Exploring Biomarkers in Breast Cancer: Hallmarks of Diagnosis, Treatment, and Follow-Up in Clinical Practice.

Breast cancer is a prevalent malignancy in the present day, particularly affecting women as one of the most common forms of cancer. A significant portion of patients initially present with localized disease, for which curative treatments are pursued. Conversely, another substantial segment is diagnosed with metastatic disease, which has a worse prognosis. Recent years have witnessed a profound transformation in the prognosis for this latter group, primarily due to the discovery of various biomarkers and the emergence of targeted therapies. These biomarkers, encompassing serological, histological, and genetic indicators, have demonstrated their value across multiple aspects of breast cancer management. They play crucial roles in initial diagnosis, aiding in the detection of relapses during follow-up, guiding the application of targeted treatments, and offering valuable insights for prognostic stratification, especially for highly aggressive tumor types. Molecular markers have now become the keystone of metastatic breast cancer diagnosis, given the diverse array of chemotherapy options and treatment modalities available. These markers signify a transformative shift in the arsenal of therapeutic options against breast cancer. Their diagnostic precision enables the categorization of tumors with elevated risks of recurrence, increased aggressiveness, and heightened mortality. Furthermore, the existence of therapies tailored to target specific molecular anomalies triggers a cascade of changes in tumor behavior. Therefore, the primary objective of this article is to offer a comprehensive review of the clinical, diagnostic, prognostic, and therapeutic utility of the principal biomarkers currently in use, as well as of their clinical impact on metastatic breast cancer. In doing so, our goal is to contribute to a more profound comprehension of this complex disease and, ultimately, to enhance patient outcomes through more precise and effective treatment strategies.

Enhanced Membrane Incorporation of H289Y Mutant GluK1 Receptors from the Audiogenic Seizure-Prone GASH/Sal Model: Functional and Morphological Impacts on Xenopus Oocytes.

Epilepsy is a neurological disorder characterized by abnormal neuronal excitability, with glutamate playing a key role as the predominant excitatory neurotransmitter involved in seizures. Animal models of epilepsy are crucial in advancing epilepsy research by faithfully replicating the diverse symptoms of this disorder. In particular, the GASH/Sal (genetically audiogenic seizure-prone hamster from Salamanca) model exhibits seizures resembling human generalized tonic-clonic convulsions. A single nucleotide polymorphism (SNP; C9586732T, p.His289Tyr) in the Grik1 gene (which encodes the kainate receptor GluK1) has been previously identified in this strain. The H289Y mutation affects the amino-terminal domain of GluK1, which is related to the subunit assembly and trafficking. We used confocal microscopy in Xenopus oocytes to investigate how the H289Y mutation, compared to the wild type (WT), affects the expression and cell-surface trafficking of GluK1 receptors. Additionally, we employed the two-electrode voltage-clamp technique to examine the functional effects of the H289Y mutation. Our results indicate that this mutation increases the expression and incorporation of GluK1 receptors into an oocyte's membrane, enhancing kainate-evoked currents, without affecting their functional properties. Although further research is needed to fully understand the molecular mechanisms responsible for this epilepsy, the H289Y mutation in GluK1 may be part of the molecular basis underlying the seizure-prone circuitry in the GASH/Sal model.

Oxidative Stress, Inflammatory, Angiogenic, and Apoptotic molecules in Proliferative Diabetic Retinopathy and Diabetic Macular Edema Patients.

