Validation of a male-specific DNA marker confirms XX/XY-type sex determination in several Hungarian strains of African catfish (Clarias gariepinus).

African catfish (Clarias gariepinus) is a promising food fish species with significant potential and growing mass of production in freshwater aquaculture. Male African catfish possess improved production characteristics over females, therefore the use of monosex populations could be advantageous for aquaculture production. However, our knowledge about the sex determination mechanism of this species is still limited and controversial. A previously isolated male-specific DNA marker (CgaY1) was validated using offspring groups from targeted crosses (n = 630) and it was found to predict the sex of 608 individuals correctly (96.43% accuracy). Using the proportion of recombinants, we estimated the average genetic distance between the potential sex determination locus and the sex-specific marker to be 3.57 cM. As an earlier study suggested that both XX/XY and ZZ/ZW systems coexist in this species, we tested the applicability of their putative 'moderately sex-linked loci' and found that no sex-specific amplification could be detected for any of them. In addition, temperature-induced masculinization suggested by others was also tested, but no such effect was detected in our stocks when the published parameters were used for heat treatment. Altogether, our results support an exclusive XX/XY sex determination system in our African catfish stock and indicate a good potential for the future use of this male-specific DNA marker in research and commercial production.

The Role of Silence in Verbal Fluency Tasks - A New Approach for the Detection of Mild Cognitive Impairment.

OBJECTIVE: Most recordings of verbal fluency tasks include substantial amounts of task-irrelevant content that could provide clinically valuable information for the detection of mild cognitive impairment (MCI). We developed a method for the analysis of verbal fluency, focusing not on the task-relevant words but on the silent segments, the hesitations, and the irrelevant utterances found in the voice recordings. METHODS: Phonemic ('k', 't', 'a') and semantic (animals, food items, actions) verbal fluency data were collected from healthy control (HC; n = 25; Mage = 67.32) and MCI (n = 25; Mage = 71.72) participants. After manual annotation of the voice samples, 10 temporal parameters were computed based on the silent and the task-irrelevant segments. Traditional fluency measures, based on word count (correct words, errors, repetitions) were also employed in order to compare the outcome of the two methods. RESULTS: Two silence-based parameters (the number of silent pauses and the average length of silent pauses) and the average word transition time differed significantly between the two groups in the case of all three semantic fluency tasks. Subsequent receiver operating characteristic (ROC) analysis showed that these three temporal parameters had classification abilities similar to the traditional measure of counting correct words. CONCLUSION: In our approach for verbal fluency analysis, silence-related parameters displayed classification ability similar to the most widely used traditional fluency measure. Based on these results, an automated tool using voiced-unvoiced segmentation may be developed enabling swift and cost-effective verbal fluency-based MCI screening.

Temporal Speech Parameters Detect Mild Cognitive Impairment in Different Languages: Validation and Comparison of the Speech-GAP Test® in English and Hungarian.

BACKGROUND: The development of automatic speech recognition (ASR) technology allows the analysis of temporal (time-based) speech parameters characteristic of mild cognitive impairment (MCI). However, no information has been available on whether the analysis of spontaneous speech can be used with the same efficiency in different language environments. OBJECTIVE: The main goal of this international pilot study is to address the question of whether the Speech-Gap Test® (S-GAP Test®), previously tested in the Hungarian language, is appropriate for and applicable to the recognition of MCI in other languages such as English. METHODS: After an initial screening of 88 individuals, English-speaking (n = 33) and Hungarianspeaking (n = 33) participants were classified as having MCI or as healthy controls (HC) based on Petersen's criteria. The speech of each participant was recorded via a spontaneous speech task. Fifteen temporal parameters were determined and calculated through ASR. RESULTS: Seven temporal parameters in the English-speaking sample and 5 in the Hungarian-speaking sample showed significant differences between the MCI and the HC groups. Receiver operating characteristics (ROC) analysis clearly distinguished the English-speaking MCI cases from the HC group based on speech tempo and articulation tempo with 100% sensitivity, and on three more temporal parameters with high sensitivity (85.7%). In the Hungarian-speaking sample, the ROC analysis showed similar sensitivity rates (92.3%). CONCLUSION: The results of this study in different native-speaking populations suggest that changes in acoustic parameters detected by the S-GAP Test® might be present across different languages.

