Insight into the aroma quality of 'Callista' cultivar of hop (Humulus lupulus L.): Impact of harvest timing, year, and location.

Hops (Humulus lupulus L.) are essential ingredients in brewing, contributing to beer's flavor, aroma, and stability. This study pioneers an in-depth analysis of the 'Callista' cultivar, aiming to unravel how harvest timing, annual variations, and cultivation location synergistically influence its molecular profile, sensory perception, and biochemistry. Leveraging high-performance liquid chromatography and gas chromatography-mass spectrometry-olfactometry, we identified significant year-to-year and location-based fluctuations in bitter acids-the quintessential aroma constituents in hops. Our comprehensive aroma profiling discerned 55 volatile compounds, marking the first-ever sensory detection of 2-butanone in hops, with its presence showing remarkable interannual variability. This study showed significant differences among the three years tested, whereas hops were perceived "fruitier" and more "citrusy" in 2021, even though the bitter acid and aroma analysis showed that 2022 sticks out due to extremely high lupulone values up to 10% dry cone weight and 78% ß-myrcene in the oil fraction compared to 60% and 45% in 2020 and 2021, respectively. Molecular analysis of key enzymes involved in hop aroma biosynthesis revealed no significant associations with location, but a strong diurnal pattern for all genes. The results indicated that especially the hot temperatures of 2022 may have induced significant changes of cone quality, while 2021 was more interesting from the sensory evaluations, which may justify the usage of viticultural terms such as "vintage" for hop marketing. These findings contribute to a better understanding of the factors influencing hop aroma and quality.

The glycine decarboxylase complex is not essential for the cyanobacterium Synechocystis sp. strain PCC 6803.

In order to investigate the metabolic importance of glycine decarboxylase (GDC) in cyanobacteria, mutants were generated defective in the genes encoding GDC subunits and the serine hydroxymethyl-transferase (SHMT). It was possible to mutate the genes for GDC subunits P, T, or H protein in the cyanobacterial model strain Synechocystis sp. PCC 6803, indicating that GDC is not necessary for cell viability under standard conditions. In contrast, the SHMT coding gene was found to be essential. Almost no changes in growth, pigmentation, or photosynthesis were detected in the GDC subunit mutants, regardless of whether or not they were cultivated at ambient or high CO2 concentrations. The mutation of GDC led to an increased glycine/serine ratio in the mutant cells. Furthermore, supplementation of the medium with low glycine concentrations was toxic for the mutants but not for wild type cells. Conditions stimulating photorespiration in plants, such as low CO2 concentrations, did not induce but decrease the expression of the GDC and SHMT genes in Synechocystis. It appears that, in contrast to heterotrophic bacteria and plants, GDC is dispensable for Synechocystis and possibly other cyanobacteria.

Heterosides--compatible solutes occurring in prokaryotic and eukaryotic phototrophs.

The acclimation to osmotic and/or salt stress conditions induces an integrated response at different cellular levels. One acclimation strategy relies on the massive accumulation of low molecular mass compounds, so-called compatible solutes, to balance osmotic gradients and to directly protect critical macromolecules. Heterosides are compounds composed of a sugar and a polyol moiety that represent one chemical class of compatible solutes with interesting features. Well-investigated examples are glucosylglycerol, which is found in many cyanobacteria, and galactosylglycerols (floridoside and isofloridoside), which are accumulated by eukaryotic algae under salt stress conditions. Here, we review knowledge on physiology, biochemistry and genetics of heteroside accumulation in pro- and eukaryotic photoautotrophic organisms.

Transcriptional analysis of the isiAB operon in salt-stressed cells of the cyanobacterium Synechocystis sp. PCC 6803.

Expression of the isiA and isiB genes was analysed in the cyanobacterium Synechocystis sp. PCC 6803 grown in high salt or in iron-deficient medium. The detection of a 2.3-knt transcript in Northern blot experiments indicated cotranscription of isiAB in an operon, which was confirmed by reverse transcriptase PCR. The abundance of a monocistronic 1.25-knt isiA-specific mRNA was about 10-fold higher than the dicistronic message. The isiAB-specific transcripts were most abundant in cells adapted to 342 mM NaCl and under iron deficiency. The promoter of the operon was mapped to 211 bp upstream of the translational start. A putative Fur binding site was detected immediately upstream of the GTG start codon. A preliminary transcript of about 0.2 knt was detected in cells grown in conditions in which the isiAB operon was not transcribed. This indicates that a repressor binds to the identified Fur binding site and thus inhibits isiAB transcription under low salt and iron replete conditions.

