Aluminum induced proteome changes in tomato cotyledons

Cotyledons of tomato seedlings that germinated in a 20 µM AlK(SO₄)₂ solution remained chlorotic while those germinated in an aluminum free medium were normal (green) in color. Previously, we have reported the effect of aluminum toxicity on root proteome in tomato seedlings (Zhou et al.¹). Two dimensional DIGE protein analysis demonstrated that Al stress affected three major processes in the chlorotic cotyledons: antioxidant and detoxification metabolism (induced), glyoxylate and glycolytic processes (enhanced), and the photosynthetic and carbon fixation machinery (suppressed).Key words: aluminum, cotyledons, proteome, tomatoDifferent biochemical processes occur depending on the developmental stages of cotyledons. During early seed germination, before the greening of the cotyledons, glyoxysomes enzymes are very active. Fatty acids are converted to glucose via the gluconeogenesis pathway.^2,3 In greening cotyledons, chloroplast proteins for photosynthesis and leaf peroxisomal enzymes in the glycolate pathway for photorespiration are metabolized.^2–4 Enzymes involved in regulatory mechanisms such as protein kinases, protein phosphatases, and mitochondrial enzymes are highly expressed.^3,5,6The chlorotic cotyledons are similar to other chlorotic counterparts in that both contains lower levels of chlorophyll, thus the photosynthetic activities are not as active. In order to understand the impact of Al on tomato cotyledon development, a comparative proteome analysis was performed using 2D-DIGE following the as previously described procedure.¹ Some proteins accumulated differentially in Al-treated (chlorotic) and untreated cotyledons (Fig. 1). Mass spectrometry of tryptic digestion fragments of the proteins followed by database search has identified some of the differentially expressed proteins (Open in a separate windowFigure 1Image of protein spots generated by Samspot analysis of Al treated and untreated tomato cotyledons proteomes separated on 2D-DIGE.

Table 1

Proteins identified from tomato cotyledons of seeds germinating in Al-solution

Diminished Susceptibility to Cefoperazone/Sulbactam and Piperacillin/Tazobactam in Enterobacteriaceae Due to Narrow-Spectrum β-Lactamases as Well as Omp Mutation

Cefoperazone/sulbactam (CSL) and piperacillin/tazobactam (TZP) are commonly used in clinical practice in China because of their excellent antimicrobial activity. CSL and TZP-nonsusceptible Enterobacteriaceae are typically resistant to extended-spectrum cephalosporins such as ceftriaxone (CRO). However, 11 nonrepetitive Enterobacteriaceae strains, which were resistant to CSL and TZP yet susceptible to CRO, were collected from January to December 2020. Antibiotic susceptibility tests and whole-genome sequencing were conducted to elucidate the mechanism for this rare phenotype. Antibiotic susceptibility tests showed that all isolates were amoxicillin/clavulanic-acid resistant and sensitive to ceftazidime, cefepime, cefepime/tazobactam, cefepime/zidebactam, ceftazidime/avibactam, and ceftolozane/tazobactam. Whole-genome sequencing revealed three of seven Klebsiella pneumoniae strains harbored bla_SHV-1 only, and four harbored bla_SHV-1 and bla_TEM-1B. Two Escherichia coli strains carried bla_TEM-1B only, while two Klebsiella oxytoca isolates harbored bla_OXY-1-3 and bla_OXY-1-1, respectively. No mutation in the β-lactamase gene and promoter sequence was found. Outer membrane protein (Omp) gene detection revealed that numerous missense mutations of OmpK36 and OmpK37 were found in all strains of K. pneumoniae. Numerous missense mutations of OmpK36 and OmpK35 and OmpK37 deficiency were found in one K. oxytoca strain, and no OmpK gene was found in the other. No Omp mutations were found in E. coli isolates. These results indicated that narrow spectrum β-lactamases, TEM-1, SHV-1, and OXY-1, alone or in combination with Omp mutation, contributed to the resistance to CSL and TZP in CRO-susceptible Enterobacteriaceae.Antibiotic susceptibility tests

Use of methotrexate therapy is not associated with decreased prevalence of metabolic syndrome

With great interest, we read the article by Toms and colleagues [1] in the previous issue of Arthritis Research & Therapy, in which they assessed prevalences of metabolic syndrome (MetS) in rheumatoid arthritis (RA) patients. Moreover, they identified demographic and clinical factors that may be associated with MetS. Toms and colleagues found prevalences of up to 45% of MetS and demonstrated older age and health status (health assessment questionnaire) to be associated with MetS irrespectively of the definition used. Of most interest, an association between methotrexate (MTX) use and decreased presence of MetS was observed in patients more than 60 years of age. The investigators hypothesized that this may be attributed to a drug-specific effect (and not to an anti-inflammatory effect) either by changing levels of adenosine, which is known to interact with glucose and lipid metabolism, or by an indirect effect mediated through concomitant folic acid administration, thereby decreasing homocysteine levels.Recently, we also examined the prevalence of MetS in (a subgroup of) RA patients in the CARRÉ investigation, a prospective cohort study on prevalent and incident cardiovascular disease and its underlying cardiovascular risk factors [2]. The findings of Toms and colleagues stimulated us to perform additional analyses in our total study population (n = 353).The prevalences of MetS were 35% and 25% (Table ​(Table1)1) according to criteria of National Cholesterol Education Program (NCEP) 2004 and NCEP 2001, respectively. In multivariate backward regression analyses, we found significant associations between body mass index, pulse rate, creatinine levels, hypothyroidism and diabetes mellitus and the presence of MetS independently of the criteria used (Table ​(Table2).2). However, an independent association between single use of MTX or use of MTX in combination with other disease-modifying antirheumatic drugs, on the one hand, and a decreased prevalence of MetS, on the other hand, could not be demonstrated (even in the subgroup of patients over the age of 60).

