In vitro higher-order oligomeric assembly of the respiratory syncytial virus M2-1 protein with longer RNAs.

The respiratory syncytial virus (RSV) M2-1 protein is a transcriptional antitermination factor crucial for efficiently synthesizing multiple full-length viral mRNAs. During RSV infection, M2-1 exists in a complex with mRNA within cytoplasmic compartments called inclusion body-associated granules (IBAGs). Prior studies showed that M2-1 can bind along the entire length of viral mRNAs instead of just gene-end (GE) sequences, suggesting that M2-1 has more sophisticated RNA recognition and binding characteristics. Here, we analyzed the higher oligomeric complexes formed by M2-1 and RNAs in vitro using size exclusion chromatography (SEC), electrophoretic mobility shift assays (EMSA), negative stain electron microscopy (EM), and mutagenesis. We observed that the minimal RNA length for such higher oligomeric assembly is about 14 nucleotides for polyadenine sequences, and longer RNAs exhibit distinct RNA-induced binding modality to M2-1, leading to enhanced particle formation frequency and particle homogeneity as the local RNA concentration increases. We showed that particular cysteine residues of the M2-1 cysteine-cysteine-cystine-histidine (CCCH) zinc-binding motif are essential for higher oligomeric assembly. Furthermore, complexes assembled with long polyadenine sequences remain unaffected when co-incubated with ribonucleases or a zinc chelation agent. Our study provided new insights into the higher oligomeric assembly of M2-1 with longer RNA.IMPORTANCERespiratory syncytial virus (RSV) causes significant respiratory infections in infants, the elderly, and immunocompromised individuals. The virus forms specialized compartments to produce genetic material, with the M2-1 protein playing a pivotal role. M2-1 acts as an anti-terminator in viral transcription, ensuring the creation of complete viral mRNA and associating with both viral and cellular mRNA. Our research focuses on understanding M2-1's function in viral mRNA synthesis by modeling interactions in a controlled environment. This approach is crucial due to the challenges of studying these compartments in vivo. Reconstructing the system in vitro uncovers structural and biochemical aspects and reveals the potential functions of M2-1 and its homologs in related viruses. Our work may contribute to identifying targets for antiviral inhibitors and advancing RSV infection treatment.

Use of high-frequency electrical stimulation in gastrocutaneous fistula closure: a case report.

BACKGROUND: Gastrocutaneous fistula is a rare complication following Roux-en-Y gastric bypass, a commonly performed bariatric surgery. While most ECFs respond to conservative management, some do not close despite adequate nutritional support, infection source control, and drainage management. As such, the chronicity of these difficult-to-treat wounds can be physically and economically costly to patients. CASE REPORT: A 53-year-old female with a history of Roux-en-Y gastric bypass developed a gastrocutaneous fistula secondary to a perforated gastrojejunal ulcer, requiring immediate surgical intervention. After being discharged from the hospital, 37 days of conservative management and NPWT did not reduce the size of the fistula tract. To help control the patient's chronic abdominal pain and increase the rate of wound healing, the patient underwent treatment with HFES (20 kHz) delivered using a handheld transcutaneous electrical nerve stimulator. This electrotherapy was found to reduce the majority of the patient's pain within the first treatment session. The patient's fistula also began to decrease in size within 1 week of initiating treatment. CONCLUSION: This case report details the successful closure of a gastrocutaneous fistula after administration of HFES 3 times a week over the course of 25 days. The mechanism of action of HFES and its role in the wound healing process are also discussed.

Quantitative Volumetric Measurements of Bone Grafting Sites Within the Lower Extremity on Computed Tomography Scans.

The anterior iliac crest is one of the most used options; however, pain and other complications have been reported. Other options for bone harvest in the lower extremity, such as the proximal tibia and calcaneus, can be useful sites for bone grafting. Computed tomography angiography images of the lower extremity were analyzed using 3-D Slicer™ medical imaging software, creating an advanced 3-dimensional model. Bone volume (cm3) and bone mineral density (Hounsfield units) were measured from the cancellous bone in the anterior iliac crest, posterior iliac crest, proximal tibia, and the calcaneus. Fifteen studies were included. The total volume measured it was of 61.88 ± 14.15 cm3, 19.35 ± 4.16 cm3, 32.48 ± 7.49 cm3, 26.40 ± 7.18 cm3, for the proximal tibia, anterior and posterior iliac crest, and calcaneus, respectively. Regarding Hounsfield units, the densities were 116 ± 58.77, 232.4 ± 68.65, 214.4 ± 74.45, 170.5 ± 52.32, for proximal tibia, anterior and posterior iliac crest, and calcaneus. The intraclass correlation coefficients were in average >0.94. In conclusion, the proximal tibia has more cancellous bone than the anterior and posterior iliac crest. The calcaneus has more cancellous bone than the anterior iliac crest. Bone mineral density was highest in the anterior iliac crest and in proximal tibia was the lowest value.