The aim of this study is to evaluate molecules involved in oxidative stress (OS), inflammation, angiogenesis, and apoptosis, and discern which of these are more likely to be implicated in proliferative diabetic retinopathy (PDR) and diabetic macular edema (DME) by investigating the correlation between them in the plasma (PLS) and vitreous body (VIT), as well as examining data obtained from ophthalmological examinations. Type 2 diabetic (T2DM) patients with PDR/DME (PDRG/DMEG; n = 112) and non-DM subjects as the surrogate controls (SCG n = 48) were selected according to the inclusion/exclusion criteria and programming for vitrectomy, either due to having PDR/DME or macular hole (MH)/epiretinal membrane (ERM)/rhegmatogenous retinal detachment. Blood samples were collected and processed to determine the glycemic profile, total cholesterol, and C reactive protein, as well as the malondialdehyde (MDA), 4-hydroxynonenal (4HNE), superoxide dismutase (SOD), and catalase (CAT) levels and total antioxidant capacity (TAC). In addition, interleukin 6 (IL6), vascular endothelial growth factor (VEGF), and caspase 3 (CAS3) were assayed. The VITs were collected and processed to measure the expression levels of all the abovementioned molecules. Statistical analyses were conducted using the R Core Team (2022) program, including group comparisons and correlation analyses. Compared with the SCG, our findings support the presence of molecules involved in OS, inflammation, angiogenesis, and apoptosis in the PLS and VIT samples from T2DM. In PLS from PDRG, there was a decrease in the antioxidant load (p < 0.001) and an increase in pro-angiogenic molecules (p < 0.001), but an increase in pro-oxidants (p < 0.001) and a decline in antioxidants (p < 0.001) intravitreally. In PLS from DMEG, pro-oxidants and pro-inflammatory molecules were augmented (p < 0.001) and the antioxidant capacity diminished (p < 0.001), but the pro-oxidants increased (p < 0.001) and antioxidants decreased (p < 0.001) intravitreally. Furthermore, we found a positive correlation between the PLS-CAT and the VIT-SOD levels (rho = 0.5; p < 0.01) in PDRG, and a negative correlation between the PSD-4HNE and the VIT-TAC levels (rho = 0.5; p < 0.01) in DMEG. Integrative data of retinal imaging variables showed a positive correlation between the central subfield foveal thickness (CSFT) and the VIT-SOD levels (rho = 0.5; p < 0.01), and a negative correlation between the CSFT and the VIT-4HNE levels (rho = 0.4; p < 0.01) in PDRG. In DMEG, the CSFT displayed a negative correlation with the VIT-CAT (rho = 0.5; p < 0.01). Exploring the relationship of the abovementioned potential biomarkers between PLS and VIT may help detecting early molecular changes in PDR/DME, which can be used to identify patients at high risk of progression, as well as to monitor therapeutic outcomes in the diabetic retina.

Energy drives the dynamic localization of cyanobacterial nitrogen regulators during diurnal cycles.

Cyanobacteria, phototrophic organisms performing oxygenic photosynthesis, must adapt their metabolic processes to the challenges imposed by the succession of days and nights. Two conserved cyanobacterial proteins, PII and PipX, function as hubs of the nitrogen interaction network, forming complexes with a variety of diverse targets. While PII proteins are found in all three domains of life as integrators of signals of the nitrogen and carbon balance, PipX proteins are unique to cyanobacteria, where they provide a mechanistic link between PII signalling and the control of gene expression by the global nitrogen regulator NtcA. Here we demonstrate that PII and PipX display distinct localization patterns during diurnal cycles, co-localizing into the same foci at the periphery and poles of the cells during dark periods, a circadian-independent process requiring a low ATP/ADP ratio. Genetic, cellular biology and biochemical approaches used here provide new insights into the nitrogen regulatory network, calling attention to the roles of PII as energy sensors and its interactions with PipX in the context of essential signalling pathways. This study expands the contribution of the nitrogen regulators PII and PipX to integrate and transduce key environmental signals that allow cyanobacteria to thrive in our planet.

Cross-talk and regulatory interactions between the essential response regulator RpaB and cyanobacterial circadian clock output.

The response regulator RpaB (regulator of phycobilisome associated B), part of an essential two-component system conserved in cyanobacteria that responds to multiple environmental signals, has recently been implicated in the control of cell dimensions and of circadian rhythms of gene expression in the model cyanobacterium Synechococcus elongatus PCC 7942. However, little is known of the molecular mechanisms that underlie RpaB functions. In this study we show that the regulation of phenotypes by RpaB is intimately connected with the activity of RpaA (regulator of phycobilisome associated A), the master regulator of circadian transcription patterns. RpaB affects RpaA activity both through control of gene expression, a function requiring an intact effector domain, and via altering RpaA phosphorylation, a function mediated through the N-terminal receiver domain of RpaB. Thus, both phosphorylation cross-talk and coregulation of target genes play a role in the genetic interactions between the RpaA and RpaB pathways. In addition, RpaB∼P levels appear critical for survival under light:dark cycles, conditions in which RpaB phosphorylation is environmentally driven independent of the circadian clock. We propose that the complex regulatory interactions between the essential and environmentally sensitive NblS-RpaB system and the SasA-RpaA clock output system integrate relevant extra- and intracellular signals to the circadian clock.

