World Health Organization (WHO) defines high blood pressure as systolic blood pressure of ≥ 140 mmHg or diastolic blood pressure of ≥ 90 mmHg or receiving medication for high blood pressure . UncontrIn 2011, we established the CKD.QLD Registry for the surveillance, practice improvement and research of CKD. The CKD.QLD Registry, which has been described elsewhere , is a neThis cross sectional study includes all patients from three major public renal services in Queensland who gave consent to join the CKD.QLD registry from May 2011 to Dec 2015 and had complete records of BP readings and anti-hypertensive medications at time of consent. We defined hypertension as a recorded co-morbidity in the medical records and/or prescription of anti-hypertensive medications recorded when participants consented to join the Registry.Recruitment into the CKD.QLD registry is described elsewhere . BrieflyThe present study selected adult (≥18 years) participants who met the inclusion criteria of a history of hypertension at baseline and who had CKD stage 3A to 5, who were under public nephrology care from three main study sites in the CKD.QLD Registry. Patients age < 18 years and those receiving dialysis or with a kidney transplant were excluded.2 or ≥ 30 kg/m2. Smoking status was recorded as dichotomous variable (yes/no) indicating whether the participant currently smoked cigarettes or not. The four groups of eGFR were CKD stage 3A (45 ≤ eGFR < 60), 3B (30 ≤ eGFR < 45) and 4 (≥15 to < 30) and 5(< 15). The three groups of ACR/PCR were normal (ACR < 30 mg/g or PCR < 150 mg/g), mild (30 ≤ ACR < 300 mg/g or 150 ≤ PCR < 500 mg/g) and severe (ACR ≥300 mg/g or PCR ≥500 mg/g).Participant demographic information , health conditions , smoking status, laboratory markers  and anti-hypertensive medications were extracted from the CKD.QLD registry. A history of CVD is a binary variable derived from the patient record. If the participants had coronary artery disease, prior revascularisation, heart failure, stroke or peripheral vascular diseases, then they assigned as having had cardiovascular disease. Diabetes is a binary variable indicating the history of diabetes at baseline. It includes serum fasting glucose ≥7.0 mmol/L (≥126 mg/dL), non-fasting glucose ≥11.1 mmol/L (≥200 mg/dL), glycated hemoglobin A1c ≥6.5%, and/or use of glucose lowering drugs, and is also derived from a renal diagnosis of diabetic nephropathy +/− a comorbidity of diabetes. Obesity is a binary variable indicating whether BMI was < 30 kg/mWe stratified the study population blood pressures as uncontrolled versus controlled, where lack of control was defined by either of two international targets. The first is the WHO definition of measured systolic blood pressure (SBP) of ≥140 mmHg, and/or diastolic blood pressure (DBP) of ≥90 mmHg. The second is the more complex KDIGO guideline for blood pressure control for kidney disease taking into account albuminuria and consists of measured SBP ≥130 mmHg, and/or DBP ≥80 mmHg , 17, 18.We evaluated patterns of drug use firstly by the number of anti-hypertensive drug classes, as defined by the Australian Medicines Handbook . MedicatWe compared participant characteristics at consent according to BP control status at the two target levels. Bivariate relationships between BP control and each of the variables of interest, and number of anti-hypertensive drug classes and each of the variables of interest were explored using Chi-square analyses. Generalised linear models (GLMs), with binomial distribution and log link , were peMultivariable modelling was performed to explore the factors associated with BP control at two levels, including age, sex, smoking, other health conditions  and laboratory markers (eGFR and ACR/PCR). Due to the small numbers, indigenous status was not included in the models. There were also missing data in smoking status, obesity and ACR/PCR category. Only participants with the complete set of covariates were included in the multivariable analyses. In addition, multivariable modelling was performed to examine the prevalence ratio of uncontrolled BP according to anti-hypertensive drug classes adjusting for age and sex.p value < 0.05 (two-tail).Preliminary adjusted GLMs  tested t2 body surface area. Of 1719 participants with available data, 31.9 and 40.3% had mildly or severely elevated levels of ACR/PCR.Of the 1814 age- and CKD stage-eligible participants from these three sites enrolled in CKD.QLD at the time of this study, 1750, or 96%, had a history of hypertension. Table Overall, 61.7 and 36.3% of participants had BP controlled to SBP/DBP < 140/90 and < 130/80 mmHg respectively at time of enrolment to the registry.p < 0.05 except eGFR< 15 with BP control < 130/80 as p = 0.067), irrespective of which method was used to define target. Table p > 0.05).Bivariate analyses show that BP control to SBP/DBP < 140/90 or < 130/80 mmHg in participants without CVD (56.5% or 30.8%), or with eGFR < 15 (50.0% or 32.2%), or with severe ACR/PCR elevations (54.8 and 30.1%) was worse than the respective referent groups . There were no differences in the number of drug classes used to control BP to WHO compared to KDIGO targets. Rates of control of BP were lower in groups prescribed more classes of antihypertensive drugs.Table p < 0.001) . There was no significant difference among eGFR group in terms of number of drug class use (p = 0.138).Among all participants, RAAS inhibitors were the most frequently prescribed medication group (69.1% of participants), followed by beta-blockers and CCB (48% of participants each) and diuretics (45.6% of participants). Mineralocorticoid receptor antagonists, included as diuretics, were used in 3.2% of the cohort (or 7% of those on diuretics). Table Figure p = 0.192), and to 130/80 mmHg , which is a bottom line statistically significant (p = 0.049). CCB and other anti-hypertensive drugs were significantly associated with more likelihood of uncontrolled BP to 140/90 mmHg , and to 130/80 mmHg .Table p < 0.01). Participants with CVD were less likely to have uncontrolled BP  compared to those without CVD (p < 0.001). Participants with severe ACR/PCR elevations were 1.58  more likely to have uncontrolled BP to < 140/90 mmHg compared to those without significant ACR/PCR.Table p < 0.01). Participants who had CVD were less likely to have uncontrolled BP  compared to those without CVD (p < 0.001). Participants with severe ACR/PCR elevations were 1.28  more likely to have uncontrolled BP compared to normal group.The associations of these variables with BP control was similar when the target was 130/80 mmHg. Participants aged ≥65 years were 1.12 times more likely to have uncontrolled BP (1.03–1.22) compared those < 65 years old  were more likely to have uncontrolled BP at both targets of 140/90 mmHg and 130/80 mmHg compared to those aged< 65 years, which is consistent with the literature 5 years w, 22. NotIn this cohort, and in contrast to a previous study , particiThe group in our study population with uncontrolled BP tended to be using more classes of anti-hypertensive drugs, suggesting that a stepped care or multidrug response has been taken to difficult-to-management hypertension. They were also older (≥65 years) and more often had diabetes, CVD, obesity and severe ACR/PCR elevations. The highest prevalence of hypertension control was in participants on only one class of antihypertensive medications. This group also had less CVD co-morbidity (39.6%), less diabetes (43.1%) and better kidney function, with eGFR> 30 in 69.7%. The most common first line drug was either an ACE inhibitor (35.3%) or ARB (22.3%). We are unable to determine whether the drug was initiated for treatment of proteinuria rather than hypertension or for both reasons.Physicians appeared at least partly to be following international recommendations for the management of CKD. RAAS inhibitors (ACE inhibitors and ARBs) were the most frequently used medications, in line with their reported renoprotective effect in addition to their anti-hypertensive properties. Where only one anti-hypertensive drug was listed, an ACE inhibitor was the most common class, followed by ARBs. The efficacy of these drugs is well established . HoweverOur study is one of the few examining BP management in real world people with non-dialysis CKD. Data were derived from three main study sites across Queensland and the enrolment representation of patients with CKD in these three sites at that time was 55, 86 and 95% respectively.We acknowledge limitations in this study. The study sampled BP management at one time point, when patients consented to the CKD.QLD registry, and we know that BP varies over time and according to the measurement environment. There is the risk of mislabelling . The selThe WHO has established a specific target to reduce prevalence of elevated BP (≥140/90 mmHg) by 25% by 2025 and has recommended that healthcare professionals measure BP at all relevant clinical encounters . The WorOur findings show considerable success in achieving BP targets set by international guidelines in people with CKD. However, we must acknowledge that BP control does not meet international benchmarks yet. The data also reveal variations in the patterns of hypertension management. Health care providers commonly used several classes of antihypertensive drugs, and, appropriately, used more classes of anti-hypertensive drugs in those who had uncontrolled BP. Factors associated with use of more classes of antihypertensive medicines among people with uncontrolled BP (> 140/90 mmHg) were older age, diabetes, CVD, obesity and severe albuminuria/proteinuria. We also found additional risk factors for poor BP control, such as older age (≥ 65 years) and/or severe ACR/PCR elevations. Better BP control has important benefits for people with CKD. As evidenced, salt restriction also improves BP control in many patients with CKD (Stage 1–4) . Therefo


