A model industrial workhorse: Bacillus subtilis strain 168 and its genome after a quarter of a century.

The vast majority of genomic sequences are automatically annotated using various software programs. The accuracy of these annotations depends heavily on the very few manual annotation efforts that combine verified experimental data with genomic sequences from model organisms. Here, we summarize the updated functional annotation of Bacillus subtilis strain 168, a quarter century after its genome sequence was first made public. Since the last such effort 5 years ago, 1168 genetic functions have been updated, allowing the construction of a new metabolic model of this organism of environmental and industrial interest. The emphasis in this review is on new metabolic insights, the role of metals in metabolism and macromolecule biosynthesis, functions involved in biofilm formation, features controlling cell growth, and finally, protein agents that allow class discrimination, thus allowing maintenance management, and accuracy of all cell processes. New 'genomic objects' and an extensive updated literature review have been included for the sequence, now available at the International Nucleotide Sequence Database Collaboration (INSDC: AccNum AL009126.4).

The absence of the queuosine tRNA modification leads to pleiotropic phenotypes revealing perturbations of metal and oxidative stress homeostasis in Escherichia coli K12.

Queuosine (Q) is a conserved hypermodification of the wobble base of tRNA containing GUN anticodons but the physiological consequences of Q deficiency are poorly understood in bacteria. This work combines transcriptomic, proteomic and physiological studies to characterize a Q-deficient Escherichia coli K12 MG1655 mutant. The absence of Q led to an increased resistance to nickel and cobalt, and to an increased sensitivity to cadmium, compared to the wild-type (WT) strain. Transcriptomic analysis of the WT and Q-deficient strains, grown in the presence and absence of nickel, revealed that the nickel transporter genes (nikABCDE) are downregulated in the Q- mutant, even when nickel is not added. This mutant is therefore primed to resist to high nickel levels. Downstream analysis of the transcriptomic data suggested that the absence of Q triggers an atypical oxidative stress response, confirmed by the detection of slightly elevated reactive oxygen species (ROS) levels in the mutant, increased sensitivity to hydrogen peroxide and paraquat, and a subtle growth phenotype in a strain prone to accumulation of ROS.

Cysteine Dealkylation in Bacillus subtilis by a Novel Flavin-Dependent Monooxygenase.

In this paper, we describe the biochemical reconstitution of a cysteine salvage pathway and the biochemical characterization of each of the five enzymes involved. The salvage begins with amine acetylation of S-alkylcysteine, followed by thioether oxidation. The C-S bond of the resulting sulfoxide is cleaved using a new flavoenzyme catalytic motif to give N-acetylcysteine sulfenic acid. This is then reduced to the thiol and deacetylated to complete the salvage pathway. We propose that this pathway is important in the catabolism of alkylated cysteine generated by proteolysis of alkylated glutathione formed in the detoxification of a wide range of electrophiles.

Temporal evolution of master regulator Crp identifies pyrimidines as catabolite modulator factors.

The evolution of microorganisms often involves changes of unclear relevance, such as transient phenotypes and sequential development of multiple adaptive mutations in hotspot genes. Previously, we showed that ageing colonies of an E. coli mutant unable to produce cAMP when grown on maltose, accumulated mutations in the crp gene (encoding a global transcription factor) and in genes involved in pyrimidine metabolism such as cmk; combined mutations in both crp and cmk enabled fermentation of maltose (which usually requires cAMP-mediated Crp activation for catabolic pathway expression). Here, we study the sequential generation of hotspot mutations in those genes, and uncover a regulatory role of pyrimidine nucleosides in carbon catabolism. Cytidine binds to the cytidine regulator CytR, modifies the expression of sigma factor 32 (RpoH), and thereby impacts global gene expression. In addition, cytidine binds and activates a Crp mutant directly, thus modulating catabolic pathway expression, and could be the catabolite modulating factor whose existence was suggested by Jacques Monod and colleagues in 1976. Therefore, transcription factor Crp appears to work in concert with CytR and RpoH, serving a dual role in sensing both carbon availability and metabolic flux towards DNA and RNA. Our findings show how certain alterations in metabolite concentrations (associated with colony ageing and/or due to mutations in metabolic or regulatory genes) can drive the evolution in non-growing cells.

Fetal Cardiac Interventions-Are They Safe for the Mothers?