PipX, the coactivator of NtcA, is a global regulator in cyanobacteria.

To modulate the expression of genes involved in nitrogen assimilation, the cyanobacterial PII-interacting protein X (PipX) interacts with the global transcriptional regulator NtcA and the signal transduction protein PII, a protein found in all three domains of life as an integrator of signals of the nitrogen and carbon balance. PipX can form alternate complexes with NtcA and PII, and these interactions are stimulated and inhibited, respectively, by 2-oxoglutarate, providing a mechanistic link between PII signaling and NtcA-regulated gene expression. Here, we demonstrate that PipX is involved in a much wider interaction network. The effect of pipX alleles on transcript levels was studied by RNA sequencing of S. elongatus strains grown in the presence of either nitrate or ammonium, followed by multivariate analyses of relevant mutant/control comparisons. As a result of this process, 222 genes were classified into six coherent groups of differentially regulated genes, two of which, containing either NtcA-activated or NtcA-repressed genes, provided further insights into the function of NtcA-PipX complexes. The remaining four groups suggest the involvement of PipX in at least three NtcA-independent regulatory pathways. Our results pave the way to uncover new regulatory interactions and mechanisms in the control of gene expression in cyanobacteria.

A nomogram for predicting complications in patients with solid tumours and seemingly stable febrile neutropenia.

BACKGROUND: We sought to develop and externally validate a nomogram and web-based calculator to individually predict the development of serious complications in seemingly stable adult patients with solid tumours and episodes of febrile neutropenia (FN). PATIENTS AND METHODS: The data from the FINITE study (n=1133) and University of Salamanca Hospital (USH) FN registry (n=296) were used to develop and validate this tool. The main eligibility criterion was the presence of apparent clinical stability, defined as events without acute organ dysfunction, abnormal vital signs, or major infections. Discriminatory ability was measured as the concordance index and stratification into risk groups. RESULTS: The rate of infection-related complications in the FINITE and USH series was 13.4% and 18.6%, respectively. The nomogram used the following covariates: Eastern Cooperative Group (ECOG) Performance Status ⩾2, chronic obstructive pulmonary disease, chronic cardiovascular disease, mucositis of grade ⩾2 (National Cancer Institute Common Toxicity Criteria), monocytes <200/mm(3), and stress-induced hyperglycaemia. The nomogram predictions appeared to be well calibrated in both data sets (Hosmer-Lemeshow test, P>0.1). The concordance index was 0.855 and 0.831 in each series. Risk group stratification revealed a significant distinction in the proportion of complications. With a ⩾116-point cutoff, the nomogram yielded the following prognostic indices in the USH registry validation series: 66% sensitivity, 83% specificity, 3.88 positive likelihood ratio, 48% positive predictive value, and 91% negative predictive value. CONCLUSIONS: We have developed and externally validated a nomogram and web calculator to predict serious complications that can potentially impact decision-making in patients with seemingly stable FN.

Application of eligibility criteria from DAPA-HF, EMPEROR-Reduced, and PARADIGM-HF trials to a population with heart failure with reduced ejection fraction at a specialized cardiology Clinic in Medellin, Colombia: A retrospective cohort study.

INTRODUCTION: The evidence supporting pharmacological heart failure treatment relies on randomized clinical trials with stringent inclusion and exclusion criteria. OBJECTIVES: Assess the eligibility of outpatients with chronic heart failure for the trials DAPA-HF, EMPEROR-reduced, and PARADIGM-HF, while exploring potential differences among study populations. METHODS: By reviewing medical records, we determined the eligibility rate for each study and evaluated the incidence of heart failure hospitalizations and all-cause mortality during this period. RESULTS: A total of 446 patients were included in the cohort. Approximately 75% would be ineligible for the trials, mainly because of their comorbidities. Ineligible patients had a higher all-cause mortality, but a similar incidence of hospitalization. CONCLUSION: Approximately 1 in 4 patients from a heart failure clinic in Medellin, Colombia would meet the eligibility criteria for the DAPA-HF, EMPEROR-reduced, and PARADIGM-HF trials. These findings highlight the need to complement randomized clinical trials with real-world data.

