Histological analysis of direct somatic embryogenesis in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> (L.) Heynh

In Arabidopsis the in vitro culture of immature zygotic embryos (IZEs) at a late stage of development, on the solid medium containing synthetic auxin, leads to formation of somatic embryos via direct somatic embryogenesis (DSE). The presented results provide evidence that in IZE cells competent for DSE are located in the protodermis and subprotodermis of the adaxial side of cotyledons and somatic embryos displayed a single- or multicellular origin. Transgenic Arabidopsis lines expressing the GUS reporter gene, driven by the DR5 and LEC2 promoters, were used to analyse the distribution of auxin to mark embryogenic cells in cultured explants and develop somatic embryos. The analysis showed that at the start of the culture auxin was accumulated in all explant tissues, but from the fourth day onwards its location shifted to the protodermis and subprotodermis of the explant cotyledons. In globular somatic embryos auxin was detected in all cells, with a higher concentration in the protodermis, and in the heart stage its activity was mainly displayed in the shoot, root pole and cotyledon primordia. The embryogenic nature of dividing protodermal and subprotodermal cells accumulating auxin was confirmed by high expression of promoter activity of LEC2 in these cells. Analysis of symplasmic tracer (CFDA) distribution indicated symplasmic isolation between tissues engaged in DSE and other parts of an explant. Symplasmic isolation of somatic embryos from the explant was also detected.     

Interaction between cadmium, zinc and silver-substituted plastocyanin and cytochrome b 6 f complex — heavy metals toxicity towards photosynthetic apparatus

This paper reports the results of research on the interaction between the cytochrome f of the active cytochrome b ₆ f complex (incubated with Cd-, Zn-, and Ag-substituted plastocyanins) and Cu-plastocyanin. The presented studies show, that the metal derivatives of plastocyanin can have an influence on the photosynthetic electron transfer path: cytochrome b ₆ f complex — photosystem I. The metal-substituted plastocyanins occupy the plastocyanin electron transfer site of the cytochrome f. The stopped-flow measurements show, that although the metal derivatives of plastocyanin do not influence the rate of cyt f- Pc electron transfer, creation of the non-electron-transfer complexes characterised by a strong binding between the cyt f and substituted plastocyanins and their slow release, dependent on the redox state of the substituted metal, results in the decrease of a turnover of the cytochrome complex. The research was done in the Department of Plant Biochemistry, Freiburg University, Sch?nzlestrasse 1, 79 104 Freiburg, Germany     

Significant genetic differentiation between Poland and Germany follows present-day political borders, as revealed by Y-chromosome analysis

To test for human population substructure and to investigate human population history we have analysed Y-chromosome diversity using seven microsatellites (Y-STRs) and ten binary markers (Y-SNPs) in samples from eight regionally distributed populations from Poland (n=913) and 11 from Germany (n=1,215). Based on data from both Y-chromosome marker systems, which we found to be highly correlated (r=0.96), and using spatial analysis of the molecular variance (SAMOVA), we revealed statistically significant support for two groups of populations: (1) all Polish populations and (2) all German populations. By means of analysis of the molecular variance (AMOVA) we observed a large and statistically significant proportion of 14% (for Y-SNPs) and 15% (for Y-STRs) of the respective total genetic variation being explained between both countries. The same population differentiation was detected using Monmoniers algorithm, with a resulting genetic border between Poland and Germany that closely resembles the course of the political border between both countries. The observed genetic differentiation was mainly, but not exclusively, due to the frequency distribution of two Y-SNP haplogroups and their associated Y-STR haplotypes: R1a1*, most frequent in Poland, and R1*(xR1a1), most frequent in Germany. We suggest here that the pronounced population differentiation between the two geographically neighbouring countries, Poland and Germany, is the consequence of very recent events in human population history, namely the forced human resettlement of many millions of Germans and Poles during and, especially, shortly after World War II. In addition, our findings have consequences for the forensic application of Y-chromosome markers, strongly supporting the implementation of population substructure into forensic Y chromosome databases, and also for genetic association studies.     

