The Effect of Short- and Long-Term Cryopreservation on Chicken Primordial Germ Cells.

Primordial germ cells (PGCs) are the precursors of functional gametes and the only cell type capable of transmitting genetic and epigenetic information from generation to generation. These cells offer valuable starting material for cell-based genetic engineering and genetic preservation, as well as epigenetic studies. While chicken PGCs have demonstrated resilience in maintaining their germness characteristics during both culturing and cryopreservation, their handling remains a complex challenge requiring further refinement. Herein, the study aimed to compare the effects of different conditions (freezing-thawing and in vitro cultivation) on the expression of PGC-specific marker genes. Embryonic blood containing circulating PGCs was isolated from purebred Green-legged Partridgelike chicken embryos at 14-16 Hamburger-Hamilton (HH) embryonic development stage. The blood was pooled separately for males and females following sex determination. The conditions applied to the blood containing PGCs were as follows: (1) fresh isolation; (2) cryopreservation for a short term (2 days); and (3) in vitro culture (3 months) with long-term cryopreservation of purified PGCs (~2 years). To characterize PGCs, RNA isolation was carried out, followed by quantitative reverse transcription polymerase chain reaction (RT-qPCR) to assess the expression levels of specific germ cell markers (SSEA1, CVH, and DAZL), as well as pluripotency markers (OCT4 and NANOG). The investigated genes exhibited consistent expression among PGCs maintained under diverse conditions, with no discernible differences observed between males and females. Notably, the analyzed markers demonstrated higher expression levels in PGCs when subjected to freezing than in their freshly isolated counterparts.

Characterization of alcohol-related seizures in withdrawal syndrome.

OBJECTIVE: Alcohol-related seizures (ARS) are one of the most important consequences of alcohol withdrawal syndrome (AWS). However, demographic and clinical characteristics, and furthermore, the relationship of ARS with delirium tremens (DT), have not yet been evaluated in detail. Therefore, the aim of the present study was to reveal the correlates of ARS and examine the interaction of ARS with the occurrence of DT and with the severity of AWS. METHODS: In the retrospective study (Study 1) 2851 medical charts of inpatient admissions characterized by AWS and DT were listed. Demographic and clinical variables of ARS were assessed. In the follow-up study (Study 2), patients admitted with AWS without (N = 28) and with (N = 18) ARS were enrolled. Study 1 was performed between 2008 and 2023, and Study 2 was performed in 2019 in Hungary. To determine the severity of AWS, the Clinical Institute Withdrawal Assessment Scale for Alcohol, Revised (CIWA-Ar) was used. ARS is a provoked, occasional seizure; therefore, patients with epilepsy syndrome were excluded from the two studies. Statistical analyses were performed by the means of chi-square tests, multinomial logistic regressions, mixed ANOVA, and derivation. RESULTS: The occurrence of DT, the history of ARS, and somatic co-morbidities were found to be risk factors for the appearance of ARS. ARS was proved to be a risk factor for the development of DT. In the follow-up study, there was no difference in the decrease of CIWA-Ar scores between the groups. SIGNIFICANCE: Our present findings support the likelihood of kindling, which is one of the most important mechanisms underlying the development of ARS, but do not directly prove its presence. Additionally, our results revealed that the severity of AWS is not influenced by the presence of ARS. PLAIN LANGUAGE SUMMARY: Provoked, occasional seizures during AWS are defined as ARS. In the present study, predictors and interactions of these seizures with DT-the most severe form of withdrawal-and with the severity of withdrawal were examined in retrospective and follow-up studies. The present study shows that a history of withdrawal seizures, the occurrence of DT, and somatic comorbidities are predictors of the development of seizures. Furthermore, our findings suggest that the presence of seizures does not influence the severity of withdrawal.

Unidirectional hybridization between American paddlefish Polyodon spathula (Walbaum, 1792) and sterlet Acipenser ruthenus (Linnaeus, 1758).

