Conserved autophagy and diverse cell wall composition: unifying features of vascular tissues in evolutionarily distinct plants.

BACKGROUND AND AIMS: The formation of multifunctional vascular tissues represents a significant advancement in plant evolution. Differentiation of conductive cells is specific, involving two main pathways, namely protoplast clearance and cell wall modification. In xylogenesis, autophagy is a crucial process for complete protoplast elimination in tracheary elements, whose cell wall also undergoes strong changes. Knowledge pertaining to living sieve elements, which lose most of their protoplast during phloemogenesis, remains limited. We hypothesized that autophagy plays a crucial role, not only in complete cytoplasmic clearance in xylem but also in partial degradation in phloem. Cell wall elaborations of mature sieve elements are not so extensive. These analyses performed on evolutionarily diverse model species potentially make it possible to understand phloemogenesis to an equal extent to xylogenesis. METHODS: We investigated the distribution of ATG8 protein, which is an autophagy marker, and cell wall components in the roots of ferns, gymnosperms and angiosperms (monocots, dicot herbaceous plants and trees). Furthermore, we conducted a bioinformatic analysis of complete data on ATG8 isoforms for Ceratopteris richardii. KEY RESULTS: The presence of ATG8 protein was confirmed in both tracheary elements and sieve elements; however, the composition of cell wall components varied considerably among vascular tissues in the selected plants. Arabinogalactan proteins and ß-1,4-galactan were detected in the roots of all studied species, suggesting their potential importance in phloem formation or function. In contrast, no evolutionary pattern was observed for xyloglucan, arabinan or homogalacturonan. CONCLUSIONS: Our findings indicate that the involvement of autophagy in plants is universal during the development of tracheary elements that are dead at maturity and sieve elements that remain alive. Given the conserved nature of autophagy and its function in protoplast degradation for uninterrupted flow, autophagy might have played a vital role in the development of increasingly complex biological organizations, including the formation of vascular tissues. However, different cell wall compositions of xylem and phloem in different species might indicate diverse functionality and potential for substance transport, which is crucial in plant evolution.

Virulence and resistance patterns of Vibrio cholerae non-O1/non-O139 acquired in Germany and other European countries.

Global warming has caused an increase in the emergence of Vibrio species in marine and estuarine environments as well as fresh water bodies. Over the past decades, antimicrobial resistance (AMR) has evolved among Vibrio species toward various antibiotics commonly used for the treatment of Vibrio infections. In this study, we assessed virulence and resistance patterns of Vibrio cholerae non-O1/non-O139 strains derived from Germany and other European countries. A total of 63 clinical and 24 environmental Vibrio cholerae non-O1/non-O139 strains, collected between 2011 and 2021, were analyzed. In silico antibiotic resistances were compared with resistance phenotypes according to EUCAST breakpoints. Additionally, genetic relatedness between isolates was assessed by two cgMLST schemes (SeqSphere +, pubMLST). Both cgMLST schemes yielded similar results, indicating high genetic diversity among V. cholerae non-O1/non-O139 isolates. Some isolates were found to be genetically closely related (allelic distance < 20), which suggests an epidemiological link. Thirty-seven virulence genes (VGs) were identified among 87 V. cholerae non-O1/non-O139 isolates, which resulted in 38 virulence profiles (VPs). VPs were similar between clinical and environmental isolates, with the exception of one clinical isolate that displayed a higher abundance of VGs. Also, a cluster of 11 environmental isolates was identified to have the lowest number of VGs. Among all strains, the predominant virulence factors were quorum sensing protein (luxS), repeats-in-toxins (rtxC/rtxD), hemolysin (hlyA) and different type VI secretion systems (T6SS) genes. The genotypic profiles revealed antibiotic resistance genes (ARGs) associated with resistance to beta-lactams, quinolones, macrolides, tetracycline, antifolate, aminoglycosides, fosfomycin, phenicols and sulfonamide. Carbapenemase gene VCC-1 was detected in 10 meropenem-resistant V. cholerae non-O1/non-O139 isolates derived from surface water in Germany. The proportion of resistance among V. cholerae non-O1/non-O139 species isolates against first line treatment (3rd generation cephalosporin, tetracycline and fluoroquinolone) was low. Empirical treatment would likely have been effective for all of the clinical V. cholerae non-O1/non-O139 isolates examined. Nevertheless, carbapenem-resistant isolates have been present in fresh water in Germany and might represent a reservoir for ARGs. Monitoring antimicrobial resistance is crucial for public health authorities to minimize the risks for the human population.