Ratiometric Sensing of Glyphosate in Water Using Dual Fluorescent Carbon Dots.

Glyphosate is a broad-spectrum pesticide used in crops and is found in many products used by industry and consumers. Unfortunately, glyphosate has been shown to have some toxicity toward many organisms found in our ecosystems and has been reported to have carcinogenic effects on humans. Hence, there is a need to develop novel nanosensors that are more sensitive and facile and permit rapid detection. Current optical-based assays are limited as they rely on changes in signal intensity, which can be affected by multiple factors in the sample. Herein, we report the development of a dual emissive carbon dot (CD) system that can be used to optically detect glyphosate pesticides in water at different pH levels. The fluorescent CDs emit blue and red fluorescence, which we exploit as a ratiometric self-referencing assay. We observe red fluorescence quenching with increasing concentrations of glyphosate in the solution, ascribed to the interaction of the glyphosate pesticide with the CD surface. The blue fluorescence remains unaffected and serves as a reference in this ratiometric approach. Using fluorescence quenching assays, a ratiometric response is observed in the ppm range with detection limits as low as 0.03 ppm. Our CDs can be used to detect other pesticides and contaminants in water, as cost-effective and simple environmental nanosensors.

Clinical-radiomic model in advanced kidney cancer predicts response to tyrosine kinase inhibitors.

Renal cancer has a global incidence and mortality of 2.2 and 1.8%, respectively. Up to 30% of these patients are intrinsically resistant to tyrosine kinase inhibitors (TKI). The National Comprehensive Cancer Network guidelines do not include any predictive factors regarding response to systemic therapy with TKI in recurrent and advanced diseases. The present study aimed to explore whether a model based on radiomics could predict treatment response in patients with advanced kidney cancer treated with TKIs. The current study included 62 patients with advanced kidney cancer (stages 3 and 4) that underwent a CT scan in the arterial phase from March 2016 to November 2020. Texture analysis was run on the largest cross-sectional area of the primary tumor from each CT scan. A total of three different models were built from radiomics features and clinical data to analyze them by logistic regression and determine whether they correlated with the response to TKI. A receiver operating characteristic curve analysis was performed in each model to calculate the area under the curve (AUC) and the 95% confidence interval (CI). Significant radiomics features and clinical variables were identified and then a clinical model was created (AUC=0.90; sensitivity 75%; specificity 82.35%; CI 95%, 0.78-1.00), a radiomic model (AUC=0.66; sensitivity 16.67%; specificity 89.47%, CI 95%, 0.45-0.87) and a combined model (AUC=0.94; sensitivity 83.33%; specificity 94.12%; CI 95%, 0.84-1.00). Overall, models based on clinical data and radiomics could anticipate response to systemic therapy with TKI in patients with advanced kidney cancer.

Derivation of a score to predict infection due to multidrug-resistant Pseudomonas aeruginosa: a tool for guiding empirical antibiotic treatment.

