Mutational loss of carotenoids in alkaliphilic Bacillus pseudofirmus OF4 results in sensitivity to oxidative stress and growth at high pH.

Alkaliphilic Bacillus pseudofirmus OF4, which has a broad pH growth range of 7.5 to above 10.5, is yellow-pigmented due to carotenoids. Carotenoids contribute to membrane rigidity and can alleviate cellular oxidative stress. This study was undertaken to gain insight into the roles carotenoids play in alkaliphile physiology. Carotenoid content was high in stationary phase and in cells grown nonfermentatively at pH 10.5 A colourless mutant was isolated by the in-frame deletion of a key carotenogenic gene, crtM. In cells grown to stationary phase in a pH 10.5 medium with a suboptimal concentration of Na+, the ∆crtM strain exhibited lower resistance to paraquat and hydrogen peroxide. Preincubation of the mutant in a nutrient-free pH 10.5 buffer revealed a pronounced sensitivity to hydrogen peroxide in growth at pH 7.5. In growth curves in media with optimal or suboptimal nutrient concentrations conducted at 37°, the mutant grew identically to the wild-type at pH 7.5 but its lag time was longer than the wild-type at pH 10.5 and growth was slower when the carbon source, malate, was limiting. When the temperature of the growth curves was lowered to 25°, the mutant no longer had a pH 10.5 phenotype, implicating the effect of carotenoids on membrane rigidity for the pH 10.5 growth phenotype. These results suggest that carotenoids in B. pseudofirmus OF4 play a role in managing oxidative stress when cells are adapting to other stressful conditions such as nutrient limitation while also helping to maintain membrane fluidity/rigidity balance for membrane-linked functions.

The c-ring stoichiometry of ATP synthase is adapted to cell physiological requirements of alkaliphilic Bacillus pseudofirmus OF4.

The c-rings of ATP synthases consist of individual c-subunits, all of which harbor a conserved motif of repetitive glycine residues (GxGxGxG) important for tight transmembrane α-helix packing. The c-ring stoichiometry determines the number of ions transferred during enzyme operation and has a direct impact on the ion-to-ATP ratio, a cornerstone parameter of cell bioenergetics. In the extreme alkaliphile Bacillus pseudofirmus OF4, the glycine motif is replaced by AxAxAxA. We performed a structural study on two mutants with alanine-to-glycine changes using atomic force microscopy and X-ray crystallography, and found that mutants form smaller c12 rings compared with the WT c13. The molar growth yields of B. pseudofirmus OF4 cells on malate further revealed that the c12 mutants have a considerably reduced capacity to grow on limiting malate at high pH. Our results demonstrate that the mutant ATP synthases with either c12 or c13 can support ATP synthesis, and also underscore the critical importance of an alanine motif with c13 ring stoichiometry for optimal growth at pH >10. The data indicate a direct connection between the precisely adapted ATP synthase c-ring stoichiometry and its ion-to-ATP ratio on cell physiology, and also demonstrate the bioenergetic challenges and evolutionary adaptation strategies of extremophiles.

Cardiolipin is dispensable for oxidative phosphorylation and non-fermentative growth of alkaliphilic Bacillus pseudofirmus OF4.

Cardiolipin (CL), a membrane phospholipid in bacteria and mitochondria, has been hypothesized to facilitate movement of protons on the outer surface of membranes in support of respiration-dependent ATP synthesis, oxidative phosphorylation (OXPHOS). If so, the high levels of membrane CL found in alkaliphilic bacteria, such as Bacillus pseudofirmus OF4, might facilitate its robust OXPHOS at pH 10.5, where the bulk protonmotive (PMF) force is low. To address the role of CL in Bacillus pseudofirmus OF4, we studied strains in which genes (cls) potentially encoding a CL synthase (CLs) were deleted: three single (ΔclsA, ΔclsB, and ΔclsC), one double (ΔclsA/B), and one triple (ΔclsA/B/C) mutant. Two-dimensional thin layer chromatography analyses of lipid extracts from (32)P-labeled strains showed that the wild-type CL content was 15% of total phospholipids at pH 10.5 versus 3% at pH 7.5 during log phase. The % CL was higher (28-33%) at both pH values during stationary phase. The clsA gene plays a major role in CL biosynthesis as no detectable CL was found in ΔclsA-containing mutants, whereas the CL precursor phosphatidylglycerol was elevated. The ΔclsB mutant exhibited no significant reduction in CL, but clsB expression was up-regulated and appeared to support growth at pH 7.5. In the absence of detectable CL, the alkaliphile showed no significant deficits in non-fermentative growth, respiration-dependent ATP synthesis, or salt tolerance. Minor deficits in respiration and ATP synthase assembly were noted in individual mutants. In long term survival experiments, significant growth defects were found in ΔclsA strains and the ΔclsC strain at pH 10.5.

Lobectomy or Total Thyroidectomy-Where Is the Pendulum now for Differentiated Thyroid Cancer?