Artificial Intelligence Supports Automated Characterization of Differentiated Human Pluripotent Stem Cells.

Revolutionary advances in AI and deep learning in recent years have resulted in an upsurge of papers exploring applications within the biomedical field. Within stem cell research, promising results have been reported from analyses of microscopy images to, that is, distinguish between pluripotent stem cells and differentiated cell types derived from stem cells. In this work, we investigated the possibility of using a deep learning model to predict the differentiation stage of pluripotent stem cells undergoing differentiation toward hepatocytes, based on morphological features of cell cultures. We were able to achieve close to perfect classification of images from early and late time points during differentiation, and this aligned very well with the experimental validation of cell identity and function. Our results suggest that deep learning models can distinguish between different cell morphologies, and provide alternative means of semi-automated functional characterization of stem cell cultures.

Widening Aging Eyes and Ending Outdoor Tearing with Retrobulbar Injections of Dermal Fillers.

As people age, the intraorbital fat may diminish and palpebral fissures may narrow, causing the eyes to increasingly tear outside in cold weather. As the bulbus recedes from the conjunctiva, a "wind trap" is formed in the lateral corner of the eye. This wind trap appears to irritate the adjacent lacrimal gland. In this article, an 84-year-old patient experienced annoying outdoor tearing despite having undergone three tarsal strip canthopexies during the past 20 years. Methods: Retrobulbar injections of 3.5-mL high-viscous dermal fillers (Bellafill or Radiesse) pushed the eyeballs forward, aligned the bulbus with the conjunctiva, and closed the wind trap behind the lateral canthus. Magnetic resonance imaging confirmed the filler material in the posterior lateral corner of the orbit. Results: The effect was immediate: the patient's constant outdoor tearing had resolved after the first treatment of his senile enophthalmos. In addition, the narrow palpebral fissure had widened by 2 mm and rejuvenated his aging eyes. Conclusions: An eyeball that has receded with age can be pushed forward with a retrobulbar injection of a long lasting dermal filler to reattach to the eyelids.

Microbiomes of Urine and the Prostate Are Linked to Human Prostate Cancer Risk Groups.

BACKGROUND: Bacteria play a suspected role in the development of several cancer types, and associations between the presence of particular bacteria and prostate cancer have been reported. OBJECTIVE: To provide improved characterisation of the prostate and urine microbiome and to investigate the prognostic potential of the bacteria present. DESIGN, SETTING, AND PARTICIPANTS: Microbiome profiles were interrogated in sample collections of patient urine (sediment microscopy: n = 318, 16S ribosomal amplicon sequencing: n = 46; and extracellular vesicle RNA-seq: n = 40) and cancer tissue (n = 204). OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS: Microbiomes were assessed using anaerobic culture, population-level 16S analysis, RNA-seq, and whole genome DNA sequencing. RESULTS AND LIMITATIONS: We demonstrate an association between the presence of bacteria in urine sediments and higher D'Amico risk prostate cancer (discovery, n = 215 patients, p < 0.001; validation, n = 103, p < 0.001, χ2 test for trend). Characterisation of the bacterial community led to the (1) identification of four novel bacteria (Porphyromonas sp. nov., Varibaculum sp. nov., Peptoniphilus sp. nov., and Fenollaria sp. nov.) that were frequently found in patient urine, and (2) definition of a patient subgroup associated with metastasis development (p = 0.015, log-rank test). The presence of five specific anaerobic genera, which includes three of the novel isolates, was associated with cancer risk group, in urine sediment (p = 0.045, log-rank test), urine extracellular vesicles (p = 0.039), and cancer tissue (p = 0.035), with a meta-analysis hazard ratio for disease progression of 2.60 (95% confidence interval: 1.39-4.85; p = 0.003; Cox regression). A limitation is that functional links to cancer development are not yet established. CONCLUSIONS: This study characterises prostate and urine microbiomes, and indicates that specific anaerobic bacteria genera have prognostic potential. PATIENT SUMMARY: In this study, we investigated the presence of bacteria in patient urine and the prostate. We identified four novel bacteria and suggest a potential prognostic utility for the microbiome in prostate cancer.

