A versatile transposon-based technology to generate loss- and gain-of-function phenotypes in the mouse liver.

BACKGROUND: Understanding the contribution of gene function in distinct organ systems to the pathogenesis of human diseases in biomedical research requires modifying gene expression through the generation of gain- and loss-of-function phenotypes in model organisms, for instance, the mouse. However, methods to modify both germline and somatic genomes have important limitations that prevent easy, strong, and stable expression of transgenes. For instance, while the liver is remarkably easy to target, nucleic acids introduced to modify the genome of hepatocytes are rapidly lost, or the transgene expression they mediate becomes inhibited due to the action of effector pathways for the elimination of exogenous DNA. Novel methods are required to overcome these challenges, and here we develop a somatic gene delivery technology enabling long-lasting high-level transgene expression in the entire hepatocyte population of mice. RESULTS: We exploit the fumarylacetoacetate hydrolase (Fah) gene correction-induced regeneration in Fah-deficient livers, to demonstrate that such approach stabilizes luciferase expression more than 5000-fold above the level detected in WT animals, following plasmid DNA introduction complemented by transposon-mediated chromosomal gene transfer. Building on this advancement, we created a versatile technology platform for performing gene function analysis in vivo in the mouse liver. Our technology allows the tag-free expression of proteins of interest and silencing of any arbitrary gene in the mouse genome. This was achieved by applying the HADHA/B endogenous bidirectional promoter capable of driving well-balanced bidirectional expression and by optimizing in vivo intronic artificial microRNA-based gene silencing. We demonstrated the particular usefulness of the technology in cancer research by creating a p53-silenced and hRas G12V-overexpressing tumor model. CONCLUSIONS: We developed a versatile technology platform for in vivo somatic genome editing in the mouse liver, which meets multiple requirements for long-lasting high-level transgene expression. We believe that this technology will contribute to the development of a more accurate new generation of tools for gene function analysis in mice.

High average daily temperature in summer and the incidence of thrombolytic treatment for acute ischemic stroke.

INTRODUCTION: Meteorological factors can increase stroke risk; however, their impact is not precisely understood. Heat waves during summer increase total mortality. Therefore, we hypothesized that the average daily temperature in summer may correlate with the incidence of thrombolytic treatment for acute ischemic stroke in Budapest and Pest County, Hungary. METHODS: We analyzed the relationship between the average daily temperature in summer months and the daily number of thrombolytic treatments (TT) performed with the indication of acute ischemic stroke between 1st June and 31st August each year from 2007 to 2016. The analysis was also performed after the omission of the data of the last day of the months due to possible psychosocial impact reported in our previous study. Spearman's correlation was used for statistical analysis. RESULTS: No significant correlation was found between the average summer daily temperature and the number of TT in the entire sample of the 10-year period. When omitting the data of the last day of each month, positive correlations were suspected in 2014 (r=0.225, P=0.034) and 2015 (r=0.276, P=0.009). CONCLUSION: Our findings did not confirm an association between the average daily temperature in summer and the daily number of TT throughout the examined 10-year period. However, importantly, in 2014 and 2015, the years with the highest average daily temperatures in this period, a positive correlation was found. The level of correlation is modest, indicating that risk factors, both meteorological and non-meteorological, other than the average temperature, play equally important roles in determining the incidence of thrombolytic treatment for acute ischemic stroke on the population level.

[The new target population of stroke awareness campaign: Kindergarten students]. / "Stroke-ovi". A lakossági felvilágosítás új célközönsége: az óvodások.

Background and purpose:

 Thrombolysis and/or thrombectomy have been proven effective in the treatment of acute ischemic stroke. Due to the narrow time window, the number of patients suitable for these treatments is low. The main limitation is the pre-hospital stage, few people call an ambulance in time. The delay may be caused by the population’s insufficient health knowledge, but also by the loneliness and isolation of the population most prone to stroke. Among the latter, there are many grandparents who spend considerable time with their grandchildren. This gave rise to the idea of educating even younger children about the symptoms of a stroke, enabling them to call an ambulance if necessary. To this end, we adapted the Angels Initiative project previously tested in Greece. The Hungarian pilot study Budapest District XII. took place in district kindergartens. The Angels’ original role-playing program could not be implemented due to the COVID epidemic, so the necessity called for a new, Hungarian version: the online “Stroke Ovi” program. We introduced this in several stages, and in the third we also carried out an impact study.

