Comparing subtotal parathyroidectomy and total parathyroidectomy with autotransplantation in renal transplant recipients.

Background: Both subtotal parathyroidectomy (SPTX) and total parathyroidectomy with autotransplantation (TPTX + AT) are considered acceptable surgical approaches for renal patients. It is common that parathyroid surgery is performed in patients before they undergo kidney transplantation and there is currently no evidence considering the best surgical approach in this subset of patients. Methods: Two cohorts were identified of consecutive patients who underwent parathyroidectomy for renal hyperparathyroidism by two surgeons at a single institution over equivalent time periods (SPTX and TPTX + AT). A retrospective chart review was performed to assess these techniques, including outcomes following kidney transplantation. Results: There were 125 patients analysed, with 56 patients who underwent SPTX and 69 who underwent TPTX + AT. Both cohorts effectively reduced PTH post operatively. There were 22 patients in the SPTX cohort and 26 in the TPTX + AT cohort that subsequently received kidney transplants. There were no cases of recurrent hyperparathyroidism and one of hypoparathyroidism (4.5%) in the SPTX patients post-transplant. There was one case of recurrent hyperparathyroidism (3.8%) and four of persistent hypoparathyroidism (15.4%) in the TPTX + AT patients post-transplant. Conclusions: Surgery for renal hyperparathyroidism requires a careful balance of the extent of parathyroid resection to prevent persistent/recurrent disease and avoid permanent hypoparathyroidism. SPTX may be a more appropriate option in kidney transplant candidates in order to minimise the risk of long-term hypoparathyroidism.

Listeria monocytogenes GlmR Is an Accessory Uridyltransferase Essential for Cytosolic Survival and Virulence.

The cytosol of eukaryotic host cells is an intrinsically hostile environment for bacteria. Understanding how cytosolic pathogens adapt to and survive in the cytosol is critical to developing novel therapeutic interventions against these pathogens. The cytosolic pathogen Listeria monocytogenes requires glmR (previously known as yvcK), a gene of unknown function, for resistance to cell-wall stress, cytosolic survival, inflammasome avoidance, and, ultimately, virulence in vivo. In this study, a genetic suppressor screen revealed that blocking utilization of UDP N-acetylglucosamine (UDP-GlcNAc) by a nonessential wall teichoic acid decoration pathway restored resistance to lysozyme and partially restored virulence of ΔglmR mutants. In parallel, metabolomic analysis revealed that ΔglmR mutants are impaired in the production of UDP-GlcNAc, an essential peptidoglycan and wall teichoic acid (WTA) precursor. We next demonstrated that purified GlmR can directly catalyze the synthesis of UDP-GlcNAc from GlcNAc-1P and UTP, suggesting that it is an accessory uridyltransferase. Biochemical analysis of GlmR orthologues suggests that uridyltransferase activity is conserved. Finally, mutational analysis resulting in a GlmR mutant with impaired catalytic activity demonstrated that uridyltransferase activity was essential to facilitate cell-wall stress responses and virulence in vivo. Taken together, these studies indicate that GlmR is an evolutionary conserved accessory uridyltransferase required for cytosolic survival and virulence of L. monocytogenes. IMPORTANCE Bacterial pathogens must adapt to their host environment in order to cause disease. The cytosolic bacterial pathogen Listeria monocytogenes requires a highly conserved protein of unknown function, GlmR (previously known as YvcK), to survive in the host cytosol. GlmR is important for resistance to some cell-wall stresses and is essential for virulence. The ΔglmR mutant is deficient in production of an essential cell-wall metabolite, UDP-GlcNAc, and suppressors that increase metabolite levels also restore virulence. Purified GlmR can directly catalyze the synthesis of UDP-GlcNAc, and this enzymatic activity is conserved in both Bacillus subtilis and Staphylococcus aureus. These results highlight the importance of accessory cell wall metabolism enzymes in responding to cell-wall stress in a variety of Gram-positive bacteria.

Sulfur Availability Impacts Accumulation of the 2-Thiouridine tRNA Modification in Bacillus subtilis.