Modelling the Effects of Weather Conditions on Cereal Grain Contamination with Deoxynivalenol in the Baltic Sea Region.

Fusarium head blight (FHB) is one of the most serious diseases of small-grain cereals worldwide, resulting in yield reduction and an accumulation of the mycotoxin deoxynivalenol (DON) in grain. Weather conditions are known to have a significant effect on the ability of fusaria to infect cereals and produce toxins. In the past 10 years, severe outbreaks of FHB, and grain DON contamination exceeding the EU health safety limits, have occurred in countries in the Baltic Sea region. In this study, extensive data from field trials in Sweden, Poland and Lithuania were analysed to identify the most crucial weather variables for the ability of Fusarium to produce DON. Models were developed for the prediction of DON contamination levels in harvested grain exceeding 200 µg kg-1 for oats, spring barley and spring wheat in Sweden and winter wheat in Poland, and 1250 µg kg-1 for spring wheat in Lithuania. These models were able to predict high DON levels with an accuracy of 70-81%. Relative humidity (RH) and precipitation (PREC) were identified as the weather factors with the greatest influence on DON accumulation in grain, with high RH and PREC around flowering and later in grain development and ripening correlated with high DON levels. High temperatures during grain development and senescence reduced the risk of DON accumulation. The performance of the models, based only on weather variables, was relatively accurate. In future studies, it might be of interest to determine whether inclusion of variables such as pre-crop, agronomic factors and crop resistance to FHB could further improve the performance of the models.

Allies or Enemies: The Role of Reactive Oxygen Species in Developmental Processes of Black Cottonwood (Populus trichocarpa).

In contrast to aboveground organs (stems and leaves), developmental events and their regulation in underground organs, such as pioneer and fine roots, are quite poorly understood. The objective of the current study was to achieve a better understanding of the physiological and molecular role of reactive oxygen species (ROS) and ROS-related enzymes in the process of stem and pioneer root development in black cottonwood (Populus trichocarpa), as well as in the senescence of leaves and fine roots. Results of a transcriptomic analysis revealed that primary/secondary growth and senescence are accompanied by substantial changes in the expression of genes related to oxidative stress metabolism. We observed that some mechanisms common for above- and under-ground organs, e.g., the expression of superoxide dismutase (SOD) genes and SOD activity, declined during stems' and pioneer roots' development. Moreover, the localization of hydrogen peroxide (H2O2) and superoxide (O2•-) in the primary and secondary xylem of stems and pioneer roots confirms their involvement in xylem cell wall lignification and the induction of programmed cell death (PCD). H2O2 and O2•- in senescing fine roots were present in the same locations as demonstrated previously for ATG8 (AuTophaGy-related) proteins, implying their participation in cell degradation during senescence, while O2•- in older leaves was also localized similarly to ATG8 in chloroplasts, suggesting their role in chlorophagy. ROS and ROS-related enzymes play an integral role in the lignification of xylem cell walls in Populus trichocarpa, as well as the induction of PCD during xylogenesis and senescence.

Abscisic Acid and Jasmonate Metabolisms Are Jointly Regulated During Senescence in Roots and Leaves of Populus trichocarpa.

Plant senescence is a highly regulated process that allows nutrients to be mobilized from dying tissues to other organs. Despite that senescence has been extensively studied in leaves, the senescence of ephemeral organs located underground is still poorly understood, especially in the context of phytohormone engagement. The present study focused on filling this knowledge gap by examining the roles of abscisic acid (ABA) and jasmonate in the regulation of senescence of fine, absorptive roots and leaves of Populus trichocarpa. Immunohistochemical (IHC), chromatographic, and molecular methods were utilized to achieve this objective. A transcriptomic analysis identified significant changes in gene expression that were associated with the metabolism and signal transduction of phytohormones, especially ABA and jasmonate. The increased level of these phytohormones during senescence was detected in both organs and was confirmed by IHC. Based on the obtained data, we suggest that phytohormonal regulation of senescence in roots and leaves is organ-specific. We have shown that the regulation of ABA and JA metabolism is tightly regulated during senescence processes in both leaves and roots. The results were discussed with respect to the role of ABA in cold tolerance and the role of JA in resistance to pathogens.