Interspecific hybridizations among sturgeon species are feasible and often bidirectional. The American paddlefish (Polyodon spathula) from Family Polyodontidae and sturgeon species from Family Acipenseridae were reported capable of hybridization, but viable hybrids have been described only in crosses with the American paddlefish as paternal parents. In the reciprocal cross, the hybrids were not viable however embryos start to develop and reach late gastrula and early neurula stages. The goal of this study was to examine the hybridization between the sterlet sturgeon (Acipenser ruthenus) and the American paddlefish. Hybrid and purebred crosses were produced by artificial fertilization. Viable hybrid offspring were harvested (three month old) and verified in the families produced by female sterlet crossing with male American paddlefish. In the reciprocal hybrid crosses with female American paddlefish and male sterlet, the embryos development did not pass over 120 h post fertilization, indicating the unidirectional hybridization between American paddlefish and sterlet. Chromosome counting showed for the same ploidy level of viable hybrid and parent species. Analysis of three microsatellite markers confirmed the unidirectional hybridization between the American paddlefish and the sterlet species. Overall, the inferred genetic cause suggests that unidirectional hybridization between American paddlefish and sterlet may be the case not only for these two species but likely also between American paddlefish and other sturgeon species.

The Impact of the COVID-19 Pandemic on the Severity of Alcohol Use Disorder: Significance of Dual Disorders.

The coronavirus disease 2019 (COVID-19) pandemic has been assumed to impact patients diagnosed with alcohol use disorder (AUD). The severity of the influence that the COVID-19 pandemic had on the symptoms of AUD has not yet been revealed in detail. The aim of this study was to examine the impact of the COVID-19 pandemic on patients diagnosed with AUD. This retrospective study was conducted between 11 March 2017 and 31 May 2022 in Hungary. Medical charts (N = 1082) of inpatients with the diagnosis of AUD were reviewed. Based on the dates of admissions, two groups were created: the 'before COVID-19' group (11 March 2017-10 March 2020) and the 'during COVID-19' group (11 March 2020-31 May 2022). Chi-square tests, independent-sample t-tests, and multinomial logistic regressions were performed. The occurrence of delirium tremens (DT) and psychiatric co-morbidities was significantly higher during the pandemic. Our results showed that the occurrence of DT and psychiatric co-morbidities significantly increased during the pandemic. Our results revealed that the pandemic enhanced the severe consequences of AUD, and the development of AUD might have increased in frequency among individuals previously diagnosed with mental illness during the pandemic. These findings indicate the significance of dual disorders in the post-pandemic period.

[Assessment of alcohol withdrawal syndrome: new perspectives]. / Az alkoholmegvonásos szindróma ellátása: új perspektívák.

Alcohol withdrawal syndrome is one of the most important consequences of alcohol use disorder, a complex neuropsychiatric disorder, which is firstly treated in non-specific and secondly in psychiatric/addictive in- or outpatient units. On the other hand, alcohol withdrawal syndrome is one of the most important outcomes of the severity of alcohol use disorder, further, it can lead to the development of alcohol-related seizure and delirium tremens. Hence, early recognition and optimal treatment of alcohol withdrawal syndrome have a critical importance. Therefore, the main goal of the present review was - by systematically summarizing the scientific data published during the past two decades - to form a unique diagnostic and therapeutic algorithm. During the recognition and the course of alcohol withdrawal syndrome, the Clinical Institute Withdrawal Assessment for Alcohol, Revised scale, while in the risk assessment the Prediction of Alcohol Withdrawal Severity Scale are the recommended psychometric tools. Benzodiazepines are the key elements of the pharmacotherapy of alcohol withdrawal syndrome. Many studies have evaluated that diazepam, chlordiazepoxide, lorazepam and oxazepam with distinct indications have sufficient evidence in the treatment of alcohol withdrawal syndrome. However, in the past few years some authors have recommended the importance of non-benzodiazepine medications. The efficacy of propofol, phenobarbital, carbamazepin, oxcarbamazepin and alpha-2 receptor agonists in the treatment of alcohol withdrawal syndrome have been revealed. Furthermore, it has been evaluated that benzodiazepines are recommended in the treatment of alcohol-related seizure and delirium tremens. In the present review, our aim was to construct a unique, up-to-date diagnostic and therapeutic algorithm by summarizing the related papers published during the past two decades. Hence this scheme may be useful in the optimal treatment of patients diagnosed with alcohol use disorder and it could help to conduct further clinical researches. Orv Hetil. 2023; 164(38): 1487-1496.

Pluripotency-Associated microRNAs in Early Vertebrate Embryos and Stem Cells.

MicroRNAs (miRNAs), small non-coding RNA molecules, regulate a wide range of critical biological processes, such as proliferation, cell cycle progression, differentiation, survival, and apoptosis, in many cell types. The regulatory functions of miRNAs in embryogenesis and stem cell properties have been extensively investigated since the early years of miRNA discovery. In this review, we will compare and discuss the impact of stem-cell-specific miRNA clusters on the maintenance and regulation of early embryonic development, pluripotency, and self-renewal of embryonic stem cells, particularly in vertebrates.

