Selective mineral transport barriers at Cuscuta-host infection sites.

The uptake of inorganic nutrients by rootless parasitic plants, which depend on host connections for all nutrient supplies, is largely uncharted. Using X-ray fluorescence spectroscopy (XRF), we analyzed the element composition of macro- and micronutrients at infection sites of the parasitic angiosperm Cuscuta reflexa growing on hosts of the genus Pelargonium. Imaging methods combining XRF with 2-D or 3-D (confocal) microscopy show that most of the measured elements are present at similar concentrations in the parasite compared to the host. However, calcium and strontium levels drop pronouncedly at the host/parasite interface, and manganese appears to accumulate in the host tissue surrounding the interface. Chlorine is present in the haustorium at similar levels as in the host tissue but is decreased in the stem of the parasite. Thus, our observations indicate a restricted uptake of calcium, strontium, manganese and chlorine by the parasite. Xylem-mobile dyes, which can probe for xylem connectivity between host and parasite, provided evidence for an interspecies xylem flow, which in theory would be expected to carry all of the elements indiscriminately. We thus conclude that inorganic nutrient uptake by the parasite Cuscuta is regulated by specific selective barriers whose existence has evaded detection until now.

The evolution of the plastid phosphate translocator family.

MAIN CONCLUSION: The plastid phosphate translocators evolved in algae but diversified into several groups, which adopted different physiological functions by extensive gene duplications and losses in Streptophyta. The plastid phosphate translocators (pPT) are a family of transporters involved in the exchange of metabolites and inorganic phosphate between stroma and cytosol. Based on their substrate specificities, they were divided into four subfamilies named TPT, PPT, GPT and XPT. To analyse the occurrence of these transporters in different algae and land plant species, we identified 652 pPT genes in 101 sequenced genomes for phylogenetic analysis. The first three subfamilies are found in all species and evolved before the split of red and green algae while the XPTs were derived from the duplication of a GPT gene at the base of Streptophyta. The analysis of the intron-exon structures of the pPTs corroborated these findings. While the number and positions of introns are conserved within each subfamily, they differ between the subfamilies suggesting an insertion of the introns shortly after the three subfamilies evolved. During angiosperm evolution, the subfamilies further split into different groups (TPT1-2, PPT1-3, GPT1-6). Angiosperm species differ significantly in the total number of pPTs, with many species having only a few, while several plants, especially crops, have a higher number, pointing to the importance of these transporters for improved source-sink strength and yield. The differences in the number of pPTs can be explained by several small-scale gene duplications and losses in plant families or single species, but also by whole genome duplications, for example, in grasses. This work could be the basis for a comprehensive analysis of the molecular and physiological functions of this important family of transporters.

Identification of tomato introgression lines with enhanced susceptibility or resistance to infection by parasitic giant dodder (Cuscuta reflexa).

The parasitic flowering plant genus Cuscuta (dodder) is a parasitic weed that infects many important crops. Once it winds around the shoots of potential host plants and initiates the development of penetration organs, called haustoria, only a few plant species have been shown to deploy effective defense mechanisms to ward off Cuscuta parasitization. However, a notable exception is Solanum lycopersicum (tomato), which exhibits a local hypersensitive reaction when attacked by giant dodder (Cuscuta reflexa). Interestingly, the closely related wild desert tomato, Solanum pennellii, is unable to stop the penetration of its tissue by the C. reflexa haustoria. In this study, we observed that grafting a S. pennellii scion onto the rootstock of the resistant S. lycopersicum did not change the susceptibility phenotype of S. pennellii. This suggests that hormones, or other mobile substances, produced by S. lycopersicum do not induce a defense reaction in the susceptible tissue. Screening of a population of introgression lines harboring chromosome fragments from S. pennellii in the genome of the recurrent parent S. lycopersicum, revealed that most lines exhibit the same defense reaction as shown by the S. lycopersicum parental line. However, several lines showed different responses and exhibited either susceptibility, or cell death that extended considerably beyond the infection site. These lines will be valuable for the future identification of key loci involved in the perception of, and resistance to, C. reflexa and for developing strategies to enhance resistance to infection in crop species.

[Long-term oncological outcomes after radical prostatectomy in a non-university teaching hospital]. / Onkologische Langzeitergebnisse nach radikaler Prostatektomie in einer nicht-universitären Ausbildungsklinik.