Seasonal senescence of leaves and roots of Populus trichocarpa-is the scenario the same or different?

The remobilization and resorption of plant nutrients is considered as a crucial aspect of the seasonal senescence of plant organs. In leaves, the mechanisms responsible for the relocation of valuable compounds are well understood while the related processes in roots are still being debated. Some research indicates that remobilization in roots occurs, while other studies have not found evidence of this process. Considering that the total biomass of fine roots is equal to or greater than that of leaves, clarifying the conflicting reports and ambiguities may provide critical information on the circulation of chemical elements in forest ecosystems. This study provides new information concerning the basis for remobilization processes in roots by combining physiological data with gene expression and protein levels. We suggest that, as in leaves, molecular mechanisms involved in nitrogen (N) resorption are also activated in senescent roots. An analysis of N concentration indicated that N levels decreased during the senescence of both organs. The decrease was associated with an increase in the expression of a glutamine synthetase (GS) gene and a concomitant elevation in the amount of GS-one of the most important enzymes in N metabolism. In addition, significant accumulation of carbohydrates was observed in fine roots, which may represent an adaptation to unfavorable weather conditions that would allow remobilization to occur rather than a rapid death in response to ground frost or cold. Our results provide new insights into the senescence of plant organs and clarify contentious topics related to the remobilization process in fine roots.

Country-Wide qPCR Based Assessment of Plasmodiophora brassicae Spread in Agricultural Soils and Recommendations for the Cultivation of Brassicaceae Crops in Poland.

Clubroot is a damaging disease of oilseed rape and vegetable brassicas worldwide, caused by the soil-borne protist Plasmodiophora brassicae Wor. Due to the long life of resting spores, the assessment of the pathogen abundance in agricultural fields can serve as a guideline for disease control at the country-wide level or the regional scale. Between 2013 and 2019, we collected 431 soil samples from fields cultivated with Brassicaceae crops throughout 16 provinces of Poland. The samples were subjected to qPCR based analysis of P. brassicae DNA concentration. From these data, the spore loads and gene copies g-1 soil were calculated and used to produce an assessment of the current clubroot risk potential at a country-wide and regional scale. The country-wide map, showing the spread of the pathogen in agricultural soils, was made using ArcGis software package implementing the interpolation with the Inverse Distance Weight method. The calculation of gene copies specific to P. brassicae helped to formulate the recommendations for farmers in respect to the cultivation guidelines. It showed a high risk of yield losses in defined regions of north, south-west and central Poland and an urgent need to undertake intensive preventative measures.

Xylem Cell Wall Formation in Pioneer Roots and Stems of Populus trichocarpa (Torr. & Gray).

Regulation of gene expression, as determined by the genetics of the tree species, is a major factor in determining wood quality. Therefore, the identification of genes that play a role in xylogenesis is extremely important for understanding the mechanisms shaping the plant phenotype. Efforts to develop new varieties characterized by higher yield and better wood quality will greatly benefit from recognizing and understanding the complex transcriptional network underlying wood development. The present study provides a detailed comparative description of the changes that occur in genes transcription and the biosynthesis of cell-wall-related compounds during xylogenesis in Populus trichocarpa pioneer roots and stems. Even though results of microarray analysis indicated that only approximately 10% of the differentially expressed genes were common to both organs, many fundamental mechanisms were similar; e.g. the pattern of expression of genes involved in the biosynthesis of cell wall proteins, polysaccharides, and lignins. Gas chromatography time-of-flight mass spectrometry (GC-TOF-MS) shows that the composition of monosaccharides was also very similar, with an increasing amount of xylose building secondary cell wall hemicellulose and pectins, especially in the stems. While hemicellulose degradation was typical for stems, possibly due to the intensive level of cell wall lignification. Notably, the main component of lignins in roots were guiacyl units, while syringyl units were dominant in stems, where fibers are especially needed for support. Our study is the first comprehensive analysis, at the structural and molecular level, of xylogenesis in under- and aboveground tree parts, and clearly reveals the great complexity of molecular mechanisms underlying cell wall formation and modification during xylogenesis in different plant organs.