The aim of fetal cardiac interventions (FCI), as other prenatal therapeutic procedures, is to bring benefit to the fetus. However, the safety of the mother is of utmost importance. The objective of our study was to evaluate the impact of FCI on maternal condition, course of pregnancy, and delivery. 113 mothers underwent intrauterine treatment of their fetuses with critical heart defects. 128 percutaneous ultrasound-guided FCI were performed and analyzed. The patients were divided into four groups according to the type of FCI: balloon aortic valvuloplasty (fBAV), balloon pulmonary valvuloplasty (fBPV), interatrial stent placement (IAS), and balloon atrioseptoplasty (BAS). Various factors: maternal parameters, perioperative data, and pregnancy complications, were analyzed. There was only one major complication-procedure-related placental abruption (without need for blood products transfusion). There were no cases of: procedure-related preterm prelabor rupture of membranes (pPROM), chorioamnionitis, wound infection, and anesthesia associated complications. Tocolysis was only necessary only in two cases, and it was effective in both. None of the patients required intensive care unit admission. The procedure was effective in treating polyhydramnios associated with fetal heart failure in six out of nine cases. Deliveries occurred at term in 89%, 54% were vaginal. The results showed that FCI had a negligible impact on a further course of pregnancy and delivery.

Fixed Dose versus Height-Adjusted Conventional Dose of Intrathecal Hyperbaric Bupivacaine for Caesarean Delivery: A Prospective, Double-Blinded Randomised Trial.

The optimal intrathecal dose of local anaesthetic for caesarean section (CS) anaesthesia is still being debated. We performed a study to compare the effectiveness and safety of spinal anaesthesia with 12.5 mg of hyperbaric bupivacaine and a dosing regimen of conventional doses adjusted to parturient height. One hundred and forty parturients scheduled for elective CS were enrolled. The fixed-dose group (FD) received a spinal block with 12.5 mg of hyperbaric bupivacaine with fentanyl, whereas the adjusted-dose group (AD) received a height-adjusted dose of bupivacaine (9-13 mg) with fentanyl. Sensory block ≥ T5 dermatome within 10 min and no need for supplementary analgesia were set as the composite primary outcome (success). Rates of successful blocks and complications were compared. Complete data were available for 134 cases. Spinal anaesthesia was successful in 58 out of 67 patients in the FD group and 57 out of 67 in the AD group (p > 0.05). Eight spinals in each group failed to produce a block ≥ T5 in 10 min, and one patient in the FD group and two in the AD group required i.v. analgesics despite sensory block ≥ T5. No differences were noted in terms of hypotension, bradycardia and nausea between the FD and AD groups. Compared to the height-adjusted dose regimen based on conventional doses of hyperbaric bupivacaine, the fixed dose regimen of 12.5 mg was equally effective and did not increase the risk of spinal block-related complications.

Fetal Cardiac Interventions-Polish Experience from "Zero" to the Third World Largest Program.

This article presents the technical aspects of the Polish fetal cardiac interventions (FCI) program, including preparation of the team and modifications in the technique of the procedure that aim to increase its safety for the mother and the fetus. Over 9 years, 128 FCI in 113 fetuses have been performed: 94 balloon aortic valvuloplasties (fBAV), 14 balloon atrioseptoplasties (fBAS) with stent (BAS+), 5 balloon atrioseptoplasties without stent placement (BAS-), and 15 fetal pulmonary valvuloplasties (fBPS). The technical success rate ranged from 80% (BAS-) to 89% (fBAV), while the procedure-related death rate (defined as death within 72 hours following the procedure) ranged from 7% (fBAV and fBPV) to 20% (BAS). There were 98 live births after all FCI (3 pregnancies continue). Median gestational age at delivery was 39 weeks in our center and 38 weeks in other centers.

One-carbon metabolism, folate, zinc and translation.

The translation process, central to life, is tightly connected to the one-carbon (1-C) metabolism via a plethora of macromolecule modifications and specific effectors. Using manual genome annotations and putting together a variety of experimental studies, we explore here the possible reasons of this critical interaction, likely to have originated during the earliest steps of the birth of the first cells. Methionine, S-adenosylmethionine and tetrahydrofolate dominate this interaction. Yet, 1-C metabolism is unlikely to be a simple frozen accident of primaeval conditions. Reactive 1-C species (ROCS) are buffered by the translation machinery in a way tightly associated with the metabolism of iron-sulfur clusters, zinc and potassium availability, possibly coupling carbon metabolism to nitrogen metabolism. In this process, the highly modified position 34 of tRNA molecules plays a critical role. Overall, this metabolic integration may serve both as a protection against the deleterious formation of excess carbon under various growth transitions or environmental unbalanced conditions and as a regulator of zinc homeostasis, while regulating input of prosthetic groups into nascent proteins. This knowledge should be taken into account in metabolic engineering.

