Metabolic changes contribute to maladaptive right ventricular hypertrophy in pulmonary hypertension beyond pressure overload: an integrative imaging and omics investigation.

Right ventricular (RV) failure remains the strongest determinant of survival in pulmonary hypertension (PH). We aimed to identify relevant mechanisms, beyond pressure overload, associated with maladaptive RV hypertrophy in PH. To separate the effect of pressure overload from other potential mechanisms, we developed in pigs two experimental models of PH (M1, by pulmonary vein banding and M2, by aorto-pulmonary shunting) and compared them with a model of pure pressure overload (M3, pulmonary artery banding) and a sham-operated group. Animals were assessed at 1 and 8 months by right heart catheterization, cardiac magnetic resonance and blood sampling, and myocardial tissue was analyzed. Plasma unbiased proteomic and metabolomic data were compared among groups and integrated by an interaction network analysis. A total of 33 pigs completed follow-up (M1, n = 8; M2, n = 6; M3, n = 10; and M0, n = 9). M1 and M2 animals developed PH and reduced RV systolic function, whereas animals in M3 showed increased RV systolic pressure but maintained normal function. Significant plasma arginine and histidine deficiency and complement system activation were observed in both PH models (M1&M2), with additional alterations to taurine and purine pathways in M2. Changes in lipid metabolism were very remarkable, particularly the elevation of free fatty acids in M2. In the integrative analysis, arginine-histidine-purines deficiency, complement activation, and fatty acid accumulation were significantly associated with maladaptive RV hypertrophy. Our study integrating imaging and omics in large-animal experimental models demonstrates that, beyond pressure overload, metabolic alterations play a relevant role in RV dysfunction in PH.

Identification of SYNJ1 in a Complex Case of Juvenile Parkinsonism Using a Multiomics Approach.

This study aimed to elucidate the genetic causes underlying the juvenile parkinsonism (JP) diagnosed in a girl with several family members diagnosed with spinocerebellar ataxia type 2 (SCA2). To achieve this, whole-exome sequencing, analysis of CAG repeats, RNA sequencing analysis on fibroblasts, and metabolite identification were performed. As a result, a homozygous missense mutation SNP T>C (rs2254562) in synaptojamin 1 (SYNJ1), which has been implicated in the regulation of membrane trafficking in the synaptic vesicles, was identified. Additionally, we observed overexpression of L1 cell adhesion molecule (L1CAM), Cdc37, GPX1, and GPX4 and lower expression of ceruloplasmin in the patient compared to the control. We also found changes in sphingolipid, inositol, and inositol phosphate metabolism. These findings help to clarify the mechanisms of JP and suggest that the etiology of JP in the patient may be multifactorial. This is the first report of the rs2254562 mutation in the SYNJ gene identified in a JP patient with seizures and cognitive impairment.

Recent developments in data acquisition, treatment and analysis with ion mobility-mass spectrometry for lipidomics.

Lipids are involved in many biological processes and their study is constantly increasing. To identify a lipid among thousand requires of reliable methods and techniques. Ion Mobility (IM) can be coupled with Mass Spectrometry (MS) to increase analytical selectivity in lipid analysis of lipids. IM-MS has experienced an enormous development in several aspects, including instrumentation, sensitivity, amount of information collected and lipid identification capabilities. This review summarizes the latest developments in IM-MS analyses for lipidomics and focuses on the current acquisition modes in IM-MS, the approaches for the pre-treatment of the acquired data and the subsequent data analysis. Methods and tools for the calculation of Collision Cross Section (CCS) values of analytes are also reviewed. CCS values are commonly studied to support the identification of lipids, providing a quasi-orthogonal property that increases the confidence level in the annotation of compounds and can be matched in CCS databases. The information contained in this review might be of help to new users of IM-MS to decide the adequate instrumentation and software to perform IM-MS experiments for lipid analyses, but also for other experienced researchers that can reconsider their routines and protocols.

Combined Genome, Transcriptome and Metabolome Analysis in the Diagnosis of Childhood Cerebellar Ataxia.

Ataxia in children is a common clinical sign of numerous neurological disorders consisting of impaired coordination of voluntary muscle movement. Its most common form, cerebellar ataxia, describes a heterogeneous array of neurologic conditions with uncountable causes broadly divided as acquired or genetic. Numerous genetic disorders are associated with chronic progressive ataxia, which complicates clinical management, particularly on the diagnostic stage. Advances in omics technologies enable improvements in clinical practice and research, so we proposed a multi-omics approach to aid in the genetic diagnosis and molecular elucidation of an undiagnosed infantile condition of chronic progressive cerebellar ataxia. Using whole-exome sequencing, RNA-seq, and untargeted metabolomics, we identified three clinically relevant mutations (rs141471029, rs191582628 and rs398124292) and an altered metabolic profile in our patient. Two POLR1C diagnostic variants already classified as pathogenic were found, and a diagnosis of hypomyelinating leukodystrophy was achieved. A mutation on the MMACHC gene, known to be associated with methylmalonic aciduria and homocystinuria cblC type, was also found. Additionally, preliminary metabolome analysis revealed alterations in our patient's amino acid, fatty acid and carbohydrate metabolism. Our findings provided a definitive genetic diagnosis reinforcing the association between POLR1C mutations and hypomyelinating leukodystrophy and highlighted the relevance of multi-omics approaches to the disease.