Posttranscriptional modifications to tRNA are critical elements for the folding and functionality of these adaptor molecules. Sulfur modifications in tRNA are installed by specialized enzymes that act on cognate tRNA substrates at specific locations. Most studied organisms contain a general cysteine desulfurase to mobilize sulfur for the synthesis of S-tRNA and other thio-cofactors. Bacillus subtilis and other Gram-positive bacteria encode multiple cysteine desulfurases that partner with specific sulfur acceptors in the biosynthesis of thio-cofactors. This metabolic layout suggests an alternate mode of regulation in these biosynthetic pathways. In this study, tRNA modifications were exploited as a readout for the functionality of pathways involving cysteine desulfurases. These analyses showed that the relative abundance of 2-thiouridine-modified tRNA (s2U) responds to sulfur availability in the growth medium in a dose-dependent manner. This study found that low sulfur concentrations lead to decreased levels of the s2U cysteine desulfurase YrvO and thiouridylase MnmA, without altering the levels of other cysteine desulfurases, SufS, NifS, and NifZ. Analysis of pathway metabolites that depend on the activity of cysteine desulfurases indicates that sulfur nutrient availability specifically impacts s2U accumulation while having no effect on the levels of other S-modified tRNA or activity levels of Fe-S enzymes. Collectively, these results support a model in which s2U tRNA serves as a marker for sulfur availability in B. subtilis. IMPORTANCE The 2-thiouridine (s2U) tRNA modification is found ubiquitously across all domains of life. YrvO and MnmA, the enzymes involved in this modification, are essential in B. subtilis, confirming the well-established role of s2U in maintaining translational efficiency and, consequently, cellular viability. Herein, we show that in the model Gram-positive organism Bacillus subtilis, the levels of s2U are responsive to sulfur availability. Downregulation of the s2U biosynthetic components leads to lower s2U levels, which may serve as a signal for the slowing of the translational apparatus during cellular nutrient insufficiency. Our findings provide the basis for the identification of a potential bacterial mode of regulation during S-metabolite depletion that may use s2U as a marker of suboptimal metabolic status.

Selective laryngoscopy before thyroidectomy: a risk assessment.

BACKGROUND: Patients presenting for thyroidectomy may have an unrecognized pre-existing vocal cord palsy (VCP). This raises the danger of bilateral VCP if a patient sustains an injury to the RLN on the sole functioning side. Part of the rationale for routine preoperative laryngoscopy is to eliminate such a risk. This paper endeavours to quantify the relevant potential risk. METHODS: Patients who underwent laryngoscopy prior to thyroid or parathyroid surgery in an endocrine surgical unit over a 5 year period were identified. Literature review revealed four papers in which VCP prevalence in patients without risk factors was reported. Using our data, combined with that of these other authors, the background rate of pre-existing VCP was ascertained, and the subsequent risk of bilateral VCP estimated. RESULTS: Of our 632 patients who underwent preoperative laryngoscopy, there were four patients (0.63%) who were found to have a unilateral VCP, but all had voice symptoms or previous neck surgery. When patients with these risk factors are excluded, our data combined with the published data provides a pre-existing VCP rate of 0.2%. Calculations estimate that if preoperative laryngoscopy is omitted in patients with no risk factors, the risk of bilateral VCP, due to the nerve on the sole functioning side being injured, would be between 1/50000 and 1/150000, depending on an individual surgeon's level of experience. CONCLUSION: Selective use of laryngoscopy prior to thyroidectomy would result in an acceptably low statistical risk of bilateral VCP. Routine laryngoscopy for all patients is not necessary.

Growth of Mycobacterium tuberculosis at acidic pH depends on lipid assimilation and is accompanied by reduced GAPDH activity.

Acidic pH arrests the growth of Mycobacterium tuberculosis in vitro (pH < 5.8) and is thought to significantly contribute to the ability of macrophages to control M. tuberculosis replication. However, this pathogen has been shown to survive and even slowly replicate within macrophage phagolysosomes (pH 4.5 to 5) [M. S. Gomes et al., Infect. Immun. 67, 3199-3206 (1999)] [S. Levitte et al., Cell Host Microbe 20, 250-258 (2016)]. Here, we demonstrate that M. tuberculosis can grow at acidic pH, as low as pH 4.5, in the presence of host-relevant lipids. We show that lack of phosphoenolpyruvate carboxykinase and isocitrate lyase, two enzymes necessary for lipid assimilation, is cidal to M. tuberculosis in the presence of oleic acid at acidic pH. Metabolomic analysis revealed that M. tuberculosis responds to acidic pH by altering its metabolism to preferentially assimilate lipids such as oleic acid over carbohydrates such as glycerol. We show that the activity of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is impaired in acid-exposed M. tuberculosis likely contributing to a reduction in glycolytic flux. The generation of endogenous reactive oxygen species at acidic pH is consistent with the inhibition of GAPDH, an enzyme well-known to be sensitive to oxidation. This work shows that M. tuberculosis alters its carbon diet in response to pH and provides a greater understanding of the physiology of this pathogen during acid stress.