Mutations in the Global Transcription Factor CRP/CAP: Insights from Experimental Evolution and Deep Sequencing.

The Escherichia coli cyclic AMP receptor protein (CRP or catabolite activator protein, CAP) provides a textbook example of bacterial transcriptional regulation and is one of the best studied transcription factors in biology. For almost five decades a large number of mutants, evolved in vivo or engineered in vitro, have shed light on the molecular structure and mechanism of CRP. Here, we review previous work, providing an overview of studies describing the isolation of CRP mutants. Furthermore, we present new data on deep sequencing of different bacterial populations that have evolved under selective pressure that strongly favors mutations in the crp locus. Our new approach identifies more than 100 new CRP mutations and paves the way for a deeper understanding of this fascinating bacterial master regulator.

Do drugs used in obstetric anesthesia interfere with early breastfeeding? What determines drug safety in lactation? Part 1.

The issues concerning the transfer of drugs into mothers' milk and their influence on breastfed babies have not been fully studied. Assessment of the situation should include such aspects as drug transfer into fetal blood and into mother's milk, the real risk of inhibiting lactogenesis 2 in women after birth, as well as the psychological consequences for the mother of suspending breastfeeding. The risk of feeding a newborn with formula based on cow's milk is another fateful issue. The following paper presents the pharmacokinetic characteristics of drugs which determine their transfer level through the placenta and into mother's milk during the perinatal period and lactation.

Do drugs used in obstetric anesthesia interfere with early breastfeeding? Characteristics of the pharmacodynamic and pharmacokinetic properties of certain drugs. Part 2.

Cesarean section requires the administration of drugs that should be limited to specific medical indications. It is important to remember that most of the available and currently administered anesthetics can affect the fetus and the newborn. In obstetric anesthesia, only such medication that demonstrates a beneficial pharmacokinetic profile and maximum effectiveness should be administered. In this article, the authors reviewed the pharmacodynamic and pharmacokinetic properties of the drugs used during anesthesia in obstetric procedures. The analysis of the influence of these drugs on the clinical condition of the newborn at birth and during breastfeeding was also presented. Drug safety was determined in the aspect of lactation and natural feeding.

Revisiting the methionine salvage pathway and its paralogues.

Methionine is essential for life. Its chemistry makes it fragile in the presence of oxygen. Aerobic living organisms have selected a salvage pathway (the MSP) that uses dioxygen to regenerate methionine, associated to a ratchet-like step that prevents methionine back degradation. Here, we describe the variation on this theme, developed across the tree of life. Oxygen appeared long after life had developed on Earth. The canonical MSP evolved from ancestors that used both predecessors of ribulose bisphosphate carboxylase oxygenase (RuBisCO) and methanethiol in intermediate steps. We document how these likely promiscuous pathways were also used to metabolize the omnipresent by-products of S-adenosylmethionine radical enzymes as well as the aromatic and isoprene skeleton of quinone electron acceptors.

Bacillus subtilis, the model Gram-positive bacterium: 20 years of annotation refinement.

Genome annotation is, nowadays, performed via automatic pipelines that cannot discriminate between right and wrong annotations. Given their importance in increasing the accuracy of the genome annotations of other organisms, it is critical that the annotations of model organisms reflect the current annotation gold standard. The genome of Bacillus subtilis strain 168 was sequenced twenty years ago. Using a combination of inductive, deductive and abductive reasoning, we present a unique, manually curated annotation, essentially based on experimental data. This reveals how this bacterium lives in a plant niche, while carrying a paleome operating system common to Firmicutes and Tenericutes. Dozens of new genomic objects and an extensive literature survey have been included for the sequence available at the INSDC (AccNum AL009126.3). We also propose an extension to Demerec's nomenclature rules that will help investigators connect to this type of curated annotation via the use of common gene names.

The revisited genome of Pseudomonas putida KT2440 enlightens its value as a robust metabolic chassis.