PD diagnosis is based primarily on clinical criteria and can be inaccurate. Biological markers, such as α‐synuclein aggregation, that reflect ongoing pathogenic processes may increase diagnosis accuracy and allow disease progression monitoring. Though α‐synuclein aggregation assays have been published, reproducibility, standardization, and validation are key challenges for their development as clinical biomarkers.To cross‐validate two α‐synuclein seeding aggregation assays developed to detect pathogenic oligomeric α‐synuclein species in CSF using samples from the same PD patients and healthy controls from the BioFIND cohort.CSF samples were tested by two independent laboratories in a blinded fashion. BioFIND features standardized biospecimen collection of clinically typical moderate PD patients and nondisease controls. α‐synuclein aggregation was measured by protein misfolding cyclic amplification (Soto lab) and real‐time quaking‐induced conversion (Green lab). Results were analyzed by an independent statistician.Measuring 105 PD and 79 healthy control CSF samples, these assays showed 92% concordance. The areas under the curve from receiver operating characteristic curve analysis for the diagnosis of PD versus healthy controls were 0.93 for protein misfolding cyclic amplification, 0.89 for real‐time quaking‐induced conversion, and 0.95 when considering only concordant assay results. Clinical characteristics of false‐positive and ‐negative subjects were not different from true‐negative and ‐positive subjects, respectively.Movement Disorders published by Wiley Periodicals, Inc. on behalf of International Parkinson and Movement Disorder Society.These α‐synuclein seeding aggregation assays are reliable and reproducible for PD diagnosis. Assay parameters did not correlate with clinical parameters, including disease severity or duration. This assay is highly accurate for PD diagnosis and may impact clinical practice and clinical trials. © 2019 The Authors.  Parkinson's disease (PD) diagnosis relies primarily on clinical identification of cardinal motor symptoms . However, clinical manifestation of PD is heterogeneous and overlaps with other related neurodegenerative disorders, which can hinder accurate diagnosis.SNCA gene encoding α‐synuclein cause genetic forms of PD.α‐Synuclein dysfunction plays a prominent role in PD pathogenesis. Aggregated α‐synuclein protein is a major constituent of Lewy bodies, and mutations in the All study protocols were approved by the institutional review board of the individual sites and for the University of Rochester Clinical Trials Coordination Center (CTCC) as outlined in the original BioFIND manuscript.on state (1–3 hours after the last PD medication dose) and V2 was performed in the practically defined off state (early morning before PD medications and approximately 12 hours after the last dose the night before). During V1, blood for DNA and plasma were collected and clinical assessments were made including the International Parkinson and Movement Disorder Society–sponsored revision of the UPDRS (MDS‐UPDRS) Parts I (nonmotor experiences of daily living), II (motor experiences of daily living), III (motor examination), and IV (motor complications) for PD subjects and only Part III for HCs. During V2, blood for RNA and plasma, CSF, and saliva  were collected, and the MDS‐UPDRS Part III was administered to PD subjects. All subjects either fasted or consumed a low‐fat diet on the morning of V2 at the time of biospecimen collection. CSF was immediately frozen in a –80°C freezer and shipped to the biorepository on dry ice at a later date.Clinical evaluation and biospecimen collection occurred during two visits, one to establish the baseline (V1) and a follow‐up 2 weeks later (V2). For PD subjects, V1 was performed during the Two independent laboratories performed their respective assays on separate aliquots of CSF from the same subjects. Both assays are based on the ability of pathological α‐synuclein oligomers to seed aggregation at the expense of exogenously supplied monomeric α‐synuclein protein. Cyclic amplification of protein misfolding (PMCA) adapted for α‐synuclein oligomer formation was performed by the Soto group,The RT‐QuIC assay was performed as previously described.The PMCA assay was performed as previously described.50), indicating removal of the majority of the seeds. When the same samples were used for assay at a later time and when measured after one versus two freeze‐thaw cycles, a good reproducibility of data was demonstrated previously. Varying seed concentrations resulted in corresponding changes in T50.50 .The PMCA assays for α‐synuclein showed specificity toward α‐synuclein seed compared to β‐amyloid (1‐42) and tau proteins.Differences between cases and controls were compared using percent and frequency for categorical data and median and range for continuous data. Categorical data were assessed using chi‐square or Fisher's exact test, where appropriate, and differences in continuous data was assessed using Wilcoxon rank sum. Sensitivity, specificity, negative predictive value (NPV), and positive predictive value (PPV) were calculated for comparisons between biomarker results and clinical diagnosis. Correlations were calculated for biomarker results between the two techniques as well as between biomarker results and clinical characteristics. Logistic regression was used to generate receiver operating characteristic (ROC) curves to assess the relationship between biomarker results and clinical diagnosis of PD. A two‐tailed alpha of 0.05 was used.http://biofind.loni.usc.edu/). The data are publically available upon registration by the Michael J. Fox Foundation for Parkinson's Research. The study protocol is available . The statistical analysis is available upon request from the authors.Individual de‐identified participant data consisting of clinical phenotypes, demographics, and assay data are available online . T50 values in PD subjects from two assays were only weakly correlated  of ROC curves were 0.94 when considering only the samples with concordant results between both assays. Separately, AUCs for the PMCA and RT‐QuIC assays were 0.93 and 0.89, respectively Table . Sensiti50 did not correlate with any clinical parameter, including disease duration, age, sex, or UPDRS scores  and leucine repeat‐rich kinase 2 (LRRK2) variants were present in a few PD patients and HCs without PD.Development of new biomarkers assays often face significant challenges in replicability in additional patient cohorts. Because of the complexity of obtaining and assaying human samples, variability may arise in many areas, including from differences in patient enrollment, biospecimen collection and processing, and assay execution. Thus, independent replication and cross‐validation of assays is a critical step that is often lacking in the literature. Therefore, we utilized the BioFIND cohort, a standardized benchmark cohort of PD patients, for blinded assay validation of two α‐synuclein aggregation assays based on the sample principle. These assays use CSF to seed the aggregation of monomeric α‐synuclein protein and are among the most promising biomarkers for PD diagnosis. Our results validate the preliminary studies and show robust power to confirm PD diagnosis from the same CSF samples at the individual level.50 times in one assay lacking aggregation in the other). Thus, discordant results may be attributed to the slight differences in the conditions, reagents, and procedures for each assay ” to refer to these tests in order to highlight the basic principle behind them. Based on their high predictive power, these α‐synuclein SAAs tested in our study have the potential to revolutionize PD diagnosis for clinical practice and clinical trials.(1) Research Project: A. Conception and Design; B. Acquisition of Data; C. Analysis and Interpretation of Data; (2) Manuscript: A. Writing of the First Draft, B. Review and Critique; (3) Other: A. Statistical Analysis; B. Obtaining Funding; C. Technical Support; D. Supervision of Data Collection.U.J.K.: 1A, 1C, 2A, 2B, 3BA.K.B.: 1C, 2B, 3AG.F.: 1BM.S.: 1BT.C.M.: 1C, 2BS.J.H.: 1C, 2B, 3BA.G.: 1A, 1C, 2B, 3B, 3DC.S.: 1A, 1C, 2B, 3B, 3DNothing to report.Figure S1: Supporting informationClick here for additional data file.Supplementary Table 1. Comparison of PMCA and RT‐QuIC methodologiesSupplementary Table 2. Correlation of assay results with clinical parameters and CSF analytes.Click here for additional data file.


Status epilepticus requires immediate treatment because treatment delay can cause permanent neurologic complications. Dexmedetomidine may be an option for the treatment of status epilepticus although its effect remains unclear with conflicting reports.A 64-year-old woman with epilepsy with complex partial seizures underwent total knee arthroplasty. After emergence from general anesthesia, she developed status epilepticus and was transferred to the intensive care unit. Following initial treatment using benzodiazepines, phenytoin, and levetiracetam, dexmedetomidine (0.37 μg/kg loading in 10 min followed by 0.6 μg/kg/h) was administered and seizures terminated in 20 min. Color density spectral array using Root® with SedLine®  showed an increase in power in high frequency band of the electroencephalogram during the seizure attacks.We described a case of status epilepticus which was treated with dexmedetomidine and monitored using color density spectral array. Status epilepticus, considered the most extreme form of seizure, requires immediate treatment, usually with benzodiazepines, because treatment delay can cause permanent neurologic complications . EpilepsHere, we describe a case of status epilepticus after emergence of general anesthesia in which dexmedetomidine was administered and the effect was observed using CDSA.2), capnometry, and non-invasive blood pressure. Patient state index was also monitored using Root® with SedLine® . After femoral nerve block with 20 mL of 0.375% ropivacaine, general anesthesia with tracheal intubation was induced with propofol 80 mg, remifentanil 0.19 μg/kg/min, and rocuronium 50 mg, and maintained with sevoflurane 1.3% and remifentanil 0.14–0.23 μg/kg/min. During the anesthesia, SpO2 was maintained at 97% or more and end-tidal CO2 was kept between 36 and 41 mmHg (more than 39 mmHg at most time points during the last hour). Patient state index were between 22 and 27. For postoperative analgesia, acetaminophen 1 g and flurbiprofen axetil 50 mg were intravenously administered. The surgery was completed without incident, and after the recovery of consciousness and spontaneous respiration, patient’s trachea was extubated. Tonic muscular contraction was observed in the upper limbs, body trunk, neck, and face along with respiratory depression. SpO2 decreased from 100 to 88%. Although these symptoms were temporarily relieved by midazolam 3 mg iv, they recurred in a few minutes. Additional midazolam 6 mg and phenytoin 125 mg were administered in the recovery room, but the symptoms did not improve, and the patient was transferred to the intensive care unit (ICU). Levetiracetam 500 mg was administered by intravenous drip. In addition, a total of 15 mg diazepam was administered intermittently during the first hour and the patient often required respiratory assistance with jaw thrust. However, convulsions persisted. CDSA showed a spread of warmer colors  in a wide frequency band of the EEG during the convulsions  with a history of epilepsy with complex partial seizures developed right knee osteoarthritis and was scheduled for total knee arthroplasty. One year previously, she underwent the same operation on the contralateral side and developed tonic-clonic convulsions after emergence of general anesthesia in the operating room and the ward. She was then treated with repeated diazepam administration; however, her postoperative management became difficult due to repeated respiratory arrests. Her epilepsy control improved subsequently although seizure attacks occurred approximately once a week, according to her medical record. She was under medication of valproate sodium, carbamazepine, and levetiracetam, each of which blood concentrations was in a therapeutic range. Preoperative examinations revealed no other abnormal findings except for an increase in γ-glutamyltransferase (61 U/L) probably due to medication. On the morning of surgery, she took usual anticonvulsants and received no anesthetic premedication. Patient monitoring included continuous electrocardiography, pulse oximetry  to dark red (maximum power) (see figure). Seizure evolution may involve increases in frequency and amplitude in EEG and thus may appear on CDSA images as upward arches on the y-axis (increased frequency) in warmer colors (increased power) [CDSA shows time on the d power) . In the d power) . Furtherd power)  or burstd power)  may contIn this case report, we presented a case of status epilepticus which was treated with dexmedetomidine and monitored using CDSA. This strategy may be an option for the management of patients with status epilepticus.