BACKGROUND: The evaluation of oncological outcomes after radical prostatectomy (RP) is a major component of quality control in prostate cancer (PCa) centres. OBJECTIVES: To evaluate the oncological outcomes after RP in a non-university teaching hospital and compare them with other high-volume PCa centres. MATERIAL UND METHODS: This study included 1,161 patients after RP who were divided into two risk groups. Low-risk: localised PCa (pT2) and prostate-specific antigen (PSA)≤ 20 ng/ml, Gleason score (GS) 6-7b and pN0. High-risk: locally advanced PCa (≥pT3a) and/or PSA >20 ng/ml and/or GS≥ 8 and/or pN1. Risk groups and clinicopathological features were correlated to biochemical recurrence (BCR)-free survival, cancer-specific survival (CSS) und overall survival (OS). RESULTS: The 10-year BCR-free survival, CSS und OS were 68.4%, 47.0% and 100% in the low-risk group and 87.4%, 89.0% and 73.9% in the high-risk group, respectively; the outcomes between risk groups were statistically significant (p<0.05). A multivariate Cox regression analysis was performed. GS was the most significant prognostic factor for CSS (p=0.00001) und BCR-free survival (p=0.00036). Nodal involvement (pN1) was strongly associated with CSS (p=0.00004). Age was the most important factor for overall survival in the high-risk group (p=0.0011). CONCLUSIONS: RP could be a curative treatment option for advanced PCa in selected cases. GS is the most important prognostic factor. Good oncological outcomes can also be achieved in non-university teaching hospitals.

Fast-Track Discovery of SARS-CoV-2-Neutralizing Antibodies from Human B Cells by Direct Functional Screening.

As the COVID-19 pandemic revealed, rapid development of vaccines and therapeutic antibodies are crucial to guarantee a quick return to the status quo of society. In early 2020, we deployed our droplet microfluidic single-cell-based platform DROPZYLLA® for the generation of cognate antibody repertoires of convalescent COVID-19 donors. Discovery of SARS-CoV-2-specific antibodies was performed upon display of antibodies on the surface of HEK293T cells by antigen-specific sorting using binding to the SARS-CoV-2 spike and absence of binding to huACE2 as the sort criteria. This efficiently yielded antibodies within 3-6 weeks, of which up to 100% were neutralizing. One of these, MTX-COVAB, displaying low picomolar neutralization IC50 of SARS-CoV-2 and with a neutralization potency on par with the Regeneron antibodies, was selected for GMP manufacturing and clinical development in June 2020. MTX-COVAB showed strong efficacy in vivo and neutralized all identified clinically relevant variants of SARS-CoV-2 at the time of its selection. MTX-COVAB completed GMP manufacturing by the end of 2020, but clinical development was stopped when the Omicron variant emerged, a variant that proved to be detrimental to all monoclonal antibodies already approved. The present study describes the capabilities of the DROPZYLLA® platform to identify antibodies of high virus-neutralizing capacity rapidly and directly.

[Retrovesical recurrence of renal cell carcinoma 14 years after laparoscopic nephrectomy: a case report]. / Retrovesikales Rezidiv eines Nierenzellkarzinoms 14 Jahre nach laparoskopischer Nephrektomie. Ein Fallbericht.

Local recurrence or metachronous distant metastasis of renal cell carcinoma mostly occurs in the first five years after radical or partial nephrectomy. Nevertheless, late local recurrences or metachronous metastases are regularly observed. The most common metastatic sites are lung and bones, although unusual metastatic sites are also possible. We present the case of a 70-year-old patient with a solitary recurrence of a clear renal cell carcinoma in retrovesical location after a relapse-free survival of 14 years. We performed cystoprostatectomy with an ileal conduit, resecting the tumour completely (in sano). The long-term prognosis of the patient cannot be estimated as yet.Despite the fact that a solitary and very late recurrence of a renal cell carcinoma in retrovesical location is extremely rare, late recurrence is a specific characteristic of renal cell carcinoma. Therefore, long-term follow-up for at least 15 years is required. Curative surgical resection is the treatment of choice for such late solitary recurrences.

Apoptosis facilitates antigen presentation to T lymphocytes through MHC-I and CD1 in tuberculosis.