The iron-regulated isiA gene of Fischerella muscicola strain PCC 73103 is linked to a likewise regulated gene encoding a Pcb-like chlorophyll-binding protein.

The expression of the chlorophyll a-binding, iron stress-induced protein IsiA is part of the cyanobacterial response to iron deficiency. A new isiA gene from the filamentous heterocystous cyanobacterial strain, Fischerella muscicola PCC 73103, was identified using standard and inverse PCR. While in unicellular cyanobacterial strains isiA is organized in an operon with isiB (encoding flavodoxin), in Fischerella not an isiB gene but another chlorophyll-binding protein encoding gene was identified downstream of isiA, which shows significant similarities to Pcb-like protein encoding genes known from prochlorophytes. The expression of both genes was clearly activated under iron deficiency. Although isiA and pcbC were independently transcribed, the size of the pcbC transcript indicates a large iron-regulated operon. Beside a 10-fold increase of isiA transcript content iron-starved cells of Fischerella showed a blue-shift in the red chlorophyll a absorption peak. In addition, chlorophyll fluorescence at 77 K was dominated by an emission peak at 685 nm. These features are in accordance with the characteristics of IsiA accumulation in iron-starved unicellular cyanobacteria, suggesting identical IsiA function in heterocystous strains in spite of different genetic organization.

Glucosylglycerol-phosphate synthase: target for ion-mediated regulation of osmolyte synthesis in the cyanobacterium synechocystis sp. strain PCC 6803

The response of cyanobacteria to a changing osmotic environment includes the accumulation of organic osmolytes such as glucosylglycerol. The activation of the enzymes involved in glucosylglycerol synthesis [glucosylglycerol-phosphate synthase (GGPS) and glucosylglycerol-phosphate phosphatase (GGPP)] in Synechocystis sp. strain PCC 6803 by various salts and salt concentrations was investigated in vitro. GGPS seemed to be the target for salt-mediated regulation of glucosylglycerol synthesis in vitro. GGPS activation was dependent on the concentration of NaCl, and a sigmoidal plot was obtained. Sensitivity to NaCl was markedly enhanced by low Mg+2 concentrations (optimal at 4 mM), but Mg2+ was not absolutely necessary for the Na+ stimulation. As in the case of NaCl, other salts (including MgCl2) stimulated GGPS. The relative order of GGPS activation in the presence of chloride by the cations at constant ionic strength was Li+ > Na+ > K+, Mg2+ Mn2+. No absolute dependence on ionic strength was observed in Mg2+/Na+-exchange experiments. The degree of activation by ions at various concentrations was positively related to the increasing destabilizing properties of the cations according to the Hofmeister rule, where chaotropic cations are most efficient. Cations were responsible for activation since chaotropic anions counteracted the activating effect of cations.

Construction of promoter probe vectors for Synechocystis sp. PCC 6803 using the light-emitting reporter systems Gfp and LuxAB.

Two promoter probe vectors were constructed for the cyanobacterium Synechocystis sp. strain PCC 6803 using reporter genes, which can be easily detected and quantified in vivo by the ability of their encoded proteins to emit light. The vectors allow the transcriptional fusion of promoter sequences with the gfp and luxAB genes, respectively, and their stable integration into a neutral site of the Synechocystis chromosome. Functionality of these vectors was demonstrated by cloning the promoter of the isiAB operon into both promoter probe vectors and analyzing the stress-dependent emission of light by the obtained reporter strains. As was found before for the isiAB operon, the P(isiAB) reporter gene fusions were induced by iron starvation and high salt stress. Induction rates of mRNA of the wild type operon and the reporter gene fusions were found to be essentially the same, indicating that a promoter fragment containing all necessary regulatory elements has been cloned. However, using the gfp gene a slow increase of protein and fluorescence was found, while the luxAB reporter gene constructs led to a rapid increase in luminescence. The same was found after retransfer of cells back into control media, in which the Gfp protein disappeared slowly, while the LuxAB-based luminescence decreased rapidly. These experiments show that both reporter genes can be used in Synechocystis: the luxAB system seems to be favourable regarding reaction time, while the gfp system has the advantage of being independent from any substrate.

