Tolerability and efficacy of the cancer vaccine UV1 in patients with recurrent or metastatic PD-L1 positive head and neck squamous cell carcinoma planned for first-line treatment with pembrolizumab - the randomized phase 2 FOCUS trial.

Background: Globally, head and neck squamous cell carcinoma (HNSCC) is the seventh most common malignancy. Despite aggressive multimodal treatment approaches, recurrent and/or metastatic (R/M) disease develops in >50% of patients. In this setting, pembrolizumab was approved for patients with PD-L1 expression. However, response rates with checkpoint inhibitor monotherapy remain limited and strategies to strengthen tumor-directed immune responses are needed. Objective: The FOCUS trial is designed to estimate the effectiveness of UV1 vaccination in combination with pembrolizumab versus pembrolizumab as a single agent in patients with R/M HNSCC. Methods and analysis: The FOCUS trial is a two-armed, randomized, multicenter phase II study which was designed to evaluate the efficacy and feasibility of the hTERT-targeted cancer vaccine UV1 as add-on to pembrolizumab in the 1st line treatment of patients with R/M PD-L1 positive (combined positive score ≥1) HNSCC. Secondary objectives are the exploration of patient subgroups most likely deriving benefit from this novel combination and the establishment of liquid biopsy tumor monitoring in HNSCC. Ethics and dissemination: This clinical study was designed and will be conducted in compliance with Good Clinical Practice and in accordance with the Declaration of Helsinki. It is intended to publish the results of this study in peer-reviewed scientific journals and to present its content at academic conferences. Conclusions: A significant number of patients with R/M HNSCC are frail and may not tolerate chemotherapy, these patients may only be suitable for pembrolizumab monotherapy. However, long term disease stabilizations remain the exception and there is a need for the development of efficacious combination regimens for this patient population. The FOCUS study aims to optimize treatment of R/M HNSCC patients with this promising new treatment approach. Clinical Trial Registration: https://clinicaltrials.gov/study/NCT05075122, identifier NCT05075122.

Adipose biopsy techniques for studies in human exercise physiology.

Adipose biopsy techniques are relatively undefined for exercise physiology research in individuals at or near normal weight. The purpose of this study was to compare the influence of two adipose biopsy techniques on tissue quality through measurements of adipocyte cell size, as well as mRNA and protein levels of select pro- and anti-inflammatory cytokines and adipokines. Thirteen participants (9 M, 4 W; 28 ± 4 yr; 27 ± 3 kg·m-2; V̇o2max: 3.3 ± 0.7 L·min-1) underwent subcutaneous adipose biopsies on either side of the umbilicus (incision: ∼8 cm lateral, sampling area: ∼5 cm lateral) using 1) a 6-mm Bergström biopsy needle and 2) a mini-liposuction approach with a 4-mm Mercedes biopsy needle that used prebiopsy tumescent delivery (∼30 mL 0.9% NaCl solution) into the sampling area (i.e., 'wet' technique). Tissue obtained was processed identically for analysis and both techniques returned high-quality tissue for histology (similar % intact adipocytes), mRNA (RNA integrity numbers >7.0), and protein. Adipocyte size was similar (P > 0.05) between both techniques (Bergström: 6,116 ± 1,652 µm2, 554-23,522 µm2; Mercedes: 6,517 ± 952 µm2, 926-21,969 µm2). There were also no differences (P > 0.05) between the two techniques for the measured cytokines (pro- and anti-inflammatory) and adipokines at the mRNA and protein levels. Adipocyte size was positively correlated with body mass index and body fat percentage, and negatively correlated with V̇o2max (P < 0.05). These results suggest both adipose biopsy techniques used in the current investigation are appropriate for histological, transcriptional, and translational level measurements in exercise physiology studies of nonobese women and men.NEW & NOTEWORTHY This study provides investigators with useful information related to adipose biopsy sampling approaches that can be used when planning studies that use measurements of adipose histology, as well as measurements at the mRNA and protein level. Adipose periumbilical sampling with the Bergström biopsy needle and the Mercedes wet mini-liposuction technique are both appropriate options for studies in exercise physiology and in nonobese individuals.

Multitissue responses to exercise: a MoTrPAC feasibility study.