ALDH7A1 deficiency, PNPO deficiency, and others, usually causing neonatal onset seizures resistant to treatment with common antiepileptic drugs. Recently, biallelic mutations in PLPBP were shown to be a novel cause of vitamin B6‐dependent epilepsy with a variable phenotype. The different vitamin B6‐responsive epilepsies can be detected and distinguished by their respective biomarkers and genetic analysis. Unfortunately, metabolic biomarkers for early detection and prognosis of PLPBP deficiency are currently still lacking. Here, we present data from two further patients with vitamin B6‐dependent seizures caused by variants in PLPBP, including a novel missense variant, and compare their genotype and phenotypic presentation to previously described cases. Hyperglycinemia and hyperlactatemia are the most consistently observed biochemical abnormalities in pyridoxal phosphate homeostasis protein (PLPHP) deficient patients and were present in both patients in this report within the first days of life. Lactic acidemia, the neuroradiological, and clinical presentation led to misdiagnosis of a mitochondrial encephalopathy in two previously published cases with an early fatal course. Similarly, on the background of glycine elevation in plasma, glycine encephalopathy was wrongly adopted as diagnosis for a patient in our report. In this regard, lactic acidemia as well as hyperglycinemia appear to be diagnostic pitfalls in patients with vitamin B6‐responsive epilepsies, including PLPHP deficiency.Vitamin B6‐responsive epilepsies are a group of genetic disorders including In vitamin B6‐responsive epilepsies, including PLPHP deficiency, there are several diagnostic pitfalls, including lactic acidemia as well as hyperglycinemia, highlighting the importance of a pyridoxine trial, and genetic testing. They are important to recognize as they can be treated with vitamin B6 supplied as pyridoxine (PN) or as the biologically active form of vitamin B6, pyridoxal 5′‐phosphate (PLP). Enzymes utilizing PLP as a cofactor are found in all organisms and catalyze diverse reactions mostly associated with amino acid as well as neurotransmitter metabolism among other pathways.ALDH7A1 gene (MIM# 266100) and pyridoxal 5′‐phosphate responsive epilepsy caused by mutations in the PNPO gene (MIM# 610090) are prototypic vitamin B6 dependent epilepsies. In the latter, deficiency of pyridox(am)ine 5′‐phosphate oxidase impairs PLP synthesis and recycling, while variants affecting ALDH7A1 function lead to accumulation of piperideine‐6‐carboxylate (P6C) and alpha‐amino adipic semialdehyde (α‐AASA) from lysine degradation pathways. P6C may immobilize PLP by formation of a condensation product resulting in PLP deficiency. Other PN‐ or PLP‐responsive seizure disorders include neonatal hypophosphatasia (MIM# 241500), hyperphosphatasia with mental retardation syndrome , and hyperprolinemia type 2 (MIM# 239510).Pyridoxine‐dependent epilepsy (PDE) caused by mutations in the PLPBP gene  were shown to be a novel cause of vitamin B6‐dependent epilepsy (MIM# 617290).Recently, biallelic mutations in the y MIM# 61290.7 butPLPBP variants.Different vitamin B6‐dependent epilepsies can be detected and distinguished by their respective biomarkers and genetic analysis. In PDE, for example, elevated α‐AASA, P6C, and pipecolic acid concentrations can be detected in the cerebrospinal fluid (CSF), urine and plasma, and serve as biomarkers.PLPBP variants have been described.So far, 28 cases of vitamin B6‐dependent epilepsy caused by PLPBP mutations, compare their genotype and phenotypic presentation to previously described cases and highlight diagnostic pitfalls.Here, we present data from two further unrelated patients with vitamin B6‐dependent seizures caused by 1.1Detailed information regarding pregnancy, birth, clinical, biochemical, and metabolic features as well as EEG findings and semiology for the two cases described below are provided in Table 1.1.1This boy was the fourth child of consanguineous parents of Turkish descent. There were no known cases of epilepsy in the family. Within the first 5 hours seizures were noted. On day 1, the boy responded well to treatment with phenobarbital. However, on day 2, seizures continued also after treatment with midazolam and the addition of levetiracetam. Seizures stopped on day 4 when PN treatment at an initial dose of 100 mg (i.v.) was administered and PDE was suspected. Measurement of pipecolic acid in plasma and CSF however, revealed normal levels . Ion Chef was used to prepare libraries which were sequenced using the Ion Proton system . Base calling, preprocessing of the reads, short read alignment and variant calling was performed using the Torrent Suite including the Torrent Variant Caller  (Life Technologies Corporation).Variant prioritization was performed with a cascade of filtering steps in VarSeq  using following parameters: a frequency <1% in gnomAD,2.1.2Whole genome analysis of the patient and his parents was performed with a 30X coverage using HiSeq X, PCR‐free library preparation method. The resulting sequences were analyzed using an in‐house mutation identification pipeline (MIP) as previously described.33.1PLPBP gene (NM_007198.3). The variant was validated by PCR and Sanger sequencing and subsequent sequencing of the parents showed that both are heterozygous for the variant. The variant exists at a frequency of 0.0001 in the gnomAD database and is in silico predicted to change the normal splicing of PLPBP mRNA . Further, the variant was previously reported as compound heterozygous with another variant to cause recessive inherited vitamin B6‐dependent epilepsy (MIM #617290).After filtering, exome sequencing revealed a homozygous splice‐site variant, c.207+1G>A in the 3.2PLPBP gene. The variant was verified by Sanger sequencing and both parents shown to be heterozygous carriers. The variant c.121C>T is found in one of 246.000 alleles in the gnomAD database and has previously not been reported in patients. The resulting protein change p.Arg41Trp is predicted probably damaging by PolyPhen2 and deleterious by SIFT. Following the ACMG guidelines the variant is classified as likely pathogenic.Analysis from whole genome data revealed as the only potentially pathogenic finding a homozygous missense variant, c.121C>T (p.Arg41Trp) in a conserved region of the 4PLPBP‐associated vitamin B6‐dependent epilepsy caused by homozygous variants in PLPBP, including a novel missense variant, c.121C>T, p.(Arg41Trp) and a splice‐site variant c.207+1G>A, which was previously identified in compound heterozygous state with another splice‐site variant.PLPBP variants .PLPBP have been identified.Here, we describe two additional patients with >T, p.Arg1Trp and >T, p.Arg1Trp and To date, 30 patients with PLPHP deficiency have been described, including this report, with four patients with early demise  deficiency (MIM#608643) and might contribute to the phenotype.Unfortunately, a metabolic biomarker for early detection and prognosis of PLPHP deficiency is currently still lacking. As the clinical and metabolic presentation is mostly unspecific, diagnostic confirmation is relying on vitamin B6 treatment trial in patients with early epileptic encephalopathy, and on genetic testing. CSF homocarnosine deficiency was previously suggested as a possible biomarker for PLPHP deficiency, as it was undetectable in one patient at two occasions (3 weeks and 17 months of age).cy MIM#60643 and mplpbp−/− zebrafish larvae showed a more remarkable effect of PN on the epileptic phenotype and survival with a clear dose‐dependency.From the first PLPHP deficient patients reported, it was concluded that treatment with PLP produced better seizure control than treatment with PN.In conclusion, we would like to suggest that PLPHP deficient vitamin B6‐dependent epilepsy is an important differential diagnosis to congenital lactic acidosis of mitochondrial origin and possibly attenuated glycine encephalopathy. Patients presenting with these features should be given a trial with vitamin B6 until a final diagnosis has been achieved.Clinical patient care and diagnostic: K.V.J., M.F., M.B., N.D., S.G. Evaluation and interpretation of metabolic, genetic, and neuroradiological results: T.S., M.B., A.W., M.C., M.B., J.E., C.G.M., N.D., S.G. Drafting and writing of the manuscript: K.V.J., T.S., N.D., S.G.Kristian Vestergaard Jensen, Maria Frid, Tommy Stödberg, Michela Barbaro, Anna Wedell, Mette Christensen, Mads Bak, Jakob Ek, Camilla Gøbel Madsen, Niklas Darin, and Sabine Grønborg declare that they have no conflict of interest.Informed consent was obtained from both patients' families for publication of case reports including publications of pictures of cerebral imaging.The authors' institutions do not require ethics approval for publication of case histories.


