Polyphosphate is a primordial chaperone.

Composed of up to 1,000 phospho-anhydride bond-linked phosphate monomers, inorganic polyphosphate (polyP) is one of the most ancient, conserved, and enigmatic molecules in biology. Here we demonstrate that polyP functions as a hitherto unrecognized chaperone. We show that polyP stabilizes proteins in vivo, diminishes the need for other chaperone systems to survive proteotoxic stress conditions, and protects a wide variety of proteins against stress-induced unfolding and aggregation. In vitro studies reveal that polyP has protein-like chaperone qualities, binds to unfolding proteins with high affinity in an ATP-independent manner, and supports their productive refolding once nonstress conditions are restored. Our results uncover a universally important function for polyP and suggest that these long chains of inorganic phosphate may have served as one of nature's first chaperones, a role that continues to the present day.

Bone Marrow Adipocyte Developmental Origin and Biology.

PURPOSE OF REVIEW: This review explores how the relationships between bone marrow adipose tissue (BMAT) adipogenesis with advancing age, obesity, and/or bone diseases (osteopenia or osteoporosis) contribute to mechanisms underlying musculoskeletal pathophysiology. RECENT FINDINGS: Recent studies have re-defined adipose tissue as a dynamic, vital organ with functions extending beyond its historic identity restricted solely to that of an energy reservoir or sink. "State of the art" methodologies provide novel insights into the developmental origin, physiology, and function of different adipose tissue depots. These include genetic tracking of adipose progenitors, viral vectors application, and sophisticated non-invasive imaging modalities. While constricted within the rigid bone cavity, BMAT vigorously contributes to local and systemic metabolic processes including hematopoiesis, osteogenesis, and energy metabolism and undergoes dynamic changes as a function of age, diet, bone topography, or sex. These insights will impact future research and therapies relating to osteoporosis.

A NAC for regulating metabolism: the nitrogen assimilation control protein (NAC) from Klebsiella pneumoniae.

The nitrogen assimilation control protein (NAC) is a LysR-type transcriptional regulator (LTTR) that is made under conditions of nitrogen-limited growth. NAC's synthesis is entirely dependent on phosphorylated NtrC from the two-component Ntr system and requires the unusual sigma factor σ54 for transcription of the nac gene. NAC activates the transcription of σ70-dependent genes whose products provide the cell with ammonia or glutamate. NAC represses genes whose products use ammonia and also represses its own transcription. In addition, NAC also subtly adjusts other cellular functions to keep pace with the supply of biosynthetically available nitrogen.

Properties of the NAC (nitrogen assimilation control protein)-binding site within the ureD promoter of Klebsiella pneumoniae.

The nitrogen assimilation control protein (NAC) of Klebsiella pneumoniae is a LysR-type transcriptional regulator that activates transcription when bound to a DNA site (ATAA-N5-TnGTAT) centered at a variety of distances from the start of transcription. The NAC-binding site from the hutU promoter (NBShutU) is centered at -64 relative to the start of transcription but can activate the lacZ promoter from sites at -64, -54, -52, and -42 but not from sites at -47 or -59. However, the NBSs from the ureD promoter (ureDp) and codB promoter (codBp) are centered at -47 and -59, respectively, and NAC is fully functional at these promoters. Therefore, we compared the activities of the NBShutU and NBSureD within the context of ureDp as well as within codBp. The NBShutU functioned at both of these sites. The NBSureD has the same asymmetric core as the NBShutU. Inverting the NBSureD abolished more than 99% of NAC's ability to activate ureDp. The key to the activation lies in the TnG segment of the TnGTAT half of the NBSureD. Changing TnG to GnT, TnT, or GnG drastically reduced ureDp activation (to 0.5%, 6%, or 15% of wild-type activation, respectively). The function of the NBSureD, like that of the NBShutU, requires that the TnGTAT half of the NBS be on the promoter-proximal (downstream) side of the NBS. Taken together, our data suggest that the positional specificity of an NBS is dependent on the promoter in question and is more flexible than previously thought, allowing considerable latitude both in distance and on the face of the DNA helix for the NBS relative to that of RNA polymerase.

Expanded role for the nitrogen assimilation control protein in the response of Klebsiella pneumoniae to nitrogen stress.

Klebsiella pneumoniae is able to utilize many nitrogen sources, and the utilization of some of these nitrogen sources is dependent on the nitrogen assimilation control (NAC) protein. Seven NAC-regulated promoters have been characterized in K. pneumoniae, and nine NAC-regulated promoters have been found by microarray analysis in Escherichia coli. So far, all characterized NAC-regulated promoters have been directly related to nitrogen metabolism. We have used a genome-wide analysis of NAC binding under nitrogen limitation to identify the regions of the chromosome associated with NAC in K. pneumoniae. We found NAC associated with 99 unique regions of the chromosome under nitrogen limitation. In vitro, 84 of the 99 regions associate strongly enough with purified NAC to produce a shifted band by electrophoretic mobility shift assay. Primer extension analysis of the mRNA from genes associated with 17 of the fragments demonstrated that at least one gene associated with each fragment was NAC regulated under nitrogen limitation. The large size of the NAC regulon in K. pneumoniae indicates that NAC plays a larger role in the nitrogen stress response than it does in E. coli. Although a majority of the genes with identifiable functions that associated with NAC under nitrogen limitation are involved in nitrogen metabolism, smaller subsets are associated with carbon and energy acquisition (18 genes), and growth rate control (10 genes). This suggests an expanded role for NAC regulation during the nitrogen stress response, where NAC not only regulates genes involved in nitrogen metabolism but also regulates genes involved in balancing carbon and nitrogen pools and growth rate.

