BACKGROUND:Linezolid is a key component of treatment regimens for rifampicin-resistant or multidrug-resistant tuberculosis. Toxicity-related linezolid interruption is not uncommon, but little is known about its effect on treatment-related outcomes. Furthermore, there are no data on the frequency of successful rechallenge and its associated predictors. This study aimed to ascertain the relationship between linezolid discontinuation and treatment outcome, and the likelihood of successful rechallenge in patients who discontinued linezolid. METHODS:We conducted an observational cohort study to review programmatic data from a South African national database on which all patients with drug-resistant tuberculosis are required to be registered. Data were extracted between Jan 1, 2018, and March 16, 2022. Data capture occurs at primary, secondary, and tertiary centres. We included adults 18 years or older with rifampicin-resistant or multidrug-resistant tuberculosis who had received linezolid and had recorded treatment outcomes. We excluded patients with extrapulmonary tuberculosis, those who were not exposed to a bedaquiline-containing regimen, and patients who died before completing their course of linezolid. The frequency of linezolid interruption and subsequent linezolid rechallenge was evaluated from clinical data for patients in the database. The primary outcome measure was the proportion of patients with an unfavourable outcome (death, treatment failure, or loss to follow-up) in those in whom linezolid was interrupted compared with those who continued treatment. Outcomes were ascertained using logistic regression to estimate adjusted odds ratios (aORs). FINDINGS:10 102 (83·7%) of 12 064 patients registered on the database between Jan 1, 2018, and Dec 31, 2020, met the study inclusion criteria and were included in the analysis. 6142 (60·8%) patients were male and 3960 (39·2%) were female. Treatment with linezolid was interrupted in 1974 (19·5%) patients, and the median time to interruption was 4·6 weeks (IQR 2·0-8·6). After adjusting for potentially confounding variables, linezolid interruption was associated with greater odds of an unfavourable outcome compared with completion of treatment (aOR 4·24 [95% CI 3·79-4·75], p<0·0001). Of the 1974 patients who interrupted their course of linezolid, only 65 (3·3%) were rechallenged and, of these, 47 (72·3%) successfully completed their linezolid course. Those successfully rechallenged were more likely to have a favourable outcome than were those in whom rechallenge failed (OR 4·03 [1·08-15·0], p=0·038). A shorter time to initial linezolid interruption was associated with reduced rechallenge success (OR 0·26 [0·08-0·85], p=0·022) and a higher likelihood of unfavourable outcome (OR 1·59 [1·33-1·93]; p<0·0001). INTERPRETATION:Linezolid interruption was common and associated with unfavourable clinical outcomes. Linezolid rechallenge was frequently successful and associated with better clinical outcomes. These findings inform clinical practice and have implications for the management of patients receiving oral, short-course regimens for multidrug-resistant tuberculosis. FUNDING:The South African Medical Research Council, the European and Developing Countries Clinical Trials Partnership, and the National Research Foundation.