OBJECTIVES: Multidrug-resistant Pseudomonas aeruginosa (MDR-PSA) constitutes an emerging health problem. A predictive score of MDR-PSA infection would allow an early adaptation of empirical antibiotic therapy. METHODS: We performed a single-centre case-control (1:2) retrospective study including 100 patients with MDR-PSA and 200 with a non-MDR-PSA infection. Cases and controls were matched by site of infection, clinical characteristics and immunosuppression. A point risk score for prediction of MDR-PSA infection was derived from a logistic regression model. Secondary outcomes (clinical improvement, complications and discharge) were also compared. RESULTS: Cases with MDR-PSA infection were younger than controls (67.5 vs. 73.0 y; P = 0.031) and have more frequent cirrhosis (9% vs. 2%; P = 0.005). Independent risk factors for MDR-PSA infection were prior antibiotic treatment (80% vs. 50.5%; P < 0.001), prior colonisation with MDR bacteria (41% vs. 13.5%; P < 0.001), hospital-acquired infection (63% vs. 47%; P = 0.009) and septic shock at diagnosis (33% vs. 14%; P < 0.001). Adequate therapy was less frequent in MDR-PSA infections (31% vs. 66.5% for empirical therapy; P < 0.001). The risk score included: previous MDR-PSA isolation (11 points), prior antibiotic use (3 points), hospital-acquired infection (2 points) and septic shock at diagnosis (2 points). It showed an area under the curve of 0.755 (95% CI: 0.70-0.81) and allowed to classify individual risk into various categories: 0-2 points (<20%), 3-5 points (25%-45%), 7-11 points (55%-60%), 13-16 points (75%-87%) and a maximum of 18 points (93%). CONCLUSION: Infections due to MDR-PSA have a poorer prognosis than those produced by non-MDR-PSA. Our score could guide empirical therapy for MDR-PSA when P. aeruginosa is isolated.

Environmental exposure to cadmium reduces the primary antibody-mediated response of wood mice (Apodemus sylvaticus) from differentially polluted locations in the Netherlands.

The Wood mouse (Apodemus sylvaticus) is a widespread mammalian species that acts as a reservoir host for multiple infections, including zoonotic diseases. Exposure to immunotoxins, like for instance trace metals, may reduce the ability of the host to mount proper responses to pathogens, potentially increasing the transmission and prevalence of infections. Antibody-mediated responses are crucial in preventing and limiting infections, and the quantification of the primary antibody response is considered a sensitive predictor of immunosuppression. The current study aims to investigate effects of cadmium exposure on the antibody-mediated responses of wood mice inhabiting polluted and non-polluted areas in the Netherlands. Wood mice were captured alive at different locations and immunized to sheep red blood cells (SRBC) to induce a primary antibody response. SRBC-specific antibody-producing cells, or plaque forming cells (PFC), were quantified and related to kidney cadmium levels. Differential circulating main leukocyte populations were also characterised. Cadmium concentrations in mice kidneys differed between mice captured at different locations, and increased with individual body mass, likely associated with age-related time of exposure. Effect of cadmium was apparent on the percentages of B cell counts in blood. Because of potential natural immune heterogeneity between wild rodent populations, mice immune responses were analysed and compared grouped by captured locations. Capture location had significant effect on the total counts of white blood cells. Increasing cadmium exposure in wood mice captured from polluted sites was associated with a decrease of splenic PFC counts. This field research shows that wood mice antibody responses can be impaired by cadmium exposure, even at low environmental levels, by affecting B cell functioning mainly. Impaired B cell function can make exposed mice more susceptible to infections, potentially increasing the reservoir function of their populations. It also shows that immunomodulatory effects in the field should be assessed site specifically.

Modulatory Effects of Mercury (II) Chloride (HgCl2 ) on Chicken Macrophage and B-Lymphocyte Cell Lines with Viral-Like Challenges In Vitro.

Mercury (Hg) is a toxic trace metal ubiquitously distributed in the environment. Inorganic mercury (as HgCl2 ) can cause immunotoxicity in birds, but the mechanisms of action are still not fully resolved, especially with respect to responses to viral infections. To investigate the potential immunomodulatory effects of Hg2+ on specific cell types of the avian immune system, chicken macrophage (HD-11) and B-lymphocyte (DT40) cell lines were applied as in vitro models for the innate and adaptive immune systems, respectively. The cells were stimulated with synthetic double-stranded RNA, which can be recognized by toll-like receptor-3 to mimic a viral infection. The Hg2+ showed concentration-dependent cytotoxicity in both cell lines, with similar median effect concentrations at 30 µM. The cytotoxicity of Hg2+ was closely related to glutathione (GSH) depletion and reactive oxygen species induction, whereas the de novo synthesis of GSH acted as a primary protective strategy. Nitric oxide produced by activated macrophages was strongly inhibited by Hg2+ , and was also influenced by cellular GSH levels. Cell proliferation, gene expression of microRNA-155, and cellular IgM levels in B cells were decreased at noncytotoxic Hg2+ concentrations. The secretion of antiviral interferon-α was induced by Hg2+ in both cell lines. Overall, our results suggest that Hg2+ exposure can cause immunomodulatory effects in birds by disrupting immune cell proliferation and cytokine production, and might result in disorders of the avian immune system. Environ Toxicol Chem 2021;40:2813-2824. © 2021 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC.