The Effects of Freezing Media on the Characteristics of Male and Female Chicken Primordial Germ Cell Lines.

Recently, in vitro gene preservation has gained ground thanks to its lower cost and higher stability compared to in vivo techniques. One of the methods that can preserve female-specific W chromosome-linked genes is primordial germ cell (PGC) freezing. PGCs can be isolated from Hamburger-Hamilton stage 14-16 embryos via blood sampling. In our experiment, we used two newly established Black Transylvanian naked neck chicken cell lines and four cell lines from our gene bank. We compared two different freezing media (FAM1 and FAM2) in this study. The cell number and viability of the PGCs were measured before freezing (BF) and after thawing on Day 0, Day 1, and Day 7 of cultivation. We analyzed the germ cell-specific chicken vasa homologue (CVH) expression profile in PGCs using RT-qPCR. We found that on Day 0, immediately after thawing, the cell number in cell lines frozen with the FAM2 medium was significantly higher than in the FAM1-treated ones. On Day 1 and Day 7, the cell number and viability were also higher in most cell lines frozen with FAM2, but the difference was insignificant. The freezing also affected the chicken vasa homologue gene expression in male lines treated with both freezing media.

Seasonality and Delirium Tremens in Hospitalized Patients with Alcohol Dependence Syndrome.

INTRODUCTION: Due to the high rate of mortality, recognizing the contributing factors of alcohol-related delirium tremens (DT), which is the most severe form of alcohol withdrawal state (AWS) is pivotal in clinical settings. Previous studies suggested relationship between seasonality and other types of delirium; however, to our knowledge, this is the first empirical study which examined the role of seasonality in DT in alcohol dependence syndrome (ADS). METHODS: A retrospective study was undertaken between 2008 and 2015; medical records of 1,591 patients were included, which yielded 2,900 hospital appearances. Three groups were formed based on the ICD-10 diagnoses: ADS, AWS, and DT. The characteristics of the groups were analysed with one-way ANOVA and χ2 tests. Multinomial logistic regression was used to explore the potential predictors of DT, including seasonality. RESULTS: The highest incidence of DT was in spring (36.8%; χ2 (3) = 27.666; p < 0.001), especially in March (13.9%; χ2 (11) = 33.168; p < 0.001). Spring, higher mean age, higher presence of comorbid somatic disorders, and lower occurrence of comorbid psychiatric disorders were significant predictive variables for DT with the control of socio-demographic and clinical variables. CONCLUSIONS: The present study revealed that spring, especially March is a critical period in temperate climate zone regarding DT. This can be interpreted as a late winter effect since the temperature is lower in this month compared to other spring months. Furthermore, higher age and the occurrence of comorbid somatic disorders can be considered as risk factors in case of DT. These results support the need of further clinical studies to better understand the impact of seasonality on DT.

Effects of Hydrostatic Pressure Treatment of Newly Fertilized Eggs on the Ploidy Level and Karyotype of Pikeperch Sander lucioperca (Linnaeus, 1758).

We studied the effect of different magnitudes (7000 PSI (48.26 MPa), 8000 PSI (55.16 MPa), and 9000 PSI (62.05 MPa)) of hydrostatic pressure on the ploidy of pikeperch larvae. Pressure shock was applied 5 min after the fertilization of eggs at a water temperature of 14.8 ± 1 °C. A 7000 PSI pressure shock was applied for 10 or 20 min, while 8000 and 9000 PSI treatments lasted for 10 min. Each treatment with its respective control was completed in triplicate, where different females' eggs served as a replicate. In the treatment groups exposed to 7000 PSI for 10 min, only diploid and triploid larvae were identified, while 2n/3n mosaic individuals were found after a 20-min exposure to a 7000 PSI pressure shock. The application of 8000 or 9000 PSI pressure shocks resulted in only triploid and mosaic individuals. Among larvae from eggs treated with 8000 PSI, three mosaic individuals with 2n/3n karyotype were identified (4.0 ± 6.9%), while a single (2.0 ± 3.5%) 1n/3n mosaic individual was found in the 9000 PSI-treated group. To our knowledge, this is the first report that demonstrates the induction of a haplo-triploid karyotype by hydrostatic pressure shock in teleost fish. The dominance of triploid individuals with a reasonable survival rate (36.8 ± 26.1%) after 8000 PSI shock supports the suitability of the hydrostatic pressure treatment of freshly fertilized eggs for triploid induction in pikeperch.