ABSTRACTRationaleIn the upper respiratory tract replicating (culturable) SARS-CoV-2 is recoverable for ∼ 4 to 8 days after symptom onset, however, there is paucity of data about the frequency or duration of replicating virus in the lower respiratory tract (i.e. the human lung).ObjectivesWe undertook lung tissue sampling (needle biopsy), shortly after death, in 42 mechanically ventilated decedents during the Beta and Delta waves. An independent group of 18 ambulatory patents served as a control group.MethodsLung biopsy cores from decedents underwent viral culture, histopathological analysis, electron microscopy, transcriptomic profiling and immunohistochemistry.Results38% (16/42) of mechanically ventilated decedents had culturable virus in the lung for a median of 15 days (persisting for up to 4 weeks) after symptom onset. Lung viral culture positivity was not associated with comorbidities or steroid use. Delta but not Beta variant lung culture positivity was associated with accelerated death and secondary bacterial infection (p<0.05). Nasopharyngeal culture was negative in 23.1% (6/26) of decedents despite lung culture positivity. This, hitherto, undescribed bio-phenotype of lung-specific persisting viral replication was associated with an enhanced transcriptomic pulmonary pro-inflammatory response but with concurrent viral culture positivity.ConclusionsConcurrent, rather than sequential active viral replication continues to drive a heightened pro-inflammatory response in the human lung beyond the second week of illness and was associated with variant-specific increased mortality and morbidity. These findings have potential implications for the design of interventional strategies and clinical management of patients with severe COVID-19 disease.At a Glance CommentaryScientific Knowledge on the SubjectInvestigations to understand SARS-CoV-2 viral shedding (determined by PCR or antigen testing) have extensively focused on samples from the upper respiratory tract. The widely accepted view is that acute severe SARS-CoV-2 infection is characterised by a viral replicative phase in the first week of symptomatic illness followed by a pro-inflammatory immunopathologic phase peaking in the second and third weeks of illness. However, it remains unclear whether detection of SARS-CoV-2 beyond 2 weeks after symptom onset in published studies represent active replication competent virus because it may represent residual genomic or antigenic material in the tissue.What This Study Adds to the FieldWe have identified a, hitherto, undescribed bio-phenotype of acute severe COVID-19 characterised by persisting viral replication in the lung for up to 4 weeks after symptom onset. ∼40% of acute severe COVID-19 intensive care unit (ICU) decedents (n=42) had nasopharyngeal swab culture positivity at ∼2 weeks post-symptom onset versus only ∼5% in a group of ambulatory control patients (n=18). There was compartment-specific (nasopharynx versus lung) discordance. The phenotype of lung-specific persisting viral replication was associated with variant-specific accelerated death, an exaggerated inflammatory response, and attenuated T-cell immunity in the lung (based on histopathological and transcriptomic studies). This challenges the traditional view that viral replication occurs during the first 5 to 10 days of illness, which is followed by an effector or hyperinflammatory phase. This is the first study, to our knowledge, to systematically culture virus from the human lung and map out its related clinical determinants, and which describes the human lung transcriptomic profile of culture-positive versus culture-negative patients with severe COVID-19 disease.

The pathogenesis of coronavirus disease 2019 (COVID-19) pneumonia remains poorly understood. The urine proteome of hospitalized patients with severe COVID-19 pneumonia, compared with severe non-COVID-19 pneumonia controls, was distinct and associated with lower abundance of several host proteins. Protein-specific machine learning analysis outlined biomarker combinations able to differentiate COVID-19 pneumonia from non-COVID-19 pneumonia controls.

Two in every five patients with active tuberculosis (TB) remain undiagnosed or unreported. Therefore community-based, active case-finding strategies require urgent implementation. However, whether point-of-care (POC), portable battery-operated, molecular diagnostic tools deployed at a community level, compared with conventionally used POC smear microscopy, can shorten time-to-treatment initiation, thus potentially curtailing transmission, remains unclear. To clarify this issue, we performed an open-label, randomized controlled trial in periurban informal settlements of Cape Town, South Africa, where we TB symptom screened 5,274 individuals using a community-based scalable mobile clinic. Some 584 individuals with HIV infection or symptoms of TB underwent targeted diagnostic screening and were randomized (1:1) to same-day smear microscopy ( n = 296) or on-site DNA-based molecular diagnosis ( n = 288; GeneXpert). The primary aim was to compare time to TB treatment initiation between the arms. Secondary aims included feasibility and detection of probably infectious people. Of participants who underwent targeted screening, 9.9% (58 of 584) had culture-confirmed TB. Time-to-treatment initiation occurred significantly earlier in the Xpert versus the smear-microscopy arm (8 versus 41 d, P = 0.002). However, overall, Xpert detected only 52% of individuals with culture-positive TB. Notably, Xpert detected almost all of the probably infectious patients compared with smear microscopy (94.1% versus 23.5%, P = <0.001). Xpert was associated with a shorter median time to treatment of probably infectious patients (7 versus 24 d, P = 0.02) and a greater proportion of infectious patients were on treatment at 60 d compared with the probably noninfectious patients (76.5% versus 38.2%, P < 0.01). Overall, a greater proportion of POC Xpert-positive participants were on treatment at 60 d compared with all culture-positive participants (100% versus 46.5%, P < 0.01). These findings challenge the traditional paradigm of a passive case-finding, public health strategy and argues for the implementation of portable DNA-based diagnosis with linkage to care as a community-oriented, transmission-interruption strategy. The study was registered with the South African National Clinical Trials Registry (application ID 4367; DOH-27-0317-5367) and ClinicalTrials.gov (NCT03168945).