Table 1

Characteristics of the study population

Allelic frequency and genotypes of prion protein at codon 136 and 171 in Iranian Ghezel sheep breeds

PrP genotypes at codons 136 and 171 in 120 Iranian Ghezel sheep breeds were studied using allele-specific PCR amplification and compared with the well-known sheep breeds in North America, the United States and Europe. The frequency of V allele and VV genotype at codon 136 of Ghezel sheep breed was significantly lower than AA and AV. At codon 171, the frequency of allele H was significantly lower than Q and R. Despite the similarities of PrP genotypes at codons 136 and 171 between Iranian Ghezel sheep breeds and some of the studied breeds, significant differences were found with others. Planning of effective breeding control and successful eradication of susceptible genotypes in Iranian Ghezel sheep breeds will not be possible unless the susceptibility of various genotypes in Ghezel sheep breeds to natural or experimental scrapie has been elucidated.Key words: scrapie, Ghezel sheep breed, PrP genotyping, allele specific amplification, codon 136, codon 171Scrapie was first described in England in 1732,¹ and it is an infectious neurodegenerative fatal disease of sheep and goats belonging to the group of transmissible subacute spongiform encephalopathies (TSEs), along with bovine spongiform encephalopathy (BSE), chronic wasting disease and Creutzfeldt-Jakob disease.^2,3 The term prion, proteinaceous infectious particles, coined by Stanley B. Prusiner, was introduced, and he presents the idea that the causal agent is a protein.⁴ Prion proteins are discovered in two forms, the wild-type form (PrP^c) and the mutant form (PrP^Sc).⁵ Although scrapie is an infectious disease, the susceptibility of sheep is influenced by genotypes of the prion protein (PrP) gene.^2,6 Researchers have found that the PrP allelic variant alanine/arginine/arginine (ARR) at codons 136, 154 and 171 is associated with resistance to scrapie in several breeds.^7–14 Most of the sheep populations in the Near East and North African Region (84% of the total population of 255 million) are raised in Iran, Turkey, Pakistan, Sudan, Algeria, Morocco, Afghanistan, Syria and Somalia.¹⁵ In 2003, the Iranian sheep population was estimated at 54,000,000 head. The Ghezel sheep breed, which also is known as Kizil-Karaman, Mor-Karaman, Dugli, Erzurum, Chacra, Chagra, Chakra, Gesel, Gezel, Kazil, Khezel, Khizel, Kizil, Qezel, Qizil and Turkish Brown, originated in northwestern Iran and northeastern Turkey. By considering sheep breeds as one of the main sources of meat, dairy products and related products, a global screening attempt is started in different areas. In compliance with European Union Decision 2003/100/EC, each member state has introduced a breeding program to select for resistance to TSEs in sheep populations to increase the frequency of the ARR allele. A similar breeding program is established in United States and Canada. The Near East and North African Region still needs additional programs to help the global plan of eradication of scrapie-susceptible genotypes. The current study was the first to assess the geographical and molecular variation of codons 136 and 171 polymorphism between Iranian Ghezel sheep breed and well-known sheep breeds.Polymorphism at codon 136 is associated with susceptibility to scrapie in both experimental and natural models.^10,11,13,16 17 and Austrian Carynthian sheep.¹⁸ Swiss White Alpine showed higher frequency of allele V at position 136 than Swiss Oxford Down, Swiss Black-Brown Mountain and Valais Blacknose.¹⁹ Comparison of polymorphism at codon 136 in the current study with some of other breeds (20 some flock of Hampshire sheep²¹ with current study, but the frequency of it is higher than that of some other breeds.

Table 1

Comparison of PrP allelic and genotype frequencies at codon 136 in different breeds