Two patients are described with large stones in the common bile duct. Standard ERCP was not possible. Both patients were successfully treated with percutaneous access and use of the ureteroscope with the holmium laser. Gallbladder stones are a common diagnosis in general practice. Patients may also develop stones in the common bile duct. In addition, these can occur many years after previous cholecystectomy. The treatment of choice, especially since the introduction of the laparoscopic cholecystectomy is endoscopic retrograde cholangiography (ERC) with papillotomy and removal of stones. This procedure is successful in a high percentage of cases.Sometimes local anatomy has changed and it is impossible to perform an ERC. A percutaneous procedure with canulation of the bile ducts, dilatation of the papilla, and pushing the stones towards the duodenum can be done.In the present report, we describe two patients with large stones in the common bile duct and changed local anatomy, in whom stones were successfully removed using the urologic ureteroscope.µmol/l (reference < 21 µmol/l); GGT 328 U/l (reference < 40 U/l); Alkaline Phosphatase 128 U/l (reference < 120 U/l), in addition there was a leucocytosis of 24.3 × 10E9/l (reference 4–10 × 10E9/l) and a CRP of 60 mg/l (reference < 5 mg/l).The first patient is a woman of 77 years. At the age of 17 years, she underwent a Billroth II resection because of a bleeding peptic ulcer. Her medical history revealed breast cancer and cholecystectomy. One year before the present presentation, she was admitted because of colics and nausea. Blood analysis showed cholestatic liver tests: ASAT 507 U/l (reference < 30 U/l); ALAT 479 U/l (reference < 35 U/l); Total bilirubin 24 E. coli, and she was treated accordingly with antibiotics. Percutaneous cholangiography (PTC) showed no stones (anymore!). Nevertheless balloon dilatation of the papilla was done.CT‐scan showed dilated intra‐ and extra‐hepatic bile ducts. The diameter of the common bile duct was 1.6 cm. ERC failed because of the changed anatomy. It was impossible to introduce the endoscope in the proximal part of the duodenum. Blood culture was positive for µmol/l. CT‐scan again showed dilated intra‐ and extra‐hepatic bile ducts. Blood cultures were positive for E. coli and K. pneumoniae. Because of good clinical response and decrease of the bile duct dilatation, a conservative approach with antibiotics was chosen. However, after less than one month cholangitis recurred and a PTC was done. Several small stones in the common bile duct were present. An internal biliary drain was placed for drainage. Balloon dilatation of the papilla was successful, but the stones could not be pushed through the papilla. Because of the impossibility to perform ERC  and the relative failure of the PTC, a percutaneous approach with a flexible ureteroscope was performed. During this procedure several stones were visualized and fragmented with the help of the holmium laser. The stone fragments were successfully removed through the dilated papilla via flushing with saline 0.9% . A cholangiography several days later showed a small residual stone, which was pushed into the duodenum with a balloon. The patient recovered uneventfully and was doing well without complaints and normal blood work two months later.One year later, she was admitted because of recurrent cholangitis. This time the bilirubin level was 131 E9/l; ALAT 163 U/l; total bilirubin 105 µmol/l; conjugated bilirubin 95 µmol/l; GGT 547 U/l; Alkaline Phosphatase 141 U/l; and CRP 120.8 mg/l. Ultrasound showed minimal dilated bile ducts with stones in the common bile duct. ERC was done. This time the procedure was impossible because the diverticulum had increased in size and, despite the previous papillotomy, the papilla could not be canulated anymore. However, due to the manipulations with the canula drainage of bile was restored. The patient was treated with antibiotics because of positive blood cultures with K. oxytoca and S. aureus. A PTC was done and an internal drain was placed. After improvement of the clinical condition, PTC with assistance of the ureteroscope and laser was performed. During two consecutive procedures large stones . The percutaneous transhepatic endoscopic approach is a combined procedure with interventional radiology. Antegrade biliary access is achieved, and a wire is placed into the common bile duct. A 12/14 F ureteral access sheath is then placed under fluoroscopic guidance. A flexible ureteroscope is guided through the access sheath into the biliary system .The urologist has great experience with this instrument in the removal of kidney stones. The stones can be fragmented under direct endoscopic vision, and the fragments are flushed or pushed into the duodenum.It can be concluded that percutaneous endoscopic holmium laser lithotripsy is a minimally invasive alternative to all other techniques published in the literature, especially for open salvage surgery for complex biliary calculi. This treatment is both safe and efficacious. Of course, success depends on a multidisciplinary approach , 10.


Evidence suggests the possible impact of ambient high temperature on fetal growth and birth outcomes. However, little is known about the relative impact of exposure to heat and cold and the possible vulnerable window during pregnancy.10, SO2, NO2, and O3, were obtained from relevant government agencies. This study was to examine the associations of maternal exposure to ambient temperature  with the duration of gestation and birth weight.Data on a total of 237,585 pregnant women from January 1st, 2001 to December 31st, 2010 were acquired from the Queensland Health, Australia. Daily data on meteorological factors, including ambient temperature, relative humidity, barometric pressure, and air pollutants, such as PMA J-shaped association between minimum temperature at conception and duration of gestation was observed after adjusting for seasonality and other confounders. Compared to women who were exposed to the minimum temperature of 15–20 °C in the first gestational week, exposure to the minimum temperature of > 20 °C significantly increased the duration of gestation by 0.029 weeks . A cumulative effect was found when exposure across the first four weeks was examined. There was an inverted U-shaped relationship between minimum temperature at delivery and the duration of gestation. Compared to women exposed to 15–20 °C, exposure to minimum temperature of > 20 °C and ≤ 10 °C was associated with a shortened gestation by 0.030 weeks  and 0.018 weeks , respectively. By contrast, an inverse relationship between maximum temperature and birth weight was observed. Compared to exposure to the maximum temperature of > 30 °C in the last week of pregnancy, maternal exposure to 20–25 °C and < 20 °C significantly increased birth weight by 0.011 kg  and 0.018 kg , respectively. Similarly, a mild cumulative effect was observed when maximum temperature exposure across the four weeks before delivery was evaluated.The finding emphasized the importance of keeping an optimal temperature range during pregnancy for reducing the risk of preterm birth and low birthweight.The online version of this article (10.1186/s12884-018-2100-y) contains supplementary material, which is available to authorized users. Climate has frequently been changing throughout the Earth’s history. However, the recent pace of warming far exceeds that of any previous warming episode in the past 10,000 years , 3. GlobPrevious studies have explored the short-term and long-term effects of maternal exposure to heat stress on preterm birth, stillbirth, and birth weight –17. In gThe effect of ambient temperature on birth outcomes may depend on the stage of gestation, with a possible susceptible period during pregnancy , 21. ForBased on above, we speculated the possibility that high temperature and cold condition could have varied health effect, and, meanwhile, the effect may depended on different exposure period of gestation. This study focused on two important birth outcomes, duration of gestation and birth weight, aimed to address two issues: 1) what is the overall impact of both hot and cold temperatures on duration of gestation and birth weight? 2) is there any possible susceptible period to temperature exposure for different pregnant outcomes?Participants in this study were all the singleton births born in Brisbane, Australia, between January 1st, 2001 and December 31st, 2010. Data were collected from the Perinatal Data Collection Unit (PDCU) of the Queensland Health Statistics Centre. All births were recorded according to the following criteria: 1) at least 20 weeks of gestation; or 2) at least 400 g in birth weight; 3) born in Queensland including all public hospitals, private hospitals, and private midwifery and medical practices.In this study only live birth was eligible. Among all live births, those whose last menstrual date was earlier than January 1st 2001 were excluded to match our environmental exposure records that covered from January 1st 2001 to December 31st 2010. The final sample consisted of 237,585 singleton live births. The study profile and participants selection process are shown in Fig. The following variables were collected based on standardized maternal medical records: duration of gestation, date of last menstrual period, date of birth, gender of baby, birth weight, mode of labor onset , maternal age group, marital status , Indigenous status, and parity.Meteorological factors, including daily maximum and minimum temperatures, relative humidity at 9:00 am and 15:00 pm, and ambient barometric pressure at 9:00 am and 15:00 pm, were recorded in nine monitoring stations in Brisbane during the study period.3), nitrogen dioxide (NO2), and sulfur dioxide (SO2), were recorded in five monitoring stations in Brisbane.Air pollutants, including particulate matter with a diameter < 10 μm (PM10), ozone  based on the original daily data. Since the date of conception and birth varied among different pregnant women, we, then, fitted weekly data of these environment indicators to each pregnant woman, using their mean levels in the first/last four weeks of the corresponding gestation. To compare the possible acute and cumulative effects of temperature exposure, the exposures in the first one gestational week vs. in the first four gestational weeks and in the last gestational week vs. in the last four gestational weeks were examined, respectively, in the subsequent analyses. Descriptive statistics for meteorological and pollutant indicators at conception/delivery are shown in . Generalized cross validation was used to automatically select the degree of smoothing for spines. Based on the distribution of temperature and overall effect of temperature on duration of gestation/birth weight in GAM, maximum temperature was recorded into following four groups as > 30 °C, 25–30 °C, 20–25 °C, and ≤ 20 °C, while minimum temperature was grouped as > 20 °C, 15–20 °C, 10–15 °C, and ≤ 10 °C.10, O3, NO2, and SO2) around conception and delivery, were further controlled. Accumulating studies documented that other seasonal factors , independent of ambient temperature, may affect fetal development and birth outcomes [a), and temperature around conception were simultaneously taken into account when examining the health effect of temperature exposure at delivery (Model 3b).Generalized linear regression models were further applied to estimate the crude and adjusted associations between maximum/minimum temperature groups  and duration of gestation/birth weight . Adjustments were made following a three-step procedure. Model 1 was adjusted for all maternal and perinatal variables (as listed in Table outcomes –24. Therwww.r-project.org). In the presentation of the results, the statistical significance was set at p value < 0.05 (two tailed).All analyses were performed using the Statistical Analysis System (SAS) for Windows, version 9.2  and R version 2.15.1 , and the mean birth weight was 3.42 kg . Table p < 0.001). The smoothed plots of nonlinear associations of duration of gestation with minimum temperature at the first/last gestational week are shown in Fig. , both p < 0.001). By contrast, approximate linear trend or wave curves, without clear correlates, were observed between maximum temperature, either at concept or at delivery, with duration of gestation .The association between ambient temperature and duration of gestation/birth weight was explored by penalized splines in generalized additive models. We observed a J-shaped association of duration of gestation with both minimum