Microsatellite instability in colorectal cancer

BACKGROUND: Colorectal cancer (CRC) is the third most common cancer in the world. In 75% CRC develops sporadically, in 25% hereditary or as a consequence of inflammatory bowel disease. CRC carcinogenesis develops over many years. The cause of CRC in 85% is chromosomal instability (CIN) and in 15% microsatellite instability (MSI-H), where hereditary nonpolyposis colorectal cancer (HNPCC) represents 10-20%. Microsatellite sequences (MS) are repeated sequences of short stretches of DNA all over the genome. Microsatellite stability (MSS) means MS are the same in each cell of an individual, whereas microsatellite instability (MSI-H) means MS differ in normal and cancer cells of an individual. The cause of MSI-H is a damaged mismatch repair mechanism (MMR), with the most important MMR proteins being MSH2, MLH1 and MSH6. CONCLUSIONS: MSI-H seems to be an important prognostic factor in CRC and an important predictive factor of CRC chemotherapeutic treatment efficacy. Clinical trials conducted until now have shown contradictory findings in different chemotherapeutic settings, adjuvant and palliative; therefore MSI-H is going to be the object of the future research. The future of cancer treatment is in the individualized therapy based on molecular characteristics of the tumour, such as MSI-H in CRC.     

Pipette tip with integrated electrodes for gene electrotransfer of cells in suspension: a feasibility study in CHO cells

Background

Gene electrotransfer is a non-viral gene delivery method that requires successful electroporation for DNA delivery into the cells. Changing the direction of the electric field during the pulse application improves the efficacy of gene delivery. In our study, we tested a pipette tip with integrated electrodes that enables changing the direction of the electric field for electroporation of cell suspension for gene electrotransfer.

Materials and methods

A new pipette tip consists of four cylindrical rod electrodes that allow the application of electric pulses in different electric field directions. The experiments were performed on cell suspension of CHO cells in phosphate buffer. Plasmid DNA encoding for green fluorescent protein (GFP) was used and the efficiency of gene electrotransfer was determined by counting cells expressing GFP 24 h after the experiment.

Results

Experimental results showed that the percentage of cells expressing GFP increased when the electric field orientation was changed during the application. The GFP expression was almost two times higher when the pulses were applied in orthogonal directions in comparison with single direction, while cell viability was not significantly affected.

Conclusions

We can conclude that results obtained with the described pipette tip are comparable to previously published results on gene electrotransfer using similar electrode geometry and electric pulse parameters. The tested pipette tip, however, allows work with small volumes/samples and requires less cell manipulation.     

Effect of changes in left ventricular anatomy and conduction velocity on the QRS voltage and morphology in left ventricular hypertrophy: a model study

The increased QRS voltage is considered to be a specific electrocardiogram (ECG) sign of left ventricular hypertrophy (LVH), and it is expected that the QRS voltage reflects the increase in left ventricular mass (LVM). However, the increased QRS voltage is only one of QRS patterns observed in patients with LVH. According to the solid angle theory, the resultant QRS voltage is influenced not only by spatial (anatomic) but also by nonspatial (electrophysiologic) determinants. In this study, we used a computer model to evaluate the effect of changes in anatomy and conduction velocity of the left ventricle on QRS complex characteristics.

Material and Methods

The model defines the geometry of cardiac ventricles analytically as parts of ellipsoids and allows to change dimensions of the ventricles, as well as the conduction velocity in the individual layers of myocardium. Three types of anatomic changes were simulated: concentric hypertrophy, eccentric hypertrophy, and dilatation. The conduction velocity was slowed in the inner layer of the left ventricle representing the Purkinje fiber mesh and in the layers representing the working myocardium. The outcomes of the model are presented as the time course of the spatial QRS vector magnitude, the vectorcardiographic QRS loops (VCGs) in horizontal, left sagittal, and frontal planes, as well as derived 12-lead ECGs. The following indicators of the 12-lead ECG were evaluated: the left axis deviation, the intrinsicoid deflection in V6, Cornell voltage, Cornell voltage-duration product, and Sokolow-Lyon index.

Results

The increase in LVM did not affect the QRS voltage proportionally, and the LVM and type of hypertrophy were not the only determinants of the QRS patterns. The conduction velocity slowing resulted in a spectrum of QRS patterns including increased QRS voltage and duration, left axis deviation, prolonged intrinsicoid deflection, VCG patterns of left bundle branch block, as well as pseudo-normal VCG/ECG patterns. The anatomic changes and conduction velocity slowing affected differently Sokolow-Lyon index and Cornell criteria.

Conclusion

We showed that the LVM is not the only determinant of the QRS complex changes in LVH, but it is rather a combination of anatomic and electric remodeling that creates the whole spectrum of the QRS complex changes seen in LVH patients. The slowed conduction velocity in the model heart produced QRS patterns consistent with changes described in LVH, even if the LVM was not changed.     