 We adapted the Angels Initiative’s international program and its Hungarian translation to our program. We prepared the original, live role-playing form, with a parent meeting in the selected “test kindergarten”. Due to the uncertainly lingering impact of the COVID epidemic, we reevaluated our plan, using the Hungarian storybook and take-home workbook created in the meantime, we developed our own online version in several kindergartens in Budapest. We held 10 and then 25 minute sessions a week for 5 weeks. In the third educational cycle, which always targets new groups, we already examined the impact of the program by taking pre- and post-tests, in which not only the children but also their parents participated. In addition to neurologists and kindergarten teachers, we also included psychologists and speech therapists in our work, because we believed that in a social environment that includes parents and children, results can only be achieved through multidisciplinary cooperation.

 In the third cycle of the program, tests were taken before (pre-test) and after (post-test) among children and their parents. We only took into account those answers where we received an evaluable answer in the survey before and after the program. Our most important results: 1. there was no negative change in any question, so it was not the case that the total score of any question in the pre-test was higher than in the post-test. 2. The children learned that not only adults can call the ambulance. 3. Before the program, all children were already aware that if “someone is very ill”, the ambulance should be called. 4. Among the questions about stroke symptoms, it is important that hemiparesis, facial paresis and speech/language disorder are clear symptoms for children. Based on the parental questionnaires, the knowledge of the adults can be judged to be very good. The same number of correct answers were received during the pre-test and the post-test, on the basis of which we could not calculate a transfer effect. However, it is important that the parents considered the program useful, motivating and important for the children, so cooperation can be expected in the future.

The Hungarian “Stroke Ovi” program has so far proven to be clearly effective. This was proven by the impact assessments even if, instead of the original role-playing game, we implemented it “only” online due to the COVID epidemic. This constraint also forced and created a new “Hungarian version”. Despite the small number of samples caused by the circumstances, we consider this positive effect to be measurable. However, as the main result and evidence, we evaluated the children’s reaction, which took shape in spontaneous drawings and displayed professional values in addition to positive emotional reactions, such as the drawing of ambulances, the recurring representation of the 112 number. With the involvement of the media, we think online education is also a good option in the series of stroke campaigns, but we think the original role-playing form is really effective. At the same time, we can see that the application of the new method requires great caution due to the education of developing children. For this reason, results can only be achieved through social and multidisciplinary cooperation involving neurologists, psychologists, kindergarten teachers, and parents.

Mutations in Rht-B1 Locus May Negatively Affect Frost Tolerance in Bread Wheat.

The wheat semi-dwarfing genes Rht (Reduced height) are widely distributed among the contemporary wheat varieties. These genes also exert pleiotropic effects on plant tolerance towards various abiotic stressors. In this work, frost tolerance was studied in three near-isogenic lines of the facultative variety 'April Bearded' (AB), carrying the wild type allele Rht-B1a (tall phenotype), and the mutant alleles Rht-B1b (semi-dwarf) and Rht-B1c (dwarf), and was further compared with the tolerance of a typical winter type variety, 'Mv Beres'. The level of freezing tolerance was decreasing in the order 'Mv Beres' > AB Rht-B1a > AB Rht-B1b > AB Rht-B1c. To explain the observed differences, cold acclimation-related processes were studied: the expression of six cold-related genes, the phenylpropanoid pathway, carbohydrates, amino acids, polyamines and compounds in the tricarboxylic acid cycle. To achieve this, a comprehensive approach was applied, involving targeted analyses and untargeted metabolomics screening with the help of gas chromatography/liquid chromatography­mass spectrometry setups. Several cold-related processes exhibited similar changes in these genotypes; indeed, the accumulation of eight putrescine and agmatine derivatives, 17 flavones and numerous oligosaccharides (max. degree of polymerization 18) was associated with the level of freezing tolerance in the 'April Bearded' lines. In summary, the mutant Rht alleles may further decrease the generally low frost tolerance of the Rht-B1a, and, based on the metabolomics study, the mechanisms of frost tolerance may differ for a typical winter variety and a facultative variety. Present results point to the complex nature of frost resistance.