We assessed the feasibility of the Molecular Transducers of Physical Activity Consortium (MoTrPAC) human adult clinical exercise protocols, while also documenting select cardiovascular, metabolic, and molecular responses to these protocols. After phenotyping and familiarization sessions, 20 subjects (25 ± 2 yr, 12 M, 8 W) completed an endurance exercise bout (n = 8, 40 min cycling at 70% V̇o2max), a resistance exercise bout (n = 6, ∼45 min, 3 sets of ∼10 repetition maximum, 8 exercises), or a resting control period (n = 6, 40 min rest). Blood samples were taken before, during, and after (10 min, 2 h, and 3.5 h) exercise or rest for levels of catecholamines, cortisol, glucagon, insulin, glucose, free fatty acids, and lactate. Heart rate was recorded throughout exercise (or rest). Skeletal muscle (vastus lateralis) and adipose (periumbilical) biopsies were taken before and ∼4 h following exercise or rest for mRNA levels of genes related to energy metabolism, growth, angiogenesis, and circadian processes. Coordination of the timing of procedural components (e.g., local anesthetic delivery, biopsy incisions, tumescent delivery, intravenous line flushes, sample collection and processing, exercise transitions, and team dynamics) was reasonable to orchestrate while considering subject burden and scientific objectives. The cardiovascular and metabolic alterations reflected a dynamic and unique response to endurance and resistance exercise, whereas skeletal muscle was transcriptionally more responsive than adipose 4 h postexercise. In summary, the current report provides the first evidence of protocol execution and feasibility of key components of the MoTrPAC human adult clinical exercise protocols. Scientists should consider designing exercise studies in various populations to interface with the MoTrPAC protocols and DataHub.NEW & NOTEWORTHY This study highlights the feasibility of key aspects of the MoTrPAC adult human clinical protocols. This initial preview of what can be expected from acute exercise trial data from MoTrPAC provides an impetus for scientists to design exercise studies to interlace with the rich phenotypic and -omics data that will populate the MoTrPAC DataHub at the completion of the parent protocol.

Circulating Tumor DNA in Head and Neck Squamous Cell Carcinoma.

Tumors shed cell-free DNA (cfDNA) into the plasma. "Liquid biopsies" are a diagnostic test to analyze cfDNA in order to detect minimal residual cancer, profile the genomic tumor landscape, and monitor cancers non-invasively over time. This technique may be useful in patients with head and neck squamous cell carcinoma (HNSCC) due to genetic tumor heterogeneity and limitations in imaging sensitivity. However, there are technical challenges that need to be overcome for the widespread use of liquid biopsy in the clinical management of these patients. In this review, we discuss our current understanding of HNSCC genetics and the role of cfDNA genomic analyses as an emerging precision diagnostic tool.

A significant proportion of patients with primary central nervous system lymphoma harbor clonal bone marrow B-cells.

Clonal bone marrow (BM) B-cell populations are a common finding in patients with suspected primary central nervous system lymphoma (PCNSL). To assess their clinical significance, benign monoclonal B-cell lymphocytosis (MBL) needs to be differentiated from concomitant BM involvement, since patients with secondary central nervous system lymphoma (SCNSL) generally require more intensive treatment directed also at the component outside the central nervous system (CNS). Here, we retrospectively analyzed BM samplings in 51 patients with suspected PCNSL. We found clonal B cell populations in 8 of 51 cases (16%) by flow cytometry. None of these eight cases had BM involvement by high-grade lymphoma. No lymphoma relapses outside the CNS were recorded. Together, our data points at a significant percentage of patients with suspected PCNSL harboring clonal BM B-cells, which in this patient group always represented benign MBL. Failure to differentiate these populations from systemic lymphoma involvement may result in overtreatment of patients.

Effects of silymarin on angiogenesis and oxidative stress in streptozotocin-induced diabetes in mice.

The present study evaluated the effects of acute treatment with silymarin, an extract that is obtained from Silybum marianum, on angiogenesis, oxidative stress, and inflammation in normoglycemic and diabetic mice. Diabetes was induced by streptozotocin (80 mg/kg, intraperitoneal) in male Swiss mice, 6 weeks of age. A polyether-polyurethane sponge was surgically implanted in the back of the mice as a model of healing in both diabetic and normoglycemic animals that were treated with oral silymarin or water for 10 days. The pancreas, liver, kidneys, blood, and sponges were collected and analyzed. Diabetes led to impairments of antioxidant defenses, reflected by a reduction of pancreatic superoxide dismutase and hepatic and renal catalase and an increase in pancreatic lipoperoxidation. An inflammatory process was observed in diabetic mice, reflected by an increase in pancreatic tumor necrosis factor α (TNF-α) and the infiltration of inflammatory cells in islets. The number of vessels was lower in the implanted sponges in diabetic mice. Silymarin treatment attenuated this damage, restoring antioxidant enzymes and reducing pancreatic TNF-α and inflammatory infiltration. However, silymarin treatment did not restore angiogenesis or glycemia. In conclusion, treatment with silymarin red uced oxidative stress and inflammation that were induced in the model of streptozotocin-induced diabetes in several organs, without apparent toxicity. Silymarin may be a promising drug for controlling diabetic complications.