Thyroid surgery remains an essential treatment of thyroid cancer. The historical one-size-fits-all approach to differentiated (papillary and follicular) thyroid carcinoma of total thyroidectomy with central lymph node dissection has been shown to be overtreatment with associated risk of perioperative complications including nerve palsy and hypoparathyroidism. Furthermore, thyroid lobectomy may obviate life-long thyroid hormone replacement. Low-risk thyroid cancers have a low risk of recurrence and those that do recur can be salvaged with reoperation without compromising prognosis. Perioperative risk stratification for recurrence and death greatly influence the need for total thyroidectomy.

Adrenal schwannoma can be FDG-Avid on PET/CT: case report and review of historic institutional pathology.

Schwannomas are benign, generally indolent tumors of neural crest origin and comprise the most common histologic tumor of peripheral nerves. Schwannomas are a rare histology for retroperitoneal tumors and very rare histologic findings for tumors of the adrenal gland with fewer than 50 cases in the reported literature. Here we present a case report of a non-hormonally functional but metabolically active adrenal tumor with indeterminate imaging characteristics with final pathology showing a 6.1 cm adrenal schwannoma as well as historical institutional pathology review revealing two additional cases.

Long-term effects of primary hyperparathyroidism and parathyroidectomy on kidney function.

IMPORTANCE: Limited evidence supports kidney dysfunction as an indication for parathyroidectomy in asymptomatic primary hyperparathyroidism (PHPT). OBJECTIVE: To investigate the natural history of kidney function in PHPT and whether parathyroidectomy alters renal outcomes. DESIGN: Matched control study. SETTING: A vertically integrated health care system serving 4.6 million patients in Southern California. PARTICIPANTS: 6058 subjects with PHPT and 16 388 matched controls, studied from 2000 to 2016. EXPOSURES: Biochemically confirmed PHPT with varying serum calcium levels. MAIN OUTCOMES: Estimated glomerular filtration rate (eGFR) trajectories were compared over 10 years, with cases subdivided by severity of hypercalcemia: serum calcium 2.62-2.74 mmol/L (10.5-11 mg/dL), 2.75-2.87 (11.1-11.5), 2.88-2.99 (11.6-12), and >2.99 (>12). Interrupted time series analysis was conducted among propensity-score-matched PHPT patients with and without parathyroidectomy to compare eGFR trajectories postoperatively. RESULTS: Modest rates of eGFR decline were observed in PHPT patients with serum calcium 2.62-2.74 mmol/L (−1.0 mL/min/1.73 m2/year) and 2.75-2.87 mmol/L (−1.1 mL/min/1.73 m2/year), comprising 56% and 28% of cases, respectively. Compared with the control rate of −1.0 mL/min/1.73 m2/year, accelerated rates of eGFR decline were observed in patients with serum calcium 2.88-2.99 mmol/L (−1.5 mL/min/1.73 m2/year, P < .001) and >2.99 mmol/L (−2.1 mL/min/1.73 m2/year, P < .001), comprising 9% and 7% of cases, respectively. In the propensity score­matched population, patients with serum calcium >2.87 mmol/L exhibited mitigation of eGFR decline after parathyroidectomy (−2.0 [95% CI: −2.6 to −1.5] to −0.9 [95% CI: −1.5 to 0.4] mL/min/1.73 m2/year). CONCLUSIONS AND RELEVANCE: Compared with matched controls, accelerated eGFR decline was observed in the minority of PHPT patients with serum calcium >2.87 mmol/L (11.5 mg/dL). Parathyroidectomy was associated with mitigation of eGFR decline in patients with serum calcium >2.87 mmol/L.

Mutations in a helix-1 motif of the ATP synthase c-subunit of Bacillus pseudofirmus OF4 cause functional deficits and changes in the c-ring stability and mobility on sodium dodecyl sulfate-polyacrylamide gel electrophoresis.

The ATP synthase of the alkaliphile Bacillus pseudofirmus OF4 has a tridecameric c-subunit rotor ring. Each c-subunit has an AxAxAxA motif near the center of the inner helix, where neutralophilic bacteria generally have a GxGxGxG motif. Here, we studied the impact of four single and six multiple Ala-to-Gly chromosomal mutations in the A16xAxAxA22 motif on the capacity for nonfermentative growth and, for most of the mutants, on ATP synthesis by ADP- and P(i)-loaded membrane vesicles at pH 7.5 and 10.5. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analyses of the holo-ATP synthases were used to probe stability of the mutant c-rotors and mobility properties of the c-rotors as well as the monomeric c-subunits that are released from them by trichloroacetic acid treatment. Mutants containing an Ala16-to-Gly mutation exhibited the most severe functional defects. Via SDS-PAGE, most of the mutant c-monomers exhibited increased mobility relative to the wild-type (WT) c-subunit, but among the intact c-rings, only Ala16-to-Gly mutants exhibited significantly increased mobility relative to that of the WT c-ring. The hypothesis that these c-rings have a decreased c-subunit stoichiometry is still untested, but the functional impact of an Ala16-to-Gly mutation clearly depended upon additional Ala-to-Gly mutation(s) and their positions. The A16/20G double mutant exhibited a larger functional deficit than both the A16G and A16/18G mutants. Most of the mutant c-rings showed in vitro instability relative to that of the WT c-ring. However, the functional deficits of mutants did not correlate well with the extent of c-ring stability loss, so this property is unlikely to be a major factor in vivo.