A new method to determine the optimal orientation of Slim Modiolar cochlear implant electrode array insertion.

BACKGROUND AND PURPOSE: Our goal was to determine the optimal orientation of insertion of the Slim Modiolar electrode and develop an easy-to-use method to aid implantation surgery. In some instances, the electrode arrays cannot be inserted in their full length. This can lead to buckling, interscalar dislocation or tip fold-over. In our opinion, one of the possible reasons of tip fold-over is unfavourable orientation of the electrode array. Our goal was to determine the optimal orientation of the Slim Modiolar electrode array relative to clear surgical landmarks and present our method in one specified case. METHODS: For the measurement, we used the preoperative CT scan of one of our cochlear implant patients. These images were processed by an open source and free image visualization software: 3D Slicer. In the first step we marked the tip of the incus short process and then created the cochlear view. On this view we drew two straight lines: the first line represented the insertion guide of the cochlear implant and the second line was the orientation marker (winglet). We determined the angle enclosed by winglet and the line between the tip of the incus short process and the cross-section of previously created two lines. For the calculation we used a self-made python code. RESULTS: The result of our algorithm for the angle was 46.6055°. To validate this result, we segmented, from the CT scan, the auditory ossicles and the membranaceous labyrinth. From this segmentation we generated a 3D reconstruction. On the 3D view, we can see the position of the previous lines relative to the anatomical structures. After this we rotated the 3D model together with the lines so that the insertion guide forms a dot. In this view, the angle was measured with ImageJ and the result was 46.599°. CONCLUSION: We found that our method is easy, fast, and time-efficient. The surgery can be planned individually for each patient, based on their routine preoperative CT scan of the temporal bone, and the implantation procedure can be made safer. In the future we plan to use this method for all cochlear implantation surgeries, where the Slim Modiolar electrode is used.

[The first Hungarian patient with Guillain-Barre syndrome after COVID-19]. / Covid-19-asszociált Guillain­Barré-szindróma elsô hazai esete.

Guillain-Barré syndrome (GBS) is a sporadic, relatively rare disease. In serious cases, it can lead to respiratory failure and death. The correlation between SARS-CoV-2 and the risk of GBS is not yet known. COVID-19-associated prolonged pulmonary complications could be worsened by the potential airway interference caused by GBS. The literature is inconsistent whether SARS-CoV-2 virus has direct or indirect effect on the onset of GBS. The authors describe the medical history of the first published GBS patient in Hungary with a preceding confirmed COVID-19 infection. The trigger role of COVID-19 infection is assumed because of the subsequent development of GBS after COVID-19 infection. So far none of the patients in the literature (including this patient) had positive PCR of SARS-CoV-2 virus from the cerebrospinal fluid.

[Dysphagiafelmérések akut stroke-ban]. / Akut stroke-betegeken validált ágy melletti dysphagiafelmérések az elmúlt húsz évben ­ szisztematikus irodalmi áttekintés.

BACKGROUND AND PURPOSE: Stroke associated dysphagia can have serious consequences such as aspiration pneumonia. The Hungarian guideline on nutritional therapy for stroke patients recommends dysphagia assessment, as early screening can optimize disease outcome and hospital cost. Thus far, this may be the first study in Hungarian that has documented a systematic review about the available validated dysphagia assessments of acute stroke. Purpose - The aim of this study was to summarize the instrumentally validated bedside dysphagia screening tools for acute stroke patients, which were published in the last twenty years. Our objective was to describe the characteristics of the validation studies, examine their study design, and sample the sub-tests and the diagnostic accuracy of the assessments. METHODS: A systematic research was carried out of the literature between 2001 and 2021 in eight scientific databases with search terms appropriate to our objectives. Subjects of the study - 652 articles were found and were reduced to eight. We made a comparative analysis of these. RESULTS: The GUSS test reached a high level of sensitivity compared to the others. In our study sample, the prevalence of instrumentally confirmed dysphagia among acute stroke patients was 56.1%. CONCLUSION: The focus and the composition of the analyzed studies differed and posed problems such as the ambiguity of the concept of dysphagia, the difference in outcome indicators, or the timing of screening. The GUSS test, which offers domestic management, is a suitable tool for the Hungarian clinical use.