Successful cryopreservation and regeneration of a partridge colored Hungarian native chicken breed using primordial germ cells.

Primordial germ cells (PGCs) are the precursors of germline cells that generate sperm and ova in adults. Thus, they are promising tools for gene editing and genetic preservation, especially in avian species. In this study, we established stable male and female PGC lines from 6Hungarian indigenous chicken breeds with derivation rates ranging from 37.5 to 50 percent. We characterized the PGCs for expression of the germ cell-specific markers during prolonged culture in vitro. An in vivo colonization test was performed on PGCs from four Hungarian chicken breeds and the colonization rates were between 76 and 100%. Cryopreserved PGCs of the donor breed (Partridge color Hungarian) were injected into Black Transylvanian Naked Neck host embryos to form chimeric progeny that, after backcrossing, would permit reconstitution of the donor breed. For 24 presumptive chimeras 13 were male and 11 were female. In the course of backcrossing, 340 chicks were hatched and 17 of them (5%) were pure Partridge colored. Based on the backcrossing 1 hen and 3 roosters of the 24 presumptive chimeras (16.6%) have proven to be germline chimeras. Therefore, it was proven that the original breed can be recovered from primordial germ cells which are stored in the gene bank. To our knowledge, our study is a first that applied feeder free culturing conditions for both male and female cell lines successfully and used multiple indigenous chicken breeds to create a gene bank representing a region (Carpathian Basin).

Effect of Post-Hatch Heat-Treatment in Heat-Stressed Transylvanian Naked Neck Chicken.

Although numerous studies reported the effects of heat stress in chickens, it was not investigated in the Transylvanian Naked Neck breed. In our research, Transylvanian Naked Neck chickens, 24 h after hatching, were heat-treated at 38.5 °C for 12 h. We compared the control and heat-treated adult chickens' productivity parameters following 12 weeks of heat-stress at 30 °C. We found that the heat-treated layers had significantly higher egg production in heat stress, but in cockerels, the sperm quality did not differ significantly between the two groups. To detect the effect of heat-treatment on a molecular level, the expression of two heat-shock proteins and four heat-shock factors were analysed in the gonads of control and heat-treated chickens. We found that the expression level of HSP90 and HSF4 increased significantly in heat-treated female chicken gonads. Still, in adult females, the expression of HSF2 and HSF3 were substantially lower compared to the control. In adult heat-treated males, the HSP70, HSF1 and HSF3 expression levels showed a significant increase in both gonads compared to the control. We think that the presented significant differences in egg production might be related to the increased expression level of HSP90 and HSF4 in heat-treated female gonads.

Evaluation of the course and treatment of Alcohol Withdrawal Syndrome with the Clinical Institute Withdrawal Assessment for Alcohol - Revised: A systematic review-based meta-analysis.

BACKGROUND: Although the Clinical Institute Withdrawal Assessment for Alcohol - Revised (CIWA-Ar) is a gold standard tool for the clinical evaluation of alcohol withdrawal syndrome (AWS), a systematic analysis using the total scores of the CIWA-Ar as a means of an objective follow-up of the course and treatment of AWS is missing. The aims of the present study were to systematically evaluate scientific data using the CIWA-Ar, to reveal whether the aggregated CIWA-Ar total scores follow the course of AWS and to compare benzodiazepine (BZD) and non-benzodiazepine (nBZD) therapies in AWS. METHODS: 1054 findings were identified with the keyword "ciwa" from four databases (PubMed, ScienceDirect, Web of Science, Cochrane Registry). Articles using CIWA-Ar in patients treated with AWS were incorporated and two measurement intervals (cumulative mean data of day 1-3 and day 4-9) of the CIWA-Ar total scores were compared. Subgroup analysis based on pharmacotherapy regimen was conducted to compare the effectiveness of BZD and nBZD treatments. RESULTS: The random effects analysis of 423 patients showed decreased CIWA-Ar scores between the two measurement intervals (BZD: d = -1.361; CI: -1.829 < δ < -0.893; nBZD: d = -0.858; CI: -1.073 < δ < -0.643). Sampling variances were calculated for the BZD (v1 = 0.215) and the nBZD (v2 = 0.106) groups, which indicated no significant group difference (z = -1.532). CONCLUSIONS: Our findings support that the CIWA-Ar follows the course of AWS. Furthermore, nBZD therapy has a similar effectiveness compared to BZD treatment based on the CIWA-Ar total scores.