Early onset Alexander disease: a case report with evidence for manifestation of the disorder in neurohypophyseal pituicytes

We report the first case of Alexander disease diagnosed and published in the region of former Czechoslovakia. The case was characterized by early (late infantile) onset, the absence of megacephaly but with extensive internal hydrocephaly, despite a patent aqueduct. Neuropathology revealed severe depletion ofoligodendroglia and myelin, loss of axons, prominent astrocytosis with massive intracellular, dense globular GFAP aggregates which differed from typical Rosenthal fibers. Additionally, many large aggregates of GFAP were located extracellularly. Globular GFAP aggregates were also identified in neurohypophyseal pituicytes. DNA analysis disclosed a heterozygous mutation c.1117G>A in the GFAP, which is predicted to lead to the amino acid exchange p.Glu-373Lys (E373K) in the C-terminal tail of the GFAP protein. The parents and a healthy sister did not show any variation in GFAP in somatic cells.     

Analgesic treatment and predictors of satisfaction with analgesia in patients with acute undifferentiated abdominal pain.

The objectives of this prospective, observational cohort study were to examine current practice of analgesia in adults with acute abdominal pain presenting to emergency department (ED), to assess patient-physician agreement on pain severity, and to measure patients' satisfaction with pain management. METHODS: Emergency room nurse assessed initial patient's and physician's ranking on a 0-100mm visual analogue scale (VAS) as well as patient's VAS rankings 5min, 15min, and 60min after starting treatment for pain, and on discharge. In patients who received no therapy VAS scores were assessed 5min, 15min, and 60min after initial examination, and on discharge. Patient's satisfaction with pain management on discharge from ED was assessed using a four-point categorical scale in which 1=completely unsatisfied, 2=mostly unsatisfied, 3=mostly satisfied, and 4=completely satisfied. RESULTS: A total of 185 patients were enrolled. Patients' mean initial VAS was higher than physicians' (76+/-20mm, vs. 59+/-20mm, p<0.001). Physician's VAS > or = 60mm was the sole independent predictor of receiving analgesic therapy (p<0.001). On discharge from ED, 111 patients (60%) were predominantly satisfied with analgesia (satisfaction score > or = 3). Drug titration (p=0.026) and decrease in VAS score > or = 20mm between initial and discharge score (p<0.001) independently predicted patients' satisfaction. CONCLUSIONS: Patients with acute abdominal pain rated pain significantly higher than physicians who's pain estimation in turn tailored analgesia. Only 60% of patients were satisfied with analgesia. Analgesic drug titration and a decrease of > or = 20mm on VAS predicted patients' satisfaction.     

Prevalence and incidence of attention-deficit/hyperactivity disorder in Slovenian children and adolescents: a database study from a national perspective

Aim

To estimate prevalence and incidence of attention deficit hyperactivity disorder (ADHD) in children and adolescents in Slovenia using different epidemiological models.

Methods

Data from the National Institute of Public Health of the Republic of Slovenia for the period 1997-2012 were analyzed. The database includes the annual number of newly diagnosed outpatients with ADHD in Slovenia. The evaluation for ADHD diagnoses was done in accordance with the Tenth Revision of the International Classification of Diseases (ICD-10) outpatient data codes. In model 1, a linear increase was proposed to fit the data in the period from 1997 to 2003 in order to extrapolate the data before 1997. In model 2 and 3, an exponential increase in the annual incidence rate was proposed.

Results

The incidence rate of ADHD diagnosis in 1997 was 0.032% and in 2012 it increased to 0.082%. Mean prevalence rate was 750 (95% confidence interval: 660-840) per 100 000 children and adolescents. It was estimated that the prevalence rate in 2020 would be 1% (95% confidence interval: 0.875-1.125), which is 6.3-fold higher than in 1997.

Conclusions

ADHD is a common mental health disorder among Slovenian children and adolescents, but it remained underdiagnosed compared with Western countries. Our results indicated a need for improved timely interventions in Slovenia, not only in child and adolescent psychiatry but also in primary settings and adult psychiatry, where ADHD should be more efficiently recognized.Attention deficit hyperactivity disorder (ADHD) is one of the most common psychiatric disorders in childhood and adolescence, characterized by developmentally inappropriate inattention, hyperactivity, and/or impulsivity (1). It entails economic costs, causes family stress and academic and vocational adversity, and has a negative effect on the patient’s self-esteem (2). Although it is seen as a problem that takes place predominantly in childhood and adolescence, clinical and epidemiological research has shown that in 30–50% of patients ADHD persists into adulthood (3,4).Children exhibiting symptoms of inattention, hyperactivity, and impulsivity have been described previously (5). In 1980, the third edition of Diagnostic and Statistical Manual of Mental Disorders (DSM-III) introduced the term “ADD (Attention-Deficit Disorder) with or without hyperactivity.” DSM-III-R (1987) replaced this term by the term ADHD and DSM-IV presented the subtypes of ADHD (6-9).ADHD affects 3% to 9% of children worldwide (10). In European countries, it is identified as a hyperkinetic disorder according to the 10th Revision of the International Statistical Classification of Diseases and Related Health Problems (ICD-10) and in North America as ADHD (DSM-IV). DSM-IV criteria include a broader group of subjects than the ICD-10 criteria (11,12).While the popular press across Europe frequently comments on increased rates of ADHD diagnosis, questioning whether ADHD is overdiagnosed and overtreated, reviews of clinical practice suggest that in Europe ADHD is underdiagnosed and undertreated (13,14). ADHD is underdiagnosed especially in adults; which is why it is important to better understand the factors that contribute to accurate diagnosis (15). Diagnosis and management of ADHD include nonpharmacological treatment, including behavioral therapy, and pharmacological treatment with stimulants and nonstimulants (16). In many European countries including Slovenia, the incidence and prevalence of this disorder are not well researched. In Slovenia, there are also no national guidelines for ADHD treatment and diagnosis and no published data on the prevalence of ADHD (17,18). The primary aim of this study was to calculate the incidence and prevalence of ADHD diagnosis among children and adolescents in Slovenia in 2012, based on national data from 1997 to 2012. For this purpose, several different epidemiological models were developed. The secondary aim was to predict the number of Slovenian children and adolescents that will be diagnosed with ADHD in 2020.     