Monitoring multiple myeloma by next-generation sequencing of V(D)J rearrangements from circulating myeloma cells and cell-free myeloma DNA.

Recent studies suggest that circulating tumor cells and cell-free DNA may represent powerful non-invasive tools for monitoring disease in patients with solid and hematologic malignancies. Here, we conducted a pilot study in 27 myeloma patients to explore the clonotypic V(D)J rearrangement for monitoring circulating myeloma cells and cell-free myeloma DNA. Next-generation sequencing was used to define the myeloma V(D)J rearrangement and for subsequent peripheral blood tracking after treatment initiation. Positivity for circulating myeloma cells/cell-free myeloma was associated with conventional remission status (P<0.001) and 91% of non-responders/progressors versus 41% of responders had evidence of persistent circulating myeloma cells/cell-free myeloma DNA (P<0.001). About half of the partial responders showed complete clearance of circulating myeloma cells/cell-free myeloma DNA despite persistent M-protein, suggesting that these markers are less inert than the M-protein, rely more on cell turnover and, therefore, decline more rapidly after initiation of effective treatment. Positivity for circulating myeloma cells and for cell-free myeloma DNA were associated with each other (P=0.042), but discordant in 30% of cases. This indicates that cell-free myeloma DNA may not be generated entirely by circulating myeloma cells and may reflect overall tumor burden. Prospective studies need to define the predictive potential of high-sensitivity determination of circulating myeloma cells and DNA in the monitoring of multiple myeloma.

T-cell diversification reflects antigen selection in the blood of patients on immune checkpoint inhibition and may be exploited as liquid biopsy biomarker.

Cancer immunotherapy with antibodies targeting immune checkpoints, such as programmed cell death protein 1 (PD-1), shows encouraging results, but reliable biomarkers predicting response to this costly and potentially toxic treatment approach are still lacking. To explore an immune signature predictive for response, we performed liquid biopsy immunoprofiling in 18 cancer patients undergoing PD-1 inhibition before and shortly after initiation of treatment by multicolor flow cytometry and next-generation T- and B-cell immunosequencing (TCRß/IGH). Findings were correlated with clinical outcomes. We found almost complete saturation of surface PD-1 on all T-cell subsets after the first dose of the antibody. Both T- and B-cell compartments quantitatively expanded during treatment. These expansions were mainly driven by an increase in the activated T-cell compartments, as well as of naïve B- and plasma cells. Deep immunosequencing revealed a clear diversification pattern of the clonal T-cell space indicative of antigenic selection in 47% of patients, while the remaining patients showed stable repertoires. 43% of the patients with a diversification pattern showed disease control in response to the PD-1 inhibitor. No disease stabilizations were observed without clonal T-cell space diversification. Our data show for the first time a clear impact of PD-1 targeting not only on circulating T-cells, but also on B-lineage cells, shedding light on the complexity of the anti-tumor immune response. Liquid biopsy T-cell next-generation immunosequencing should be prospectively evaluated as part of a composite response prediction biomarker panel in the context of clinical studies.

Resistance to anti-CD19/CD3 BiTE in acute lymphoblastic leukemia may be mediated by disrupted CD19 membrane trafficking.

The CD19 antigen is a promising target for immunotherapy of acute lymphoblastic leukemia (ALL), but CD19- relapses remain a major challenge in about 10% to 20% of patients. Here, we analyzed 4 CD19- ALL relapses after treatment with the CD19/CD3 bispecific T-cell engager (BiTE) blinatumomab. Three were on-drug relapses, with the CD19- escape variant first detected after only 2 treatment courses. In 1 patient, the CD19- clone appeared as a late relapse 19 months after completion of blinatumomab treatment. All 4 cases showed a cellular phenotype identical to the primary diagnosis except for CD19 negativity. This argued strongly in favor of an isolated molecular event and against a common lymphoid CD19- progenitor cell or myeloid lineage shift driving resistance. A thorough molecular workup of 1 of the cases with early relapse confirmed this hypothesis by revealing a disrupted CD19 membrane export in the post-endoplasmic reticulum compartment as molecular basis for blinatumomab resistance.