Utilization of acute vascular imaging and neurointervention for acute ischaemic stroke patients in 20 Hungarian stroke centers.

BACKGROUND AND PURPOSE: Acute mortality rate of stroke in Hungary is significantly higher than in Western Europe, which is likely to be partially attributable to suboptimal treatment. METHODS: We examined the use of acute vascular imaging and mechanical thrombectomy for acute ischaemic stroke patients. We collected data on 20 consecutive patients from Hungarian stroke centers before 31st August 2016. RESULTS: Out of the reported 410 patients, 166 (40.4%) underwent CT angiography and 44 (10.7%) had mechanical thrombectomy. CONCLUSION: Only about 1/3 of acute ischaemic stroke patients eligible for thrombectomy actually had it. The underlying reasons include long onset-to-door time, low utilization of acute vessel imaging and a limited neuro-intervention capacity needing improvement.

Repeatability, reproducibility and interocular difference in the assessments of optic nerve OCT in children- a Swedish population-based study.

BACKGROUND: The aim was, first, to collect normative data of the optic nerve head and the peripapillary retinal nerve fibre layer (RNFL) thickness assessed with Cirrus SD-OCT, in healthy children in a population-based study; second, using these data, to examine repeatability, reproducibility and the interocular difference. METHODS: One-hundred and ten eyes from 57 children aged 6-15 born at term, were examined. Best-corrected visual acuity and refraction were assessed. Both eyes were examined and the interocular difference was calculated. Repeatability was calculated by one examiner performing three assessments. Thereafter, a second examiner repeated the assessments to calculate reproducibility. RESULTS: Mean RNFL thickness was 99.2 (SD 8.8) µm, mean disc area 1.89 (SD 0.37) mm2 and mean rim area 1.52 (SD 0.26) mm2. No significant correlations with age, gender or refraction were found. Repeatability and reproducibility were good overall. There was interocular symmetry between the eyes. CONCLUSIONS: Normal values for optic nerve head and RNFL thickness assessed with Cirrus SD-OCT were gathered to obtain a normal material in children. High repeatability and reproducibility indicated reliability of assessments performed by different examiners on different occasions. Overall, good correlation between right and left eyes was found.

CENTRAL MACULAR THICKNESS IN 6.5-YEAR-OLD CHILDREN BORN EXTREMELY PRETERM IS STRONGLY ASSOCIATED WITH GESTATIONAL AGE EVEN WHEN ADJUSTED FOR RISK FACTORS.

PURPOSE: To assess the macular thickness in 6.5-year-old children born extremely preterm (EPT) in comparison with children born at term and to investigate risk factors associated with the macular thickness in the preterm group. METHODS: A population-based study of 6.5-year-old children born before the gestational age of 27 weeks and age-matched control subjects. Macular assessments with optical coherence tomography were performed, and the results were compared with neonatal risk factors and sex. RESULTS: Adequate optical coherence tomography measurements were obtained from 134 children born EPT (mean gestational age of 25 weeks [range 23-26]) and 145 control subjects. The mean (range) of central macula thickness was significantly increased (P < 0.001) in the EPT group (right eyes: 282 µm [238-356], left eyes: 283 µm [229-351]), compared with the control group (right eyes: 249 µm [208-293], left eyes: 248 µm [207-290]). A multiple linear mixed model analysis of the EPT group revealed gestational age, retinopathy of prematurity, and male gender as important risk factors for an increased macular thickness. The macular thickness decreased by 3.9 µm per gestational week, when adjusted for retinopathy of prematurity and sex. CONCLUSION: Extremely preterm birth constitutes a substantial risk factor for a thick central macula, even when adjusted for retinopathy of prematurity and male gender.

[Role of the homograft bypass in extremity inferior's reconstructions]. / Homograftok szerepe az alsó végtagi érrekonstrukciókban.