Insights into the mechanism of activation of the phosphorylation-independent response regulator NblR. Role of residues Cys69 and Cys96.

Cyanobacteria respond to environmental stress conditions by adjusting their photosynthesis machinery. In Synechococcus sp. PCC 7942, phycobilisome degradation and other acclimation responses after nutrient or high light stress require activation by the phosphorylation-independent response regulator NblR. Structural modelling of its receiver domain suggested a role for Cys69 and Cys96 on activation of NblR. Here, we investigate this hypothesis by engineering Cys to Ala substitutions. In vivo and in vitro analyses indicated that mutations Cys69Ala and/or Cys96Ala have a minor impact on NblR function, structure, size, or oligomerization state of the protein, and that Cys69 and Cys96 do not seem to form disulphide bridges. Our results argue against the predicted involvement of Cys69 and Cys96 on NblR activation by redox sensing.

Structural basis for the regulation of NtcA-dependent transcription by proteins PipX and PII.

PII, an ancient and widespread signaling protein, transduces nitrogen/carbon/energy abundance signals through interactions with target proteins. We clarify structurally how PII regulates gene expression mediated by the transcription factor NtcA, the global nitrogen regulator of cyanobacteria, shedding light on NtcA structure and function and on how NtcA is activated by 2-oxoglutarate (2OG) and coactivated by the nonenzymatic PII target, protein PipX. We determine for the cyanobacteria Synechococcus elongatus the crystal structures of the PII-PipX and PipX-NtcA complexes and of NtcA in active and inactive conformations (respective resolutions, 3.2, 2.25, 2.3, and 3.05 A). The structures and the conclusions derived from them are consistent with the results of present and prior site-directed mutagenesis and functional studies. A tudor-like domain (TLD) makes up most of the PipX structure and mediates virtually all the contacts of PipX with PII and NtcA. In the PII-PipX complex, one PII trimer sequesters the TLDs of three PipX molecules between its body and its extended T loops, preventing PipX activation of NtcA. Changes in T loop conformation triggered by 2OG explain PII-PipX dissociation when 2OG is bound. The structure of active dimeric NtcA closely resembles that of the active cAMP receptor protein (CRP). This strongly suggests that with these proteins DNA binding, transcription activation, and allosteric regulation occur by common mechanisms, although the effectors are different. The PipX-NtcA complex consists of one active NtcA dimer and two PipX monomers. PipX coactivates NtcA by stabilizing its active conformation and by possibly helping recruit RNA polymerase but not by providing extra DNA contacts.

Metastatic Multifocal Malignant Peripheral Nerve Sheath Tumour in the Cervicothoracic Spinal Cord of a Dog Initially Mimicking Meningomyelitis.

A nine year old cross-breed dog was presented with a two week history of ambulatory tetraparesis and proprioceptive ataxia affecting all four limbs. Meningomyelitis of Unknown Origin (MUO) was presumptively diagnosed based on the magnetic resonance imaging (MRI) findings and cerebrospinal fluid (CSF) analysis. The dog received a tapering dose of glucocorticoids and cyclosporine, showing significant improvement and the stabilization of the clinical signs for seven months. After this period, the dog showed an acute clinical deterioration and a follow-up MRI revealed new multiple lesions affecting different spinal nerve roots along the cervicothoracic spinal cord. Following euthanasia, a final diagnose of multiple malignant peripheral nerve sheath tumors (MPNSTs) was made based on the histopathological examination. MPNSTs can affect the cranial nerves, spinal nerves or the associated nerve roots at any location and can lead to secondary spinal cord compression. The aim of the present case report is to describe the clinical presentation and atypical MRI findings of a dog with histologically confirmed multiple MPNSTs. According to the reviewed literature, this is the first reported case of simultaneous MPNSTs in the cervicothoracic spinal cord of a dog.

The effect of obesity on chronic diseases in USA: a flexible copula approach.

We analyze the effect of obesity on the incidence of hypertension, hyperlipidemia and diabetes in USA using a health production theoretical framework along with a bivariate flexible semi-parametric recursive copula model that account for endogeneity. In this approach, the effects of control variables are flexibly determined using additive predictors that allow for a variety of effects. Our findings suggest that there exist a positive and significant effect of obesity on the prevalence of all chronic diseases examined. In particular, after endogeneity is accounted for, the probability of having hypertension, hyperlipidemia and diabetes for obese individuals are, respectively, 35%, 28% and 11% higher than those under the obesity threshold. These findings suggest that lowering obesity rates could lead to significant reductions in the morbidity and mortality associated with these diseases.