Assessment of S100 protein expression in the epididymis of juvenile and adult European bison

In our study, we decided to compare S100 protein expression in the material obtained from the epididymes of 5- and 12-month-old calves, and adult European bison, and to detect any differences in S100 expression according to the animal age and size of the organ examined. We used the epididymes obtained from 6 adult European bison aged 6-12 years, from 6 at the age of 12 months and 6 calves aged 5 months. Immunocytochemical reactions were performed using the avidin-biotinylated-peroxidase (ABC) technique according to HSU. Specific polyclonal rabbit antiserum against bovine S100 protein (Bio Genex Laboratories) at a dilution at 1:400 was applied. We found the expression of S100 protein in endothelial cells of arteries, veins and lymphatic vessels in all the study animals. At the same time, we found no differences in the expression of S100 protein in vascular endothelial cells. Our observations seem to indicate that S100 expression in endothelial cells of European bison epididymis is not correlated with age or maturity of the organ tested. We found S100 protein in smooth muscle cells of arteries and veins in all European bison specimens examined. Interestingly in the current study, in young 5-month-old sexually immature European bison specimens we observed weaker expression of S100 protein in smooth muscle cells of small vessels as compared to the same cell type both in large vessels in these animals and in small vessels in adult specimens.     

In vitro selection of Remdesivir resistance suggests evolutionary predictability of SARS-CoV-2

Remdesivir (RDV), a broadly acting nucleoside analogue, is the only FDA approved small molecule antiviral for the treatment of COVID-19 patients. To date, there are no reports identifying SARS-CoV-2 RDV resistance in patients, animal models or in vitro. Here, we selected drug-resistant viral populations by serially passaging SARS-CoV-2 in vitro in the presence of RDV. Using high throughput sequencing, we identified a single mutation in RNA-dependent RNA polymerase (NSP12) at a residue conserved among all coronaviruses in two independently evolved populations displaying decreased RDV sensitivity. Introduction of the NSP12 E802D mutation into our SARS-CoV-2 reverse genetics backbone confirmed its role in decreasing RDV sensitivity in vitro. Substitution of E802 did not affect viral replication or activity of an alternate nucleoside analogue (EIDD2801) but did affect virus fitness in a competition assay. Analysis of the globally circulating SARS-CoV-2 variants (>800,000 sequences) showed no evidence of widespread transmission of RDV-resistant mutants. Surprisingly, we observed an excess of substitutions in spike at corresponding sites identified in the emerging SARS-CoV-2 variants of concern (i.e., H69, E484, N501, H655) indicating that they can arise in vitro in the absence of immune selection. The identification and characterisation of a drug resistant signature within the SARS-CoV-2 genome has implications for clinical management and virus surveillance.     

A comparative study of hourly and daily relationships between selected meteorological parameters and airborne fungal spore composition

Air sampling was conducted in Szczecin (Poland) throughout April–September 2013. The final data set included 177 daily and 4248 hourly samples. The total of 21 types of spores, which occurred in a number >10 in the season, were taken into account. The following meteorological parameters were analyzed: air temperature, relative humidity, precipitation and wind speed. Effects of individual weather parameters on hourly and daily concentrations of different fungal spore types were examined using Spearman’s rank association test, whereas effects of complex of meteorological factors on hourly and daily compositions of spore were assessed using detrended correspondence analysis (DCA) and redundancy analysis (RDA). Airborne fungal spore distribution patterns in relation to meteorological variables were determined by RDA, after DCA results detected a linear structure of the spore data. The RDA results obtained indicated that all the applied variables accounted for 20 and 22% of the total variance in the hourly and daily spore data, respectively. The results of stepwise forward selection of variables revealed all included hourly and daily meteorological variables were statistically significant. The largest amount of the total variance in the spore composition was explained by the air temperature in both cases (16%). Multivariate ordination did not show large differences between the hourly and daily relationships (with exception of wind speed impact), while the differences between simple hourly and daily correlations were more clear. Correlations between daily values of variables were in most cases higher than between hourly values of variables.     