Metabolic bifunctionality of Rv0812 couples folate and peptidoglycan biosynthesis in Mycobacterium tuberculosis.

Comparative sequence analysis has enabled the annotation of millions of genes from organisms across the evolutionary tree. However, this approach has inherently biased the annotation of phylogenetically ubiquitous, rather than species-specific, functions. The ecologically unusual pathogen Mycobacterium tuberculosis (Mtb) has evolved in humans as its sole reservoir and emerged as the leading bacterial cause of death worldwide. However, the physiological factors that define Mtb's pathogenicity are poorly understood. Here, we report the structure and function of a protein that is required for optimal in vitro fitness and bears homology to two distinct enzymes, Rv0812. Despite diversification of related orthologues into biochemically distinct enzyme families, rv0812 encodes a single active site with aminodeoxychorismate lyase and D-amino acid transaminase activities. The mutual exclusivity of substrate occupancy in this active site mediates coupling between nucleic acid and cell wall biosynthesis, prioritizing PABA over D-Ala/D-Glu biosynthesis. This bifunctionality reveals a novel, enzymatically encoded fail-safe mechanism that may help Mtb and other bacteria couple replication and division.

Bacillus subtilis PgcA moonlights as a phosphoglucosamine mutase in support of peptidoglycan synthesis.

Phosphohexomutase superfamily enzymes catalyze the reversible intramolecular transfer of a phosphoryl moiety on hexose sugars. Bacillus subtilis phosphoglucomutase PgcA catalyzes the reversible interconversion of glucose 6-phosphate (Glc-6-P) and glucose 1-phosphate (Glc-1-P), a precursor of UDP-glucose (UDP-Glc). B. subtilis phosphoglucosamine mutase (GlmM) is a member of the same enzyme superfamily that converts glucosamine 6-phosphate (GlcN-6-P) to glucosamine 1-phosphate (GlcN-1-P), a precursor of the amino sugar moiety of peptidoglycan. Here, we present evidence that B. subtilis PgcA possesses activity as a phosphoglucosamine mutase that contributes to peptidoglycan biosynthesis. This activity was made genetically apparent by the synthetic lethality of pgcA with glmR, a positive regulator of amino sugar biosynthesis, which can be specifically suppressed by overproduction of GlmM. A gain-of-function mutation in a substrate binding loop (PgcA G47S) increases this secondary activity and suppresses a glmR mutant. Our results demonstrate that bacterial phosphoglucomutases may possess secondary phosphoglucosamine mutase activity, and that this dual activity may provide some level of functional redundancy for the essential peptidoglycan biosynthesis pathway.

The Thioredoxin System Reduces Protein Persulfide Intermediates Formed during the Synthesis of Thio-Cofactors in Bacillus subtilis.

The biosynthesis of Fe-S clusters and other thio-cofactors requires the participation of redox agents. A shared feature in these pathways is the formation of transient protein persulfides, which are susceptible to reduction by artificial reducing agents commonly used in reactions in vitro. These agents modulate the reactivity and catalytic efficiency of biosynthetic reactions and, in some cases, skew the enzymes' kinetic behavior, bypassing sulfur acceptors known to be critical for the functionality of these pathways in vivo. Here, we provide kinetic evidence for the selective reactivity of the Bacillus subtilis Trx (thioredoxin) system toward protein-bound persulfide intermediates. Our results demonstrate that the redox flux of the Trx system modulates the rate of sulfide production in cysteine desulfurase assays. Likewise, the activity of the Trx system is dependent on the rate of persulfide formation, suggesting the occurrence of coupled reaction schemes between both enzymatic systems in vitro. Inactivation of TrxA (thioredoxin) or TrxR (thioredoxin reductase) impairs the activity of Fe-S enzymes in B. subtilis, indicating the involvement of the Trx system in Fe-S cluster metabolism. Surprisingly, biochemical characterization of TrxA reveals that this enzyme is able to coordinate Fe-S species, resulting in the loss of its reductase activity. The inactivation of TrxA through the coordination of a labile cluster, combined with its proposed role as a physiological reducing agent in sulfur transfer pathways, suggests a model for redox regulation. These findings provide a potential link between redox regulation and Fe-S metabolism.