temperatures at conception and at delivery (both p < 0.05). Associations of birth weight with maximum temperature at concept or minimum temperature at concept/delivery were also explored. Generally, there were wavy curves without clear correlates (not shown).The smoothed plots of association between birth weight and maximum temperature at the last gestational week is shown in Fig. Table p = 0.006) and 0.049 weeks (p < 0.001).Table p < 0.001), 0.030 weeks (p = 0.007) and 0.018 weeks (p = 0.041) of the shortened duration of gestation, respectively.Minimum temperature at delivery was also related to the duration of gestation. Compared to minimum temperature exposure of 15–20 °C in the last week, > 20 °C and 10–15 °C could significantly shorten the duration of gestation, respectively. Similarly, a mild cumulative effect was found. During the process of three-step adjustment, the associations generally maintained. In the final full model (Model 3), exposure to ambient temperature > 20 °C in the last gestational week, as well as > 20 °C and ≤ 10 °C in the last four gestational weeks, were associated with a decreased gestation age, when compared to 15–20 °C, and there were 0.042 weeks  and ≥ 37 weeks (full-term birth) to examine the relationship between exposure to ambient temperature and risks of preterm birth  and 0.018 kg (p = 0.024), respectively. A slightly cumulative effect was identified.Table Results of this study indicate a complex spectrum about the association between ambient temperature exposure and birth outcomes. For the first time, we reported that the duration of gestation seemed to be sensitive to maternal exposure to minimum temperature, while birth weight was susceptible to maximum temperature exposure.Most previous studies have focused on the relationship between high temperature and birth outcomes –17. HoweTo our knowledge, this is the first study that has investigated the relation between ambient low temperature and duration of gestation. However, two recent studies examined the impact of ambient low temperature on preterm birth. The one in Uppsala, Sweden set up a retrospective birth cohort among almost 14,000 deliveries from 1915 to 1929 and, in which, it was found that extreme cold exposure adversely affected preterm birth . HoweverAn almost linear relationship between maximum temperature and birth weight was observed. Compared to exposure to higher than 30 °C at the last week of pregnancy, birth weight significantly increased by 11 g and 18 g for those who exposed to 20–25 °C, and < 20 °C, respectively. Similarly, a mild cumulative effect was observed when maximum temperature exposure across the four weeks before delivery was evaluated. By contrast, minimum temperature was not found to be related to birth weight. The findings indicated that the period before delivery could be a susceptible window for fetal weight growth when high ambient temperature exposure exists.r = − 0.22, P < 0.01) [r = − 0.59, P < 0.01) between heat index and birth weight, where it was found that a one unit increase in heat index was associated with 2.7% decrease in birth weight [Among the previous studies on the topic of maternal temperature exposure and birth weight, there was a decrease in birth weight associated with increasing temperature at delivery , 28, 32. < 0.01) . An ecolh weight . The resh weight . Our resh weight , this fih weight . In addih weight . Pregnanh weight –8. The iA few studies have explored the possible impact of cold ambient temperature on birth weight , 25, 36,It is crucial to identify the possible vulnerable exposure window for a specific birth outcome. However, a methodological challenge arises when dealing with preterm birth, when pregnancy doesn’t consist of three full trimesters. The common compromised approach was to focus on exposure during the period before delivery , 26–30 oSeveral limitations should be acknowledged in interpreting the results. The principal limitation lies in ecological assessment of exposure, both for meteorological indicators and air pollutants. Personal exposure may be modified or attenuated by the duration spent indoors. In addition, the present study relied on the mothers’ report of the date of the last menstrual period to determine gestational age at birth, gestational weeks were used as an analytic scale in this study. A 24 h time frame may be not sufficiently accurate to quantify the difference of several hours, but this bias is likely to be non-differential and to lead the estimates towards null hypothesis. Moreover, as a population-based study, even a small reduction in mean gestational age can lead to a considerable increase in the occurrence of preterm birth. Finally, although we controlled several possible confounding factors, other factors, such as health condition and lifestyle behaviors, may be significantly associated with birth outcomes.This study provided new insights and enriched the understanding of the relationship between ambient temperature and birth outcomes through the following findings: (I) There were different pregnancy periods vulnerable to ambient temperature exposure for different birth outcomes. For the duration of gestation, both the early and late pregnant periods were important; while for birth weight, the late pregnant period was more important than the early period. (II) Duration of gestation seemed to be sensitive to minimum temperature, while birth weight was susceptible to maximum temperature. (III) A J-shaped and an inverted U-shaped associations were observed between the duration of gestation and maternal minimum temperature exposure at conception and delivery, respectively. Meanwhile, there appeared to be an almost linear relationship between maternal exposure to maximum temperature and birth weight.Additional file 1:Table S1. Summary distribution of meteorological and pollutant exposure of 237,585 births in Brisbane, 2000–2010. Summary distribution of meteorological and pollutant exposure. (DOCX 27 kb)Additional file 2:Figure S1. The associations between ambient minimum temperature and gestational age after adjustment for maternal and perinatal factors, air pollutants, and meteorological exposure. The associations between ambient minimum temperature and gestational age. (PDF 115 kb)Additional file 3:Figure S2. The associations between ambient maximum temperature and gestational age after adjustment for maternal and perinatal factors, air pollutants, and meteorological exposure. The associations between ambient maximum temperature and gestational age. (PDF 106 kb)Additional file 4:Figure S3. The associations between ambient maximum temperature and gestational age after adjustment for maternal and perinatal factors, air pollutants, and meteorological exposure. The associations between ambient maximum temperature and gestational age. (PDF 109 kb)Additional file 5:Figure S4. The associations between ambient maximum temperature and birth weight after adjustment for maternal and perinatal factors, air pollutants, and meteorological exposure. The associations between ambient maximum temperature and birth weight. (PDF 82 kb)Additional file 6:Table S2. The description of full-term birth and preterm birth by different ambient temperature category in Brisbane, 2000–2010 . The description of full-term birth and preterm birth. (DOCX 23 kb)Additional file 7:Table S3. The associations of ambient temperature at conception/delivery with preterm birth in Brisbane, 2000–2010 . Association of ambient temperature with preterm birth. (DOCX 31 kb)


It was an incidental observation in our tertiary outpatient dizziness center that patients with acquired bilateral vestibulopathy (BVP) rarely complain about being anxious about falling. This is surprising, since a bilateral vestibular deficit impairs postural stability and thus causes frequent falls , in partThis raised the question of whether intact vestibular function is relevant for distressing anxiety related to the particular type of vertigo or—in other words—whether loss of vestibular function reduces the liability to anxious behavior. This might be possible since Balaban and co-workers showed that there are neurological bases of links between balance control and anxiety. They described pathways that mediate autonomic control, vestibulo-autonomic interactions, and anxiety within a circuitry including a parabrachial nucleus network and its reciprocal connections with the central amygdaloid nucleus, the infralimbic and insular cortex, and the hypothalamus –10.N = 687) and 2015 to 2017 . All patients completed the Vertigo Handicap Questionnaire , Munich, Germany, in the years from 2010 to 2012  to measire (VHQ ) as furtire (VHQ ) to asse®9-based software combined with a MySQL® Database by automatically analyzing medical documents. These documents were the result of outpatient routines at the DSGZ, including clinical neurological and neurootological examination, comprehensive neurophysiological diagnostics . All patients were finally seen by an experienced consultant supervisor from the center. Keywords for the common vestibular disorders, such as bilateral vestibulopathy (BVP), benign paroxysmal positional vertigo (BPPV), vestibular neuritis or unilateral vestibulopathy (UVP), vestibular paroxysmia (VP), Menière’s disease (MD), vestibular migraine (VM), and functional vertigo and dizziness (FD), were used for automatic filtering of the VHQ-linked forms. Statistical analysis was performed using R Core Team (2017), R-Statistics 3.4.3 [Linking diagnoses to VHQ forms was done with custom Javacs 3.4.3 . Groups n = 575 patients with BVP, n = 672 with BPPV, n = 453 with UVP, n = 314 with VP, n = 514 with MD, n = 886 with VM, and n = 416 patients with FD for the total number of 3830 patients (groups 1 and 2) with consolidated data sets. The VHQ anxiety scores of the 687 patients of group 1 were lowest for patients with chronic BVP and UVP and highest for patients with FD  patients fulfilled a diagnosis according to SCID-I criteria. Of those, 28.9% (n = 158) were diagnosed with an anxiety disorder, 24.9% (n = 136) with a somatoform disorder, 19.0% (n = 104) with an affective disorder, 2.9% (n = 16) with a substance abuse disorder, and 0.7% (n = 4) with an eating disorder. These numbers were also reported by Lahmann and co-workers [This resulted in  FD Fig. . Signifi FD Fig. . RegardiThe present data strongly support the view that a functioning peripheral vestibular system is the prerequisite for the development of anxiety related to vertigo and explains why anxiety scores were low in BVP patients. In contrast, all episodic vestibular syndromes as well as chronic functional dizziness revealed increased VHQ anxiety scores with the highest for MD, VM, and FD. One cannot simply compare our current data with the former epidemiological studies on psychiatric comorbidity in dizzy patients based on SCID-I diagnoses . In the It is well acknowledged in affective neuroscience that the vestibular and also the cerebellar systems are reciprocally connected to various anxiety and fear brain structures (for review: ). Such a