Two in every five patients with active tuberculosis (TB) remain undiagnosed or unreported. Therefore community-based, active case-finding strategies require urgent implementation. However, whether point-of-care (POC), portable battery-operated, molecular diagnostic tools deployed at a community level, compared with conventionally used POC smear microscopy, can shorten time-to-treatment initiation, thus potentially curtailing transmission, remains unclear. To clarify this issue, we performed an open-label, randomized controlled trial in periurban informal settlements of Cape Town, South Africa, where we TB symptom screened 5,274 individuals using a community-based scalable mobile clinic. Some 584 individuals with HIV infection or symptoms of TB underwent targeted diagnostic screening and were randomized (1:1) to same-day smear microscopy (n = 296) or on-site DNA-based molecular diagnosis (n = 288; GeneXpert). The primary aim was to compare time to TB treatment initiation between the arms. Secondary aims included feasibility and detection of probably infectious people. Of participants who underwent targeted screening, 9.9% (58 of 584) had culture-confirmed TB. Time-to-treatment initiation occurred significantly earlier in the Xpert versus the smear-microscopy arm (8 versus 41 d, P = 0.002). However, overall, Xpert detected only 52% of individuals with culture-positive TB. Notably, Xpert detected almost all of the probably infectious patients compared with smear microscopy (94.1% versus 23.5%, P = < 0.001). Xpert was associated with a shorter median time to treatment of probably infectious patients (7 versus 24 d, P = 0.02) and a greater proportion of infectious patients were on treatment at 60 d compared with the probably noninfectious patients (76.5% versus 38.2%, P < 0.01). Overall, a greater proportion of POC Xpert-positive participants were on treatment at 60 d compared with all culture-positive participants (100% versus 46.5%, P < 0.01). These findings challenge the traditional paradigm of a passive case-finding, public health strategy and argues for the implementation of portable DNA-based diagnosis with linkage to care as a community-oriented, transmission-interruption strategy. The study was registered with the South African National Clinical Trials Registry (application ID 4367; DOH-27-0317-5367) and (NCT03168945).

Mycobacterium tuberculosis (M.tb) causes tuberculosis (TB) and remains one of the leading causes of mortality due to an infectious pathogen. Host immune responses have been implicated in driving the progression from infection to severe lung disease. We analyzed longitudinal RNA sequencing (RNAseq) data from the whole blood of 74 TB progressors whose samples were grouped into four six-month intervals preceding diagnosis (the GC6-74 study). We additionally analyzed RNAseq data from an independent cohort of 90 TB patients with positron emission tomography-computed tomography (PET-CT) scan results which were used to categorize them into groups with high and low levels of lung damage (the Catalysis TB Biomarker study). These groups were compared to non-TB controls to obtain a complete whole blood transcriptional profile for individuals spanning from early stages of M.tb infection to TB diagnosis. The results revealed a steady increase in the number of genes that were differentially expressed in progressors at time points closer to diagnosis with 278 genes at 13-18 months, 742 at 7-12 months and 5,131 detected 1-6 months before diagnosis and 9,205 detected in TB patients. A total of 2,144 differentially expressed genes were detected when comparing TB patients with high and low levels of lung damage. There was a large overlap in the genes upregulated in progressors 1-6 months before diagnosis (86%) with those in TB patients. A comprehensive pathway analysis revealed a potent activation of neutrophil and platelet mediated defenses including neutrophil and platelet degranulation, and NET formation at both time points. These pathways were also enriched in TB patients with high levels of lung damage compared to those with low. These findings suggest that neutrophils and platelets play a critical role in TB pathogenesis, and provide details of the timing of specific effector mechanisms that may contribute to TB lung pathology.