Microfluidic Breadboard Approach to Capillary Electrophoresis.

A breadboard approach for electrophoretic separations with contactless conductivity detection is presented. This is based on miniature off-the-shelf components such as syringe pumps, valves, and pressure controllers which could be set up in a very compact overall arrangement. It has a high flexibility for different tasks at hand, and the common operations of hydrodynamic injection and capillary flushing are automated. For demonstration of the versatility of the proposition, several very diverse configurations and modes of electrophoresis were successfully implemented, namely, standard capillary zone electrophoresis, pressure assisted zone electrophoresis, the simultaneous separation of cations and anions by dual-capillary zone electrophoresis, the separation of cationic amino acids by isotachophoresis, as well as the separation of small carboxylic acids by gradient elution moving boundary electrophoresis. The system also allows fast separations, as demonstrated by the analysis of six inorganic cations within 35 s. The approach addresses respective limitations of either conventional capillary electrophoresis instruments as well as electrophoretic lab-on-chip devices, while maintaining a performance in terms of detection limits and reproducibility comparable to standard instrumentation.

A new CE with contactless conductivity detection method for the determination of complex cationic compositions: Application to the analysis of pen inks.

A CE with contactless conductivity detection methodology using a novel background electrolyte for the separation and determination of 17 metal cations (Cs+ , Rb+ , K+ , Ca2+ , Na+ , Mg2+ , Mn2+ , Sr2+ , Li+ , Ba2+ , Fe2+ , Pb2+ , Cd2+ , Zn2+ , Co2+ , Cu2+ and Ni2+ ) and ammonium has been investigated. The buffer, based on lactic acid and ß-alanine, was experimentally compared with other two commonly used electrolytes, showing important improvements, such as shorter analysis times (<11 min), better electrophoretic resolutions and higher detectabilities for certain analytes, such as Fe2+ and Pb2+ . The inclusion of other additives such as 18-Crown-6 and α-hydroxyisobutyric acid was studied in order to obtain the best separation of the analytes of interest. The optimised method was applied to the analysis of 11 water-based pen inks and the determination of their metal composition. The methodology was demonstrated for the comparison and differentiation of pen inks.

Micro-injector for capillary electrophoresis.

A novel micro-injector for capillary electrophoresis for the handling of samples with volumes down to as little as 300 nL was designed and built in our laboratory for analyses in which the available volume is a limitation. The sample is placed into a small cavity located directly in front of the separation capillary, and the injection is then carried out automatically by controlled pressurization of the chamber with compressed air. The system also allows automated flushing of the injection chamber as well as of the capillary. In a trial with a capillary electrophoresis system with contactless conductivity detector, employing a capillary of 25 µm diameter, the results showed good stability of migration times and peak areas. To illustrate the technique, the fast separation of five inorganic cations (Na(+) , K(+) , NH4 (+) , Ca(2+) , and Mg(2+) ) was set up. This could be achieved in less than 3 min, with good limits of detection (10 µM) and linear ranges (between about 10 and 1000 µM). The system was demonstrated for the determination of the inorganic cations in porewater samples of a lake sediment core.

Instrumentino: An Open-Source Software for Scientific Instruments.

Scientists often need to build dedicated computer-controlled experimental systems. For this purpose, it is becoming common to employ open-source microcontroller platforms, such as the Arduino. These boards and associated integrated software development environments provide affordable yet powerful solutions for the implementation of hardware control of transducers and acquisition of signals from detectors and sensors. It is, however, a challenge to write programs that allow interactive use of such arrangements from a personal computer. This task is particularly complex if some of the included hardware components are connected directly to the computer and not via the microcontroller. A graphical user interface framework, Instrumentino, was therefore developed to allow the creation of control programs for complex systems with minimal programming effort. By writing a single code file, a powerful custom user interface is generated, which enables the automatic running of elaborate operation sequences and observation of acquired experimental data in real time. The framework, which is written in Python, allows extension by users, and is made available as an open source project.

Screening determination of four amphetamine-type drugs in street-grade illegal tablets and urine samples by portable capillary electrophoresis with contactless conductivity detection.

A simple and inexpensive method for the identification of four substituted amphetamines, namely, 3,4-methylenedioxy methamphetamine (MDMA), methamphetamine (MA), 3,4-methylenedioxy amphetamine (MDA) and 3,4-methylenedioxy-N-ethylamphetamine (MDEA) was developed using an in-house constructed semi-automated portable capillary electrophoresis instrument (CE) with capacitively coupled contactless conductivity detection (C(4)D). Arginine 10mM adjusted to pH4.5 with acetic acid was found to be the optimal background electrolyte for the CE-C(4)D determination of these compounds. The best detection limits achieved with and without a sample preconcentration process were 10ppb and 500ppb, respectively. Substituted amphetamines were found in different seized illicit club drug tablets and urine samples collected from different suspected users. Good agreement between results from CE-C(4)D and those with the confirmation method (GC-MS) was achieved, with correlation coefficients for the two pairs of data of more than 0.99.