Molecular analysis of the coronavirus-receptor function of aminopeptidase N.

Aminopeptidase N (APN) is a major cell surface for coronaviruses of the serogroup I. By using chimeric APN proteins assembled from human, porcine and feline APN we have identified determinants which are critically involved in the coronavirus-APN interaction. Our results indicate that human coronavirus 229E (HCV 229E) is distinct from the other serogroup I coronaviruses in that determinants located within the N-terminal parts of the human and feline APN proteins mediate the infection of HCV 229E, whereas determinants located within the C-terminal parts of porcine, feline and canine APN mediate the infection of transmissible gastro-enteritis virus (TGEV), feline infectious peritonitis virus (FIPV) and canine coronavirus (CCV), respectively. A further analysis of the mapped amino acid segments by site directed mutagenesis revealed that a short stretch of 8 amino acids in the hAPN protein plays a decisive role in mediating HCV 229E reception.

Does the Cyanophora paradoxa genome revise our view on the evolution of photorespiratory enzymes?

In the present-day O2 -rich atmosphere, the photorespiratory pathway is essential for organisms performing oxygenic photosynthesis; i.e. cyanobacteria, algae and land plants. The presence of enzymes for the plant-like 2-phosphoglycolate cycle in cyanobacteria indicates that, together with oxygenic photosynthesis, genes for photorespiratory enzymes were endosymbiotically conveyed from ancient cyanobacteria to photosynthetic eukaryotes. The genome information for Cyanophora paradoxa, a member of the Glaucophyta representing the first branching group of primary endosymbionts, and for many other eukaryotic algae was used to shed light on the evolutionary relationship of photorespiratory enzymes among oxygenic phototrophs. For example, it became possible to analyse the phylogenies of 2-phosphoglycolate phosphatase, serine:glyoxylate aminotransferase and hydroxypyruvate reductase. Analysis of the Cyanophora genome provided clear evidence that some photorespiratory enzymes originally acquired from cyanobacteria were lost, e.g. glycerate 3-kinase, while others were replaced by the corresponding enzymes from the α-proteobacterial endosymbiont, e.g. serine:glyoxylate aminotransferase. Generally, our analysis supports the view that many C2 cycle enzymes in eukaryotic phototrophs were obtained from the cyanobacterial endosymbiont, but during the subsequent evolution of algae and land plants multiple losses and replacements occurred, which resulted in a reticulate provenance of photorespiratory enzymes with different origins in different cellular compartments.

Evolution of the biochemistry of the photorespiratory C2 cycle.

Oxygenic photosynthesis would not be possible without photorespiration in the present day O2 -rich atmosphere. It is now generally accepted that cyanobacteria-like prokaryotes first evolved oxygenic photosynthesis, which was later conveyed via endosymbiosis into a eukaryotic host, which then gave rise to the different groups of algae and streptophytes. For photosynthetic CO2 fixation, all these organisms use RubisCO, which catalyses both the carboxylation and the oxygenation of ribulose 1,5-bisphosphate. One of the reaction products of the oxygenase reaction, 2-phosphoglycolate (2PG), represents the starting point of the photorespiratory C2 cycle, which is considered largely responsible for recapturing organic carbon via conversion to the Calvin-Benson cycle (CBC) intermediate 3-phosphoglycerate, thereby detoxifying critical intermediates. Here we discuss possible scenarios for the evolution of this process toward the well-defined 2PG metabolism in extant plants. While the origin of the C2 cycle core enzymes can be clearly dated back towards the different endosymbiotic events, the evolutionary scenario that allowed the compartmentalised high flux photorespiratory cycle is uncertain, but probably occurred early during the algal radiation. The change in atmospheric CO2 /O2 ratios promoting the acquisition of different modes for inorganic carbon concentration mechanisms, as well as the evolutionary specialisation of peroxisomes, clearly had a dramatic impact on further aspects of land plant photorespiration.

Perspectives on plant photorespiratory metabolism.