Evidence suggests that shared pathophysiological mechanisms in neuropsychiatric disorders (NPDs) may contribute to risk and resilience. We used single-gene and network-level transcriptomic approaches to investigate shared and disorder-specific processes underlying posttraumatic stress disorder (PTSD), Parkinson’s disease (PD) and schizophrenia in a South African sample. RNA-seq was performed on blood obtained from cases and controls from each cohort. Gene expression and weighted gene correlation network analyses (WGCNA) were performed using DESeq2 and CEMiTool, respectively. Significant differences in gene expression were limited to the PTSD cohort. However, WGCNA implicated, amongst others, ribosomal expression, inflammation and ubiquitination as key players in the NPDs under investigation. Differential expression in ribosomal-related pathways was observed in the PTSD and PD cohorts, and focal adhesion and extracellular matrix pathways were implicated in PD and schizophrenia. We propose that, despite different phenotypic presentations, core transdiagnostic mechanisms may play important roles in the molecular aetiology of NPDs.

Rationale: Improving treatment outcomes while reducing drug toxicity and shortening the treatment duration to similar to 6 months remains an aspirational goal for the treatment of multidrug-resistant/rifampicin-resistant tuberculosis (MDR/RR-TB). Objectives: To conduct a multicenter randomized controlled trial in adults with MDR/RR-TB (i.e., without resistance to fluoroquinolones or aminoglycosides). Methods: Participants were randomly assigned (1:1 ratio) to a similar to 6-month all-oral regimen that included levofloxacin, bedaquiline, and linezolid, or the standard-of-care (SOC) >= 9-month World Health Organization (WHO)-approved injectable-based regimen. The primary endpoint was a favorable WHO-defined treatment outcome (which mandates that prespecified drug substitution is counted as an unfavorable outcome) 24 months after treatment initiation. The trial was stopped prematurely when bedaquiline-based therapy became the standard of care in South Africa. Measurements and Main Results: In total, 93 of 111 randomized participants (44 in the comparator arm and 49 in the interventional arm) were included in the modified intention-to-treat analysis; 51 (55%) were HIV coinfected (median CD4 count, 158 cells/ml). Participants in the intervention arm were 2.2 times more likely to experience a favorable 24-month outcome than participants in the SOC arm (51% [25 of 49] vs. 22.7% [10 of 44]; risk ratio, 2.2 [1.2-4.1]; P= 0.006}. Toxicity-related drug substitution occurred more frequently in the SOC arm (65.9% [29 of 44] vs. 34.7% [17 of 49]; P= 0.001)], 82.8% (24 of 29) owing to kanamycin (mainly hearing loss; replaced by bedaquiline) in the SOC arm, and 64.7% (11 of 17) owing to linezolid (mainly anemia) in the interventional arm. Adverse event-related treatment discontinuation in the safety population was more common in the SOC arm (56.4% [31 of 55] vs. 32.1% [17 of 56]; P= 0.007). However, grade 3 adverse events were more common in the interventional arm (55.4% [31 of 56] vs. 32.7 [18 of 55]; P = 0.022). Culture conversion was significantly better in the intervention arm (hazard ratio, 2.6 [1.4-4.9]; P = 0.003) after censoring those with bedaquiline replacement in the SOC arm (and this pattern remained consistent after censoring for drug replacement in both arms; P= 0.01). Conclusions: Compared with traditional injectable-containing regimens, an all-oral 6-month levofloxacin, bedaquiline, and linezolid-containing MDR/RR-TB regimen was associated with a significantly improved 24-month WHO-defined treatment outcome (predominantly owing to toxicity-related drug substitution). However, drug toxicity occurred frequently in both arms. These findings inform strategies to develop future regimens for MDR/RR-TB.