Temporal Speech Parameters Indicate Early Cognitive Decline in Elderly Patients With Type 2 Diabetes Mellitus.

INTRODUCTION: The earliest signs of cognitive decline include deficits in temporal (time-based) speech characteristics. Type 2 diabetes mellitus (T2DM) patients are more prone to mild cognitive impairment (MCI). The aim of this study was to compare the temporal speech characteristics of elderly (above 50 y) T2DM patients with age-matched nondiabetic subjects. MATERIALS AND METHODS: A total of 160 individuals were screened, 100 of whom were eligible (T2DM: n=51; nondiabetic: n=49). Participants were classified either as having healthy cognition (HC) or showing signs of MCI. Speech recordings were collected through a phone call. Based on automatic speech recognition, 15 temporal parameters were calculated. RESULTS: The HC with T2DM group showed significantly shorter utterance length, higher duration rate of silent pause and total pause, and higher average duration of silent pause and total pause compared with the HC without T2DM group. Regarding the MCI participants, parameters were similar between the T2DM and the nondiabetic subgroups. CONCLUSIONS: Temporal speech characteristics of T2DM patients showed early signs of altered cognitive functioning, whereas neuropsychological tests did not detect deterioration. This method is useful for identifying the T2DM patients most at risk for manifest MCI, and could serve as a remote cognitive screening tool.

Mitochondrial sequence diversity reveals the hybrid origin of invasive gibel carp (Carassius gibelio) populations in Hungary.

BACKGROUND: Invasive gibel carp, Carassius gibelio (Bloch, 1782) has become well-established in the Hungarian waters and now are spreading in the European waters. On major concern now is the potential hybridization between gibel carp and the other invasive species in the Carassius auratus complex (CAC), which may further accelerate the spread of the whole invasive species complex. The identification of gibel carp and their hybrids is difficult because of its morphological similarity to the other species in CAC. Here we carry out a genomic assessment to understand the history of gibel carp invasion and its phylogenetic relationship with the other species in CAC. Three loci of the mitochondrial genome (D-loop, CoI, Cytb) were used to determine the phylogenetic origin of individuals and relarionship among six gibel carp populations and the other species in the CAC. METHODOLGY: A total of 132 gibel carp samples from six locations in Southern Transdanubia (Hungary) were collected after phenotypic identification to measure the genetic diversity within and among gibel carp populations of Southern Transdanubia (Hungary). The genetic background was examined by the sequences of the mitochondrial genome: D-loop, Cytochrome c oxidase I (CoI) and Cytochrome b (Cytb). Mitochondrial genetic markers are excellent tools for phylogenetic studies because they are maternally inherited. Successfully identified haplotypes were aligned and with reference sequences in nucleotide databases (i.e., NCBI-BLAST: National Centre for Biotechnology Information and BOLD: Barcode of Life Data System). The phylogenetic relationships among gibel carp populations were then analyzed together with the reference sequences to understand the relationship and the level of hybridization with the species in CAC. RESULTS: Among the 132 aligned D-loop sequences 22 haplotypes were identified. Further examination of representative individuals of the 22 haplotypes, six Cytb and four CoI sequences were detected. The largest number of haplotypes of all three loci were found in Lake Balaton, the largest shallow lake in Central Europe. Based on the NCBI-BLAST alignment of the D-loop, haplotypes of Carassius auratus auratus and Carassius a. buergeri in CAC were identified in the C. gibelio samples. Further analysis of haplotypes with the other two mitochondrial markers confirmed the occurrence of intragenus hybridization of C. gibelio in the Hungarian waters. CONCLUSION: By using three mitochondrial markers (D-loop, Cytb, CoI), we genomically characterized a gibel carp-complex in Hungarian waters and assessed the C. gibelio phylogenetic status between them. Hybrid origin of locally invasive Carassius taxon was detected in Hungary. It points out that invasive species are not only present in Hungary but reproduce with each other in the waters, further accelerating their spread.