Amputation in crush syndrome: A case report.

INTRODUCTION: Crush syndrome (CS) is a condition with a high morbidity and mortality due to severe electrolyte disorders, circulatory dysfunction and multiple organ failure, secondary to severe rhabdomyolysis and reperfusion injuries. There is controversy about the role of fasciotomy in the treatment of compartment syndromes due to crush injuries and it is still unknown if early amputation has patient-centered benefits. CASE PRESENTATION: This is a 29-year-old patient whose lower body was trapped for 50 h under a 40-meter landslide. Upon admission the left thigh was edematous and painful. Laboratories revealed a creatinine of 1.58 mg/dL, hyperkalemia, metabolic acidosis, hyperlactatemia and creatinine phosphokinase (CPK) of 88,700 U/L, suggesting CS. Despite fluid and bicarbonate infusion his renal function worsened, CPK rose and left thigh became more tense, so a fasciotomy was performed. He developed a distributive shock refractory to vasopressors, steroids and methylene blue so amputation was proposed. Two hours after amputation the vasopressor support was nearly withdrawn. DISCUSSION: This case suggests a potential benefit of amputation in patients with CS and progressive deterioration despite aggressive resuscitation. It also invites to think if this is a decision that should be considered before the establishment or in the initial stages of the syndrome, even if the viability of the extremity is still questionable. CONCLUSION: The presence of risk factors for poor prognosis can favor amputation despite the apparent viability of the limb and the morbidity of losing an extremity.

Modulatory Effects of Pb2+ on Virally Challenged Chicken Macrophage (HD-11) and B-Lymphocyte (DT40) Cell Lines In Vitro.

Elevated levels of lead have been found in waterfowl, due to human activities. Lead may cause immunomodulatory effects, but the mechanisms are largely unknown, especially after viral challenges. To characterize avian immunomodulatory hazards of lead (Pb)2+ , we used chicken macrophage (HD-11) and B-lymphocyte (DT40) cell lines, as in vitro models for the innate and adaptive immune systems, respectively. The cells were activated via toll-like receptor-3 by polyinosinic-polycytidylic acid sodium salt (poly I:C), mimicking viral infections. Our results indicate that Pb2+ is cytotoxic to both cell lines, macrophages being more sensitive. De novo synthesis of glutathione plays an important role in protecting macrophages from Pb2+ intoxication, which might also be closely involved in the induction of nitric oxide after Pb2+ exposure. Stimulatory effects on cell proliferation were noticed at noncytotoxic Pb2+ concentrations as well. Exposure to Pb2+ could also affect the inflammatory status by inhibiting the pro-inflammatory interferon (IFN)-γ while promoting the production of anti-inflammatory type I IFNs in both macrophages and B-cells, and increasing intracellular IgM levels in B-cells. These results suggest that the immunomodulatory effects of Pb2+ in birds are probably closely associated with disruption of immune cell proliferation and cytokine production, potentially causing disorders of the avian immune system. Environ Toxicol Chem 2020;39:1060-1070. © 2020 SETAC.

Cell-specific immune-modulation of cadmium on murine macrophages and mast cell lines in vitro.

Toxic trace metals are widespread contaminants that are potentially immunotoxic even at environmentally low exposure levels. They can modulate the immunity to infections, e.g., in wildlife species living in contaminated areas. The diverse immune cell types can be differentially affected by the exposure leading to the modulation of specific protective mechanisms. Macrophages and mast cells, part of the innate immune system, trigger immune responses and perform particular effector functions. The present study compared toxicological and functional effects of cadmium in two models of murine macrophages (RAW264.7 and NR8383 cell lines) and two models of murine mast cells (MC/9 and RBL-2H3 cell lines). Cadmium was selected as a model compound because its known potential to induce reactive oxygen species and its relevance as an environmental contaminant. Mechanisms of toxicity, such as redox imbalance and apoptosis induction were measured in stationary cells, while functional outcome effects were measured in activated cells. Cadmium-depleted glutathione antioxidant in all four cell lines tested although reactive oxygen species was not significantly increased. Mast cells had full dose-response depletion of glutathione below cytotoxic levels while in macrophages the depletion was not complete. Functional endpoints tumour necrosis factor-alpha and nitrite production in lipopolysaccharide-activated macrophages were increased by cadmium exposure. In contrast, mast cell lipopolysaccharide-induced tumour necrosis factor-alpha and IgE-mediated histamine release were reduced by cadmium. These data indicate potentially differential effects of cadmium among murine innate immune cell types, where mast cells would be more susceptible to oxidative stress and their function might be at a higher risk to be modulated compared to macrophages.