Genome-wide analysis of lipoxygenase gene family in Arabidopsis and rice

The enzymes called lipoxygenases (LOXs) can dioxygenate unsaturated fatty acids, which leads to lipoperoxidation of biological membranes. This process causes synthesis of signaling molecules and also leads to changes in cellular metabolism. LOXs are known to be involved in apoptotic (programmed cell death) pathway, and biotic and abiotic stress responses in plants. Here, the members of LOX gene family in Arabidopsis and rice are identified. The Arabidopsis and rice genomes encode 6 and 14 LOX proteins, respectively, and interestingly, with more LOX genes in rice. The rice LOXs are validated based on protein alignment studies. This is the first report wherein LOXs are identified in rice which may allow better understanding the initiation, progression and effects of apoptosis, and responses to bitoic and abiotic stresses and signaling cascades in plants.Key words: apoptosis, biotic and abiotic stresses, genomics, jasmonic acid, lipidsLipoxygenases (linoleate:oxygen oxidoreductase, EC 1.13.11.-; LOXs) catalyze the conversion of polyunsaturated fatty acids (lipids) into conjugated hydroperoxides. This process is called hydroperoxidation of lipids. LOXs are monomeric, non-heme and non-sulfur, but iron-containing dioxygenases widely expressed in fungi, animal and plant cells, and are known to be absent in prokaryotes. However, a recent finding suggests the existence of LOX-related genomic sequences in bacteria but not in archaea.¹ The inflammatory conditions in mammals like bronchial asthama, psoriasis and arthritis are a result of LOXs reactions.² Further, several clinical conditions like HIV-1 infection,³ disease of kidneys due to the activation of 5-lipoxygenase,^4,5 aging of the brain due to neuronal 5-lipoxygenase⁶ and atherosclerosis⁷ are mediated by LOXs. In plants, LOXs are involved in response to biotic and abiotic stresses.⁸ They are involved in germination⁹ and also in traumatin and jasmonic acid biochemical pathways.^10,11 Studies on LOX in rice are conducted to develop novel strategies against insect pests¹² in response to wounding and insect attack,¹³ and on rice bran extracts as functional foods and dietary supplements for control of inflammation and joint health.¹⁴ In Arabidopsis, LOXs are studied in response to natural and stress-induced senescence,¹⁵ transition to flowering,¹⁶ regulation of lateral root development and defense response.¹⁷The arachidonic, linoleic and linolenic acids can act as substrates for different LOX isozymes. A hydroperoxy group is added at carbons 5, 12 or 15, when arachidonic acid is the substrate, and so the LOXs are designated as 5-, 12- or 15-lipoxygenases. Sequences are available in the database for plant lipoxygenases (EC:1.13.11.12), mammalian arachidonate 5-lipoxygenase (EC:1.13.11.34), mammalian arachidonate 12-lipoxygenase (EC:1.13.11.31) and mammalian erythroid cell-specific 15-lipoxygenase (EC:1.13.11.33). The prototype member for LOX family, LOX-1 of Glycine max L. (soybean) is a 15-lipoxygenase. The LOX isoforms of soybean (LOX-1, LOX-2, LOX-3a and LOX-3b) are the most characterized of plant LOXs.¹⁸ In addition, five vegetative LOXs (VLX-A, -B, -C, -D, -E) are detected in soybean leaves.¹⁹ The 3-dimensional structure of soybean LOX-1 has been determined.^20,21 LOX-1 was shown to be made of two domains, the N-terminal domain-I which forms a β-barrel of 146 residues, and a C-terminal domain-II of bundle of helices of 693 residues²¹ (Fig. 1). The iron atom was shown to be at the centre of domain-II bound by four coordinating ligands, of which three are histidine residues.²²Open in a separate windowFigure 1Three-dimensional structure of soybean lipoxygenase L-1. The domain I (N-terminal) and domain II (C-terminal) are indicated. The catalytic iron atom is embedded in domain II (PDB ID-1YGE).²¹This article describes identification of LOX genes in Arabidopsis and rice. The Arabidopsis genome encodes for six LOX proteins²³ (www.arabidopsis.org) (LocusAnnotationNomenclatureA^*B^*C^*AT1G55020lipoxygenase 1 (LOX1)LOX185998044.45.2049AT1G17420lipoxygenase 3 (LOX3)LOX3919103725.18.0117AT1G67560lipoxygenase family proteinLOX4917104514.68.0035AT1G72520lipoxygenase, putativeLOX6926104813.17.5213AT3G22400lipoxygenase 5 (LOX5)LOX5886101058.86.6033AT3G45140lipoxygenase 2 (LOX2)LOX2896102044.75.3177Open in a separate window^*A, amino acids; B, molecular weight; C, isoelectric point.Interestingly, the rice genome (rice.plantbiology.msu.edu) encodes for 14 LOX proteins as compared to six in Arabidopsis (​and22). Of these, majority of them are composed of ∼790–950 aa with the exception for loci, LOC_Os06g04420 (126 aa), LOC_Os02g19790 (297 aa) and LOC_Os12g37320 (359 aa) (Fig. 2).Open in a separate windowFigure 2Protein alignment of rice LOXs and vegetative lipoxygenase, VLX-B,²⁸ a soybean LOX (AA B67732). The 14 rice LOCs are indicated on left and sequence position on right. Gaps are included to improve alignment accuracy. Figure was generated using ClustalX program.