In vivo features of signal transduction by the essential response regulator RpaB from Synechococcus elongatus PCC 7942.

The NblS-RpaB signalling pathway, the most conserved two-component system in cyanobacteria, regulates photosynthesis and acclimatization to a variety of environmental conditions and is involved in negative regulation of high-light-induced genes. However, relevant regulatory details of the NblS-RpaB signalling pathway remain to be elucidated. We recently showed that the response regulator RpaB is regulated by specific (de)phosphorylation from the histidine kinase NblS and that RpaB and its phosphorylatable residue Asp56 are both required for viability of Synechococcus elongatus PCC 7942. We show here that the phosphorylated form of RpaB is present in cells growing under standard laboratory conditions and that high light stress affected the ratio of phosphorylated to non-phosphorylated RpaB. It also decreased the amount of rpaB transcripts without appreciably changing the total levels of RpaB. Quantitative Western blotting and confocal microscopy analyses were consistent with RpaB being a very abundant regulator, with nucleoid localization. A genetically engineered RpaB-GFP (green fluorescent protein) fusion protein rescued lethality of the rpaB null mutant, indicating that it was functional. This is, to our knowledge, the first study demonstrating in a cyanobacterium, and for a two-component response regulator, that the in vivo ratio of phosphorylated to non-phosphorylated protein changes in response to environmental conditions.

[Assessment of the surgical risk of 1,000 consecutive episodes using the POSSUM system. Comparison between elective and emergency gastrointestinal surgery]. / Evaluación del riesgo quirúrgico de 1.000 episodios consecutivos con el sistema POSSUM. Comparación entre cirugía gastrointestinal programada y urgente.

INTRODUCTION: The aim of the present study was to assess the predictive capacity of the POSSUM system in a Spanish university hospital, and to determine its behaviour in elective gastrointestinal surgery and compare it with emergency gastrointestinal surgery (operation < 24 hours). PATIENTS AND METHOD: A total of 1,000 surgical episodes corresponding to 909 patients who required hospital admission, operated on under general or loco-regional anaesthesia, either in the elective (n= 547 episodes) or the emergency setting (n= 453), were included in the study. RESULTS: The overall morbidity was 31.9% (32.8% in elective surgery; 30.7% in emergency surgery). The discriminatory capacity of the POSSUM scale, evaluated using receiver operating characteristic (ROC) curves, was higher for the Portsmouth variant of mortality (Area Under the Curve [AUC] = 0,92) than for morbidity (AUC= 0,74). The goodness of fit between the expected values using the POSSUM scale and those observed was reduced for morbidity (Hosmer-Lemeshow [H-L] = 164.1; p< 0.05). The POSSUM scale predicted a higher number of deaths than those observed, although the Portsmouth variant was better at predicting mortality. The goodness of fit for morbidity was better for elective gastrointestinal surgery (H-L= 27.7) than emergency gastrointestinal surgery (H-L= 177.3). The logistic regression analysis identified (besides the estimated risk using the POSSUM scale itself), surgical complexity, surgery type (elective, emergency), and age of patient, as significant predictive factors of morbidity and mortality. CONCLUSIONS: In a Spanish university hospital, the POSSUM system adequately predicts morbidity risk in elective gastrointestinal surgery, and over-estimates morbidity risk in emergency gastrointestinal surgery.

First Case of a Cerebrocortical Ganglioglioma in a Dog.

Gangliogliomas are extremely rare tumors of the nervous system composed of neoplastic glial and neuronal cells. The aim of the present paper is to describe the clinical presentation, magnetic resonance imaging (MRI) findings and histopathological and immunophenotypical characteristics of a cerebral cortex ganglioglioma in a 7-year-old Border Collie. The dog presented an acute onset of tonic-clonic epileptic seizures. MRI revealed a well-defined large intra-axial mass located on the left forebrain, mainly affecting the frontal cortex. Following humane euthanasia, the histopathological examination of the mass revealed a diffuse proliferation of neoplastic glial cells mixed with anomalous neuronal bodies. Immunohistochemical analyses confirmed the presence of two different populations of neoplastic cells. Most neoplastic glial cells were immunoreactive to glial fibrillary acidic protein (GFAP) and the other subset of neoplastic cells were positive to neuronal markers such as PGP 9.5, synaptophysin (SYN) and neuron-specific enolase (NSE), suggestive of neuronal cells. These findings confirmed the diagnosis of a cerebrocortical ganglioglioma. To the authors knowledge, this is the first description of a ganglioglioma of the cerebral cortex in a dog.