Polyphenol-Rich Foods Alleviate Pain and Ameliorate Quality of Life in Fibromyalgic Women.

Objectives: The present study aimed to describe the antioxidant dietary intake of patients with fibromyalgia and explore the association of the results with glutathione status, pain, quality of life, and socioeconomic status. METHODS: 38 fibromyalgic female patients and 35 female controls (mean age = 48.6 ± 8.1 and 47.6 ± 10.0 years, respectively) were evaluated. The number of tender points, pain threshold, quality of life, physical activity, socioeconomic status, nutritional status, intake of antioxidant micronutrients and foods with high total antioxidant capacity, and total salivary glutathione were evaluated. RESULTS: The number of tender points, pain threshold, and quality of life were worse in the fibromyalgia group. The consumption of vegetable juices was more common among women with fibromyalgia and consumption of red wine and beer were more common among healthy women. The adjusted mean intakes of antioxidant vitamins as well as selenium were higher for the control group (p ≤ 0.01). There was no difference for salivary levels of glutathione between the groups and no correlation for intake of antioxidant micronutrients and pain or quality of life among fibromyalgic women. However, intake of foods rich in polyphenols was associated with lower numbers of tender points (coffee, r = - 0.346; pear, r = - 0.331) and better quality of life (red fruits, r = - 0.342; dark chocolate, r = - 0.404) in the fibromyalgic group. In these women, associations between glutathione levels and food intake, pain or quality of life were not found. CONCLUSION: This study indicated that antioxidant protection from bioactive compounds present in fruit and vegetables could have an adjuvant role in fibromyalgia treatment.

Metabolic switches during the first steps of adipogenic stem cells differentiation.

The understanding of metabolism during cell proliferation and commitment provides a greater insight into the basic biology of cells, allowing future applications. Here we evaluated the energy and oxidative changes during the early adipogenic differentiation of human adipose tissue-derived stromal cells (hASCs). hASCs were maintained under differentiation conditions during 3 and 7days. Oxygen consumption, mitochondrial mass and membrane potential, reactive oxygen species (ROS) generation, superoxide dismutase (SOD) and catalase activities, non-protein thiols (NPT) concentration and lipid peroxidation were analyzed. We observed that 7days of adipogenic induction are required to stimulate cells to consume more oxygen and increase mitochondrial activity, indicating organelle maturation and a transition from glycolytic to oxidative energy metabolism. ROS production was only increased after 3days and may be involved in the differentiation commitment. ROS source was not only the mitochondria and we suggest that NOX proteins are related to ROS generation and therefore adipogenic commitment. ROS production did not change after 7days, but an increased activity of catalase and NPT concentration as well as a decreased lipid peroxidation were observed. Thus, a short period of differentiation induction is able to change the energetic and oxidative metabolic profile of hASCs and stimulate cytoprotection processes.

Impairment of oxidative phosphorylation increases the toxicity of SYD-1 on hepatocarcinoma cells (HepG2).

Toxicity of the SYD-1 mesoionic compound (3-[4-chloro-3-nitrophenyl]-1,2,3-oxadiazolium-5-olate) was evaluated on human liver cancer cells (HepG2) grown in either high glucose (HG) or galactose (GAL) medium, and also on suspended cells kept in HG medium. SYD-1 was able to decrease the viability of cultured HepG2 cells in a dose-dependent manner, as assessed by MTT, LDH release and dye with crystal violet assays, but no effect was observed on suspended cells after 1-40 min of treatment. Respiration analysis was performed after 2 min (suspended cells) or 24 h (cultured cells) of treatment: no change was observed in suspended cells, whereas SYD-1 inhibited as well basal, leak and uncoupled states of the respiration in cultured cells with HG medium. These inhibitions were consistent with the decrease in pyruvate level and increase in lactate level. Even more extended results were obtained with HepG2 cells grown in GAL medium where, additionally, the ATP amount was reduced. Furthermore, SYD-1 appears not to be transported by the main ABC multidrug transporters. These results show that SYD-1 is able to change the metabolism of HepG2 cells, and suggest that its cytotoxicity is related to impairment of mitochondrial metabolism. Therefore, we may propose that SYD-1 is a potential candidate for hepatocarcinoma treatment.

The lectin BJcuL induces apoptosis through TRAIL expression, caspase cascade activation and mitochondrial membrane permeability in a human colon adenocarcinoma cell line.