To study why neonates have these differences, and whether these effects are linked to prenatal caffeine exposure, we had to develop an analytical method for an accurate measurement of caffeine and metabolites concentration. The analysis was carried out using fetal bovine serum (FBS) as biological matrix in a high-performance liquid chromatography with an ultraviolet detector method. This method allows acceptable chromatographic resolution between analytes in 15 minutes. It was validated and proved to be linear in the 0.1–40 µg/mL range for caffeine, paraxanthine, theobromine, and theophylline in the same chromatographic analysis. Accuracy for quality control samples for intra- and interday assays was ranged from 96.5 to 105.2% and 97.1 to 106.2%. Precision had CV no more than 10% in all concentration levels for all analytes. No differences were observed between quantification in human and FBS. This method was applied to quantify plasma drug concentration in mothers and their newborns in a Mexican northeast population. In our study, we confirmed self-reported caffeine maternal intake in 85.2% (n=23); meanwhile, in their newborn's plasma, it was detected only in 78% (n=21). Caffeine plasma concentrations in mother and newborn had a linear relationship, and no differences were observed between groups (mothers versus children). These results suggest that our analytical method and substitution of biological matrix was linear, precise, and accurate for caffeine quantification and could be used for measuring prenatal exposure and let us to study, in the future, concentration differences observed during apnea clinical treatment.Caffeine is recognized as the first-line therapeutic agent for apnea of prematurity. The dosage regimen is 10 mg/kg loading dose and 2.5 mg/kg maintenance dose. However, the plasma concentration achieved, not always, is therapeutically useful. It makes necessary to increase the doses to reach plasma concentration up to 30 or 35  Caffeine   is amongThis drug has psychoactive properties. It is a slightly dissociative and stimulant drug because of its nonselective antagonist action against adenosine receptors , 4.μg/mL [μg/mL or even higher to be able to attain therapeutic effect [Clinically, caffeine is recognized as the first-line therapeutic agent for apnea of prematurity , becauseμg/mL , 8. In sc effect , 9. It hc effect , 11, andThe presence of multiple cytochrome P-450 isoforms (CYP450) may explain the species, strain, age, tissue, and sex differences as well as the effect of inducers, nutritional status, and human drug metabolism. In humans, caffeine is metabolized through the liver microsomal drug-metabolizing enzyme system CYP450. In neonates, the expression of this enzymatic system is dependent on their prenatal and postnatal age , 12. It The analytical methods used for the quantification of caffeine and its main metabolites are immunoassay with monoclonal antibodies, which presents as main disadvantage the selectivity and sensitivity of the method , and liqAnother analytical problem is to obtain caffeine-free human plasma from volunteers. Some authors have tried to use substitutes, human plasma treated with activated charcoal to remove caffeine, or the manufacture of synthetic plasma devoid of caffeine . We evalRecent studies have reported that intake of caffeine during pregnancy (plasma concentration estimated by self-reported consumption) is associated with the development of sickness such as obesity and hypertension, besides others , 22.Our group is interested in quantifying plasma levels of caffeine and its metabolites to determine what is the prenatal exposure to caffeine in our population and if this exposure has an impact on therapeutic response (apnea) or if it increases risk or predisposition to other diseases , 23.For this reason, initially we had to develop an analytical method to quantify caffeine and its metabolites in plasma at our laboratory.In this study, we present the results on the use of fetal bovine serum (FBS) for the development and validation of an analytical method by HPLC for the quantification of caffeine and three of its main metabolites in samples of neonates and their mothers to perform the previously mentioned studies and results of its clinical application.β-hydroxyethyl)theophylline (IS) (H9006) were obtained from Sigma . Theophylline  (J1H052) was USP standard . Acetonitrile and methanol were of HPLC grade, and acetic acid was of reagent grade; all were acquired from J.T. Baker . Water used during this study was of HPLC grade . Fetal bovine serum (FBS) was used as biological matrix to prepare calibration curves and quality control samples used during method validation and quantification of newborn samples .Caffeine  (C0750), paraxanthine  (D5385), theobromine  , and 7-(µm) (Agilent Technologies). The column oven was maintained at 40°C, while the autosampler was set at room temperature. Fifteen microliters of processed sample was injected into the HPLC system.HPLC system consisted in a quaternary pump with a degasser, and it was coupled to an autosampler and DAD–UV detector (Agilent 1200 series) . Separation was performed on a reverse-phase column Zorbax® SB-Aq narrow bore RR  during 30 min. Chromatographic data were processed using Chemstation for LC systems software (Agilent Technologies).Stock solutions of Par, Theo, and Caf (4 mg/mL) were prepared separately, dissolving an appropriate amount of each drug in diluent (Milli-Q water). Theb solution was prepared at half concentration than the others (2 mg/mL) due to theobromine having the lowest aqueous solubility compared to the rest of caffeine alkaloids. This solution was prepared adding Theb to diluent (Milli-Q water) and heating and mixing the solution prior to obtain its total volume. The solution was cooled to room temperature before it was adjusted to its final volume.µg/mL.Working solutions for the different points in the calibration curve or quality control samples were prepared simultaneously for all interest drugs in water. Those solutions were obtained mixing the necessary reagent volume to attain different concentrations; 1, 3, 5, 10, 25, 50, 100, 200, 300, and 400 β-hydroxyethyl)theophylline) was prepared at 20 µg/mL in Milli-Q water. It was stored until use.IS working solution . In all cases, the biological matrix dilution did not exceed 10%. These solutions were vortexed for one min, and then 0.5 mL aliquots were transferred into 1.5 mL Eppendorf microcentrifuge tubes and stored at −60°C until use.Seven level calibration curves were constructed by spiking water or drug-free FBS with known amounts of Caf, Theo, Par, and Theb to reach concentrations of 0.1, 0.3, 1, 2.5, 10, 20, and 40 The analytical method was developed, validated, and challenged in a clinical trial. The clinical study was focused to quantify prenatal caffeine exposure, and later the analytical method will be applied to study clinical pharmacokinetics of caffeine in preterm neonates. This study reports only the results of the pilot trial of prenatal caffeine. The clinical protocol was reviewed and approved by our Institutional Research and Ethics Board registered according to Mexican law to authorize and oversee the conduction of clinical trials (13CI19039138 and CONBIOETICA-19-CEI-011-20161017). The protocol was conducted in accordance with the Declaration of Helsinki. All participants  agreed to be included in the study, by signing an informed consent form.Inclusion criteria are as follows: women 18 years old or older, pregnant with single or multiple products, gestational age less than 34 completed weeks (based on dates and confirmed with an ultrasound examination), and without a history of preeclampsia or any neonatal abnormality.We excluded women with known fetal genetic or major malformations, very critical condition, or fetal demise.Blood samples (1 mL) were collected during the 15 minutes immediately after delivery from umbilical cord (neonates) or from venous puncture (mothers). Samples were collected in heparinized Vacutainer® tubes and centrifuged for 10 min at 3500 rpm under a controlled temperature of 10°C. The plasma supernatant was carefully transferred to two polypropylene tubes and frozen at −60°C until they were analyzed.µL of plasma samples  in a 1.5 mL Eppendorf tube. To this solution, we added 350 µL of a 10% (v/v) acetic acid solution and 400 µL of water. The tube was vortexed for 20 s and then centrifuged at 12000 rpm for 10 min at 4°C. The supernatant clear layer was collected and preprocessed in a solid-phase extraction (SPE) system.One hundred microliters of the IS working solution was added to 150 The sample pretreatment procedure was carried out on plasma by means of solid-phase extraction (SPE) on polymeric 96-well plates Strata-X™  . The plate was placed in a 96-well plate vacuum manifold system . The vacuum pressure was adjusted between −15 and −20 mmHg, and each well was activated by washing with 1 mL methanol followed by 1 mL water.2 gas at 40°C. The residue was dissolved in 50 μL mobile phase and filtered through 0.22 mm filters, and an aliquot (15 μL) was injected into the HPLC.One milliliter of each sample supernatant (previous step) was placed on its corresponding well and allowed to pass through low vacuum (−5 to −6 mmHg). The wells were then washed with 1 mL 5% methanol at vacuum pressure between −15 and −20 mmHg. Caffeine and its metabolites were eluted with 1 mL methanol-2% acetic acid (70 : 30) solution. The eluent was dried under NValidation was carried out following the criteria established in the Mexican regulatory guidelines , which at-test, with a statistical significance level (α) of 0.05 and assuming normal data distribution. All statistical analyses were performed using SPSS software version 22.0.Results of caffeine and metabolites for prenatal exposure pilot study were expressed as plasma concentration for mothers and newborns. Plasma concentration differences by each mother/child pair (M/C) were calculated and used to construct a Bland–Altman plot for each compound. The differences between each M/C pair plasma concentrations were analyzed for means difference with Student's We tested different analytical methods reported for caffeine and metabolite quantification  in orderIt is well known that fluorescence is more sensitive than UV for analysis of various compounds. However, fluorescence sensitivity to detect caffeine in biological samples remains a challenge.µg/mL, and excitation wavelength (λexc) and emission wavelength (λemis) were 272 nm and 385 nm, respectively (data not shown). Recently, Weldegebreal et al. reported fluorescence caffeine wavelengths similar to those we found [At the beginning of our experimental process, we found a report that described fluorescence parameters for caffeine analysis. We obtained the same values of fluorescence spectrum by scanning a caffeine aqueous solution at 40 we found . Howeverwe found .π-stacked complexes [We believe that another problem could be related with the caffeine fluorescence in biological samples. Some researchers have described that caffeine and its metabolites (theophylline and theobromine) can quench fluorescence signals for proteins, amino acids, and hemoglobin . But, coomplexes .Recently, a “light traffic” detector has been developed to improve caffeine fluorescence detection. This device uses a compound called “caffeine orange” to conjugate caffeine and increase about 250-fold the fluorescence signal. However, the main setback is that it is not selective and can increase fluorescence for caffeine, theobromine, theophylline, and other methylxanthines. This result suggests that fluorescence intensity signal by caffeine is too low for direct detection and needs derivatization to increase response .μg/ml for each analyte in solution. We tested C8 and C18 columns with different pH values or mobile phase composition. However, retention time for each analyte was too short (losing resolution) or too long . Our analytical method had an excellent chromatographic behavior. We were able to separate Caf, Theo, Theb, Par, and IS in 15 minutes using a chromatographic column designed to retain hydrophilic compounds using highly aqueous mobile phase without “phase collapse.” This method was faster than other HPLC-UV methods that have been reported previously with a running time higher than 20 min or even up to 60 min [µg/mL) and upper (40.0 µg/mL)   and zoom0 µg/mL) ; plasma 0 µg/mL)  without 0 µg/mL)  containiµg/mL, in a first-order model for all analytes. The linear equation for each assayed analyte was as follows: for theobromine (Theb/IS) = −0.001 + 0.056 (concentration), r2 = 0.998   .