Occurrence of autophagy during pioneer root and stem development in Populus trichocarpa.

MAIN CONCLUSION: Autophagy is involved in developmentally programmed cell death and is identified during the early development of phloem, as well as xylem with a dual role, as both an inducer and executioner of cell death. The regulation of primary and secondary development of roots and stems is important for the establishment of root systems and for the overall survival of trees. The molecular and cellular basis of the autophagic processes, which are used at distinct moments during the growth of both organs, is crucial to understand the regulation of their development. To address this, we use Populus trichocarpa seedlings grown in a rhizotron system to examine the autophagy processes involved in root and stem development. To monitor the visual aspects of autophagy, transmission electron microscopy (TEM) and immunolocalization of AuTophaGy-related protein (ATG8) enabled observations of the phenomenon at a structural level. To gain further insight into the autophagy process at the protein and molecular level, we evaluated the expression of ATG gene transcripts and ATG protein levels. Alternations in the expression level of specific ATG genes and localization of ATG8 proteins were observed during the course of root or stem primary and secondary development. Specifically, ATG8 was present in the cells exhibiting autophagy, during the differentiation and early development of xylem and phloem tissues, including both xylary and extraxylary fibers. Ultrastructural observations revealed tonoplast invagination with the formation of autophagic-like bodies. Additionally, the accumulation of autophagosomes was identifiable during the differentiation of xylem in both organs, long before the commencement of cell death. Taken together, these results provide evidence in support of the dual role of autophagy in developmental PCD. A specific role of the controller of cell death, which is a committed step with the release of hydrolytic enzymes from the vacuole and final digestion of protoplast, from which there is no return once initiated, is only attributed to mega-autophagy.

Autophagy counteracts instantaneous cell death during seasonal senescence of the fine roots and leaves in Populus trichocarpa.

BACKGROUND: Senescence, despite its destructive character, is a process that is precisely-regulated. The control of senescence is required to achieve remobilization of resources, a principle aspect of senescence. Remobilization allows plants to recapture valuable resources that would otherwise be lost to the environment with the senescing organ. Autophagy is one of the critical processes that is switched on during senescence. This evolutionarily conserved process plays dual, antagonistic roles. On the one hand, it counteracts instantaneous cell death and allows the process of remobilization to be set in motion, while on the other hand, it participates in the degradation of cellular components. Autophagy has been demonstrated to occur in many plant species during the senescence of leaves and flower petals. Little is known, however, about the senescence process in other ephemeral organs, such as fine roots, whose lifespan is also relatively short. We hypothesized that, like the case of seasonal leaf senescence, autophagy also plays a role in the senescence of fine roots, and that both processes are synchronized in their timing. RESULTS: We evaluated which morphological and cytological symptoms are universal or unique in the senescence of fine roots and leaves. The results of our study confirmed that autophagy plays a key role in the senescence of fine roots, and is associated also with the process of cellular components degradation. In both organs, structures related to autophagy were observed, such as autophagic bodies and autophagosomes. The role of autophagy in the senescence of these plant organs was further confirmed by an analysis of ATG gene expression and protein detection. CONCLUSIONS: The present study is the first one to examine molecular mechanisms associated with the senescence of fine roots, and provide evidence that can be used to determine whether senescence of fine roots can be treated as another example of developmentally programmed cell death (dPCD). Our results indicate that there is a strong similarity between the senescence of fine roots and other ephemeral organs, suggesting that this process occurs by the same autophagy-related mechanisms in all plant ephemeral organs.

Combination of microbiological culture and multiplex PCR increases the range of vaginal microorganisms identified in cervical cancer patients at high risk for bacterial vaginosis and vaginitis.