The ATP synthase a-subunit of extreme alkaliphiles is a distinct variant: mutations in the critical alkaliphile-specific residue Lys-180 and other residues that support alkaliphile oxidative phosphorylation.

A lysine residue in the putative proton uptake pathway of the ATP synthase a-subunit is found only in alkaliphilic Bacillus species and is proposed to play roles in proton capture, retention and passage to the synthase rotor. Here, Lys-180 was replaced with alanine (Ala), glycine (Gly), cysteine (Cys), arginine (Arg), or histidine (His) in the chromosome of alkaliphilic Bacillus pseudofirmus OF4. All mutants exhibited octylglucoside-stimulated ATPase activity and ß-subunit levels at least as high as wild-type. Purified mutant F(1)F(0)-ATP synthases all contained substantial a-subunit levels. The mutants exhibited diverse patterns of native (no octylglucoside) ATPase activity and a range of defects in malate growth and in vitro ATP synthesis at pH 10.5. ATP synthesis by the Ala, Gly, and His mutants was also impaired at pH 7.5 in the presence of a protonophoric uncoupler. Thus Lys-180 plays a role when the protonmotive force is reduced at near neutral, not just at high pH. The Arg mutant exhibited no ATP synthesis activity in the alkaliphile setting although activity was reported for a K180R mutant of a thermoalkaliphile synthase (McMillan, D. G., Keis, S., Dimroth, P., and Cook, G. M. (2007) J. Biol. Chem. 282, 17395-17404). The hypothesis that a-subunits from extreme alkaliphiles and the thermoalkaliphile represent distinct variants was supported by demonstration of the importance of additional alkaliphile-specific a-subunit residues, not found in the thermoalkaliphile, for malate growth of B. pseudofirmus OF4. Finally, a mutant B. pseudofirmus OF4 synthase with switched positions of Lys-180 (helix 4) and Gly-212 (helix 5) retained significant coupled synthase activity accompanied by proton leakiness.

Genome of alkaliphilic Bacillus pseudofirmus OF4 reveals adaptations that support the ability to grow in an external pH range from 7.5 to 11.4.

Bacillus pseudofirmus OF4 is an extreme but facultative alkaliphile that grows non-fermentatively in a pH range from 7.5 to above 11.4 and can withstand large sudden increases in external pH. It is a model organism for studies of bioenergetics at high pH, at which energy demands are higher than at neutral pH because both cytoplasmic pH homeostasis and ATP synthesis require more energy. The alkaliphile also tolerates a cytoplasmic pH > 9.0 at external pH values at which the pH homeostasis capacity is exceeded, and manages other stresses that are exacerbated at alkaline pH, e.g. sodium, oxidative and cell wall stresses. The genome of B. pseudofirmus OF4 includes two plasmids that are lost from some mutants without viability loss. The plasmids may provide a reservoir of mobile elements that promote adaptive chromosomal rearrangements under particular environmental conditions. The genome also reveals a more acidic pI profile for proteins exposed on the outer surface than found in neutralophiles. A large array of transporters and regulatory genes are predicted to protect the alkaliphile from its overlapping stresses. In addition, unanticipated metabolic versatility was observed, which could ensure requisite energy for alkaliphily under diverse conditions.

Endocrine Surgery: Management of Postoperative Complications Following Endocrine Surgery of the Neck.

Cervical endocrine operations include parathyroidectomy, thyroid lobectomy, thyroidectomy, central neck dissection, and lateral neck dissection. The vital structures at risk include the recurrent laryngeal nerves to the intrinsic muscles of the larynx, additional cranial nerves, parathyroid glands essential for calcium homeostasis, aerodigestive structures, and great vessels. Here, the authors discuss complications of endocrine neck surgery, including cervical hematoma and other fluid collections, hypocalcemia from hypoparathyroidism, and nerve injuries, along with their prevention, mitigation, and management. Significant and permanent morbidity can result, but fortunately the overall rate of complications remains low, especially when surgery is performed by high-volume surgeons.

Parallel multiplicative target screening against divergent bacterial replicases: identification of specific inhibitors with broad spectrum potential.

Typically, biochemical screens that employ pure macromolecular components focus on single targets or a small number of interacting components. Researches rely on whole cell screens for more complex systems. Bacterial DNA replicases contain multiple subunits that change interactions with each stage of a complex reaction. Thus, the actual number of targets is a multiple of the proteins involved. It is estimated that the overall replication reaction includes up to 100 essential targets, many suitable for discovery of antibacterial inhibitors. We have developed an assay, using purified protein components, in which inhibitors of any of the essential targets can be detected through a common readout. Use of purified components allows each protein to be set within the linear range where the readout is proportional to the extent of inhibition of the target. By performing assays against replicases from model Gram-negative and Gram-positive bacteria in parallel, we show that it is possible to distinguish compounds that inhibit only a single bacterial replicase from those that exhibit broad spectrum potential.