INTRODUCTION: Vascular homografts are used for limb salvage in cases of graft infection after previous reconstructive vascular surgery or inadequate autologous veins. During multi-organ donation the thoracic aorta segment, aortic bifurcation, iliac arteries, femoral arteries, popliteal arteries, femoral veins and greater saphenous veins can be harvested. Our aim was to optimize the use of homografts by analyzing the results of previous procedures. METHODS: The patient information was processed retrospectively, using the clinical computer system. 162 procedures were performed on 144 patients between 2007 and 2014. The short- and long-term patency, hemorrhagic complication rate, amputation rate and mortality was examined in our study. The location, graft type and length of cryopreservation were taken into consideration. Aortoiliac and femoropopliteal reconstructions with arterial and venous homografts were examined. RESULTS: The mean age of the patients was 63.6 ± 10.7 years, the mean follow-up period was 36 ± 28 months. The primary patency rates at the postoperative 1, 3 and 6 months were 83.7%, 75.0% and 63.4%. In this study the arterial and deep venous homografts had better primary patency rates compared to the superficial venous homografts: at the postoperative 1, 3, 6 months the arterial homograft results were 85.6%, 78.6% and 74.3%, the greater saphenous vein homograft results were 81.4%, 70.4% and 47.7% in the same intervals. CONCLUSION: The reconstructive surgical procedures in septic area mean serious challenge for the vascular surgeons. The AB0 compatibility of the graft and the recipient did not result better long-term outcomes compared to the non-compatible grafts. According to our data the ideal choice of homogenous graft is an arterial homograft which was not cryopreserved longer than 6 months.

Rapid elimination of Carboxy-THC in a cohort of chronic cannabis users.

Urinary 11-nor-Δ(9)-tetrahydrocannabinol-9-carboxylic acid (Carboxy-THC) concentrations, normalised to creatinine output, have been demonstrated to be a useful tool in the interpretation of the results of a series of urine tests for cannabis. These tests, often termed historical data, can be used to identify potential chronic cannabis users who may present occupational health and safety risks within the workplace. Conversely, the data can also be used to support employee claims of previous regular, rather than recent, cannabis use. This study aimed at examining the mean elimination of Carboxy-THC in 37 chronic users undergoing voluntary abstinence over a 2-week period. Urine specimens were collected prior to the study and after 1 and 2 weeks of abstinence. Carboxy-THC levels in urine were measured by gas chromatography-mass spectrometry (GC-MS) following alkaline hydrolysis, organic solvent extraction and derivatisation to form its pentafluoropropionic derivative. The creatinine-normalised Carboxy-THC concentrations declined rapidly over the 2 weeks of abstinence period and the majority of chronic cannabis users (73%) reduced their urinary Carboxy-THC levels to below the 15-µg/L confirmatory cutoff within that time. The study further highlights the value of historical urinary Carboxy-THC data as a means of identifying potential occupational health and safety risks among chronic cannabis users.

Higher Incidence of Stroke on the Last Day of the Month in Hungary-a Role for Psychosocial Factors and Financial Insecurity?

BACKGROUND: The seasonal cumulation of acute ischemic stroke events is a well-known phenomenon. Critical days are determined by both biological and psychosocial factors. We hypothesized that the financial stability of those with a monthly income living in an economically unpredictable environment rises upon the arrival of their salary and decreases in the preceding days, leading to anxiety and existential insecurity, which may increase the incidence of acute ischemic stroke. METHODS: We assessed the daily average number of thrombolytic treatments due to acute ischemic stroke in Hungary between December 1, 2005, and November 30, 2013, calculating the ratio of thrombolytic treatments on the last day of the month (irrespectively whether it was the 28th-31st days) to thrombolytic treatments on the other days, and determined 95% confidence intervals. RESULTS: In this period, 7880 thrombolytic treatments were performed nationwide (2.70/day), out of which 1867 occurred on the last day of the month (19.45/day). If the 28th, 29th, or 30th was not the last day of the month, 15.8, 20.6, and 22 times less thrombolytic treatments, respectively, were performed than on the last day of that month. CONCLUSION: We propose that financial insecurity on the days prior to the receipt of a salary might play a role in the elevation of stroke incidence observed on the last day of the month in Hungary. Further analysis of this phenomenon and its psychosocial effects is needed to adequately allocate healthcare resources and to take preventive measures in the high-risk population.