BACKGROUND: Bacterial vaginosis (BV) and vaginitis in cervical cancer patients might becaused by mixed aerobic, anaerobic, and atypical bacteria. Since genital tract infections can be complicated, early and accurate identification of causal pathogens is vital. OBJECTIVES: The purpose of this study was i) to determinate if currently used aerobic culture methods are sufficiently sensitive to identify pathogens that can appear in the cervix of women after cancer treatment; ii) to investigate if molecular methods can improve the diagnostic process of BV and vaginitis, as well as broaden the range of detectable pathogens that would otherwise be difficult to cultivate. METHODS: A one-year hospital-based study was conducted in 2011/2012. Cervical swabs from 130 patients were examined by microbiological culture and multiplex PCR. RESULTS: Swab samples were positive for 107 and 93 women by microbiological culture and multiplex PCR, respectively The most common bacteria isolated from culture were: Escherichia coli, Enterococcus faecalis, Streptococcus agalactiae, and Staphylococcus aureus, and using the molecular technique were: Gardnerella vaginalis, Bacteroides fragilis, Ureoplasma ureoliticum/parvum, Mobiluncus curtisii and Atopobium vaginae. CONCLUSIONS: Multiplex PCR might contribute to the diagnosis of genital tract infections and it broadens the number of detectable microorganisms responsible for BV. Combination of these two methods may become the basis for standardized diagnosis of BV and vaginitis.

Usability application of multiplex polymerase chain reaction in the diagnosis of microorganisms isolated from urine of patients treated in cancer hospital.

BACKGROUND: THE OBJECTIVE OF THIS STUDY WAS: i) to compare the results of urine culture with polymerase chain reaction (PCR) -based detection of microorganisms using two commercially available kits, ii) to assess antimicrobial susceptibility of urine isolates from cancer patients to chosen antimicrobial drugs and, if necessary, to update the recommendation of empirical therapy. MATERIALS AND METHODS: A one-year hospital-based prospective study has been conducted in Greater Poland Cancer Centre and Genetic Medicine Laboratory CBDNA Research Centre in 2011. Urine cultures and urine PCR assay from 72 patients were examined. RESULTS: Urine cultures and urine PCR assay from 72 patients were examined. Urine samples were positive for 128 strains from which 95 (74%) were identical in both tests. The most frequently isolated bacteria in both culture and PCR assay were coliform organisms and Enterococcus spp. The Gram negative bacilli were most resistant to cotrimoxazol. 77.2% of these bacilli and 100% of E. faecalis and S. agalactiae were sensitive to amoxicillin-clavulanic acid. 4.7% of Gram positive cocci were resistant to nitrofurantoin. CONCLUSIONS: The PCR method quickly finds the causative agent of urinary tract infection (UTI) and, therefore, it can help with making the choice of the proper antimicrobial therapy at an early stage. It appears to be a viable alternative to the recommendations made in general treatment guidelines, in cases where diversified sensitivity patterns of microorganisms have been found.

Specific Fab fragments recovered by phage display technique recognizing human spermatozoa.

Human hybridoma cell lines are often unstable and loose ability for antibody production. Sometimes, they show low and varying levels of heavy and light chains synthesis. Therefore it is reasonable to preserve generated specificities of light and heavy chains by cloning them to phagemid vector and creating phage display library. The aim of this study was to construct phage display library of Fab fragments recognizing sperm surface antigens. The source of mRNA constituted seven hybridoma cell lines producing antisperm antibodies which was proved by ELISA, and agglutination test as well as by inhibition of sperm to penetrate hamster oocytes. Fragments of cDNA encoding kappa/lambda and gamma chains were cloned into pComb3HSS phagemid vector and amplified in XL-1Blue. The library was panned against whole unfixed sperm cells. Three positive clones selected after fourth round of panning showed heavy chain belonging to VH4 family, two of them (G28, K61) possessed lambda chain from VL2 family and one (H43) kappa chain from VK1 family. As these Fabs revealed similarities to antibodies against some proteins involved in sperm motility and cell fusion it can be suggested that these Fabs may be a cause of infertility. Finally, we proved that it is feasible to preserve specificities produced by human hybridomas using phage display technique and we recovered some Fabs which may be of diagnostic and research value, and may also have some value for contraceptive vaccine.