µg/mL) were prepared in FBS, in human plasma, or in mobile phase, and peak areas were compared. Mean analytical recovery for all concentration levels of Theb, Par, Theo, and Caf was 92, 96, 95, and 102%, respectively, and 95% for the IS. Inter- and intraday accuracy and precision values of the assay are presented in The optimal extraction recovery was obtained with SPE cartridges as it was described previously. To calculate absolute recovery of caffeine, metabolites, and IS, 5 sets of samples ; samples were also stable for at least two freeze and thaw cycles  and for at least 8 h on the worktable at room temperature . This was enough time according to our preparation sample method. Analytes were also stable for at least 13 h in the autosampler , which made it possible to analyze about 50 samples in a row.There were no endogenous compounds that interfered either with caffeine or its metabolites or with IS, even in hemolyzed or jaundiced samples. Other results of selectivity evaluation demonstrated that there were no interfering peaks from any of the following drugs: acetaminophen, ibuprofen, erythromycin, furosemide, or heparin, which are commonly used during neonatal pharmacotherapy.The trial was conducted from July 2013 through December 2013 in three medical centers located in Northeastern Mexico: the Hospital Regional Materno Infantil (Center 1), the Hospital Metropolitano Dr. Bernardo Sepúlveda (Center 2), and the Hospital Zambrano-Hellion (Center 3). The first two are public hospitals that harbor level II-III neonatal intensive care units and belong to the Servicios de Salud Network in Nuevo León, Mexico. The last is a private hospital with a level III neonatal intensive care unit and belongs to the academic and research health branch of Tecnológico de Monterrey in Monterrey, Nuevo León, Mexico. Seventy-three pregnant females that were admitted to the obstetric ward during labor were eligible to participate. Forty-six were excluded for different reasons: fetal death (19), malformation (1), or transfer to another unit (4), and 22 eligible mothers who declined the invitation to participate. Thus, only 27 pregnant volunteers participated in this study.n=23); meanwhile, in newborns' plasma, caffeine was only quantified in 78% (n=21). Means of plasma Caf concentration were 0.87 ± 0.22 and 0.89 ± 0.24 µg/mL in mother and newborn groups, respectively. No differences were observed in occurrence and level of caffeine from other populations [µg/mL, 0.17 ± 0.05 and 0.21 ± 0.04 µg/mL, and 0.12 ± 0.03 and 0.12 ± 0.03 µg/mL for mother and newborn groups, respectively. No differences were detected between groups assessed by t-test or Bland–Altman plot  due to their body size, as is the case in newborns, and it can be used for repeated sample procurement such as in pharmacokinetics studies as well as in therapeutic drug monitoring of caffeine and its metabolites in human neonates and perhaps also in small mammals .We developed a rapid and sensitive analytical method for the simultaneous determination of caffeine and its three principal metabolites in adult and newborn human plasma using HPLC technique after solid-phase extraction with Strata-X™ plates. This method had an acceptable accuracy (96.5–105.2% for intraday and 97.1–106.2% for interday) and precision with a CV less than 10% in all concentration levels used as quality control samples for all analytes. This method is suitable for use in individuals in which blood extraction volumes are limited (150


Body composition in infancy plays a central role in the programming of metabolic diseases. Fat mass (FM) is determined by personal and environmental factors. Anthropometric measurements allow for estimations of FM in many age groups; however, correlations of these measurements with FM in early stages of life are scarcely reported. The aim of this study was to evaluate anthropometric and clinical correlates of FM in healthy term infants at 6 months of age.n = 102) from a prospective cohort. Weight, length, skinfolds  and waist circumference (WC) were measured at 6 months. Body mass index (BMI) and WC/length ratio were computed. Type of feeding during the first 6 months of age was recorded. Air displacement plethysmography was used to asses FM (percentage -%-) and FM index (FMI) was calculated. Correlations and general linear models were performed to evaluate associations.Healthy term newborns (p < 0.001). Exclusive/predominant breastfed infants had higher FM and anthropometric measurements at 6 months. Models that showed the strongest associations with FM (% and index) were SFS + WC + sex + type of feeding.Significant correlations were observed between all anthropometric measurements and FM (% and index)(Anthropometry showed good correlations with FM at 6 months of age. Skinfolds sum and waist circumference were the strongest anthropometric variables associated to FM. Exclusive/predominant breastfeeding was strongly associated with FM. Worldwide increasing prevalence of overweight and obesity among children has highlighted the need for better strategies in its early detection and prevention. The 2013 Global Burden of Disease Study showed a rise of 47% in overweight and obesity prevalence in children since 1980 –7.Because body composition in early infancy could play a central role in the programming of metabolic diseases , 9, accuWeight-for-length (W/L) and body-mass-index for age (BMI/A) are anthropometric indices used for the diagnosis of overweight and obesity in children . The eviTherefore, the aim of this study was to evaluate how anthropometric measurements and clinical factors correlate with FM at 6 months of age in healthy term Mexican infants.This descriptive study derives from a prospective cohort of pregnant women conducted at the National Institute of Perinatology .We consecutively selected women in their first trimester of pregnancy (< 14 weeks), who did not have comorbidities , did not use medication that affected metabolism  and did not have multiple pregnancies. All adult women that accepted to participate signed an informed consent; for those < 19 years old, the consent of parents was also required. Women were followed monthly until the end of pregnancy.Newborns were selected by convenience from healthy mothers participating in the afore-mentioned cohort. We invited to participate only those mother-baby binomials that did not develop gestational diabetes or preeclampsia and whose babies were born at the institutional hospital. We included healthy term newborns (≥37 weeks). Those babies born prematurely (< 37 weeks) or with congenital/metabolic diseases were excluded. Babies that lacked body composition assessment at 6 months were eliminated from the analysis.A nutritionist with prior experience and adequately trained in anthropometric measurement standardization performed all the babies’ anthropometric procedures at birth (first 72 h) and at 6 months of age, following the methods described by Lohman .2) and sex-specific z-scores for BMI/A and W/L were calculated using the World Health Organization (WHO) Anthro software program . Risk of overweight (> 1 standard deviation scores), overweight (> 2 standard deviation scores) and obesity (> 3 standard deviation scores) was defined based on z-scores.Infants were measured without clothes. The average of two measurements was recorded, except for weight and FM. At birth, weight was recorded in grams to the nearest 0.1 kg using a pediatric scale . Low birth weight (< 2500 g) and macrosomia (> 4000 g) were recorded. At 6 months, weight was measured using the digital scale from the PEAPOD Infant Body Composition System . Recumbent length was measured to the nearest 0.1 cm using an infantometer . With the help of a nurse, the infant was laid down and her/his head was placed in the Frankfort plane  . Length Waist circumference (WC) was measured around the umbilicus to the nearest 0.1 cm with a fiberglass anthropometric measuring tape . WC-to-length ratio (WLR) was obtained dividing WC by length.Skinfolds  were measured with a Lange caliper  from the child’s left arm holding the skin between the index finger and thumb (one centimeter above the measurement site) while the baby was laid down. BSF and TSF were measured taking as reference the mid-point of the arm grasping the skin parallel to the floor. SSF was taken at a 45° to the horizontal plane. A difference > 2 mm between both measurements required a third measurement. Skinfold sum (SFS) was obtained by adding the values from the three skinfolds aforementioned.2) was computed to correct for variations in FM related to body size [The assessment of FM (percentage -%- and kilograms -kg-) and fat-free mass  was performed at 6 months using the PEAPOD Infant Body Composition System , 26. Eveody size .At 1-, 3- and 6-months-old visits, mothers were asked a series of questions regarding their infant’s feeding practices. Using the information collected from the 6-months questionnaire, infants were classified within 3 breastfeeding categories according to the WHO definition : a) exclMothers participating in the cohort study reported their age and pre-gestational weight in their first visit. Pre-gestational BMI was computed and classified according to WHO criteria .Descriptive statistics and frequencies were performed for all variables. Mean differences were analyzed using Student’s t-test for parametric variables or U Mann-Whitney for non-parametric ones. One-way ANCOVA was performed to evaluate differences in anthropometric indices and FM according to type of feeding. Pearson’s and Spearman’s correlations were used to evaluate bivariate associations between FM (% and index) and anthropometric measures.p-value < 0.05 was considered statistically significant. Statistics were performed with the IBM SPSS Software .General linear models were performed to evaluate the association of anthropometric measurements  and clinical factors with FM (% and index) at 6 months. The dependent variable was FM% or FMI; independent variables included sex and type of feeding. For FM% models, length was considered as a variable, and excluded in FMI models. A n = 94)  of newborns and no infants were classified as macrosomic. Most of the newborns had normal BMI/A at birth , and there were no infants classified with obesity during the first 6 months of age.Mothers had a mean age of 27.1 ± 8.7 years and their mean pre-gestational BMI was 24.7 ± 5.2 kg/mp < 0.05). No other anthropometric differences were observed by sex (p > 0.05).Descriptive data of mothers, newborns and infants at 6 months is presented in Table FM (% and kg) was higher and FFM (% and kg) was lower in infants classified as E/P BF when compared to BF + F or BF < 6 m + F. Except for WC, all anthropometric measurements were higher in the E/P BF group  Table .Table 2Fp < 0.001). The strongest correlation coefficients were for BMI (r = 0.691), SFS (r = 0.603) and WC (r = 0.591), followed by WLR (r = 0.589), SSF (r = 0.588) and TSF (r = 0.581). The weakest correlations were for weight (r = 0.569) and BSF (r = 0.409).Correlations of anthropometric measurements with FM% at 6 months were all statistically significant (p < 0.0001). In order of strength, correlation coefficients were as follows: BMI (r = 0.816), WLR (r = 0.693), WC (r = 0.691), weight (r = 0.668), SFS (r = 0.640), SSF (r = 0.631), TSF (r = 0.624) and BSF (r = 0.415).All anthropometric measurements had significant correlations with FMI (The strongest models of FM% included SFS + WC + length (model 2) followed by TSF + WC + length Model 4) Table . Regardi Table . This is one of the few studies measuring FM at 6 months of age using ADP in a group of healthy term infants. Our results showed that anthropometric measurements are highly correlated with FM in infants. SFS and WC were strongly associated to FM.In our study, the model that best correlated anthropometry to FM at 6 months of age included SFS + WC. We observed that when more anthropometric variables were included in the model, the strength of the association with FM improved, as other authors have reported , 22.The increase in the prevalence of childhood overweight and obesity worldwide has encouraged the study of body composition during early stages of life , 30. In On the other hand, BMI has been proposed to assess growth and nutritional status because it is known to correlate with body fatness and increases the risk of obesity-related diseases in adults , 33. HowSkinfolds are valid measures of subcutaneous fat and have been used for estimating total body fat in adults, but there is limited information about their validity in childhood and infancy. Bias may be introduced by age, measurement error and error inherent with increasing fatness , 32. In WC is a measurement used to assess central adiposity , 16. ConThe use of FMI  has been proposed as a more accurate indicator of adiposity and more sensitive to changes in body fat stores , 47, eveIn our study, infants that were exclusive/predominant breastfed during the first 6 months showed higher FM and anthropometric measurements at 6 months. Some authors have reported higher adiposity in exclusively breastfed infants when compared to those that were formula fed at 3–4 and at 6 months of age .This study has some limitations. Fat mass measurement at birth was not feasible, therefore, we have no data to correlate in this stage. It is well known that skinfold measurement may introduce a high measurement error; to decrease it, one nutritionist with previous experience and anthropometric training and standardization performed them by duplicate. The small sample and low prevalence of obesity in this group of infants might limit the associations found in this analysis. Performance of anthropometric measures may differ in the extremes of obesity, as has been shown in adults.Anthropometric measurements were good determinants of FM at 6 months of age in healthy term infants. Skinfolds sum and waist circumference were the strongest anthropometric variables associated to FM. Type of feeding and sex influence body fat in this age group. Exclusive/predominant breastfeeding was strongly associated with FM.