By the time the complete genome sequence of the soil bacterium Pseudomonas putida KT2440 was published in 2002 (Nelson et al., ) this bacterium was considered a potential agent for environmental bioremediation of industrial waste and a good colonizer of the rhizosphere. However, neither the annotation tools available at that time nor the scarcely available omics data-let alone metabolic modeling and other nowadays common systems biology approaches-allowed them to anticipate the astonishing capacities that are encoded in the genetic complement of this unique microorganism. In this work we have adopted a suite of state-of-the-art genomic analysis tools to revisit the functional and metabolic information encoded in the chromosomal sequence of strain KT2440. We identified 242 new protein-coding genes and re-annotated the functions of 1548 genes, which are linked to almost 4900 PubMed references. Catabolic pathways for 92 compounds (carbon, nitrogen and phosphorus sources) that could not be accommodated by the previously constructed metabolic models were also predicted. The resulting examination not only accounts for some of the known stress tolerance traits known in P. putida but also recognizes the capacity of this bacterium to perform difficult redox reactions, thereby multiplying its value as a platform microorganism for industrial biotechnology.

Generation of mutation hotspots in ageing bacterial colonies.

How do ageing bacterial colonies generate adaptive mutants? Over a period of two months, we isolated on ageing colonies outgrowing mutants able to use a new carbon source, and sequenced their genomes. This allowed us to uncover exquisite details on the molecular mechanism behind their adaptation: most mutations were located in just a few hotspots in the genome, and over time, mutations increasingly were consistent with the involvement of 8-oxo-guanosine, formed exclusively on the transcribed strand. This work provides strong support for retromutagenesis as a general process creating adaptive mutations during ageing.

The effectiveness of neurolytic block of sphenopalatine ganglion using zygomatic approach for the management of trigeminal neuropathy.

This study was performed to present the outcomes of trigeminal neuropathy management with the application of neurolytic block of sphenopalatine ganglion. This type of procedure is used in cases where pain is not well controlled with medical treatment. Twenty patients were treated with sphenopalatine ganglion neurolysis after their response to pharmacological management was not satisfactory. Significant pain relief was experienced by all but one patient and they were able to reduce or stop their pain medication. The time of pain relief was between a few months and 9 years during the study period. Number of procedures implemented varied as some of the patients have been under the care of our Pain Clinic for as long as 18 years, satisfied with this type of management and willing to have the procedure repeated if necessary. It appears that neurolytic block of sphenopalatine ganglion is effective enough and may be an option worth further consideration in battling the pain associated with trigeminal neuropathy.

Pharmacological treatment and regional anesthesia techniques for pain management after completion of both conservative and surgical treatment of endometriosis and pelvic adhesions in women with chronic pelvic pain as a mandated treatment strategy.

INTRODUCTION: Chronic pelvic pain syndrome occurs in 4-14% of women. Pain pathomechanism in this syndrome is complex, as it is common to observe the features of nociceptive, inflammatory, neuropathic and psychogenic pain. The common findings in women with pelvic pain are endometriosis and pelvic adhesions. OBJECTIVE: Aim of the study was to test the effectiveness of pharmacological treatment and regional anesthesia techniques for pain control as the next step of treatment after the lack of clinical results of surgical and pharmacological methods normally used in the management of endometriosis and pelvic adhesions. MATERIALS AND METHOD: 18 women were treated between January 2010 - October 2013 in the Pain Clinic of the Department of Anaesthesiology and Intensive Care at the Centre for Postgraduate Education in Warsaw due to chronic pelvic pain syndrome related to either endometriosis or pelvic adhesions. During the previous step of management, both conservative and surgical treatments were completed without achieving satisfactory results. Initial constant pain severity was 3-9 points on the Numeric Rating Scale, while the reported paroxysmal pain level was 7-10. The pharmacological treatment implemented was based on oral gabapentinoids and antidepressants, aided by neurolytic block of ganglion of Walther, pudendal nerve blocks and topical treatment (5% lidocaine, 10% amitriptyline, 10% gabapentin). RESULTS: In 17 women, a significant reduction of both constant and paroxysmal pain was achieved, of which complete and permanent cessation of pain occurred in 6 cases. One patient experienced no improvement in the severity of her symptoms. CONCLUSIONS: The combination of pain management with pharmacological treatment, pudendal nerve blocks, neurolysis of ganglion impar (Walther) and topical preparations in cases of chronic pelvic pain syndrome seems to be adequate medical conduct after failed or otherwise ineffective causative therapy.