In vitro contractile activity of porcine myometrium during luteolysis and early pregnancy: effect of oxytocin and progesterone

The present study was undertaken to elucidate the role of OT in myometrial contractility in sows. Spontaneous and OT-stimulated contractions of the inner circular (CM) and outer longitudinal (LM) layers isolated from cyclic (Days 14-16) and early pregnant (Days 14-16) sows were examined in six cyclic and six pregnant sows. In addition, the role of P(4) in the modulation of OT-induced uterine contractions was investigated. The contractile activity of the LM and CM layers was recorded in a tissue chamber filled with Krebs-Ringer solution. Myometrial contractility was expressed as area under the contractility curve (AUC) and frequency of contractions. Myometrial longitudinal and circular muscles exhibited spontaneous contractility in sows during both luteolysis and early-pregnancy. The mean AUC was higher (p<0.05) in the LM layer compared to the CM layer in both cyclic and pregnant animals. In addition, pregnant sows were characterized by higher AUC in both LM and CM layers in comparison to cyclic sows. The CM layer was unresponsive to examined treatments. Oxytocin (1-3x10(-8) and 1-3x10(-7)M) increased the AUC and frequency of contractions of the LM layer in both examined animal groups, being more effective during luteolysis (p<0.001) than early pregnancy (p<0.01). Response of the LM layer to OT appeared to be clearly related to the initial spontaneous level of contractility. This response to OT was inhibited (p<0.05) in the presence of OT antagonist (10(-6)M). Moreover, in pregnant sows, OT-stimulated contractile activity of myometrium was inhibited (p<0.05) by P(4) (10(-5)M). In conclusion, OT receptors present in myometrial cells (especially in the LM layer) are involved in the regulation of contractile activity of porcine myometrium during luteolysis and early-pregnancy. In addition, progesterone appears to be involved in this regulation.     

High glucose increases glomerular filtration barrier permeability by activating protein kinase G type Iα subunits in a Nox4-dependent manner

Hyperglycemia is a primary factor that disturbs podocyte function in the glomerular filtration process; this disturbance leads to the development of diabetic nephropathy, and ultimately, renal failure. Podocyte function may also be altered by biological agents that modify protein kinase activity, including the cGMP-activated protein kinase type Iα (PKGIα). We hypothesized that hyperglycemia-induced podocyte protein hyperpermeability was dependent on PKGIα activation, and that PKGIα was activated via dimerization induced by reactive oxygen species. This hypothesis was investigated in rat podocytes cultured in high glucose (HG, 30 mM). Protein expression was measured with Western blot and immunofluorescence. Podocyte permeability was measured with a transmembrane albumin flux assay. We found that HG increased podocyte permeability in long-term incubations (1, 3, and 5 days); permeability was increased by 66% on day 5. This effect was abolished with apocynin, a NAD(P)H inhibitor, and Rp-8-Br-cGMPS, a PKG inhibitor. It was also abolished by introducing small interfering RNAs (siRNAs) against Nox4 and PKGIα into cultured podocytes. Furthermore, HG increased PKGIα dimerization by 138% (0.23±0.04 vs. 0.54±0.09; P<0.05); this effect was abolished with a siRNA against Nox4. Our observations suggested that HG could increase albumin permeability across the podocyte filtration barrier via Nox4-dependent PKGIα dimerization.     

Accumulation of low-molecular-weight organic acids in roots and leaf segments of Zea mays plants treated with cadmium and copper

Maize seedlings grown in a nutrient solution were treated with Cd (50, 100 µM) or Cu (50, 100 µM). Roots and separated leaf sections (from the youngest––basal, through the middle––mature, to the oldest––apical) were analysed. Shoot and root fresh weight, and root net growth rates were reduced significantly after Cu application in comparison to Cd. Copper (50, 100 µM) and cadmium (100 µM) decreased the sum of chlorophyll and total carotenoid pools mostly in mature and old leaf sections. The concentration of Cu and Cd increased in the old and mature leaf sections. Analysis of organic acids showed that both metals differently influenced the low-molecular-weight organic acids (LMWOAs) content in maize leaf sections. In mature sections, the excess Cu increased the succinate and tartrate contents, whereas in the young ones mainly the tartrate level was elevated. Cadmium elevated citrate accumulation in mature and old sections, compared to the control plants. Malate, the main LMWOA in maize leaves, increased only after addition of 100 µM of Cd (mature and old sections) or 50 µM of Cu (old sections). Analysis of LMWOAs in roots showed that the excess of Cd or Cu induced higher accumulation of tartrate and malate and, additionally, copper increased the citrate content.