Protective immunity against Mycobacterium tuberculosis involves major histocompatibility complex class I (MHC-I)- and CD1-restricted CD8 T cells, but the mechanisms underlying antigen delivery to antigen-presenting molecules remain enigmatic. Macrophages, the primary host cells for mycobacteria, are CD1-negative. Here we show that M. tuberculosis phagosomes are secluded from the cytosolic MHC-I processing pathway and that mycobacteria-infected cells lose their antigen-presenting capacity. We also show that mycobacteria induce apoptosis in macrophages, causing the release of apoptotic vesicles that carry mycobacterial antigens to uninfected antigen-presenting cells (APCs). Inhibition of apoptosis reduced transfer of antigens to bystander cells and activation of CD8 T cells. Uninfected dendritic cells, which engulfed extracellular vesicles, were indispensable for subsequent cross-presentation of antigens, through MHC-I and CD1b, to T cells from mycobacteria-sensitized donors. This new 'detour' pathway for presentation of antigens from a phagosome-contained pathogen shows the functional significance of infection-induced apoptosis in the activation of CD8 T cells specific for both protein and glycolipid antigens in tuberculosis.

The Enigma of Interspecific Plasmodesmata: Insight From Parasitic Plants.

Parasitic plants live in intimate physical connection with other plants serving as their hosts. These host plants provide the inorganic and organic compounds that the parasites need for their propagation. The uptake of the macromolecular compounds happens through symplasmic connections in the form of plasmodesmata. In contrast to regular plasmodesmata, which connect genetically identical cells of an individual plant, the plasmodesmata that connect the cells of host and parasite join separate individuals belonging to different species and are therefore termed "interspecific". The existence of such interspecific plasmodesmata was deduced either indirectly using molecular approaches or observed directly by ultrastructural analyses. Most of this evidence concerns shoot parasitic Cuscuta species and root parasitic Orobanchaceae, which can both infect a large range of phylogenetically distant hosts. The existence of an interspecific chimeric symplast is both striking and unique and, with exceptions being observed in closely related grafted plants, exist only in these parasitic relationships. Considering the recent technical advances and upcoming tools for analyzing parasitic plants, interspecific plasmodesmata in parasite/host connections are a promising system for studying secondary plasmodesmata. For open questions like how their formation is induced, how their positioning is controlled and if they are initiated by one or both bordering cells simultaneously, the parasite/host interface with two adjacent distinguishable genetic systems provides valuable advantages. We summarize here what is known about interspecific plasmodesmata between parasitic plants and their hosts and discuss the potential of the intriguing parasite/host system for deepening our insight into plasmodesmatal structure, function, and development.

Footprints of parasitism in the genome of the parasitic flowering plant Cuscuta campestris.

A parasitic lifestyle, where plants procure some or all of their nutrients from other living plants, has evolved independently in many dicotyledonous plant families and is a major threat for agriculture globally. Nevertheless, no genome sequence of a parasitic plant has been reported to date. Here we describe the genome sequence of the parasitic field dodder, Cuscuta campestris. The genome contains signatures of a fairly recent whole-genome duplication and lacks genes for pathways superfluous to a parasitic lifestyle. Specifically, genes needed for high photosynthetic activity are lost, explaining the low photosynthesis rates displayed by the parasite. Moreover, several genes involved in nutrient uptake processes from the soil are lost. On the other hand, evidence for horizontal gene transfer by way of genomic DNA integration from the parasite's hosts is found. We conclude that the parasitic lifestyle has left characteristic footprints in the C. campestris genome.

Making the connections--the crucial role of metabolite transporters at the interface between chloroplast and cytosol.

Eukaryotic cells are most fascinating because of their high degree of compartmentation. This is particularly true for plant cells, due to the presence of chloroplasts, photosynthetic organelles of endosymbiotic origin that can be traced back to a single cyanobacterial ancestor. Plastids are major hubs in the metabolic network of plant cells, their metabolism being heavily intertwined with that of the cytosol and of other organelles. Solute transport across the plastid envelope by metabolite transporters is key to integrating plastid metabolism with that of other cellular compartments. Here, we review the advances in understanding metabolite transport across the plastid envelope membrane.

Cytokinin Response Factor 5 has transcriptional activity governed by its C-terminal domain.

Cytokinin Response Factors (CRFs) are AP2/ERF transcription factors involved in cytokinin signal transduction. CRF proteins consist of a N-terminal dimerization domain (CRF domain), an AP2 DNA-binding domain, and a clade-specific C-terminal region of unknown function. Using a series of sequential deletions in yeast-2-hybrid assays, we provide evidence that the C-terminal region of Arabidopsis CRF5 can confer transactivation activity. Although comparative analyses identified evolutionarily conserved protein sequence within the C-terminal region, deletion experiments suggest that this transactivation domain has a partially redundant modular structure required for activation of target gene transcription.