Biomechanical considerations in the pathogenesis of osteoarthritis of the knee

Osteoarthritis is the most common joint disease and a major cause of disability. The knee is the large joint most affected. While chronological age is the single most important risk factor of osteoarthritis, the pathogenesis of knee osteoarthritis in the young patient is predominantly related to an unfavorable biomechanical environment at the joint. This results in mechanical demand that exceeds the ability of a joint to repair and maintain itself, predisposing the articular cartilage to premature degeneration. This review examines the available basic science, preclinical and clinical evidence regarding several such unfavorable biomechanical conditions about the knee: malalignment, loss of meniscal tissue, cartilage defects and joint instability or laxity.     

Metabolomic changes in fatty liver can be modified by dietary protein and calcium during energy restriction

AIM： To characterise the effect of energy restriction （ER） on liver lipid and primary metabolite profile by using metabolomic approach. We also investigated whether the effect of energy restriction can be further enhanced by modification of dietary protein source and calcium. METHODS： Liver metabolomic profile of lean and obese C57BI/6J mice （n = 10/group） were compared with two groups of weight-reduced mice. ER was performed on control diet and whey protein-based high-calcium diet （whey ＋ Ca）. The metabolomic analyses were performed using the UPLC/MS based lipidomic platform and the HPLC/MS/MS based primary metabolite platform.
RESULTS： ER on both diets significantly reduced hepatic lipid accumulation and lipid droplet size, while only whey ＋ Ca diet significantly decreased blood glucose （P 〈 0.001） and serum insulin （P 〈 0.01）. In hepatic lipid species the biggest reduction was in the level of triacylglycerols and cerarnides while the level of cholesterol esters was significantly increased during ER. Interestingly, diacylglycerol to phospholipid ratio, an indicator of relative amount of diabetogenic diglyceride species, was increased in the control ER group, but decreased in the whey ＋ Ca ER group （P 〈 0.001, vs obese）. ER on whey ＋ Ca diet also totally reversed the obesity induced increase in the relative level of lipotoxic cerarnides （P 〈 0.001, vs obese; P 〉 0.05, vs lean）. These changes were accompanied with up-regulated TCA cycle and pentose phosphate pathway rnetabolites.
CONCLUSION： ER-induced changes on hepatic rnetabolornic profile can be significantly affected by dietary protein source. The therapeutic potential of whey protein and calcium should be further studied.     

Prediction of non-alcoholic fatty-liver disease and liver fat content by serum molecular lipids

Aims/hypothesis

We examined whether analysis of lipids by ultra-performance liquid chromatography (UPLC) coupled to MS allows the development of a laboratory test for non-alcoholic fatty-liver disease (NAFLD), and how a lipid-profile biomarker compares with the prediction of NAFLD and liver-fat content based on routinely available clinical and laboratory data.

Methods

We analysed the concentrations of molecular lipids by UPLC-MS in blood samples of 679 well-characterised individuals in whom liver-fat content was measured using proton magnetic resonance spectroscopy (¹H-MRS) or liver biopsy. The participants were divided into biomarker-discovery (n?=?287) and validation (n?=?392) groups to build and validate the diagnostic models, respectively.

Results

Individuals with NAFLD had increased triacylglycerols with low carbon number and double-bond content while lysophosphatidylcholines and ether phospholipids were diminished in those with NAFLD. A serum-lipid signature comprising three molecular lipids (‘lipid triplet’) was developed to estimate the percentage of liver fat. It had a sensitivity of 69.1% and specificity of 73.8% when applied for diagnosis of NAFLD in the validation series. The usefulness of the lipid triplet was demonstrated in a weight-loss intervention study.

Conclusions/interpretation

The liver-fat-biomarker signature based on molecular lipids may provide a non-invasive tool to diagnose NAFLD, in addition to highlighting lipid molecular pathways involved in the disease.