Polyphasic evaluation of key cyanobacteria in biocrusts from the most arid region in Europe.

Cyanobacteria are key microbes in topsoil communities that have important roles in preventing soil erosion, carbon and nitrogen fixation, and influencing soil hydrology. However, little is known regarding the identity and distribution of the microbial components in the photosynthetic assemblages that form a cohesive biological soil crust (biocrust) in drylands of Europe. In this study, we investigated the cyanobacterial species colonizing biocrusts in three representative dryland ecosystems from the most arid region in Europe (SE Spain) that are characterized by different soil conditions. Isolated cyanobacterial cultures were identified by a polyphasic approach, including 16S rRNA gene sequencing, phylogenetic relationship determination, and morphological and ecological habitat assessments. Three well-differentiated groups were identified: heterocystous-cyanobacteria (Nostoc commune, Nostoc calcicola, Tolypothrix distorta and Scytonema hyalinum), which play an important role in N and C cycling in soil; nonheterocystous bundle-forming cyanobacteria (Microcoleus steenstrupii, Trichocoleus desertorum, and Schizothrix cf. calcicola); and narrow filamentous cyanobacteria (Leptolyngbya frigida and Oculatella kazantipica), all of which are essential genera for initial biocrust formation. The results of this study contribute to our understanding of cyanobacterial species composition in biocrusts from important and understudied European habitats, such as the Mediterranean Basin, a hotspot of biodiversity, where these species are keystone pioneer organisms.

Structural determinant of functionality in acyl lipid desaturases.

Little is known about the structure-function relationship of membrane-bound lipid desaturases. Using a domain-swapping strategy, we found that the N terminus (comprising the two first transmembrane segments) region of Bacillus cereus DesA desaturase improves Bacillus subtilis Des activity. In addition, the replacement of the first two transmembrane domains from Bacillus licheniformis inactive open reading frame (ORF) BL02692 with the corresponding domain from DesA was sufficient to resurrect this enzyme. Unexpectedly, we were able to restore the activity of ORF BL02692 with a single substitution (Cys40Tyr) of a cysteine localized in the first transmembrane domain close to the lipid-water interface. Substitution of eight residues (Gly90, Trp104, Lys172, His228, Pro257, Leu275, Tyr282, and Leu284) by site-directed mutagenesis produced inactive variants of DesA. Homology modeling of DesA revealed that His228 is part of the metal binding center, together with the canonical His boxes. Trp104 shapes the hydrophobic tunnel, whereas Gly90 and Lys172 are probably involved in substrate binding/recognition. Pro257, Leu275, Tyr282, and Leu284 might be relevant for the structural arrangement of the active site or interaction with electron donors. This study reveals the role of the N-terminal region of Δ5 phospholipid desaturases and the individual residues necessary for the activity of this class of enzymes.

The stringent response plays a key role in Bacillus subtilis survival of fatty acid starvation.

The stringent response is a universal adaptive mechanism to protect bacteria from nutritional and environmental stresses. The role of the stringent response during lipid starvation has been studied only in Gram-negative bacteria. Here, we report that the stringent response also plays a crucial role in the adaptation of the model Gram-positive Bacillus subtilis to fatty acid starvation. B. subtilis lacking all three (p)ppGpp-synthetases (RelBs , RelP and RelQ) or bearing a RelBs variant that no longer synthesizes (p)ppGpp suffer extreme loss of viability on lipid starvation. Loss of viability is paralleled by perturbation of membrane integrity and function, with collapse of membrane potential as the likely cause of death. Although no increment of (p)ppGpp could be detected in lipid starved B. subtilis, we observed a substantial increase in the GTP/ATP ratio of strains incapable of synthesizing (p)ppGpp. Artificially lowering GTP with decoyinine rescued viability of such strains, confirming observations that low intracellular GTP is important for survival of nutritional stresses. Altogether, our results show that activation of the stringent response by lipid starvation is a broadly conserved response of bacteria and that a key role of (p)ppGpp is to couple biosynthetic processes that become detrimental if uncoordinated.