Characterization of AtNST-KT1, a novel UDP-galactose transporter from Arabidopsis thaliana.

Nucleotide sugar transporters (NST) mediate the transfer of nucleotide sugars from the cytosol into the lumen of the endoplasmatic reticulum and the Golgi apparatus. Because the NSTs show similarities with the plastidic phosphate translocators (pPTs), these proteins were grouped into the TPT/NST superfamily. In this study, a member of the NST-KT family, AtNST-KT1, was functionally characterized by expression of the corresponding cDNA in yeast cells and subsequent transport experiments. The histidine-tagged protein was purified by affinity chromatography and reconstituted into proteoliposomes. The substrate specificity of AtNST-KT1 was determined by measuring the import of radiolabelled nucleotide mono phosphates into liposomes preloaded with various unlabelled nucleotide sugars. This approach has the advantage that only one substrate has to be used in a radioactively labelled form while all the nucleotide sugars can be provided unlabelled. It turned out that AtNST-KT1 represents a monospecific NST transporting UMP in counterexchange with UDP-Gal but did not transport other nucleotide sugars. The AtNST-KT1 gene is ubiquitously expressed in all tissues. AtNST-KT1 is localized to Golgi membranes. Thus, AtNST-KT1 is most probably involved in the synthesis of galactose-containing glyco-conjugates in plants.

Transport of carbon in non-green plastids.

Non-green plastids are important sites for the biosynthesis of starch and fatty acids, which are essential for plant development and reproduction, and have a significant role in human nutrition. Unlike chloroplasts, all the metabolites for these processes in non-green plastids have to be imported via specific transport proteins. Recent advances in unravelling the molecular structures and substrate specificities of the transporters connecting the biochemical pathways between cytosol and stroma now make it possible to develop models for metabolic fluxes in these pathways. The basic principle of adapting the transport capacities of the plastid envelope to the physiological needs of the plant is the variable production of closely related transporters with overlapping substrate specificities.

Analysis of early morbidity and functional outcome of thulium: yttrium-aluminum-garnet laser enucleation for benign prostate enlargement: patient age and prostate size determine adverse surgical outcome.

OBJECTIVE: To evaluate complications and functional outcome and to identify patient-associated risk factors, we analyzed consecutive patients undergoing thulium:yttrium-aluminum-garnet laser enucleation of the prostate (ThuLEP) in our department. METHODS: A total of 234 patients were prospectively analyzed. Preoperative data, postoperative complications, and outcome at 6, 12, and 24 months were recorded. Individual risk factors for complications and treatment failure were assessed by univariate and multivariate analyses. RESULTS: Mean age at surgery was 72.88 ± 7.83 years. Mean preoperative prostate size was 84.8 ± 34.9 mL. Thirty-day complication rate was 19.7%. Functional treatment failure occurred in 9.0% of all patients. Decline of mean International Prostate Symptom Score was -75%, quality of life index -76%, and postvoid residual -86% at 24 months. Maximum urine flow at 24 months was improved at +231%. In univariate analysis, age >80 years and prostate size <50 mL were significant predictors of complications, which was confirmed by multivariate analysis (P = .0277 and .0409, respectively). Age >80 years, prostate size <80 mL or <50 mL, and American Society of Anesthesiologists classification were significant predictors of functional treatment failure in univariate analysis. Prostate size <80 mL or <50 mL was significantly associated with treatment failure (P < .001) in multivariate analysis. CONCLUSION: ThuLEP is a safe and efficient surgical procedure, even in a patient cohort with high prostate volumes, age, and comorbidities. However, high patient age and small prostate size were significant determinants of adverse outcomes after surgery. To address the question of optimal therapy selection for patients with prostates smaller than 80 mL, further prospective randomized evaluation of ThuLEP and alternative surgical interventions is needed.

The toxoplasma apicoplast phosphate translocator links cytosolic and apicoplast metabolism and is essential for parasite survival.

Apicomplexa are unicellular eukaryotic pathogens that carry a vestigial algal endosymbiont, the apicoplast. The physiological function of the apicoplast and its integration into parasite metabolism remain poorly understood and at times controversial. We establish that the Toxoplasma apicoplast membrane-localized phosphate translocator (TgAPT) is an essential metabolic link between the endosymbiont and the parasite cytoplasm. TgAPT is required for fatty acid synthesis in the apicoplast, but this may not be its most critical function. Further analyses demonstrate that TgAPT also functions to supply the apicoplast with carbon skeletons for additional pathways and, indirectly, with energy and reduction power. Genetic ablation of the transporter results in rapid death of parasites. The dramatic consequences of loss of its activity suggest that targeting TgAPT could be a viable strategy to identify antiparasitic compounds.