Rapamycin can restore the negative regulatory function of transforming growth factor beta 1 in high grade lymphomas.

TGF-ß1 (transforming growth factor beta 1) is a negative regulator of lymphocytes, inhibiting proliferation and switching on the apoptotic program in normal lymphoid cells. Lymphoma cells often lose their sensitivity to proapoptotic/anti-proliferative regulators such as TGF-ß1. Rapamycin can influence both mTOR (mammalian target of rapamycin) and TGF-ß signaling, and through these pathways it is able to enhance TGF-ß induced anti-proliferative and apoptotic responses. In the present work we investigated the effect of rapamycin and TGF-ß1 combination on cell growth and on TGF-ß and mTOR signalling events in lymphoma cells. Rapamycin, an inhibitor of mTORC1 (mTOR complex 1) did not elicit apoptosis in lymphoma cells; however, the combination of rapamycin with exogenous TGF-ß1 induced apoptosis and restored TGF-ß1 dependent apoptotic machinery in several lymphoma cell lines with reduced TGF-ß sensitivity in vitro. In parallel, the phosphorylation of p70 ribosomal S6 kinase (p70S6K) and ribosomal S6 protein, targets of mTORC1, was completely eliminated. Knockdown of Smad signalling by Smad4 siRNA had no influence on apoptosis induced by the rapamycin+TGF-ß1, suggesting that this effect is independent of Smad signalling. However, apoptosis induction was dependent on early protein phosphatase 2A (PP2A) activity, and in part on caspases. Rapamycin+TGF-ß1 induced apoptosis was not completely eliminated by a caspase inhibitor. These results suggest that high mTOR activity contributes to TGF-ß resistance and lowering mTORC1 kinase activity may provide a tool in high grade B-cell lymphoma therapy by restoring the sensitivity to normally available regulators such as TGF-ß1.

Macular function measured by binocular mfERG and compared with macular structure in healthy children.

PURPOSE: To create normative data in children from binocular multifocal ERG (mfERG) recordings and compare results with the macular thickness. METHODS: Forty-nine 5- to 15-year-old healthy, full-term children were examined with Espion Multifocal System, using DTL electrodes. The stimulus matrix consisted of 37 hexagonal elements. Amplitudes, implicit times and response densities (presented in three rings) of the first-order component P1 were analyzed. Measurements of macular thickness were performed with spectral-domain Cirrus OCT. RESULTS: There were no significant differences between right and left eyes regarding mfERG recordings. Median P1 implicit times of Rings 1-3 of the 46 right eyes were 30.0, 30.0 and 30.8 ms and response densities 20.5, 10.9 and 7.6 nV/deg(2), respectively. Implicit time was longer in boys than in girls (p = 0.009, 0.039, 0.005 in Rings 1-3) and was correlated with age (r s = 0.417, 0.316, 0.274 in Rings 1-3). Implicit time in Ring 1 correlated significantly with the inner circle of the OCT measurements (p = 0.014). CONCLUSION: Binocular mfERG with DTL electrodes is a reliable test of the central macular function in children and correlates with macular structure. As previously not shown, there was a significant difference in implicit time between boys and girls.

Anomalous equivalent potential temperature: an atmospheric feature predicting days with higher risk for fatal outcome in acute ischemic stroke-a preliminary study.

Acute stroke is a life-threatening condition. Fatal outcome is related to risk factors, some of these affected by climatic changes. Forecasting potentially harmful atmospheric processes may therefore be of practical importance in the acute care of stroke patients. We analyzed the history of all patients with acute ischemic stroke (N = 184) confirmed by neuroimaging including those who died (N = 35, 15 males) at our hospital department in the winter months of 2009. Patient data were anonymized, and the human meteorologists were only aware of patients' age, gender, and exact time of death. Of the meteorological parameters, equivalent potential temperature (EPT) has been chosen for analysis. EPT is generally used for forecasting thunderstorms, but in the case of synoptic scale airflow (10(6) m), it is suitable for characterizing the air mass inflowing from different regions. The behavior of measured EPT values was compared to the climatic (30 years) averages. We developed meteorological criteria for anomalous periods of EPT and tested if such periods are associated with higher rate of fatal outcome. The duration of anomalous and non-anomalous periods was nearly equal during the studied 3 months. Stroke onset distributed similarly between anomalous and non-anomalous days; however, of the 35 deaths, 27 occurred during anomalous periods: on average, 0.56 deaths occurred on anomalous days and 0.19 on non-anomalous days. Winter periods meeting the criteria of anomalous EPT may have a significant adverse human-meteorological impact on the outcome in acute ischemic stroke.