Dementia in Hungary: General practitioners' routines and perspectives regarding early recognition.

Background: Undetected dementia in primary care is a global problem. Since general practitioners (GPs) act as the first step in the identification process, examining their routines could help us to enhance the currently low recognition rates.Objectives: The study aimed to explore, for the first time in Hungary, the dementia identification practices and views of GPs.Methods: In the context of an extensive, national survey (February-November 2014) 8% of all practicing GPs in Hungary (n = 402) filled in a self-administered questionnaire. The questions (single, multiple-choice, Likert-type) analysed in the present study explored GPs' methods and views regarding dementia identification and their ideas about the optimal circumstances of case-finding.Results: The vast majority of responding GPs (97%) agreed that the early recognition of dementia would enhance both the patients' and their relatives' well-being. When examining the possibility of dementia, most GPs (91%) relied on asking the patients general questions and only a quarter of them (24%) used formal tests, even though they were mostly satisfied with both the Clock Drawing Test (69%) and the Mini-Mental State Examination (65%). Longer consultation time was chosen as the most important facet of improvement needed for better identification of dementia in primary care (81%). Half of the GPs (49%) estimated dementia recognition rate to be lower than 30% in their practice.Conclusions: Hungarian GPs were aware of the benefits of early recognition, but the shortage of consultation time in primary care was found to be a major constraint on efficient case-finding.

Analytical characterization of human milk oligosaccharides - potential applications in pharmaceutical analysis.

Human breast milk is the gold standard for infant feeding and the best possible nourishment a new-born could have. Breastfeeding is the natural way to provide optimal nutritional, immunological and emotional nurturing for the healthy growth and development of infants. Human milk is a complex and dynamic biofluid comprised of many hundreds to thousands of distinct bioactive structures, among which one of the most abundant substances are the non-conjugated complex carbohydrates referred to as human milk oligosaccharides (HMOs). Due to their structural diversity and abundance, HMOs possess many beneficial biological functions. In order to understand human milk composition and HMO functions, state-of-the-art glycomic methods are inevitable. The industrial, large scale chemoenzymatic production of the most abundant HMOs became a reality in the last years and it evokes the need for straightforward and genuine analytical procedures to monitor the synthetic process and the quality of the products. It is obvious, that HMOs represent the next breakthrough in infant nutrition, as the addition of HMOs (such as 2'-fucosyllactose or lacto-N-neotetraose) to infant- and follow-on formulas, processed cereal-based food and baby foods for infants and young children etc. will revolutionize this field. This review highlights the potential applications of HMOs in the (bio)pharmaceutical industry, also summarizes the analytical methods available for the characterization of HMOs. An overview of the structure and function of HMOs along with their determination methods in complex matrices are provided. Various separation methods including liquid- and gas chromatography and capillary electrophoresis for the characterization and novel approaches for the quantitation of HMOs are discussed.

Qualitative and quantitative analysis of N-acetyllactosamine and lacto-N-biose, the two major building blocks of human milk oligosaccharides in human milk samples by high-performance liquid chromatography-tandem mass spectrometry using a porous graphitic carbon column.

This study presents a validated, porous graphitic carbon stationary phase-based LC-MS/MS method for the identification and quantification of lacto-N-biose (LNB) and N-acetyllactosamine (LacNAc). These compounds are the major building blocks of human milk oligosaccharides, however the presence of their unbound form in human milk has not been examined so far. The separation of these highly related structures in their alditol form was accomplished by a gradient LC method and multiple reaction monitoring (MRM) analysis after appropriate sample preparation including size-exclusion chromatography and solid-phase extraction. Baseline separation of the components provides the selectivity for the method. Validation was performed according to the European Medicines Agency (EMA) Guidelines and the method was found to be precise and accurate. Using our developed and validated method we were able to identify and quantify both saccharides in human milk for the first time. Based on our results the LacNAc concentration is in the range of 6.7-31µg/mL while LNB concentration decreased from 26µg/mL below the detection limit during the first week of lactation. The presence of LNB and LacNAc in human milk also implies new biological functions which can lead us closer to the understanding of the various functions of this complex biofluid.