Soil salinization is increasing globally, driving a reduction in crop yields that threatens food security. Salinity stress reduces plant growth by exerting two stresses on plants: rapid shoot ion-independent effects which are largely osmotic and delayed ionic effects that are specific to salinity stress. In this study we set out to delineate the osmotic from the ionic effects of salinity stress. Arabidopsis thaliana plants were germinated and grown for two weeks in media supplemented with 50, 75, 100, or 125 mM NaCl (that imposes both an ionic and osmotic stress) or iso-osmolar concentrations (100, 150, 200, or 250 mM) of sorbitol, that imposes only an osmotic stress. A subsequent transcriptional analysis was performed to identify sets of genes that are differentially expressed in plants grown in (1) NaCl or (2) sorbitol compared to controls. A comparison of the gene sets identified genes that are differentially expressed under both challenge conditions (osmotic genes) and genes that are only differentially expressed in plants grown on NaCl (ionic genes, hereafter referred to as salt-specific genes). A pathway analysis of the osmotic and salt-specific gene lists revealed that distinct biological processes are modulated during growth under the two conditions. The list of salt-specific genes was enriched in the gene ontology (GO) term “response to auxin.” Quantification of the predominant auxin, indole-3-acetic acid (IAA) and IAA biosynthetic intermediates revealed that IAA levels are elevated in a salt-specific manner through increased IAA biosynthesis. Furthermore, the expression of NITRILASE 2 (NIT2), which hydrolyses indole-3-acetonitile (IAN) into IAA, increased in a salt-specific manner. Overexpression of NIT2 resulted in increased IAA levels, improved Na:K ratios and enhanced survival and growth of Arabidopsis under saline conditions. Overall, our data suggest that auxin is involved in maintaining growth during the ionic stress imposed by saline conditions.

Background: Improving treatment outcomes, reducing drug toxicity, eliminating the need for injectable agents, and shortening the treatment duration to 6-months (the same as rifampicin-susceptible tuberculosis) remains an aspirational goal for the treatment of multidrug-resistant/rifampicin-resistant tuberculosis (MDR/RR-TB).Methods: We conducted a multicentre randomised controlled trial in adults with MDR/RR-TB (i.e. without resistance to fluoroquinolones or aminoglycosides). Participants were randomly assigned (1:1 ratio) to a 6-month all-oral regimen that included levofloxacin, bedaquiline and linezolid, or the standard-of-care ≥ 9-month WHO-approved injectable-based regimen. The primary endpoint was a favourable WHO-defined treatment outcome 24 months after treatment initiation.Findings: 93 of 111 participants randomised were included in the modified intention-to-treat analysis; 51 (55%) were HIV co-infected (median CD4 count 158 cells/ml). Participants in the intervention arm were 2·2 times more likely to experience a favourable 24-month outcome ( 95% CI) than participants in the standard-of-care arm [RR 2·2 (1·2–4·1); p=0·006]. They were also less likely, over a 24-month period, to experience an unfavourable outcome event than participants in the standard-of-care arm [HR 0·4, (0·2–0·6), p<0·001]. Toxicity-related drug substitution occurred more frequently in the standard-of-care arm [(65·9% (29/44) versus 36·7% (18/49), p= 0·001)]; 79.3% (23/29) due to kanamycin (mainly hearing loss; replaced by bedaquiline) in the standard-of-care arm, and 83·3% (15/18) due to linezolid (mainly anaemia) in the interventional arm. Time to culture conversion was significantly better in the interventional arm [HR 2·6 (1·4–4·9); p= 0·003] after censoring those with bedaquiline replacement in the standard-of-care arm.Interpretation: An all-oral 6-month levofloxacin, bedaquiline and linezolid-containing MDR/RR-TB regimen was associated with significantly improved 24-month treatment outcomes compared with traditional injectable-containing regimens. However, drug toxicity occurred frequently in both arms. These findings inform strategies to develop future regimens for MDR/RR-TB.Trial Registration: The trial was registered on clinicaltrials.gov (NCT02454205).Funding: South African MRC, Pfizer, and the European and Developing Country Clinical Trial Partnership (EDCTP)Declaration of Interest: None to declare. Ethical Approval: An institutional review board or ethics committee at each participating trial site reviewed and approved the protocol and informed consent documents.