Table 2

Genes encoding lipoxygenases in rice

Genome-wide analysis of thioredoxin fold superfamily peroxiredoxins in Arabidopsis and rice

A broad range of peroxides generated in subcellular compartments, including chloroplasts, are detoxified with peroxidases called peroxiredoxins (Prx). The Prx are ubiquitously distributed in all organisms including bacteria, fungi, animals and also in cyanobacteria and plants. Recently, the Prx have emerged as new molecules in antioxidant defense in plants. Here, the members which belong to Prx gene family in Arabidopsis and rice are been identified. Overall, the Prx members constitute a small family with 10 and 11 genes in Arabidopsis and rice respectively. The prx genes from rice are assigned to their functional groups based on homology search against Arabidopsis protein database. Deciphering the Prx functions in rice will add novel information to the mechanism of antioxidant defense in plants. Further, the Prx also forms the part of redox signaling cascade. Here, the Prx gene family has been described for rice.Key words: antioxidant defense, chloroplast, gene family, oxidative stress, reactive oxygen speciesThe formation of free radicals and reactive oxygen species (ROS) occur in several enzymatic and non-enzymatic reactions during cellular metabolism. The accumulation of these reactive and deleterious intermediates is suppressed by antioxidant defense mechanism comprised of low molecular weight antioxidants and enzymes. In photosynthetic organisms, the defense against the damage from free radicals and oxidative stress is crucial. For instance, the ROS production occurs in photosystem II with generation of singlet oxygen (¹O₂) and hydrogen peroxide (H₂O₂),^1,2 photosystem I from superoxide anion radicals (O₂⁻),³ and during photorespiration with generation of H₂O₂.⁴ ROS production may exceed under environmental stress conditions like excess light, low temperature and drought.⁵The antioxidant defense mechanism is activated by antioxidant metabolities and enzymes which detoxify ROS and lipid peroxides. The detoxification of ROS can occur in various cellular compartments such as chloroplasts, mitochondria, peroxisomes and cytosol.⁶ The enzymes like ascorbate peroxidase, catalase, glutathione peroxidase and superoxide dismutase are prominent antioxidant enzymes.⁶ The peroxiredoxins (Prx) emerged as new components in the antioxidant defense network of barley.^7,8 Later, Prx were studied in other plants.^9–14Prx can be classified into four different functional groups, PrxQ, 1-Cys Prx, 2-Cys Prx and Type-2 Prx.^15,16 They are members of the thioredoxin fold superfamily.^17,18 In this study, the prx genes found in Arabidopsis and rice genomes are been identified. The Arabidopsis genome encodes 10 prx genes classified into four functional categories, 1-Cys Prx, 2-Cys Prx, PrxQ and Type-2 Prx.¹³ Of these, one each of 1-Cys Prx and PrxQ, two of 2-Cys Prx (2-Cys PrxA and 2-Cys PrxB) and six Type-2 Prx (PrxA–F) are identified¹³ (LocusAnnotationSynonymA^*B^*C^*AT1G481301-Cysteine peroxiredoxin 1 (ATPER1)1-Cys Prx21624081.36.603AT1G60740Peroxiredoxin type 2Type-2 PrxD16217471.95.2297AT1G65970Thioredoxin-dependent peroxidase 2 (TPX2)Type-2 PrxC16217413.95.2297AT1G65980Thioredoxin-dependent peroxidase 1 (TPX1)Type-2 PrxB16217427.84.9977AT1G65990Type 2 peroxiredoxin-relatedType-2 PrxA55362653.66.4368AT3G06050Peroxiredoxin IIF (PRXIIF)Type-2 PrxF20121445.29.3905AT3G116302-Cys Peroxiredoxin A (2CPA, 2-Cys PrxA)2-Cys PrxA26629091.77.5686AT3G26060ATPRX Q, periredoxin QPrxQ21623677.810.0565AT3G52960Peroxiredoxin type 2Type-2 PrxE23424684.09.572AT5G062902-Cysteine Peroxiredoxin B (2CPB, 2-Cys PrxB)2-Cys PrxB27329779.55.414Open in a separate window^*A, amino acids; B, molecular weight; C, isoelectric point.In rice (rice.plantbiology.msu.edu/), there are 11 genomic loci which encode for Prx proteins (​and33). Interestingly, a new prx gene (LOC_Os07g15670) annotated as “peroxiredoxin, putative, expressed” is identified making the tally of prx genes to eleven in rice as compared to ten in Arabidopsis (​and22). The BLAST search has identified its counterpart in Arabidopsis which has been annotated as “antioxidant/oxidoreductase” (AT1G21350) in the TAIR database (www.arabidopsis.org). The rice LOC_Os07g15670 and Arabidopsis AT1G21350 share protein homology %68/78 for 236 amino acids (ChromosomeLocus IdPutative function/AnnotationA^*B^*C^*1LOC_Os01g16152peroxiredoxin, putative, expressed19920873.68.22091LOC_Os01g24740peroxiredoxin-2E-1, chloroplast precursor, putative10711591.56.79061LOC_Os01g48420peroxiredoxin, putative, expressed16317290.85.68282LOC_Os02g09940peroxiredoxin, putative, expressed22623179.56.5352LOC_Os02g33450peroxiredoxin, putative, expressed26228096.95.77094LOC_Os04g339702-Cys peroxiredoxin BAS1, chloroplast precursor, putative, expressed12213410.24.37056LOC_Os06g09610peroxiredoxin, putative, expressed2662892610.50976LOC_Os06g42000peroxiredoxin, putative, expressed23323688.39.20597LOC_Os07g15670peroxiredoxin, putative, expressed25327684.69.85457LOC_Os07g44440peroxiredoxin, putative, expressed22124232.65.36187LOC_Os07g44430peroxiredoxin, putative25627785.36.8544Open in a separate window^*A, amino acids; B, molecular weight; C, isoelectric point.