Effect of miR-302b MicroRNA Inhibition on Chicken Primordial Germ Cell Proliferation and Apoptosis Rate.

The primordial germ cells (PGCs) are the precursors for both the oocytes and spermatogonia. Recently, a novel culture system was established for chicken PGCs, isolated from embryonic blood. The possibility of PGC long-term cultivation issues a new advance in germ cell preservation, biotechnology, and cell biology. We investigated the consequence of gga-miR-302b-5P (5P), gga-miR-302b-3P (3P) and dual inhibition (5P/3P) in two male and two female chicken PGC lines. In treated and control cell cultures, the cell number was calculated every four hours for three days by the XLS Imaging system. Comparing the cell number of control and treated lines on the first day, we found that male lines had a higher proliferation rate independently from the treatments. Compared to the untreated ones, the proliferation rate and the number of apoptotic cells were considerably reduced at gga-miR-302b-5P inhibition in all PGC lines on the third day of the cultivation. The control PGC lines showed a significantly higher proliferation rate than 3P inhibited lines on Day 3 in all PGC lines. Dual inhibition of gga-miR-302b mature miRNAs caused a slight reduction in proliferation rate, but the number of apoptotic cells increased dramatically. The information gathered by examining the factors affecting cell proliferation of PGCs can lead to new data in stem cell biology.

Modulation of Sensory Nerve Function by Insulin: Possible Relevance to Pain, Inflammation and Axon Growth.

Insulin, besides its pivotal role in energy metabolism, may also modulate neuronal processes through acting on insulin receptors (InsRs) expressed by neurons of both the central and the peripheral nervous system. Recently, the distribution and functional significance of InsRs localized on a subset of multifunctional primary sensory neurons (PSNs) have been revealed. Systematic investigations into the cellular electrophysiology, neurochemistry and morphological traits of InsR-expressing PSNs indicated complex functional interactions among specific ion channels, proteins and neuropeptides localized in these neurons. Quantitative immunohistochemical studies have revealed disparate localization of the InsRs in somatic and visceral PSNs with a dominance of InsR-positive neurons innervating visceral organs. These findings suggested that visceral spinal PSNs involved in nociceptive and inflammatory processes are more prone to the modulatory effects of insulin than somatic PSNs. Co-localization of the InsR and transient receptor potential vanilloid 1 (TRPV1) receptor with vasoactive neuropeptides calcitonin gene-related peptide and substance P bears of crucial importance in the pathogenesis of inflammatory pathologies affecting visceral organs, such as the pancreas and the urinary bladder. Recent studies have also revealed significant novel aspects of the neurotrophic propensities of insulin with respect to axonal growth, development and regeneration.

The effect of dual inhibition of Ras-MEK-ERK and GSK3ß pathways on development of in vitro cultured rabbit embryos.

Dual inhibition (2i) of Ras-MEK-ERK and GSK3ß pathways enables the derivation of embryo stem cells (ESCs) from refractory mouse strains and, for permissive strains, allows ESC derivation with no external protein factor stimuli involvement. In addition, blocking of ERK signalling in 8-cell-stage mouse embryos leads to ablation of GATA4/6 expression in hypoblasts, suggesting fibroblast growth factor (FGF) dependence of hypoblast formation in the mouse. In human, bovine or porcine embryos, the hypoblast remains unaffected or displays slight-to-moderate reduction in cell number. In this study, we demonstrated that segregation of the hypoblast and the epiblast in rabbit embryos is FGF independent and 2i treatment elicits only a limited reinforcement in favour of OCT4-positive epiblast populations against the GATA4-/6-positive hypoblast population. It has been previously shown that TGFß/Activin A inhibition overcomes the pervasive differentiation and inhomogeneity of rat iPSCs, rat ESCs and human iPSCs while prompting them to acquire naïve properties. However, TGFß/Activin A inhibition, alone or together with Rho-associated, coiled-coil containing protein kinase (ROCK) inhibition, was not compatible with the viability of rabbit embryos according to the ultrastructural analysis of preimplantation rabbit embryos by electron microscopy. In rabbit models ovulation upon mating allows the precise timing of progression of the pregnancy. It produces several embryos of the desired stage in one pregnancy and a relatively short gestation period, making the rabbit embryo a suitable model to discover the cellular functions and mechanisms of maintenance of pluripotency in embryonic cells and the embryo-derived stem cells of other mammals.