It has been demonstrated that the cytotoxic effect of BJcuL, the lectin isolated from Bothrops jararacussu venom, on human gastric carcinoma is accompanied by the inhibition of extracellular matrix adhesion, cytoskeleton disassembly and apoptosis induction. The present study aimed to evaluate the apoptosis mechanisms triggered by the BJcuL interaction with specific glycans on the surface of HT29 human colon adenocarcinoma cells. The results demonstrated that BJcuL interacts with glycoligands targets on the cell, which were inhibited in the presence of d-galactose. It shows a dose-dependently cytotoxic effect that is inhibited in the presence of d-galactose. A dose-dependent cell aggregation decrease was also observed for the HT29 cells. Analysis of cell proliferation inhibition was assessed by anti-PCNA and demonstrated that lectin diminishes PCNA expression when compared with untreated cells. Differences in apoptotic marker expression estimated by immunohistochemistry revealed that the lectin promotes an increase in TRAIL expression, leading to an increase in the expression of FADD, caspase-8 and Bax. Besides the increased expression of apoptosis-related proteins, our results revealed that the lectin promotes a mitochondrial respiration decrease and a 75% increase in the amount of cytochrome c released. Together these results suggest that the cytotoxicity of BJcuL can sensitize pro-apoptotic proteins in the cytoplasm and mitochondria, leading to the apoptotic cascade.

Sydnone SYD-1 affects the metabolic functions of isolated rat hepatocytes.

Previously, we demonstrated that sydnone SYD-1 (3-[4-chloro-3-nitrophenyl]-1,2,3-oxadiazolium-5-olate) impairs the mitochondrial functions linked to energy provision and suggested that this effect could be associated with its antitumor activity. Herein, we evaluated the effects of SYD-1 (25 and 50 µM) on rat hepatocytes to determine its cytotoxicity on non-tumor cells. SYD-1 (25 and 50 µM) did not affect the viability of hepatocytes in suspension after 1-40 min of incubation. However, the viability of the cultured hepatocytes was decreased by ∼66% as a consequence of treatment with SYD-1 (50 µM) for 18 h. Under the same conditions, SYD-1 promoted an increase in the release of LDH by ∼19%. The morphological changes in the cultured cells treated with SYD-1 (50 µM) were suggestive of cell distress, which was demonstrated by the presence of rounded hepatocytes, cell fragments and monolayer impairment. Furthermore, fluorescence microscopy showed an increase in the annexin label after treatment with SYD-1 (50 µM), suggesting that apoptosis had been induced in these cells. SYD-1 did not affect the states of respiration in the suspended hepatocytes, but the pyruvate levels were decreased by ∼36%, whereas the lactate levels were increased by ∼22% (for the 50 µM treatment). The basal and uncoupled states of respiration of the cultured hepatocytes were inhibited by ∼79% and ∼51%, respectively, by SYD-1 (50 µM). In these cells, SYD-1 (50 µM) increased the pyruvate and lactate levels by ∼84% and ∼16%, respectively. These results show that SYD-1 affects important metabolic functions related to energy provision in hepatocytes and that this effect was more pronounced on cells in culture than those in suspension.

Effects of statins on liver cell function and inflammation in septic rats.

BACKGROUND: Several studies suggest that the presence of statins may be beneficial during sepsis, but this idea is controversial. The aim of this study was to investigate the effects of long-term statin treatment in the livers of septic animals, focusing on its antioxidant, antiinflammatory, and metabolic properties. MATERIALS AND METHODS: Male Wistar rats were treated orally with simvastatin, atorvastatin, or vehicle once a d. After 30 d, sepsis was induced by cecal ligation and puncture (CLP) in Control, Simvastatin-treated, and Atorvastatin-treated groups, while the Sham group underwent only laparotomy. The Basal Simvastatin and Basal Atorvastatin groups received only their respective drugs without surgery. Twenty-four h after CLP or laparotomy, samples were collected from anesthetized rats for evaluation of hepatic oxidative stress, liver histology, hepatic mitochondria enzyme activity, leukocyte counts in blood and peritoneal cavity, gene expression of hepatic superoxide dismutase and TNF-2, and plasma biochemistry. RESULTS: Most parameters that we tested exhibited expected changes upon sepsis induction. However, statin treatment only improved liver mitochondrial enzymatic activity. In other parameters, simvastatin and atorvastatin failed to protect the liver against injuries incurred upon the CLP-induced polymicrobial sepsis model. CONCLUSIONS: Pretreatment with simvastatin or atorvastatin alone before sepsis induction improved mitochondrial activity in the liver; however, this result was not reproduced in other biomarkers of liver function and leukocyte migration during sepsis. Future studies should be performed to evaluate whether statins can be combined with other drugs to increase the efficacy of sepsis therapy.