Sexismo y concepciones de laviolencia de género contra la mujeren cuatro universidades de la ciudadde Manizales (Colombia), 2015 / Sexism and conceptions of gender violence against women in four universities in the city of Manizales (Colombia), 2015

Estudiar el sexismo y la violencia de género contra la mujer, en estudiantesde cuatro universidades de la ciudad de Manizales. Materiales y métodos: estudiode corte transversal. La población estuvo constituida por 19’042 estudiantes universitarios,y la muestra para el estudio la conformaron 1’393 estudiantes de todas lascarreras de pregrado. Se indagan variables demográficas y de violencia de génerocontra la mujer mediante 8 cuestionarios. Resultados: El 49,4% de los participantesfueron mujeres. Los 8 cuestionarios excepto calidad de la relación entre géneros tienenrelación significativa con el género. En algunos casos también entre universidades.Para mujeres y hombres respectivamente los cuestionarios presentan los siguientesresultados: calidad de las relaciones 47,7%, 47,9%; Sexismo hostil: 48,3%, 57,9%,sexismo benevolente: 46,5%, 51,5%; justificación de la violencia de género: 26,7%,29,6%; justificación del sexismo y la violencia como reacción: 23,7, 29,6; consejos depersonas adultas: 45,8%, 48,9%; violencia física de la pareja contra la mujer: 30,1%,28,5%, violencia emocional de la pareja contra la mujer: 36,1%, 31,3%. Factorescomo el nivel de autoestima, y el estrato social influencian las variables de violenciade género contra la mujer. Conclusiones: los resultados muestran que la violenciade género contra la mujer todavía está presente en forma significativa en estas 4universidades, lo que demuestra que todavía hay mucho que hacer en este aspectoen la sociedad colombiana. También en una proporción considerable esta violenciade género es aceptada por las mujeres...

Structural insights into bacterial resistance to cerulenin.

UNLABELLED: Cerulenin is a fungal toxin that inhibits both eukaryotic and prokaryotic ketoacyl-acyl carrier protein synthases or condensing enzymes. It has been used experimentally to treat cancer and obesity, and is a potent inhibitor of bacterial growth. Understanding the molecular mechanisms of resistance to cerulenin and similar compounds is thus highly relevant for human health. We have previously described a Bacillus subtilis cerulenin-resistant strain, expressing a point-mutated condensing enzyme FabF (FabF[I108F]) (i.e. FabF with isoleucine 108 substituted by phenylalanine). We now report the crystal structures of wild-type FabF from B. subtilis, both alone and in complex with cerulenin, as well as of the FabF[I108F] mutant protein. The three-dimensional structure of FabF[I108F] constitutes the first atomic model of a condensing enzyme that remains active in the presence of the inhibitor. Soaking the mycotoxin into preformed wild-type FabF crystals allowed for noncovalent binding into its specific pocket within the FabF core. Interestingly, only co-crystallization experiments allowed us to trap the covalent complex. Our structure shows that the covalent bond between Cys163 and cerulenin, in contrast to that previously proposed, implicates carbon C3 of the inhibitor. The similarities between Escherichia coli and B. subtilis FabF structures did not explain the reported inability of ecFabF[I108F] (i.e. FabF from Escherichia coli with isoleucine 108 substituted by phenylalanine) to elongate medium and long-chain acyl-ACPs. We now demonstrate that the E. coli modified enzyme efficiently catalyzes the synthesis of medium and long-chain ketoacyl-ACPs. We also characterized another cerulenin-insensitive form of FabF, conferring a different phenotype in B. subtilis. The structural, biochemical and physiological data presented, shed light on the mechanisms of FabF catalysis and resistance to cerulenin. DATABASE: Crystallographic data (including atomic coordinates and structure factors) have been deposited in the Protein Data Bank under accession codes 4LS5, 4LS6, 4LS7 and 4LS8.

A novel amidotransferase required for lipoic acid cofactor assembly in Bacillus subtilis.