Pulmonary tuberculosis (PTB) is characterized by lung granulomas, inflammation and tissue destruction. Here we used within-subject peripheral blood gene expression over time to correlate with the within-subject lung metabolic activity, as measured by positron emission tomography (PET) to identify biological processes and pathways underlying overall resolution of lung inflammation. We used next-generation RNA sequencing and [18F]FDG PET-CT data, collected at diagnosis, week 4, and week 24, from 75 successfully cured PTB patients, with the [18F]FDG activity as a surrogate for lung inflammation. Our linear mixed-effects models required that for each individual the slope of the line of [18F]FDG data in the outcome and the slope of the peripheral blood transcript expression data correlate, i.e., the slopes of the outcome and explanatory variables had to be similar. Of 10,295 genes that changed as a function of time, we identified 639 genes whose expression profiles correlated with decreasing [18F]FDG uptake levels in the lungs. Gene enrichment over-representation analysis revealed that numerous biological processes were significantly enriched in the 639 genes, including several well known in TB transcriptomics such as platelet degranulation and response to interferon gamma, thus validating our novel approach. Others not previously associated with TB pathobiology included smooth muscle contraction, a set of pathways related to mitochondrial function and cell death, as well as a set of pathways connecting transcription, translation and vesicle formation. We observed up-regulation in genes associated with B cells, and down-regulation in genes associated with platelet activation. We found 254 transcription factor binding sites to be enriched among the 639 gene promoters. In conclusion, we demonstrated that of the 10,295 gene expression changes in peripheral blood, only a subset of 639 genes correlated with inflammation in the lungs, and the enriched pathways provide a description of the biology of resolution of lung inflammation as detectable in peripheral blood. Surprisingly, resolution of PTB inflammation is positively correlated with smooth muscle contraction and, extending our previous observation on mitochondrial genes, shows the presence of mitochondrial stress. We focused on pathway analysis which can enable therapeutic target discovery and potential modulation of the host response to TB.

Background: The utility of heated and humidified high-flow nasal oxygen (HFNO) for severe COVID-19-related hypoxaemic respiratory failure (HRF), particularly in settings with limited access to intensive care unit (ICU) resources, remains unclear, and predictors of outcome have been poorly studied. Methods: We included consecutive patients with COVID-19-related HRF treated with HFNO at two tertiary hospitals in Cape Town, South Africa. The primary outcome was the proportion of patients who were successfully weaned from HFNO, whilst failure comprised intubation or death on HFNO. Findings: The median (IQR) arterial oxygen partial pressure to fraction inspired oxygen ratio (PaO2/FiO(2)) was 68 (54-92) in 293 enroled patients. Of these, 137/293 (47%) of patients [P(a)O2/FiO(2) 76 (63-93)] were successfully weaned from HFNO. The median duration of HFNO was 6 (3-9) in those successfully treated versus 2 (1-5) days in those who failed (p<0.001). A higher ratio of oxygen saturation/FiO(2) to respiratory rate within 6 h (ROX-6 score) after HFNO commencement was associated with HFNO success (ROX-6; AHR 0.43, 0.31-0.60), as was use of steroids (AHR 0.35, 95%CI 0.19-0.64). A ROX-6 score of >= 3.7 was 80% predictive of successful weaning whilst ROX-6 <= 2.2 was 74% predictive of failure. In total, 139 patents (52%) survived to hospital discharge, whilst mortality amongst HFNO failures with outcomes was 129/140 (92%). Interpretation: In a resource-constrained setting, HFNO for severe COVID-19 HRF is feasible and more almost half of those who receive it can be successfully weaned without the need for mechanical ventilation. (C) 2020 The Author(s). Published by Elsevier Ltd.

Transcriptomics profiling aims to identify and quantify all transcripts present within a cell type or tissue at a particular state, and thus provide information on the genes expressed in specific experimental settings, differentiation or disease conditions. RNA-Seq technology is becoming the standard approach for such studies, but available analysis tools are often hard to install, configure and use by users without advanced bioinformatics skills.Within reason, DEWE aims to make RNA-Seq analysis as easy for non-proficient users as for experienced bioinformaticians. DEWE supports two well-established and widely used differential expression analysis workflows: using Bowtie2 or HISAT2 for sequence alignment; and, both applying StringTie for quantification, and Ballgown and edgeR for differential expression analysis. Also, it enables the tailored execution of individual tools as well as helps with the management and visualisation of differential expression results.DEWE provides a user-friendly interface designed to reduce the learning curve of less knowledgeable users while enabling analysis customisation and software extension by advanced users. Docker technology helps overcome installation and configuration hurdles. In addition, DEWE produces high quality and publication-ready outputs in the form of tab-delimited files and figures, as well as helps researchers with further analyses, such as pathway enrichment analysis.The abilities of DEWE are exemplified here by practical application to a comparative analysis of monocytes and monocyte-derived dendritic cells, a study of clinical relevance. DEWE installers and documentation are freely available at https://www.sing-group.org/dewe.