Diverse Mechanisms of Sulfur Decoration in Bacterial tRNA and Their Cellular Functions.

Sulfur-containing transfer ribonucleic acids (tRNAs) are ubiquitous biomolecules found in all organisms that possess a variety of functions. For decades, their roles in processes such as translation, structural stability, and cellular protection have been elucidated and appreciated. These thionucleosides are found in all types of bacteria; however, their biosynthetic pathways are distinct among different groups of bacteria. Considering that many of the thio-tRNA biosynthetic enzymes are absent in Gram-positive bacteria, recent studies have addressed how sulfur trafficking is regulated in these prokaryotic species. Interestingly, a novel proposal has been given for interplay among thionucleosides and the biosynthesis of other thiocofactors, through participation of shared-enzyme intermediates, the functions of which are impacted by the availability of substrate as well as metabolic demand of thiocofactors. This review describes the occurrence of thio-modifications in bacterial tRNA and current methods for detection of these modifications that have enabled studies on the biosynthesis and functions of S-containing tRNA across bacteria. It provides insight into potential modes of regulation and potential evolutionary events responsible for divergence in sulfur metabolism among prokaryotes.

Abbreviated Pathway for Biosynthesis of 2-Thiouridine in Bacillus subtilis.

UNLABELLED: The 2-thiouridine (s(2)U) modification of the wobble position in glutamate, glutamine, and lysine tRNA molecules serves to stabilize the anticodon structure, improving ribosomal binding and overall efficiency of the translational process. Biosynthesis of s(2)U in Escherichia coli requires a cysteine desulfurase (IscS), a thiouridylase (MnmA), and five intermediate sulfur-relay enzymes (TusABCDE). The E. coli MnmA adenylates and subsequently thiolates tRNA to form the s(2)U modification. Bacillus subtilis lacks IscS and the intermediate sulfur relay proteins, yet its genome contains a cysteine desulfurase gene, yrvO, directly adjacent to mnmA. The genomic synteny of yrvO and mnmA combined with the absence of the Tus proteins indicated a potential functionality of these proteins in s(2)U formation. Here, we provide evidence that the B. subtilis YrvO and MnmA are sufficient for s(2)U biosynthesis. A conditional B. subtilis knockout strain showed that s(2)U abundance correlates with MnmA expression, and in vivo complementation studies in E. coli IscS- or MnmA-deficient strains revealed the competency of these proteins in s(2)U biosynthesis. In vitro experiments demonstrated s(2)U formation by YrvO and MnmA, and kinetic analysis established a partnership between the B. subtilis proteins that is contingent upon the presence of ATP. Furthermore, we observed that the slow-growth phenotype of E. coli ΔiscS and ΔmnmA strains associated with s(2)U depletion is recovered by B. subtilis yrvO and mnmA. These results support the proposal that the involvement of a devoted cysteine desulfurase, YrvO, in s(2)U synthesis bypasses the need for a complex biosynthetic pathway by direct sulfur transfer to MnmA. IMPORTANCE: The 2-thiouridine (s(2)U) modification of the wobble position in glutamate, glutamine, and lysine tRNA is conserved in all three domains of life and stabilizes the anticodon structure, thus guaranteeing fidelity in translation. The biosynthesis of s(2)U in Escherichia coli requires seven proteins: the cysteine desulfurase IscS, the thiouridylase MnmA, and five intermediate sulfur-relay enzymes (TusABCDE). Bacillus subtilis and most Gram-positive bacteria lack a complete set of biosynthetic components. Interestingly, the mnmA coding sequence is located adjacent to yrvO, encoding a cysteine desulfurase. In this work, we provide evidence that the B. subtilis YrvO is able to transfer sulfur directly to MnmA. Both proteins are sufficient for s(2)U biosynthesis in a pathway independent of the one used in E. coli.

Shared-intermediates in the biosynthesis of thio-cofactors: Mechanism and functions of cysteine desulfurases and sulfur acceptors.