In the companion paper we reported that Bacillus subtilis requires three proteins for lipoic acid metabolism, all of which are members of the lipoate protein ligase family. Two of the proteins, LipM and LplJ, have been shown to be an octanoyltransferase and a lipoate : protein ligase respectively. The third protein, LipL, is essential for lipoic acid synthesis, but had no detectable octanoyltransferase or ligase activity either in vitro or in vivo. We report that LipM specifically modifies the glycine cleavage system protein, GcvH, and therefore another mechanism must exist for modification of other lipoic acid requiring enzymes (e.g. pyruvate dehydrogenase). We show that this function is provided by LipL, which catalyses the amidotransfer (transamidation) of the octanoyl moiety from octanoyl-GcvH to the E2 subunit of pyruvate dehydrogenase. LipL activity was demonstrated in vitro with purified components and proceeds via a thioester-linked acyl-enzyme intermediate. As predicted, ΔgcvH strains are lipoate auxotrophs. LipL represents a new enzyme activity. It is a GcvH:[lipoyl domain] amidotransferase that probably uses a Cys-Lys catalytic dyad. Although the active site cysteine residues of LipL and LipB are located in different positions within the polypeptide chains, alignment of their structures show these residues occupy similar positions. Thus, these two homologous enzymes have convergent architectures.

Structural plasticity and catalysis regulation of a thermosensor histidine kinase.

Temperature sensing is essential for the survival of living cells. A major challenge is to understand how a biological thermometer processes thermal information to optimize cellular functions. Using structural and biochemical approaches, we show that the thermosensitive histidine kinase, DesK, from Bacillus subtilis is cold-activated through specific interhelical rearrangements in its central four-helix bundle domain. As revealed by the crystal structures of DesK in different functional states, the plasticity of this helical domain influences the catalytic activities of the protein, either by modifying the mobility of the ATP-binding domains for autokinase activity or by modulating binding of the cognate response regulator to sustain the phosphotransferase and phosphatase activities. The structural and biochemical data suggest a model in which the transmembrane sensor domain of DesK promotes these structural changes through conformational signals transmitted by the membrane-connecting two-helical coiled-coil, ultimately controlling the alternation between output autokinase and phosphatase activities. The structural comparison of the different DesK variants indicates that incoming signals can take the form of helix rotations and asymmetric helical bends similar to those reported for other sensing systems, suggesting that a similar switching mechanism could be operational in a wide range of sensor histidine kinases.

Phosphatidylethanolamine synthesis is required for optimal virulence of Brucella abortus.

The Brucella cell envelope contains the zwitterionic phospholipids phosphatidylcholine (PC) and phosphatidylethanolamine (PE). Synthesis of PC occurs exclusively via the PC synthase pathway, implying that the pathogen depends on the choline synthesized by the host cell to form PC. Notably, PC is necessary to sustain a chronic infection process, which suggests that the membrane lipid content is relevant for Brucella virulence. In this study we investigated the first step of PE biosynthesis in B. abortus, which is catalyzed by phosphatidylserine synthase (PssA). Disruption of pssA abrogated the synthesis of PE without affecting the growth in rich complex medium. In minimal medium, however, the mutant required choline supplementation for growth, suggesting that at least PE or PC is necessary for Brucella viability. The absence of PE altered cell surface properties, but most importantly, it impaired several virulence traits of B. abortus, such as intracellular survival in both macrophages and HeLa cells, the maturation of the replicative Brucella-containing vacuole, and mouse colonization. These results suggest that membrane phospholipid composition is critical for the interaction of B. abortus with the host cell.

Signalling pathways controlling fatty acid desaturation.

Microorganisms, plants and animals regulate the synthesis of unsaturated fatty acids (UFAs) during changing environmental conditions as well as in response to nutrients. Unsaturation of fatty acid chains has important structural roles in cell membranes: a proper ratio of saturated to UFAs contributes to membrane fluidity. Alterations in this ratio have been implicated in various disease states including cardiovascular diseases, immune disorders, cancer and obesity. They are also the major components of triglycerides and intermediates in the synthesis of biologically active molecules such as eicosanoids, which mediates fever, inflammation and neurotransmission. UFAs homeostasis in many organisms is achieved by feedback regulation of fatty acid desaturases gene transcription. Here, we review recently discovered components and mechanisms of the regulatory machinery governing the transcription of fatty acid desaturases in bacteria, yeast and animals.