SummaryMost metal hyperaccumulating plants accumulate nickel, yet the molecular basis of Ni hyperaccumulation is not well understood. We chose Senecio coronatus to investigate this phenomenon as this species displays marked variation in shoot Ni content across ultramafic outcrops in the Barberton Greenstone Belt (South Africa), thus allowing an intraspecific comparative approach to be employed. No correlation between soil and shoot Ni contents was observed, suggesting that this variation has a genetic rather than environmental basis. This was confirmed by our observation that the accumulation phenotype of plants from two hyperaccumulator and two non‐accumulator populations was maintained when the plants were grown on a soil mix from these four sites for 12 months. We analysed the genetic variation among 12 serpentine populations of S. coronatus, and used RNA‐seq for de novo transcriptome assembly and analysis of gene expression in hyperaccumulator versus non‐accumulator populations. Genetic analysis revealed the presence of hyperaccumulators in two well supported evolutionary lineages, indicating that Ni hyperaccumulation may have evolved more than once in this species. RNA‐Seq analysis indicated that putative homologues of transporters associated with root iron uptake in plants are expressed at elevated levels in roots and shoots of hyperaccumulating populations of S. coronatus from both evolutionary lineages. We hypothesise that Ni hyperaccumulation in S. coronatus may have evolved through recruitment of these transporters, which play a role in the iron‐deficiency response in other plant species.

BACKGROUND:Cyclic nucleotides have been shown to play important signaling roles in many physiological processes in plants including photosynthesis and defence. Despite this, little is known about cyclic nucleotide-dependent signaling mechanisms in plants since the downstream target proteins remain unknown. This is largely due to the fact that bioinformatics searches fail to identify plant homologs of protein kinases and phosphodiesterases that are the main targets of cyclic nucleotides in animals.METHODS:An affinity purification technique was used to identify cyclic nucleotide binding proteins in Arabidopsis thaliana. The identified proteins were subjected to a computational analysis that included a sequence, transcriptional co-expression and functional annotation analysis in order to assess their potential role in plant cyclic nucleotide signaling.RESULTS:A total of twelve cyclic nucleotide binding proteins were identified experimentally including key enzymes in the Calvin cycle and photorespiration pathway. Importantly, eight of the twelve proteins were shown to contain putative cyclic nucleotide binding domains. Moreover, the identified proteins are post-translationally modified by nitric oxide, transcriptionally co-expressed and annotated to function in hydrogen peroxide signaling and the defence response. The activity of one of these proteins, GLYGOLATE OXIDASE 1, a photorespiratory enzyme that produces hydrogen peroxide in response to Pseudomonas, was shown to be repressed by a combination of cGMP and nitric oxide treatment.CONCLUSIONS:We propose that the identified proteins function together as points of cross-talk between cyclic nucleotide, nitric oxide and reactive oxygen species signaling during the defence response.

Cyclic nucleotides (CNs) are intracellular second messengers that play an important role in mediating physiological responses to environmental and developmental signals, in species ranging from bacteria to humans. In response to these signals, CNs are synthesized by nucleotidyl cyclases and then act by binding to and altering the activity of downstream target proteins known as cyclic nucleotide-binding proteins (CNBPs). A number of CNBPs have been identified across kingdoms including transcription factors, protein kinases, phosphodiesterases, and channels, all of which harbor conserved CN-binding domains. In plants however, few CNBPs have been identified as homology searches fail to return plant sequences with significant matches to known CNBPs. Recently, affinity pull-down techniques have been successfully used to identify CNBPs in animals and have provided new insights into CN signaling. The application of these techniques to plants has not yet been extensively explored and offers an alternative approach toward the unbiased discovery of novel CNBP candidates in plants. Here, an affinity pull-down technique for the identification of the plant CN interactome is presented. In summary, the method involves an extraction of plant proteins which is incubated with a CN-bait, followed by a series of increasingly stringent elutions that eliminates proteins in a sequential manner according to their affinity to the bait. The eluted and bait-bound proteins are separated by one-dimensional gel electrophoresis, excised, and digested with trypsin after which the resultant peptides are identified by mass spectrometry—techniques that are commonplace in proteomics experiments. The discovery of plant CNBPs promises to provide valuable insight into the mechanism of CN signal transduction in plants.