Impact of Systemic Comorbidities on Ocular Hypertension and Open-Angle Glaucoma, in a Population from Spain and Portugal.

Open-angle glaucoma (OAG), the most prevalent clinical type of glaucoma, is still the main cause of irreversible blindness worldwide. OAG is a neurodegenerative illness for which the most important risk factor is elevated intraocular pressure (IOP). Many questions remain unanswered about OAG, such as whether nutritional or toxic habits, other personal characteristics, and/or systemic diseases influence the course of glaucoma. As such, in this study, we performed a multicenter analytical, observational, case-control study of 412 participants of both sexes, aged 40-80 years, that were classified as having ocular hypertension (OHT) or OAG. Our primary endpoint was to investigate the relationship between specific lifestyle habits; anthropometric and endocrine-metabolic, cardiovascular, and respiratory events; and commonly used psychochemicals, with the presence of OHT or OAG in an ophthalmologic population from Spain and Portugal. Demographic, epidemiological, and ocular/systemic clinical data were recorded from all participants. Data were analyzed using the R Statistics v4.1.2 and RStudio v2021.09.1 programs. The mean age was 62 ± 15 years, with 67-80 years old comprising the largest subgroup sample of participants in both study groups. The central corneal thickness (ultrasound pachymetry)-adjusted IOP (Goldman tonometry) in each eye was 20.46 ± 2.35 and 20.1 ± 2.73 mmHg for the OHT individuals, and 15.8 ± 3.83 and 16.94 ± 3.86 mmHg for the OAG patients, with significant differences between groups (both p = 0.001). The highest prevalence of the surveyed characteristics in both groups was for overweight/obesity and daily coffee consumption, followed by psychochemical drug intake, migraine, and peripheral vasospasm. Our data show that overweight/obesity, migraine, asthma, and smoking are major risk factors for conversion from OHT to OAG in this Spanish and Portuguese population.

A phase II randomised trial of abiraterone acetate plus prednisone in combination with docetaxel or docetaxel plus prednisone after disease progression to abiraterone acetate plus prednisone in patients with metastatic castration-resistant prostate cancer: The ABIDO-SOGUG trial.

BACKGROUND: We aimed to compare the efficacy and safety of maintaining or withdrawing abiraterone acetate plus prednisone (AAP) in patients with metastatic castration-resistant prostate cancer who had experienced cancer progression to this treatment and were beginning a docetaxel-based therapy. PATIENTS AND METHODS: Phase II, randomised, open-label study conducted in patients with metastatic castration-resistant prostate cancer who were asymptomatic or mildly symptomatic. After open-label treatment with AAP, patients who had experienced cancer progression to AAP were randomised to 75 mg/m2 of docetaxel plus AAP or to receive 75 mg/m2 of docetaxel plus 10 mg of prednisone orally daily. The primary outcome was the radiographic progression-free survival rate at 12 months as evaluated by the investigators in all randomised patients. RESULTS: A total of 148 patients were included in open-label treatment with AAP, and of them, 94 patients were randomised to receive either docetaxel plus AAP (intervention group; n = 47) or docetaxel plus prednisone (control group; n = 47). The 12-month radiographic progression-free survival rates did not differ between the intervention group (34.9%; 95% CI 20.7-49.2) and the control group (33.9%; 95% CI 19.5-48.3). There were no significant differences in the time to radiographic progression and the overall survival between the intervention and control groups. Grade 3-5 neutropenia with the combination of docetaxel plus prednisone and AA was more frequent than with docetaxel plus prednisone (59.6% versus 27.7%). CONCLUSION: Our results indicate that the therapeutic strategy of maintaining AAP added to docetaxel in chemotherapy-naïve patients who have experienced cancer progression to AAP treatment should not be further evaluated and should be avoided in clinical practice. CLINICAL TRIALS: NCT02036060 https://clinicaltrials.gov/ct2/show/NCT02036060.