Avaliação in vivo do efeito hipocolesterolêmico e toxicológico preliminar do extrato bruto hidroalcoólico e decocção da Vitex megapotamica (Spreng) Moldenke (V. montevidensis Cham) / Evaluation of prospective hypocholesterolemic effect and preliminary toxicology of crude extract and decoction from Vitex megapotamica (Spreng) Moldenke (V. montevidensis Cham) in vivo

As doenças cardiovasculares (DC) estão, de modo geral, associadas a elevados níveis séricos de lipídeos, atingindo homens e mulheres, sem distinção de idade. Entre as propriedades atribuídas pela medicina popular à Vitex megapotamica (Spreng) Moldenke (V. montevidensis Cham) -Tarumã estão a de reduzir os níveis séricos de colesterol e triglicerídeo. O principal objetivo do presente estudo foi avaliar o potencial hipocolesterolêmico e hipolipidêmico da V. megapotamica, bem como fazer um estudo toxicológico preliminar. Para tanto, foi realizada indução hiperlipidêmica usando um modelo que preconiza o emprego de propiltiuracil 1,25 mg/300 g de peso e colesterol 200 mg/kg de peso, aplicados via oral em ratos machos pesando 300 ± 10 g. Foi administrado, por via oral, aos animais hiperlipidêmicos previamente induzidos 300 mg/kg de extrato hidroalcoólico das folhas de V. megapotamica ou 300 mL da decocção da casca da planta. Após o final de cada tratamento, o perfil lipídico foi ensaiado, bem como os níveis de glicose, quando relevante. Nossos resultados confirmaram o efeito hipolipidêmico do extrato hidroalcoólico e da decocção pela redução dos níveis séricos de colesterol e triacilglicerol nas concentrações, via e forma utilizadas. Além disso, foi possível verificar que não houve lesão cardíaca, hepática ou renal pelo extrato e decocção utilizados nas avaliações toxicológicas preliminares ensaiadas.

The cardiovascular diseases are, in general, associated with high levels of serum lipids which have high incidence in middle-age men and women. Among other properties characterized by popular medicine, the Vitex megapotamica (Spreng) Moldenke (V. montevidensis Cham) - Tarumã, decreases the serum cholesterol and triacylglycerol levels. The main proposition of the present study was to evaluate the hypocholesterolemic and hipolipidaemic potential of V. megapotamica and to analyze the preliminary toxicity. It was an induction hyperlipidaemic model using propiltiuracil 1.25 mg/kg weight and cholesterol 200 mg/ kg weight per oros in male rats, weigthing 300 ± 10 g. It was administred to the animals 300 mg/ kg of hydroalcoholic leaves extract of V. megapotamica or 300 mL of hull decoction per oros. After the end of each treatment, the lipid profile was essayed as well as glucose, when relevant. Our results confirmed the V. megapotamica extract and decoction hypolipidaemic effect by the decrease of cholesterol and triacylglycerides serum levels in concentration, via and preparation performed. Furthermore, the toxicological preliminary assays showed there was not extract and decoction damage induction in cardiac and hepatic tissues, as well as in kidney physiology by assays performed.

Transcriptional regulation and structural modeling of the FutC subunit of an ABC-type iron transporter in Synechocystis sp. strain PCC 6803.

The futC gene encodes a subunit of an ATP-binding cassette (ABC)-type iron transporter in Synechocystis sp. strain PCC 6803. In the present study, we have focused on the environmental regulation of futC transcription in the model organism Synechocystis sp. strain PCC 6803 and, moreover, studied the transcriptional regulation of the other transporter subunits, futA1, futA2 and futB. The steady-state amounts of the futA1, futA2, futB and futC transcripts were regulated under several conditions studied including darkness, temperature, alternative nitrogen source, salt and osmotic stresses and iron deficiency. Transcription of all subunits of the FutABC-iron transporter seems to be under similar regulation, which, according to our results, may also apply to genes encoding subunits of other transporters in Synechocystis. The sequence alignment, including sequences from six different organisms, revealed the conserved nature of FutC. Based on the sequence alignment and the structural model of FutC, the monomer consists of a nucleotide-binding domain (NBD) and a regulatory domain. The NBD is well conserved indicating completely functional ATP binding.