Table 3

Identification of rice homologs of peroxiredoxins in A. thaliana

Escherichia coli O157:H7 and Other E. coli Strains Share Physiological Properties Associated with Intestinal Colonization

Escherichia coli isolates (72 commensal and 10 O157:H7 isolates) were compared with regard to physiological and growth parameters related to their ability to survive and persist in the gastrointestinal tract and found to be similar. We propose that nonhuman hosts in E. coli O157:H7 strains function similarly to other E. coli strains in regard to attributes relevant to gastrointestinal colonization.Escherichia coli is well known for its ecological versatility (15). A life cycle which includes both gastrointestinal and environmental stages has been stressed by both Savageau (15) and Adamowicz et al. (1). The gastrointestinal stage would be subjected to acid and detergent stress. The environmental stage is implicit in E. coli having transport systems for fungal siderophores (4) as well as pyrroloquinoline quinone-dependent periplasmic glucose utilization (1) because their presence indicates evolution in a location containing fungal siderophores and pyrroloquinoline quinone (1).Since its recognition as a food-borne pathogen, there have been numerous outbreaks of food-borne infection due to E. coli O157:H7, in both ground beef and vegetable crops (6, 13). Cattle are widely considered to be the primary reservoir of E. coli O157:H7 (14), but E. coli O157:H7 does not appear to cause disease in cattle. To what extent is E. coli O157:H7 physiologically unique compared to the other naturally occurring E. coli strains? We feel that the uniqueness of E. coli O157:H7 should be evaluated against a backdrop of other wild-type E. coli strains, and in this regard, we chose the 72-strain ECOR reference collection originally described by Ochman and Selander (10). These strains were chosen from a collection of 2,600 E. coli isolates to provide diversity with regard to host species, geographical distribution, and electromorph profiles at 11 enzyme loci (10).In our study we compared the 72 strains of the ECOR collection against 10 strains of E. coli O157:H7 and six strains of E. coli which had been in laboratory use for many years (Table ​(Table1).1). The in vitro comparisons were made with regard to factors potentially relevant to the bacteria''s ability to colonize animal guts, i.e., acid tolerance, detergent tolerance, and the presence of the Entner-Doudoroff (ED) pathway (Table ​(Table2).2). Our longstanding interest in the ED pathway (11) derives in part from work by Paul Cohen''s group (16, 17) showing that the ED pathway is important for E. coli colonization of the mouse large intestine. Growth was assessed by replica plating 88 strains of E. coli under 40 conditions (Table ​(Table2).2). These included two LB controls (aerobic and anaerobic), 14 for detergent stress (sodium dodecyl sulfate [SDS], hexadecyltrimethylammonium bromide [CTAB], and benzalkonium chloride, both aerobic and anaerobic), 16 for acid stress (pH 6.5, 6.0, 5.0, 4.6, 4.3, 4.2, 4.1, and 4.0), four for the ability to grow in a defined minimal medium (M63 glucose salts with and without thiamine), and four for the presence or absence of a functional ED pathway (M63 with gluconate or glucuronate). All tests were done with duplicate plates in two or three separate trials. The data are available in Tables S1 to S14 in the supplemental material, and they are summarized in Table ​Table22.

TABLE 1.

E. coli strains used in this study

Stress-induced flowering

Many plant species can be induced to flower by responding to stress factors. The short-day plants Pharbitis nil and Perilla frutescens var. crispa flower under long days in response to the stress of poor nutrition or low-intensity light. Grafting experiments using two varieties of P. nil revealed that a transmissible flowering stimulus is involved in stress-induced flowering. The P. nil and P. frutescens plants that were induced to flower by stress reached anthesis, fruited and produced seeds. These seeds germinated, and the progeny of the stressed plants developed normally. Phenylalanine ammonialyase inhibitors inhibited this stress-induced flowering, and the inhibition was overcome by salicylic acid (SA), suggesting that there is an involvement of SA in stress-induced flowering. PnFT2, a P. nil ortholog of the flowering gene FLOWERING LOCUS T (FT) of Arabidopsis thaliana, was expressed when the P. nil plants were induced to flower under poor-nutrition stress conditions, but expression of PnFT1, another ortholog of FT, was not induced, suggesting that PnFT2 is involved in stress-induced flowering.Key words: flowering, stress, phenylalanine ammonia-lyase, salicylic acid, FLOWERING LOCUS T, Pharbitis nil, Perilla frutescensFlowering in many plant species is regulated by environmental factors, such as night-length in photoperiodic flowering and temperature in vernalization. On the other hand, a short-day (SD) plant such as Pharbitis nil (synonym Ipomoea nil) can be induced to flower under long days (LD) when grown under poor-nutrition, low-temperature or high-intensity light conditions.^1–9 The flowering induced by these conditions is accompanied by an increase in phenylalanine ammonia-lyase (PAL) activity.¹⁰ Taken together, these facts suggest that the flowering induced by these conditions might be regulated by a common mechanism. Poor nutrition, low temperature and high-intensity light can be regarded as stress factors, and PAL activity increases under these stress conditions.¹¹ Accordingly, we assumed that such LD flowering in P. nil might be induced by stress. Non-photoperiodic flowering has also been sporadically reported in several plant species other than P. nil, and a review of these studies suggested that most of the factors responsible for flowering could be regarded as stress. Some examples of these factors are summarized in 12^–14