Environmental control of phosphorylation pathways in a branched two-component system.

NblS, the most conserved histidine kinase in cyanobacteria, regulates photosynthesis and acclimatization to a variety of environmental conditions. We used in silico, in vivo and in vitro approaches to identify RpaB and SrrA as the cognate response regulators of NblS and to characterize relevant interactions between components of this signalling system. While genetic analysis showed the importance of the NblS to RpaB phosphorylation branch for culture viability in Synechococcus elongatus PCC 7942, in vitro assays indicated a strong preference for NblS to phosphorylate SrrA. This apparent discrepancy can be explained by environmental insulation of the RpaB pathway, achieved by RpaB-dependent repression of srrA under standard, low light culture conditions. After a strong but transient increase in srrA expression upon high light exposure, negative regulation of srrA and other high light inducible genes takes place, suggesting cooperation between pathways under environmental conditions in which both RpaB and SrrA are present. Complex regulatory interactions between RpaB and SrrA, two response regulators with a common evolutionary origin that are controlled by a single histidine kinase, are thus emerging. Our results provide a paradigm for regulatory interactions between response regulators in a branched two-component system.

The nitrogen interaction network in Synechococcus WH5701, a cyanobacterium with two PipX and two P(II)-like proteins.

Nitrogen regulation involves the formation of different types of protein complexes between signal transducers and their transcriptional or metabolic targets. In oxygenic phototrophs, the signal integrator P(II) activates the enzyme N-acetyl-l-glutamate kinase (NAGK) by complex formation. P(II) also interacts with PipX, a protein with a tudor-like domain that mediates contacts with P(II) and with the transcriptional regulator NtcA, to which it binds to increase its activity. Here, we use a combination of in silico, yeast two-hybrid and in vitro approaches to investigate the nitrogen regulation network of Synechococcus WH5701, a marine cyanobacterium with two P(II) (GlnB_A and GlnB_B) and two PipX (PipX_I and PipX_II) proteins. Our results indicate that GlnB_A is functionally equivalent to the canonical P(II) protein from Synechococcus elongatus. GlnB_A interacted with PipX and NAGK proteins and stimulated NAGK activity, counteracting arginine inhibition. GlnB_B had only a slight stimulatory effect on NAGK activity, but its potential to bind effectors and form heterotrimers in Synechococcus WH5701 indicates additional regulatory functions. PipX_II, and less evidently PipX_I, specifically interacted with GlnB_A and NtcA, supporting a role for both Synechococcus WH5701 PipX proteins in partner swapping with GlnB_A and NtcA.

Regulatory Connections Between the Cyanobacterial Factor PipX and the Ribosome Assembly GTPase EngA.

Cyanobacteria, phototrophic organisms performing oxygenic photosynthesis, must adapt their metabolic processes to important environmental challenges, like those imposed by the succession of days and nights. Not surprisingly, certain regulatory proteins are found exclusively in this phylum. One of these unique proteins, PipX, provides a mechanistic link between signals of carbon/nitrogen and of energy, transduced by the signaling protein PII, and the control of gene expression by the global nitrogen regulator NtcA. PII, required for cell survival unless PipX is inactivated or downregulated, functions by protein-protein interactions with transcriptional regulators, transporters, and enzymes. PipX also functions by protein-protein interactions, and previous studies suggested the existence of additional interacting partners or included it into a relatively robust six-node synteny network with proteins apparently unrelated to the nitrogen regulation system. To investigate additional functions of PipX while providing a proof of concept for the recently developed cyanobacterial linkage network, here we analyzed the physical and regulatory interactions between PipX and an intriguing component of the PipX synteny network, the essential ribosome assembly GTPase EngA. The results provide additional insights into the functions of cyanobacterial EngA and of PipX, showing that PipX interacts with the GD1 domain of EngA in a guanosine diphosphate-dependent manner and interferes with EngA functions in Synechococcus elongatus at a low temperature, an environmentally relevant context. Therefore, this work expands the PipX interaction network and establishes a possible connection between nitrogen regulation and the translation machinery. We discuss a regulatory model integrating previous information on PII-PipX with the results presented in this work.