A number of studies have shown that functionally related genes are often co-expressed and that computational based co-expression analysis can be used to accurately identify functional relationships between genes and by inference, their encoded proteins. Here we describe how a computational based co-expression analysis can be used to link the function of a specific gene of interest to a defined cellular response. Using a worked example we demonstrate how this methodology is used to link the function of the Arabidopsis Wall-Associated Kinase-Like 10 gene, which encodes a functional guanylyl cyclase, to host responses to pathogens.

Over 1,000 putative secreted peptides have been identified in the Arabidopsis genome, but little is known about their functional roles. Here we demonstrate, using PROPEP2 in a worked example, how a computational-based co-expression analysis can be used to associate the function of a specific plant peptide with a specific biological process. The analysis reveals that PROPEP2 is, highly co-expressed with pathogen defense genes including, the PROPEP3 paralog and the extracellular Pep1 and Pep2 receptors. It additionally reveals that the co-expressed genes are induced in response to a broad range of pathogens and their associated elicitors. The results are completely supportive of previously known functional roles of this peptide and thus clearly demonstrate that the computational-based analysis presented can be used to associate the function of a specific plant peptide with a specific biological process and can also accurately identify functionally related genes.

BACKGROUND:The carotenoids are pure isoprenoids that are essential components of the photosynthetic apparatus and are coordinately synthesized with chlorophylls in chloroplasts. However, little is known about the mechanisms that regulate carotenoid biosynthesis or the mechanisms that coordinate this synthesis with that of chlorophylls and other plastidial synthesized isoprenoid-derived compounds, including quinones, gibberellic acid and abscisic acid. Here, a comprehensive transcriptional analysis of individual carotenoid and isoprenoid-related biosynthesis pathway genes was performed in order to elucidate the role of transcriptional regulation in the coordinated synthesis of these compounds and to identify regulatory components that may mediate this process in Arabidopsis thaliana.RESULTS:A global microarray expression correlation analysis revealed that the phytoene synthase gene, which encodes the first dedicated and rate-limiting enzyme of carotenogenesis, is highly co-expressed with many photosynthesis-related genes including many isoprenoid-related biosynthesis pathway genes. Chemical and mutant analysis revealed that induction of the co-expressed genes following germination was dependent on gibberellic acid and brassinosteroids (BR) but was inhibited by abscisic acid (ABA). Mutant analyses further revealed that expression of many of the genes is suppressed in dark grown plants by Phytochrome Interacting transcription Factors (PIFs) and activated by photoactivated phytochromes, which in turn degrade PIFs and mediate a coordinated induction of the genes. The promoters of PSY and the co-expressed genes were found to contain an enrichment in putative BR-auxin response elements and G-boxes, which bind PIFs, further supporting a role for BRs and PIFs in regulating expression of the genes. In osmotically stressed root tissue, transcription of Calvin cycle, methylerythritol 4-phosphate pathway and carotenoid biosynthesis genes is induced and uncoupled from that of chlorophyll biosynthesis genes in a manner that is consistent with the increased synthesis of carotenoid precursors for ABA biosynthesis. In all tissues examined, induction of β-carotene hydroxylase transcript levels are linked to an increased demand for ABA.CONCLUSIONS:This analysis provides compelling evidence to suggest that coordinated transcriptional regulation of isoprenoid-related biosynthesis pathway genes plays a major role in coordinating the synthesis of functionally related chloroplast localized isoprenoid-derived compounds.