Investigation of the Guinea fowl and domestic fowl hybrids as potential surrogate hosts for avian cryopreservation programmes.

In the last decade, avian gene preservation research has focused on the use of the early precursors of the reproductive cells, the primordial germ cells (PGCs). This is because avian PGCs have a unique migration route through the vascular system which offers easy accessibility. Furthermore, culturing of the cells in vitro, freezing/thawing, reintegration into a recipient embryo and the development of the germ cells can be carried out in well-defined laboratory circumstances. The efficient recovery of the donor genotype and the frequency of germline transmission from the surrogate host animals are still areas which need further development. Thus, the aim of the present study was to investigate an infertile interspecific hybrid (recipient) as an appropriate host for primordial germ cells from native poultry breeds. Guinea fowl × chicken hybrids were produced, the crossing was repeated inversely. The phenotype, the hatching time, the hatching rate, the sex ratio, the presence of own germ cells, the fertility and the phenotype of viable hybrids and the incidence of chromosomal abnormalities of dead hybrid embryos were described. 6.65% viable offspring was obtained with crossing of Guinea fowl females with domestic fowl males. Crossing of domestic fowl hens with Guinea fowl male resulted in lower fertility, 0.14% viable offspring. Based on the investigations, the observed offspring from the successful crossing were sterile male hybrids, thus an extreme form of Haldane's rule was manifested. The sterile hybrid male embryos were tested by injecting fluorescently labeled chicken PGCs. The integration rate of labeled PGCs was measured in 7.5-day, 14.5-day and 18.5-day old embryonic gonads. 50%, 5.3% and 2.4% of the injected hybrid embryos survived and 40%, 5.3% and 2.4% of the examined gonads contained fluorescent labeled donor PGCs. Therefore, these sterile hybrid males may be suitable recipients for male PGCs and possibly for female PGCs although with lower efficiency. This research work shows that the sterility of hybrids can be used in gene conservation to be a universal host for PGCs of different avian species.

[The first step towards a unified approach: validation of the Hungarian version of the Clinical Institute Withdrawal Assessment of Alcohol, Revised in Hungarian general hospital settings]. / Elso lépés egy egységes szemlélet felé: az Alkohol Megvonási Skála bevezetése a hazai betegellátási gyakorlatba.

Introduction and aim: The available literature and protocols have unequivocally suggested that the Clinical Institute Withdrawal Assessment of Alcohol, Revised is a psychometric scale for identifying and following the signs of alcohol withdrawal. However, there has not been any validated tool for the identification of withdrawal symptoms in Hungarian general hospital settings. The aim of the present study was to evaluate the validity and the reliability of the Hungarian version of this scale among patients hospitalized with alcohol withdrawal syndrome. Method: The translation of the scale into Hungarian was done by 'back translation' method, followed by testing the face validity. The empirical phase was performed in the Department of Psychiatry, University of Szeged. Patients admitted with alcohol withdrawal syndrome (n = 30) were recruited from the inpatient units of the clinic. Clinical Institute Withdrawal Assessment of Alcohol, Revised and Clinical Global Impression - Severity Scale were recorded every two days. Statistical comparisons of data were performed with repeated-measures ANOVA. Cronbach's alpha, item-total correlation, convergent and discriminant validity were determined. Results: Significant decrease of the total scores of Clinical Institute Withdrawal Assessment of Alcohol, Revised and Clinical Global Impression - Severity Scale was observed between the six measurements (F = 202.46, p<0.001; F = 503.04, p<0.001). Cronbach alpha values were above 0.7 during the first 3 measurement days. The withdrawal and severity scores recorded the same day showed positively significant correlations (>0.45). Conclusion: Our findings demonstrate that the Clinical Institute Withdrawal Assessment of Alcohol, Revised is a reliable and valid psychometric tool for the detailed analysis of withdrawal symptoms in Hungarian general hospital settings. Orv Hetil. 2019; 160(30): 1184-1192.

Enhancement of chicken primordial germ cell in vitro maintenance using an automated cell image analyser.