Relevance of novel inflammatory markers in stroke-induced immunosuppression.

BACKGROUND: Acute ischemic stroke (AIS) has a biphasic effect on the peripheral immune system. The initial inflammatory response is followed by systemic immunosuppression, referred to as stroke-induced immunosuppression (SIIS), leading to severe complications in stroke patients. We aimed to identify an inflammatory marker that best represents this biphasic immunological response after AIS. METHODS: We investigated the alteration of CRP, WBC, neutrophil count, suPAR levels, CD4+ CD25high Tregs, CD64+ and CD177+ neutrophils and monocytes in 12 acute ischemic stroke patients free of infection within 6 hours and one week after the insult. As controls, 14 age-matched healthy individuals were included. RESULTS: CRP, WBC and neutrophil count values were comparable in stroke patients within 6 hours and controls, however, they were elevated in stroke one week after the insult. suPAR levels were higher in both stroke groups compared to controls. The prevalence of CD64+ neutrophils was higher in stroke patients within 6 hours than in controls and it decreased in stroke one week after the insult below the level in controls (5.95 [5.41-8.75] % vs. 32.38 [9.21-43.93] % vs. 4.06 [1.73-6.77] %, p < 0.05). CONCLUSIONS: Our pilot study identified that the prevalence of CD64+ neutrophils may reflect a biphasic alteration of the immune response following AIS. Since its level decreases below baseline after one week of the CNS insult in stroke patients without infection, it might serve as a reliable candidate to identify the developing inflammatory response due to infection after stroke in the future.

Reduced cone photoreceptor function and subtle systemic manifestations in two siblings with loss of SCLT1.

BACKGROUND: The sodium channel and clathrin linker 1 gene (SCLT1) has been involved in the pathogenesis of various ciliopathy disorders such as Bardet-Biedl syndrome, orofaciodigital syndrome type IX, and Senior-Løken syndrome. Detailed exams are warranted to outline all clinical features. Here, we present a family with a milder phenotype of SCLT1-related disease. MATERIAL AND METHODS: Comprehensive eye examination including fundus images, OCT, color vision, visual fields and electroretinography were performed. Affected individuals were assessed by a pediatrician and a medical geneticist for systemic features of ciliopathy. Investigations included echocardiography, abdominal ultrasonography, blood work-up for diabetes, liver and kidney function. Genetic testing included NGS retinal dystrophy panel, segregation analysis and transcriptome sequencing. RESULTS: Two male children, age 10 and 8 years, were affected with attention deficit hyperactivity disorder (ADHD), obesity and mild photophobia. The ophthalmic exam revealed reduced best-corrected visual acuity (BCVA), strabismus, hyperopia, astigmatism and moderate red-green defects. Milder changes suggesting photoreceptors disease were found on retinal imaging. Electroretinogram confirmed cone photoreceptors dysfunction. Genetic testing revealed a homozygous likely pathogenic, splice-site variant in SCLT1 gene NM_144643.3: c.1439 + 1del in the proband and in the affected brother. The unaffected parents were heterozygous for the SCLT1 variant. Transcriptome sequencing showed retention of intron 16 in the proband. CONCLUSIONS: In this report, we highlight the importance of further extensive diagnostics in patients with unexplained reduced vision, strabismus, refractive errors and ADHD spectrum disorders. SCLT1-related retinal degeneration is very rare and isolated reduced function of cone photoreceptors has not previously been observed.

Disentangling the effects of terroir, season, and vintage on the grapevine fungal pathobiome.