Table 1

Some cases of stress-induced flowering

Focus on Weed Control: Herbicides as Weed Control Agents: State of the Art: I. Weed Control Research and Safener Technology: The Path to Modern Agriculture

The purpose of modern industrial herbicides is to control weeds. The species of weeds that plague crops today are a consequence of the historical past, being related to the history of the evolution of crops and farming practices. Chemical weed control began over a century ago with inorganic compounds and transitioned to the age of organic herbicides. Targeted herbicide research has created a steady stream of successful products. However, safeners have proven to be more difficult to find. Once found, the mode of action of the safener must be determined, partly to help in the discovery of further compounds within the same class. However, mounting regulatory and economic pressure has changed the industry completely, making it harder to find a successful herbicide. Herbicide resistance has also become a major problem, increasing the difficulty of controlling weeds. As a result, the development of new molecules has become a rare event today.Modern industrial herbicide research begins with the analysis and definition of research objectives. A major part of this lies in the definition of economically important weeds in major arable crops (Kraehmer, 2012). Weed associations change slowly over time. It is important, therefore, to foresee such changes. Today’s weed associations result from events in the distant past. They are associated with the history of crops and the evolution of farm management. In Europe and the Americas, some large-acre crops such as winter oilseed rape and spring oilseed rape (canola), both derived from Brassica spp., and soybean (Glycine max) have attained their current importance only within the last 100 years. Other Old World crops, such as cereals, have expanded over a very long time span and were already rather widespread in Neolithic times (Zohary et al., 2012). The dominance of crop species in agricultural habitats only left room for weed species that could adapt to cultivation technologies. Changes in crop management and the global weed infestation have happened in waves. A major early factor in Europe was presumably the grain trade in the Roman period (Erdkamp, 2005). The Romans spread their preferred crops and, unintentionally, associated weed seeds throughout Europe, Asia, and Africa. A second wave of global vegetation change started in the 16th century after the discovery of the Americas. Crops and weeds were distributed globally by agronomists and botanists. Alien species started to spread on all continents. A third phase can be seen in the 19th century with the industrialization of agriculture and the breeding of competitive crop varieties. The analysis of weed spectra in arable fields grew from this historical background. Weeds are plants interfering with the interests of people (Kraehmer and Baur, 2013), which is why they have been controlled by farmers for millennia.Chemical weed control began just about a century ago with a few inorganic compounds, such as sulfuric acid, copper salts, and sodium chlorate (Cremlyn, 1991). The herbicidal activity of 2,4-dichlorophenoxyacetic acid was detected in the 1940s (Troyer, 2001). Büchel et al. (1977) and Cremlyn (1991), Worthington and Hance (1991). Targeted herbicide research began in the 1950s. In the early days, herbicide candidates progressed from screens purely on the basis of their having biology that would satisfy farmers’ requirements. Mode of action (MoA) studies did not play a major role in the chemical industry prior to the 1970s. Analytical tools were developed and the rapid elucidation of plant pathways and in vitro-based screen assays were used from the 1980s onward. However, in the 1990s and beyond, ever-increasing regulatory and economic pressures have changed the situation of the industry completely, and to satisfy the new requirements, selection criteria beyond biological activity have needed to be applied. Herbicide resistance in weeds has developed into a more serious problem that now constrains the application of certain types of herbicides in some markets. Finally, the introduction of crops resistant to cheap herbicides and of glyphosate-resistant soybean, in particular, took value out of the market and resulted in an enormous economic pressure on the herbicide-producing industry. As a result of this changing and more difficult landscape, the development of new molecules is now a rare event.

Table I.

History of chemical weed control innovationsPost, Postemergence application; Pre, preemergence application, based on data from Cremlyn (1991), Worthington and Hance (1991), Büchel et al. (1977), Herbicide Resistance Action Committee (www.hracglobal.com), and others.

Immunoreactivity for alpha-smooth muscle actin characterizes a potentially aggressive subgroup of little basal cell carcinomas