Primordial germ cells (PGCs) were isolated from blood samples of chicken embryos. We established four PGC lines: two males (FS-ZZ-101, GFP-ZZ-4ZP) and two females (FS-ZW-111, GFP-ZW-5ZP). We could not detect a significant difference in the marker expression profile, but there was a remarkable difference between the proliferation rates of these PGC lines. We monitored the number of PGCs throughout a three-day period using a high-content screening cell imaging and analysing system (HCS). We compared three different initial cell concentrations in the wells: ~1000 cells (1×, ~4000 (4× and ~8000 (8×. For the GFPZW- 5ZP, FS-ZZ-101 and FS-ZW-111 PGC lines the lowest doubling time was observed at 4× concentration, while for GFP-ZZ-4ZP we found the lowest doubling time at 1× concentration. At 8× initial concentration, the growth rate was high during the first two days for all cell lines, but this was followed by the appearance of cell aggregates decreasing the cell growth rate. We could conclude that the difference in proliferation rate could mainly be attributed to genotypic variation in the established PGC lines, but external factors such as cell concentration and quality of the culture medium also affect the growth rate of PGCs.

Insulin Confers Differing Effects on Neurite Outgrowth in Separate Populations of Cultured Dorsal Root Ganglion Neurons: The Role of the Insulin Receptor.

Apart from its pivotal role in the regulation of carbohydrate metabolism, insulin exerts important neurotrophic and neuromodulator effects on dorsal root ganglion (DRG) neurons. The neurite outgrowth-promoting effect is one of the salient features of insulin's action on cultured DRG neurons. Although it has been established that a significant population of DRG neurons express the insulin receptor (InsR), the significance of InsR expression and the chemical phenotype of DRG neurons in relation to the neurite outgrowth-promoting effect of insulin has not been studied. Therefore, in this study by using immunohistochemical and quantitative stereological methods we evaluated the effect of insulin on neurite outgrowth of DRG neurons of different chemical phenotypes which express or lack the InsR. Insulin, at a concentration of 10 nM, significantly increased total neurite length, the length of the longest neurite and the number of branch points of cultured DRG neurons as compared to neurons cultured in control medium or in the presence of 1 µM insulin. In both the control and the insulin exposed cultures, ∼43% of neurons displayed InsR-immunoreactivity. The proportions of transient receptor potential vanilloid type 1 receptor (TRPV1)-immunoreactive (IR), calcitonin gene-related peptide (CGRP)-IR and Bandeiraea simplicifolia isolectin B4 (IB4)-binding neurons amounted to ∼61%, ∼57%, and ∼31% of DRG neurons IR for the InsR. Of the IB4-positive population only neurons expressing the InsR were responsive to insulin. In contrast, TRPV1-IR nociceptive and CGRP-IR peptidergic neurons showed increased tendency for neurite outgrowth which was further enhanced by insulin. However, the responsiveness of DRG neurons expressing the InsR was superior to populations of DRG neurons which lack this receptor. The findings also revealed that besides the expression of the InsR, inherent properties of peptidergic, but not non-peptidergic nociceptive neurons may also significantly contribute to the mechanisms of neurite outgrowth of DRG neurons. These observations suggest distinct regenerative propensity for differing populations of DRG neurons which is significantly affected through insulin receptor signaling.

Comparison of the MicroRNA Expression Profiles of Male and Female Avian Primordial Germ Cell Lines.

Primordial germ cells (PGCs) are the precursors of adult germ cells, and among the embryonic stem-like cells in the bird embryo, only they can transmit the genetic information to the next generation. Despite the wide range of applications, very little is known about the mechanism that governs primordial germ cell self-renewal and differentiation. As a first step, we compared 12 newly established chicken PGC lines derived from two different chicken breeds, performing CCK-8 proliferation assay. All of the lines were derived from individual embryos. A significant difference was found among the lines. As microRNAs have been proved to play a key role in the maintenance of pluripotency and the cell cycle regulation of stem cells, we continued with a complex miRNA analysis. We could discover miRNAs expressing differently in PGC lines with high proliferation rate, compared to PGC lines with low proliferation rate. We found that gga-miR-2127 expresses differently in female and male cell lines. The microarray analysis also revealed high expression level of the gga-miR-302b-3p strand (member of the miR-302/367 cluster) in slowly proliferating PGC lines compared to the gga-miR-302b-5p strand. We confirmed that the inhibition of miR-302b-5p significantly increases the doubling time of the examined PGC lines. In conclusion, we found that gga-miR-181-5p, gga-miR-2127, and members of the gga-miR-302/367 cluster have a dominant role in the regulation of avian primordial germ cell proliferation.