Comparative survey of plastid and mitochondrial targeting properties of transcription factors in Arabidopsis and rice.

A group of nuclear transcription factors, the Whirly proteins, were recently shown to be targeted also to chloroplasts and mitochondria. In order to find out whether other proteins might share this feature, an in silico-based screening of transcription factors from Arabidopsis and rice was carried out with the aim of identifying putative N-terminal chloroplast and mitochondrial targeting sequences. For this, the individual predictions of several independent programs were combined to a consensus prediction using a naïve Bayes method. This consensus prediction shows a higher specificity at a given sensitivity value than each of the single programs. In both species, transcription factors from a variety of protein families that possess putative N-terminal plastid or mitochondrial target peptides as well as nuclear localization sequences, were found. A search for homologues within members of the AP2/EREBP protein family revealed that target peptide-containing proteins are conserved among monocotyledonous and dicotyledonous species. Fusion of one of these proteins to GFP revealed, indeed, a dual targeting activity of this protein. We propose that dually targeted transcription factors might be involved in the communication between the nucleus and the organelles in plant cells. We further discuss how recent results on the physical interaction between the organelles and the nucleus could have significance for the regulation of the localization of these proteins.

Carbohydrate metabolism in the Toxoplasma gondii apicoplast: localization of three glycolytic isoenzymes, the single pyruvate dehydrogenase complex, and a plastid phosphate translocator.

Many apicomplexan parasites, such as Toxoplasma gondii and Plasmodium species, possess a nonphotosynthetic plastid, referred to as the apicoplast, which is essential for the parasites' viability and displays characteristics similar to those of nongreen plastids in plants. In this study, we localized several key enzymes of the carbohydrate metabolism of T. gondii to either the apicoplast or the cytosol by engineering parasites which express epitope-tagged fusion proteins. The cytosol contains a complete set of enzymes for glycolysis, which should enable the parasite to metabolize imported glucose into pyruvate. All the glycolytic enzymes, from phosphofructokinase up to pyruvate kinase, are present in the T. gondii genome, as duplicates and isoforms of triose phosphate isomerase, phosphoglycerate kinase, and pyruvate kinase were found to localize to the apicoplast. The mRNA expression levels of all genes with glycolytic products were compared between tachyzoites and bradyzoites; however, a strict bradyzoite-specific expression pattern was observed only for enolase I. The T. gondii genome encodes a single pyruvate dehydrogenase complex, which was located in the apicoplast and absent in the mitochondrion, as shown by targeting of epitope-tagged fusion proteins and by immunolocalization of the native pyruvate dehydrogenase complex. The exchange of metabolites between the cytosol and the apicoplast is likely to be mediated by a phosphate translocator which was localized to the apicoplast. Based on these localization studies, a model is proposed that explains the supply of the apicoplast with ATP and the reduction power, as well as the exchange of metabolites between the cytosol and the apicoplast.

Identification and expression analysis of twelve members of the nucleobase-ascorbate transporter (NAT) gene family in Arabidopsis thaliana.

By screening genome databases, 12 genes encoding membrane proteins homologous to nucleobase-ascorbate transporters (NATs) were identified in Arabidopsis thaliana. A similar number of genes was found in the rice genome. The plant NAT proteins split into five clades (I-V) based on protein multisequence alignments. This classification nicely correlates with the patterns of organ- and tissue-specific expression during the whole life cycle of A. thaliana. Interestingly, expression of two members of clade III, AtNAT7 and AtNAT8, was found to be up-regulated in undifferentiated tissues such as callus or tumors produced by Agrobacterium tumefaciens. Clade V comprises AtNAT12 possessing a hydrophilic N-terminal extension. Transient expression of green fluorescent protein (GFP) fusions in different systems showed that AtNAT12 along with AtNAT7 and -8 are located in the plasma membrane. Mutations in any of the AtNAT genes do not induce phenotypic alterations. The absence of obvious mutant phenotypes in single but also in double and triple mutants suggests a high degree of functional redundancy between AtNAT genes, but might also point to redundant functions provided by genes or pathways unrelated to the AtNATs.