Basal cell carcinoma (BCC) is a very common malignant skin tumor that rarely metastatizes, but is often locally aggressive. Several factors, like large size (more than 3 cm), exposure to ultraviolet rays, histological variants, level of infiltration and perineural or perivascular invasion, are associated with a more aggressive clinical course. These morphological features seem to be more determinant in mideface localized BCC, which frequently show a significantly higher recurrence rate. An immunohistochemical profile, characterized by reactivity of tumor cells for p53, Ki67 and alpha-SMA has been associated with a more aggressive behaviour in large BCCs. The aim of this study was to verify if also little (<3 cm) basal cell carcinomas can express immunohistochemical markers typical for an aggressive behaviour.Basal cell carcinoma (BCC) is a very common malignant skin tumor that rarely metastatizes, even If Is often locally aggressive. Several factors, like large size (more than 3 cm), face localization, exposure to ultraviolet rays, histological variants, infiltration level and perineural or perivascular invasion, are associated with a more aggressive clinical course. In particular, the incidence of metastasis and/or death correlates with tumors greater than 3 cm in diameter in which setting patients are said to have 1–2 % risk of metastases that increases to 20–25% in lesions greater than 5 cm and to 50% in lesions greater than 10 cm in diameter (Snow et al., 1994). Histologically morpheiform, keratotic types and infiltrative growth of BCC are also considered features of the most aggressive course (Crowson, 2006). This can be explained by the fact that both the superficial and nodular variants of BCC are surrounded by a continuous basement membrane zone comprising collagens type IV and V admixed with laminin, while the aggressive growth variants (i.e. morpheiform, metatypical, and infiltrative growth subtypes) manifest the absence of basement membrane (Barsky et al., 1987).The molecular markers which characterize aggressive BCC include: increased expression of stromolysin (MMP-3) and collagenase-1 (MMP-1) (Cribier et al., 2001), decreased expression of syndecan-1 proteoglycan (Bayer-Garner et al., 2000) and of anti-apoptotic protein bcl-2 (Ramdial et al., 2000; Staibano et al., 2001).C-ras , c-fos (Urabe et al., 1994; Van der Schroeff et al., 1990) and p53 tumor supressor gene mutations (Auepemikiate et al., 2002) are indicative of an aggressive course.Focusing upon bcl-2 and p53 expression in BCC, there have been numerous studies documenting the utility of bcl-2 as a marker of favourable clinical behaviour while p53 expression may be a feature of a more aggressive outcome (Ramdial et al., 2000; Staibano et al., 2001; Bozdogan et al., 2002).An increased expression of cytoskeletal microfilaments like α–smooth muscle actin, frequently found in invasive BCC subtypes (Jones JCR et al., 1989), may explain an enhanced tumor mobility and deep tissue invasion through the stroma. (Cristian et al., 2001; Law et al., 2003). The aim of this preliminary study was to verify if also little (<3 cm) basal cell carcinomas may express aggressive immunohistochemical markers like p53, Ki67 and alpha-SMA. We used 31 excisional BCCs with tumor size less than 2 cm (ranging from 2 up to 20 mm) and with different skin localization (19 in the face, 6 in the trunk and 6 in the body extremities). All cases were immunostained for p53, BCL2, Ki67 and alpha-smooth muscle actin (α-SMA) (AgeSexLocationHystotypeMax.DimDepthUlcEssInfp53Bcl-2Ki67AML161MExtrKeratotic10×81No+++URD+++++-261MFaceAdenoid10×94No+URD+++---364MExtrSup mult11×130.8No+DRD+---473MFaceNodular10×82Yes+DRD+++++++++584MFaceNodular9×122Yes+DRD----684MFaceAdenoid50.8No+URD+++---784MExtrNodular13×103No+DRD+++++-852FFaceNodular40.8No+URD+++-976FFaceAdenoid10×44No+DRD+++-++-1077FFaceMorph8×61Yes+++DRD+++---1186MFaceMorph81Yes+DRD+++-++1263FFaceAdenoid41No+URD+++++1376FFaceNodular71.5No+DRD++++++-1484MFaceNodular114Yes+++DRD+--+1563FFaceKeratotic10×61.8No++DRD-+++-1668FTrunkSup mult10×60.7No++URD++--1767MFaceSup mult12×60.4No+URD+-+-1867MExtrSup mult4×30.3No+URD+++++-1932FExtrSup mult1×30.4No+URD+++-2045MTrunkNodular7×52Yes+++URD+++-2162MTrunkSup mult11×70.9No++URD-++-++2265MTrunkAdenoid7×61.5No+URD+++++-2372MTrunkNodular12×61No+URD+++-++2486FFaceKeratotic20×113.1No++DRD+++-2585MFaceNodular0.51.3No++DRD++++-2674FExtrNodular4×40.9No+URD--+-2771MFaceNodular6×121.7No+DRD--+-2864FTrunkSup mult1.3×1.50.4No++URD+++---2978FFaceNodular4×31.5No++DRD+++-+++3080MFaceKeratotic4×41.6Yes+DRD--++++Open in a separate window Our data show that p53 (75%), Bcl2 (50%) and Ki67 (63%) positivity was generally diffuse in the majority of cases. On the contrary, cytoplasmatic α-SMA expression was present only in 8 out of 31 cases (25,8%). All these 8 α-SMA positive BCCs, prevalently found in the mideface (6 out of 8), were characterized by an initial invasion beyond the dermis. Among these 6 face-localized α-SMA positive BCCs, 1 showed a sclerosing aggressive histotype, 1 a keratotic type and 4 a nodular histotype.These 8 little α-SMA-positive BCCs, compared to the others 23 α-SMA negative samples, all showed a major aggressiveness features: facial location, ulceration, morpheiform histotype and deeper infiltration into the dermis (Location