Being intimately intertwined with (C3) photosynthesis, photorespiration is an incredibly high flux-bearing pathway. Traditionally, the photorespiratory cycle was viewed as closed pathway to refill the Calvin-Benson cycle with organic carbon. However, given the network nature of metabolism, it hence follows that photorespiration will interact with many other pathways. In this article, we review current understanding of these interactions and attempt to define key priorities for future research, which will allow us greater fundamental comprehension of general metabolic and developmental consequences of perturbation of this crucial metabolic process.

Ecological genomics of marine picocyanobacteria.

Marine picocyanobacteria of the genera Prochlorococcus and Synechococcus numerically dominate the picophytoplankton of the world ocean, making a key contribution to global primary production. Prochlorococcus was isolated around 20 years ago and is probably the most abundant photosynthetic organism on Earth. The genus comprises specific ecotypes which are phylogenetically distinct and differ markedly in their photophysiology, allowing growth over a broad range of light and nutrient conditions within the 45 degrees N to 40 degrees S latitudinal belt that they occupy. Synechococcus and Prochlorococcus are closely related, together forming a discrete picophytoplankton clade, but are distinguishable by their possession of dissimilar light-harvesting apparatuses and differences in cell size and elemental composition. Synechococcus strains have a ubiquitous oceanic distribution compared to that of Prochlorococcus strains and are characterized by phylogenetically discrete lineages with a wide range of pigmentation. In this review, we put our current knowledge of marine picocyanobacterial genomics into an environmental context and present previously unpublished genomic information arising from extensive genomic comparisons in order to provide insights into the adaptations of these marine microbes to their environment and how they are reflected at the genomic level.

[Salivary stones and stenosis. A comprehensive classification]. / Lithiases et sténoses salivaires. Une classification pratique des pathologies non tumorales.

INTRODUCTION: Sialendoscopy and sialo-MRI enable diagnosis of salivary gland obstructive pathologies, such as lithiasis, stenosis and dilatations. Therefore, a classification of these pathologies is needed, allowing large series comparisons, for better diagnosis and treatment of salivary pathologies. MATERIAL AND METHODS: With help from people from the European Sialendoscopy Training Center (ESTC), the results of sialographies, sialoMRI and sialendoscopies, a comprehensive classification of obstructive salivary pathologies is described, based on the absence or presence of lithiasis (L), stenosis (S) and dilatation (D) ("LSD" classification). DISCUSSION: It appears that a classification of salivary gland obstructive pathologies should be described. We hope it will be widely used and of course criticized to be improved and to compare the results of salivary gland diagnostic methods, such as sialography and sialendoscopy and also the results and indications for salivary gland therapeutic methods, such as lithotripsy, sialendoscopy and/or open surgery.

Salivary stones and stenosis. A comprehensive classification.

INTRODUCTION: Sialendoscopy and sialoMRI enables diagnosis of salivary gland obstructive pathologies, such as lithiasis, stenosis, and dilatations. Therefore, a classification of these pathologies is needed, allowing large series comparisons, for better diagnosis and treatment of salivary pathologies. MATERIAL AND METHODS: With help from people from the European Sialendoscopy Training Center (ESTC), the results of sialographies, sialoMRI and sialendoscopies, a comprehensive classification of obstructive salivary pathologies is described, based on the absence or presence of lithiasis (L), stenosis (S), and dilatation (D) ("LSD" classification). DISCUSSION: It appears that a classification of salivary gland obstructive pathologies should be described. We hope it will be widely used and of course criticized to be improved and to compare the results of salivary gland diagnostic methods, such as sialography and sialendoscopy, and also the results and indications for salivary gland therapeutic methods, such as lithotripsy, sialendoscopy, and/or open surgery.

[Tympanoplasty with adipose tissue]. / Tympanoplastik mit autologem Fettgewebe.

BACKGROUND: Among other materials it is also possible to use autologous fat tissue for closing tympanic membrane perforations. In this study we have evaluated 44 consecutive myringoplasties with adipose tissue performed between 1999 and 2001. MATERIAL AND METHOD: The indications were residual microperforations following tympanoplasty with temporalis fascia or tympanic membrane perforations due to trauma or chronic otitis media simplex. Myringoplasty with fat tissue was performed as an outpatient procedure and took about 15 minutes. The adipose tissue was harvested from the posterior side of the ear lobe in local anaesthesia. After refreshing the borders of the tympanic membrane perforation with a micro hook, the adipose tissue was positioned into the perforation by using a handheld or fixed ear speculum. The graft was covered with a silk strip soaked with Garamycine ointment. In bigger perforations a bed of gelfoam was put into the tympanic cavity in order to avoid adhesions between the graft and the promontorium. RESULTS: A permanent healing of the tympanic membrane was achieved in 40 (91 %) out of the 44 patients. In 21 patients hearing improved between 5 -10 dB. Surgical complications did not occur. CONCLUSIONS: Our results indicate that transcanal myringoplasty with adipose tissue is a simple and minimally invasive method for closing small to medium sized tympanic membrane perforations.