P = 0.0013) showed an opposite expression pattern with its target miR‐485  in PTC, indicating that the RP11‐159F24.1/miR‐485/mRNAs axis might play an important role in the development of PTC. In conclusion, this study has constructed a PTC‐related lncRNA‐miRNA‐mRNA network and identified the hub lncRNA RP11‐159F24.1 in the tumorigenesis, which provided novel insights to explore the underlying mechanism of PTC.Papillary thyroid cancer (PTC) is one of the most common cancers worldwide, and its carcinogenesis is influenced by a complex network of gene interactions. In this study, the microarray expression profile was re‐annotated into a lncRNA‐mRNA biphasic profile. LncRNA‐mRNA interactions were confirmed by established miRNA‐RNA data and hypergeometric test. Then, a PTC‐related lncRNA‐miRNA‐mRNA network (PTCRN) was constructed by integrating differentially expressed genes with the RNA‐RNA networks. The new network consisted of 21 lncRNAs, 241 mRNAs and 803 edges. To prioritize PTC‐related genes, we performed topological analysis and random walk with restart (PWR) algorithm analysis of PTCRN. Both analyses identified lncRNA RP11‐159F24.1 as a hub node in the network, which could interact with 47 mRNAs by sponging miR‐485. In functional enrichment analysis, these interacting mRNAs were associated with the pathways in cancer. In validation, RP11‐159F24.1 (up‐regulated;  Then, hub genes were identified by both topological analysis and random walk with a restart (RWR) algorithm analysis. Our findings could help to reveal the lncRNA‐miRNA‐mRNA regulatory network in PTC and provide novel insights in identifying the mechanism of PTC.22.1http://www.ncbi.nlm.nih.gov/geo/). The dataset was based on the chip platform of Affymetrix Human Genome U133 plus 2.0 Array (GPL570), and contained 44 pairs of PTC and adjacent normal tissues.Raw microarray data of GSE33630 were obtained from the Expression Omnibus (GEO) database (2.2http://www.affymetrix.com). The human genome (hg38) and related annotation file (Release 28) were obtained from the GENCODE database (https://www.gencodegenes.org). By using HISAT2, we identified probe‐matched lncRNA and protein‐coding sequences. Transcripts were included if fulfilling the following criteria: (a) detected by more than three probes; (b) each probe was mapped without mismatch; (c) each probe was matched to only one transcript in probe‐transcript pairs.The microarray probe sequences were downloaded from the Affymetrix's website  is a powerful online tool used to systematically identify RNA‐RNA and protein‐RNA interaction networks from 108 CLIP‐Seq datasets.StarBase v2.0  represented the number of edges linked to node v. It was described as follows:To analyze topological features of PTCRN, the network degree, betweenness,and closeness centrality were calculated respectively by the “igraph” R package.e was the number of edges linked to node v.where CB) represented the number of shortest paths between all pairs of nodes in the network that passed through a specific node. It was described as follows:Betweenness centrality  was the node count (v) from node s to node t.where CC) represented the mean distance between a node and all other nodes. It was described as follows:Closeness centrality  represented the average shortest paths from node v to all other nodes (t); n is the total number of nodes.where dist, while zero for nonseed nodes. Then, the RWR algorithm simulated a random walker that moved on the PTCRN starting from the three genes. In detail, let Pi be a vector representing the probability of each node after the ith moving procedure was complete. After each moving procedure, Pi was updated as follows:In this study, we selected three PTC‐related genes  as seed nodes in the RWR algorithm, which has been validated by multiple experiments. The initial probability M was the column‐wise normalized adjacency matrix of the network; r was the restart probability of returning to the seed nodes at every step (r = 0.8 in this study). When the L1‐norm of the difference between two successive vectors was <1 × 106, the vector became stable, the RWR algorithm stopped and output Pi+1 as the final vector. Each component in this vector indicated the probability of a node being a PTC‐related gene.where P value was calculated for each gene, as follows:Then, we adopted a permutation test to remove the false positives, which was performed in three steps: (a) 1000 gene sets were randomly produced, and each contained three genes; (b) for each set, the RWR algorithm was executed on the PTCRN using genes in the set as seed nodes; (c) the θ represented the number of gene sets, which yielded a higher probability of gene (g) than that yielded by the known PTC‐related genes. A P value of <0.05 was considered significant.where 2.8http://software.broadinstitute.org/gsea/msigdb) and KEGG PATHWAY Database (https://www.kegg.jp/kegg/pathway.html). False discovery rate (FDR) <0.05 was chosen as the cut‐off criteria.To investigate potential function of candidate feature genes, gene ontology (GO) and KEGG pathway analyses were performed using The Molecular Signatures Database (MSigDB) (2.9https://xenabrowser.net). Paired t test was performed, and a P value of <0.05 was considered significant.Another GEO dataset of GSE35570 which contained 34 pairs of PTC and adjacent normal samples, was used to validate candidate lncRNA expression. To validate the lncRNA‐sponged miRNA expression, RNA‐sequencing data of 53 pairs of samples were downloaded from The Cancer Genome Atlas (TCGA) database  was constructed by integrating DEGs with the RNA‐RNA networks. The new network consisted of 21 lncRNAs, 241 mRNAs and 803 edges Figure .3.2The topological features of the network were evaluated by the degree, betweenness and closeness centrality. We listed the overlap of top 30 genes with topological features in each dimension Figure . Six lnc3.3P = 0.040). In the subnetwork, RP11‐159F24.1 could interact with 47 mRNAs through sponging has‐miR‐485‐5p   .Another GEO dataset of GSE35570 which contained 34 pairs of PTC and adjacent normal samples, was used to validate RP11‐159F24.1 expression. RP11‐159F24.1 also showed a significant higher expression level in PTC  Figure . RNA‐seq4In this study, we investigated the molecular mechanism of PTC by building a lncRNA‐miRNA‐mRNA regulatory network. First, the microarray expression profile was re‐annotated into a lncRNA‐mRNA biphasic profile. Then, lncRNA‐mRNA interactions were confirmed by established miRNA‐RNA data and hypergeometric test. Finally, a PTC‐related lncRNA‐miRNA‐mRNA network (PTCRN) was constructed by integrating differentially expressed genes with the RNA‐RNA networks.To prioritize PTC‐related genes, we adopted two different methods to identify the hub nodes in PTCRN. First, topological analysis was conducted on the network, and the graph parameters indicated six lncRNAs and seven mRNAs as the hub nodes in the regulatory network. Among these genes, CAMTA1, CDC42 and RNF138 have been reported as oncogenes in multiple cancers,When performed RWR to the network, lncRNA RP11‐159F24.1 were also identified in association with the pathogenesis of PTC. In the subnetwork of RP11‐159F24.1, it could interact with 47 mRNAs though sponging the miRNA of has‐miR‐485‐5p. We supposed that the RP11‐159F24.1/miR‐485/mRNAs axis might play an import role in the development of PTC. As for RP11‐159F24.1, the validation set also indicated a higher expression in PTC. As the target of RP11‐159F24.1, miR‐485 showed a significantly lower expression in PTC, which was consistent with our hypothesis. miR‐485 (including miR‐485‐5p and miR‐485‐3p) has been reported as a tumor suppressor in multiple cancers.Although we provided a novel approach to construct a PTC‐related lncRNA‐miRNA‐mRNA network and identify hub genes in the network, the limitations should be acknowledged for our study. As the probes were re‐annotated, the network could not cover all lncRNAs. We identified a total of 4232 lncRNAs, but still missed some popular lncRNAs, like ANCR, UCA1 and POU3F3.In conclusion, this study has constructed a PTC‐related lncRNA‐miRNA‐mRNA network and identified the hub lncRNA RP11‐159F24.1 in the tumorigenesis, which provided novel insights to explore the underlying mechanism of PTC.None declared. Click here for additional data file.