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Histotype

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Local aggressiveness

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Immunohistochemistry

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FaceKeratoticMorpheiformDepht of invasion Mean value(mm)UlcerationInfiltration of the dermisP53Bcl-2Ki678 α-SMA Positive cases75%12%12%1.650%63%75%50%63%23 α-SMA Negative cases56%13%4%1.413%48%78%43%65%Open in a separate windowGiven the absence of a specific difference between α-SMA positive cases and α-SMA negative cases in the expression of aggressive immunohistochemical markers, except for a light reduction of bcl-2 in the α-SMA positive group (​and2).2). By the analysis of the data, we selected the combination that could better define an aggressive behaviour even for little BCC: α-SMA, p53, Ki67 positivity and bcl-2 negativity. We considered p53 and ki67 markers of proliferation and cell-cycle alteration, combined with a loss of apoptotic activity expressed by Bcl-2 negativity, quite characteristic of aggressiveness; moreover α-SMA positivity probably reflects invasive potential and acquired mobility by neoplastic cells.This immunohistochemical profile (α-SMA, p53, Ki67 positivity and bcl-2 negativity) in our cases of BCC is present in two of them; one is a morpheiform BCC, that is an aggressive variant, while the other one is a nodular subtype (less aggressive).Therefore, our preliminary data suggest that only α-SMA positivity should be considered as an early diagnostic marker of potential aggressiveness in little BCC: all α-SMA positive little BCC in fact showed clinical and histological features of aggressiveness. Invasive potential is probably acquired by some BCCs not only when they reach large size, but it is probably present also when they have still little size, and can be revealed by α-SMA positivity in the neoplastic cells. Open in a separate windowFigure 1BCC, nodular type, HE, 10×. Open in a separate windowFigure 2BCC, nodular type, α-SMA positivity, 10×.     

Hepatitis C Virus (HCV) Sequence Variation Induces an HCV-Specific T-Cell Phenotype Analogous to Spontaneous Resolution

Hepatitis C virus (HCV)-specific CD8⁺ T cells in persistent HCV infection are low in frequency and paradoxically show a phenotype associated with controlled infections, expressing the memory marker CD127. We addressed to what extent this phenotype is dependent on the presence of cognate antigen. We analyzed virus-specific responses in acute and chronic HCV infections and sequenced autologous virus. We show that CD127 expression is associated with decreased antigenic stimulation after either viral clearance or viral variation. Our data indicate that most CD8 T-cell responses in chronic HCV infection do not target the circulating virus and that the appearance of HCV-specific CD127⁺ T cells is driven by viral variation.Hepatitis C virus (HCV) persists in the majority of acutely infected individuals, potentially leading to chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma. The cellular immune response has been shown to play a significant role in viral control and protection from liver disease. Phenotypic and functional studies of virus-specific T cells have attempted to define the determinants of a successful versus an unsuccessful T-cell response in viral infections (10). So far these studies have failed to identify consistent distinguishing features between a T-cell response that results in self-limiting versus chronic HCV infection; similarly, the impact of viral persistence on HCV-specific memory T-cell formation is poorly understood.Interleukin-7 (IL-7) receptor alpha chain (CD127) is a key molecule associated with the maintenance of memory T-cell populations. Expression of CD127 on CD8 T cells is typically only observed when the respective antigen is controlled and in the presence of significant CD4⁺ T-cell help (9). Accordingly, cells specific for persistent viruses (e.g., HIV, cytomegalovirus [CMV], and Epstein-Barr virus [EBV]) have been shown to express low levels of CD127 (6, 12, 14) and to be dependent on antigen restimulation for their maintenance. In contrast, T cells specific for acute resolving virus infections, such as influenza virus, respiratory syncytial virus (RSV), hepatitis B virus (HBV), and vaccinia virus typically acquire expression of CD127 rapidly with the control of viremia (5, 12, 14). Results for HCV have been inconclusive. The expected increase in CD127 levels in acute resolving but not acute persisting infection has been found, while a substantial proportion of cells with high CD127 expression have been observed in long-established chronic infection (2). We tried to reconcile these observations by studying both subjects with acute and chronic HCV infection and identified the presence of antigen as the determinant of CD127 expression.Using HLA-peptide multimers we analyzed CD8⁺ HCV-specific T-cell responses and CD127 expression levels in acute and chronic HCV infection. We assessed a cohort of 18 chronically infected subjects as well as 9 individuals with previously resolved infection. In addition, we longitudinally studied 9 acutely infected subjects (5 individuals who resolved infection spontaneously and 4 individuals who remain chronically infected) (Tables ​(Tables11 and ​and2).2). Informed consent in writing was obtained from each patient, and the study protocol conformed to the ethical guidelines of the 1975 Declaration of Helsinki, as reflected in a priori approval from the local institutional review boards. HLA-multimeric complexes were obtained commercially from Proimmune (Oxford, United Kingdom) and Beckman Coulter (CA). The staining and analysis procedure was as described previously (10). Peripheral blood mononuclear cells (PBMCs) were stained with the following antibodies: CD3 from Caltag; CD8, CD27, CCR7, CD127, and CD38 from BD Pharmingen; and PD-1 (kindly provided by Gordon Freeman). Primer sets were designed for different genotypes based on alignments of all available sequences from the public HCV database (http://hcvpub.ibcp.fr). Sequence analysis was performed as previously described (8).

TABLE 1.

Patient information and autologous sequence analysis for patients with chronic and resolved HCV infection