Identification of salt-regulated genes in the genome of the cyanobacterium Synechocystis sp. strain PCC 6803 by subtractive RNA hybridization.

To identify genes transcribed preferentially under salt stress, a subtractive RNA hybridization procedure was applied to the cyanobacterium Synechocystis sp. PCC 6803. The screening of a genomic library led to the identification of several RNA species that were more abundant in salt-stressed cells than in control cells. Salt-dependent transcription of the identified genes was verified in Northern blot experiments. In addition to the previously characterized genes cpn60 (encoding GroEL; a molecular chaperone) and isiA (encoding a chlorophyll-binding protein), genes encoding a protein of unknown function (slr0082) and a putative RNA helicase (slr0083) were identified as salt-regulated genes in Synechocystis. Genes slr0082 and slr0083, located at sites adjacent to each other on the Synechocystis chromosome, were transcribed from separate promoters and showed the most significant induction 1-3 h after salt shock. The salt-regulated promoters of these two genes were mapped. Genes cpn60, slr0082, and slr0083 were also found to be induced by a cold shock. The possible role of the identified gene products for salt adaptation of Synechocystis is discussed.

Molecular analysis of the ggtBCD gene cluster of Synechocystis sp. strain PCC6803 encoding subunits of an ABC transporter for osmoprotective compounds.

Genes encoding a substrate-binding protein (ggtB) and two integral membrane proteins (ggtC and ggtD) of the binding-protein-dependent ABC transporter for glucosylglycerol were identified in the genome of Synechocystis sp. strain PCC6803. These genes are clustered on the chromosome about 220 kb away from the previously identified ggtA gene, which encodes the ATP-binding protein of this transport system. The deduced amino acid sequences show significant similarities to corresponding subunits of ABC transporters mediating uptake of maltose and other di- and oligosaccharides in bacteria and archaea. Mutants were constructed by inserting an aphII gene cassette into the coding region of the ggtB, ggtC and ggtD genes. These mutants lost the ability to take up glucosylglycerol, sucrose and trehalose, proving that these compounds are transported by the same system. A truncated ggtB gene lacking the putative signal-peptide-encoding sequence was expressed in Escherichia coli yielding a histidine-tagged soluble protein. The recombinant GgtB protein bound glucosylglycerol with a KD of 0.45 microM and exhibited a somewhat lower affinity towards sucrose and a substantially lower affinity towards trehalose. Transcript analysis by RT-PCR indicated that the genes of the ggtBCD gene cluster form an operon. The transcript level estimated by RNA slot blot analysis using a ggtC-specific probe was very low in cells grown in basal medium but increased significantly after a salt shock.

Uptake and use of the osmoprotective compounds trehalose, glucosylglycerol, and sucrose by the cyanobacterium Synechocystis sp. PCC6803.

Accumulation of exogenously supplied osmoprotective compounds was analyzed in the cyanobacterium Synechocystis sp. PCC6803, which synthesizes glucosylglycerol as the principal osmoprotective compound. Glucosylglycerol and trehalose were accumulated to high levels and protected cells of a mutant unable to synthesize glucosylglycerol against the deleterious effects of salt stress. In the wild-type, uptake of trehalose repressed the synthesis of glucosylglycerol and caused metabolic conversion of originally accumulated glucosylglycerol. Trehalose cannot be synthesized by Synechocystis and was not or only insignificantly metabolized. Sucrose, which can be synthesized in low quantities by Synechocystis, was also taken up, as indicated by its disappearance from the medium. Sucrose was not accumulated to high levels, probably due to a sucrose-degrading activity found in cells adapted to both low- and high-salt conditions. Despite its low intracellular concentration, sucrose showed a weak osmoprotective effect in salt-shocked cells of a mutant unable to synthesize glucosylglycerol.