Cysteine desulfurases utilize a PLP-dependent mechanism to catalyze the first step of sulfur mobilization in the biosynthesis of sulfur-containing cofactors. Sulfur activation and integration into thiocofactors involve complex mechanisms and intricate biosynthetic schemes. Cysteine desulfurases catalyze sulfur-transfer reactions from l-cysteine to sulfur acceptor molecules participating in the biosynthesis of thio-cofactors, including Fe-S clusters, thionucleosides, thiamin, biotin, and molybdenum cofactor. The proposed mechanism of cysteine desulfurases involves the PLP-dependent cleavage of the C-S bond from l-cysteine via the formation of a persulfide enzyme intermediate, which is considered the hallmark step in sulfur mobilization. The subsequent sulfur transfer reaction varies with the class of cysteine desulfurase and sulfur acceptor. IscS serves as a mecca for sulfur incorporation into a network of intertwined pathways for the biosynthesis of thio-cofactors. The involvement of a single enzyme interacting with multiple acceptors, the recruitment of shared-intermediates partaking roles in multiple pathways, and the participation of Fe-S enzymes denote the interconnectivity of pathways involving sulfur trafficking. In Bacillus subtilis, the occurrence of multiple cysteine desulfurases partnering with dedicated sulfur acceptors partially deconvolutes the routes of sulfur trafficking and assigns specific roles for these enzymes. Understanding the roles of promiscuous vs. dedicated cysteine desulfurases and their partnership with shared-intermediates in the biosynthesis of thio-cofactors will help to map sulfur transfer events across interconnected pathways and to provide insight into the hierarchy of sulfur incorporation into biomolecules. This article is part of a Special Issue entitled: Fe/S proteins: Analysis, structure, function, biogenesis and diseases.

Predicting date rape perceptions: the effects of gender, gender role attitudes, and victim resistance.

The effects of participant gender and victim resistance on date rape perceptions have been inconsistent. Participant gender role attitudes may contribute to these inconsistencies. We found women with traditional gender role attitudes were least likely to agree that the perpetrator was guilty of rape. Participants were less convinced of the perpetrator's guilt when the victim resisted verbally than when she resisted verbally and physically, and participants with traditional gender role attitudes were less convinced of the negative impact on the victim when she resisted verbally than when she resisted verbally and physically. Perhaps previous inconsistencies resulted from varying proportions of men and women with traditional versus liberal gender role attitudes in the samples.

Gender differences and socioeconomic status biases in judgments about blame in date rape scenarios.

Participants (80 men, 80 women) read hypothetical date rape scenarios, wherein the perpetrator's socioeconomic status (bus driver versus doctor) and the victim's level of resistance (verbal versus verbal and physical) were varied, and made judgments about who was at fault and what the consequences should be. In general, men assigned more blame to the victim and less blame to the perpetrator than did women. However, men assigned more blame to the bus driver than to the doctor. Women, on the other hand, assigned more blame to the victim who was raped by the bus driver than to the victim who was raped by the doctor. The results also indicated that participants recommended harsher punishments for the perpetrator when the victim resisted verbally than when she resisted verbally and physically. Future research on the role of the perpetrator's, the victim's, and the participants' socioeconomic status in judgments about date rape is suggested.

Practical issues in wave-front sensing by use of phase diversity.

We present the results of the phase-diversity algorithm applied to simulated and laboratory data. We show that the exact amount of defocus distance does not need to be known exactly for the phase-diversity algorithm on extended scene imaging. We determine, through computer simulation, the optimum diversity distance for various scene types. Using laboratory data, we compare the aberrations recovered with the phase-diversity algorithm and those measured with a Fizeau interferometer that uses a He-Ne laser. The two aberration sets agree with a Strehl ratio of over 0.9. The contrast of the recovered object is found to be ten times that of the raw image.

Associations between adolescent-mother and adolescent-best friend interactions.

The present study examined associations between adolescent-mother and adolescent-best friend interactions during conflict resolution tasks. Adolescents (N = 39) were videotaped while discussing unresolved problems with their mothers and then with their best friends. Mothers' behavior with adolescents and adolescents' behavior with mothers and with best friends were coded for conflict, withdrawal, communication skills, support-validation, and problem-solving. Mothers' communication and support-validation with adolescents was positively associated with adolescents' communication and support-validation with best friends, respectively. However, their behavior was not identical. Mothers were more communicative and supportive with adolescents than adolescents were with their best friends. Second, adolescents' withdrawal and support-validation with mothers was positively associated with their withdrawal and support-validation with best friends, respectively. However, they exhibited less withdrawal with their mothers than with their best friends. Possible explanations for these findings, as well as directions for future research, are discussed.