Determination and quantification of 2'-O-fucosyllactose and 3-O-fucosyllactose in human milk by GC-MS as O-trimethylsilyl-oxime derivatives.

Human milk oligosaccharides possess various biological functions by protecting the infant from several bacterial and viral infections, modulating the immune system, serving as prebiotics and also contributing to the brain development. Hence, huge effort is underway by manufacturers to produce infant formulas enriched with human milk oligosaccharides which could mimic its diverse biological role the most. For this purpose, quantification of the natural oligosaccharide composition of the human milk is a key task. This study reports a fit for purpose GC-MS method for the quantification of the TMS ether oxime derivatives of 2'-O-fucosyllactose and 3-O-fucosyllactose, the two most abundant trisaccharides in human milk. The EI fragmentation pattern of the linkage isomers is discussed in details, focusing also on specific fragment ions. The GC-MS method with external standard calibration was applied for the monitoring of concentration changes of the trisaccharides throughout the first week of lactation in human milks samples collected from two volunteers. The results showed high concentration of both 2'-FL (4525-6266µg/mL in donor A and 2694-3551µg/mL in donor B) and 3-FL (271-441µg/mL in donor A and 99-208µg/mL in donor B), while no significant change has been observed throughout the one-week lactation period. The presented GC-MS method can serve as a quality control technique for the infant formulas and also offers an alternative to existing chromatographic methods to investigate HMOs in milk samples.

Permeability test for transdermal and local therapeutic patches using Skin PAMPA method.

Using the skin as absorption site presents unique advantages that have facilitated the progression of transdermal drug delivery in the past decades. Efforts in drug research have been devoted to find a quick and reproducible model for predicting the skin permeation of molecules. The Parallel Artificial Membrane Permeability Assay (PAMPA) has been extended for prediction of transdermal permeation by developing a model with completely artificial membrane, which can mimic the permeation through the stratum corneum. The present study aims to extend the Skin PAMPA method for testing transdermal and local therapeutic patches. The original method was modified and seven commercially available transdermal and local therapeutic patches with four different active pharmaceutical ingredients (nicotine, fentanyl, rivastigmine and ketoprofen) were studied. Data were compared to the declared delivery rates that are indicated by the manufacturers. Ex vivo permeation study was also performed in order to compare the permeated amount of the released drugs obtained by the two methods. The flux across the artificial membrane as well as the human skin (ex vivo) has been calculated and compared to the in vivo flux deduced from the labelled delivery rate and the active area of the patches. The results suggest that Skin PAMPA system can serve as a useful tool for evaluation and classification of the transdermal patches.

Design and evaluation of cyclodextrin-based delivery systems to incorporate poorly soluble curcumin analogs for the treatment of melanoma.

Various analogs of curcumin show high in vitro cytotoxic activity and are potential candidates for treating a deadly skin disease, melanoma. Due to the low solubility of the drugs, a new delivery agent, namely a cationic gemini surfactant-conjugated ß-cyclodextrin, was designed to incorporate novel drug candidates of the 1,5-diaryl-3-oxo-1,4-pentadienyl family. Based on physicochemical parameters, such as particle size and zeta potential, a schematic model for the potential interaction of the drug with the delivery agent was developed. The drug formulations were highly efficient in inhibiting the growth of melanoma cells, with IC(50) values significantly lower than melphalan, the drug currently used for the treatment of in-transit melanoma. CDgemini formulations showed excellent cellular selectivity, triggering apoptosis in the A375 cell line while showing no cytotoxicity to healthy human epidermal keratinocytes. The goal is to develop this novel nanoparticle approach into a non-invasive therapy for in-transit melanoma metastasis that lacks adequate treatment to date.