5% lidocaine medicated plasters vs. sympathetic nerve blocks as a part of multimodal treatment strategy for the management of postherpetic neuralgia: a retrospective, consecutive, case-series study.

INTRODUCTION: 5% lidocaine medicated plasters (5% LMP) have been appointed as a first-line treatment for post-herpetic neuralgia (PHN), while formerly used sympathetic nerve blocks (SNBs) were recently denied their clinical efficacy. The aim of this study was to compare the results of PHN management with the use of SNBs and 5% LMP as a first-line treatment. MATERIAL AND METHODS: This study was designed as a retrospective, consecutive, case-series study. Data of 60 consecutive PHN patients with allodynia treated with the use of SNBs and 60 subsequent patients managed with 5% LMP were analyzed. Pain severity after 8 weeks was assessed to recognize the results of the implemented therapy, with numeric rating scale (NRS) score <3 or =3 considered a success. Additionally, the number of pain-free patients (NRS=0) after 8 weeks were identified in both groups and compared. RESULTS: The rate of failures in SNBs and 5% LMP group was similar (18.9% vs. 27.1% of poor treatment results, respectively), with the average change in NRS of 5.88 ± 2.41 in nerve blocks and 5.01 ± 1.67 in lidocaine group (p=0.02). Significant difference was also noted in the rates of pain-free patients: 20 patients (34.4%) treated with SNBs and 8 (13.5%) using 5% LMP were pain-free after 8 weeks of treatment. CONCLUSION: It may be concluded that SNBs may still be considered useful in PHN management, as it appears that in some cases this mode of treatment may offer some advantages over 5% LMP.

Chemical reactivity drives spatiotemporal organisation of bacterial metabolism.

In this review, we examine how bacterial metabolism is shaped by chemical constraints acting on the material and dynamic layout of enzymatic networks and beyond. These are moulded not only for optimisation of given metabolic objectives (e.g. synthesis of a particular amino acid or nucleotide) but also for curbing the detrimental reactivity of chemical intermediates. Besides substrate channelling, toxicity is avoided by barriers to free diffusion (i.e. compartments) that separate otherwise incompatible reactions, along with ways for distinguishing damaging vs. harmless molecules. On the other hand, enzymes age and their operating lifetime must be tuned to upstream and downstream reactions. This time dependence of metabolic pathways creates time-linked information, learning and memory. These features suggest that the physical structure of existing biosystems, from operon assemblies to multicellular development may ultimately stem from the need to restrain chemical damage and limit the waste inherent to basic metabolic functions. This provides a new twist of our comprehension of fundamental biological processes in live systems as well as practical take-home lessons for the forward DNA-based engineering of novel biological objects.

Sympathetic nerve blocks for the management of postherpetic neuralgia - 19 years of pain clinic experience.

BACKGROUND: Sympathetic system involvement in postherpetic neuralgia (PHN) has been targeted using peripheral sympathetic nerve blocks for a number of years with variable efficacy. The aim of this report is to present the outcomes of PHN management with concomitant use of pharmacological treatment and sympathetic nerve blocks. METHODS: We retrospectively evaluated clinical data on 563 patients with PHN symptoms treated in the pain clinic and identified cases in which sympathetic nerve blocks were implemented in the years 1992-2010. A Numeric Rating Scale was used as a pain severity assessment, with a reduction to values under 3 considered a positive therapy result. Three time intervals were considered: years 1992-1997 (I), 1998-2002 (II) and 2003-2010 (III). RESULTS: In group I, 27% of patients had poor treatment results, while in group II, the failure rate dropped to 18%. The same 18% failure rate was observed in group III as well. Treatment introduced early yielded the best results, but there was no difference among groups with a similar duration from herpes zoster onset to treatment commencement in the time periods assessed; however, from 1998 onward, the same rate of poor outcomes was also noted in the groups who started the sympathetic blockade, which aided pain clinic treatment up to 3 months and between 3 and 6 months from the onset of herpes zoster. CONCLUSION: Major progress in the pharmacological treatment of PHN appears to be an obvious factor contributing to the overall improvement in PHN management (introduction of gabapentin). Nevertheless, safely administered regional anaesthesia techniques, although performed in a very similar manner for many years, appear to provide some support as part of a multimodal approach to PHN management.