The composition, diversity and dynamics of microbial communities associated with grapevines may be influenced by various environmental factors, including terroir, vintage, and season. Among these factors, terroir stands out as a unique possible determinant of the pathobiome, the community of plant-associated pathogens. This study employed high-throughput molecular techniques, including metabarcoding and network analysis, to investigate the compositional dynamics of grapevine fungal pathobiome across three microhabitats (soil, woody tissue, and bark) using the Furmint cultivar. Samples were collected during late winter and late summer in 2020 and 2021, across three distinct terroirs in Hungary's Tokaj wine region. Of the 123 plant pathogenic genera found, Diplodia, Phaeomoniella, and Fusarium displayed the highest richness in bark, wood, and soil, respectively. Both richness and abundance exhibited significant disparities across microhabitats, with plant pathogenic fungi known to cause grapevine trunk diseases (GTDs) demonstrating highest richness and abundance in wood and bark samples, and non-GTD pathogens prevailed soil. Abundance and richness, however, followed distinct patterns Terroir accounted for a substantial portion of the variance in fungal community composition, ranging from 14.46 to 24.67%. Season and vintage also contributed to the variation, explaining 1.84 to 2.98% and 3.67 to 6.39% of the variance, respectively. Notably, significant compositional differences in fungi between healthy and diseased grapevines were only identified in wood and bark samples. Cooccurrence networks analysis, using both unweighted and weighted metrics, revealed intricate relationships among pathogenic fungal genera. This involved mostly positive associations, potentially suggesting synergism, and a few negative relationships, potentially suggesting antagonistic interactions. In essence, the observed differences among terroirs may stem from environmental filtering due to varied edaphic and mesoclimatic conditions. Temporal weather and vine management practices could explain seasonal and vintage fungal dynamics. This study provides insights into the compositional dynamics of grapevine fungal pathobiome across different microhabitats, terroirs, seasons, and health statuses. The findings emphasize the importance of considering network-based approaches in studying microbial communities and have implications for developing improved viticultural plant health strategies.

Characterization of the Sterol 24-C-Methyltransferase Genes Reveals a Network of Alternative Sterol Biosynthetic Pathways in Mucor lusitanicus.

Certain members of the order Mucorales can cause a life-threatening, often-fatal systemic infection called mucormycosis. Mucormycosis has a high mortality rate, which can reach 96 to 100% depending on the underlying condition of the patient. Mucorales species are intrinsically resistant to most antifungal agents, such as most of the azoles, which makes mucormycosis treatment challenging. The main target of azoles is the lanosterol 14α-demethylase (Erg11), which is responsible for an essential step in the biosynthesis of ergosterol, the main sterol component of the fungal membrane. Mutations in the erg11 gene can be associated with azole resistance; however, resistance can also be mediated by loss of function or mutation of other ergosterol biosynthetic enzymes, such as the sterol 24-C-methyltransferase (Erg6). The genome of Mucor lusitanicus encodes three putative erg6 genes (i.e., erg6a, erg6b, and erg6c). In this study, the role of erg6 genes in azole resistance of Mucor was analyzed by generating and analyzing knockout mutants constructed using the CRISPR-Cas9 technique. Susceptibility testing of the mutants suggested that one of the three genes, erg6b, plays a crucial role in the azole resistance of Mucor. The sterol composition of erg6b knockout mutants was significantly altered compared to that of the original strain, and it revealed the presence of at least four alternative sterol biosynthesis pathways leading to formation of ergosterol and other alternative, nontoxic sterol products. Dynamic operation of these pathways and the switching of biosynthesis from one to the other in response to azole treatment could significantly contribute to avoiding the effects of azoles by these fungi. IMPORTANCE The fungal membrane contains ergosterol instead of cholesterol, which offers a specific point of attack for the defense against pathogenic fungi. Indeed, most antifungal agents target ergosterol or its biosynthesis. Mucormycoses-causing fungi are resistant to most antifungal agents, including most of the azoles. For this reason, the drugs of choice to treat such infections are limited. The exploration of ergosterol biosynthesis is therefore of fundamental importance to understand the azole resistance of mucormycosis-causing fungi and to develop possible new control strategies. Characterization of sterol 24-C-methyltransferase demonstrated its role in the azole resistance and virulence of M. lusitanicus. Moreover, our experiments suggest that there are at least four alternative pathways for the biosynthesis of sterols in Mucor. Switching between pathways may contribute to the maintenance of azole resistance.