Genetic analysis of the nitrogen assimilation control protein from Klebsiella pneumoniae.

The nitrogen assimilation control protein (NAC) from Klebsiella pneumoniae is a typical LysR-type transcriptional regulator (LTTR) in many ways. However, the lack of a physiologically relevant coeffector for NAC and the fact that NAC can carry out many of its functions as a dimer make NAC unusual among the LTTRs. In the absence of a crystal structure for NAC, we analyzed the effects of amino acid substitutions with a variety of phenotypes in an attempt to identify functionally important features of NAC. A substitution that changed the glutamine at amino acid 29 to alanine (Q29A) resulted in a NAC that was seriously defective in binding to DNA. The H26D substitution resulted in a NAC that could bind and repress transcription but not activate transcription. The I71A substitution resulted in a NAC polypeptide that remained monomeric. NAC tetramers can bind to both long and shorter binding sites (like other LTTRs). However, the absence of a coeffector to induce the conformational change needed for the switch from the former to the latter raised a question. Are there two conformations of NAC, analogous to the other LTTRs? The G217R substitution resulted in a NAC that could bind to the longer sites but had difficulty in binding to the shorter sites, and the I222R and A230R substitutions resulted in a NAC that could bind to the shorter sites but had difficulty in binding properly to the longer sites. Thus, there appear to be two conformations of NAC that can freely interconvert in the absence of a coeffector.

The LysR-type nitrogen assimilation control protein forms complexes with both long and short DNA binding sites in the absence of coeffectors.

Most LysR-type transcriptional regulators (LTTRs) function as tetramers when regulating gene expression. The nitrogen assimilation control protein (NAC) generally functions as a dimer when binding to DNA and activating transcription. However, at some sites, NAC binds as a tetramer. Like many LTTRs, NAC tetramers can recognize sites with long footprints (74 bp for the site at nac) with a substantial DNA bend or short footprints (56 bp for the site at cod) with less DNA bending. However, unlike other LTTRs, NAC can recognize both types of sites in the absence of physiologically relevant coeffectors, suggesting that the two conformers of the NAC tetramer (extended and compact) are interchangeable without the need for any modification to induce or stabilize the change. In order for NAC to bind as a tetramer, three interactions must exist: an interaction between the two NAC dimers and an interaction between each NAC dimer and its corresponding binding site. The interaction between one dimer and its DNA site can be weak (recognizing a half-site rather than a full dimer-binding site), but the other two interactions must be strong. Since the conformation of the NAC tetramer (extended or compact) is determined by the nature of the DNA site without the intervention of a small molecule, we argue that the coeffector that determines the conformation of the NAC tetramer is the DNA site to which it binds.

Human Adipose Derived Cells in Two- and Three-Dimensional Cultures: Functional Validation of an In Vitro Fat Construct.

Obesity, defined as a body mass index of 30 kg/m2 or above, has increased considerably in incidence and frequency within the United States and globally. Associated comorbidities including cardiovascular disease, type 2 diabetes mellitus, metabolic syndrome, and nonalcoholic fatty liver disease have led to a focus on the mechanisms promoting the prevention and treatment of obesity. Commonly utilized in vitro models employ human or mouse preadipocyte cell lines in a 2-dimensional (2D) format. Due to the structural, biochemical, and biological limitations of these models, increased attention has been placed on "organ on a chip" technologies for a 3-dimensional (3D) culture. Herein, we describe a method employing cryopreserved primary human stromal vascular fraction (SVF) cells and a human blood product-derived biological scaffold to create a 3D adipose depot in vitro. The "fat-on-chip" 3D cultures have been validated relative to 2D cultures based on proliferation, flow cytometry, adipogenic differentiation, confocal microscopy/immunofluorescence, and functional assays (adipokine secretion, glucose uptake, and lipolysis). Thus, the in vitro culture system demonstrates the critical characteristics required for a humanized 3D white adipose tissue (WAT) model.

Patient advisory groups in practice improvement: sample case presentation with a discussion of best practices.

Using patient advisory groups can affect practice changes and create a patient-centric focus for a primary care practice. A successful patient advisory group has been developed for our primary care clinics. Utilizing this group, we have implemented practice improvement changes that have had a significant impact in patient care. This will be demonstrated in a case presentation involving the implementation of depression care managers at our practice sites. We will review key "best practices," as defined by the group, regarding size, composition, and meeting frequency that can be used for the development of a clinical patient advisory group.

The hpx genetic system for hypoxanthine assimilation as a nitrogen source in Klebsiella pneumoniae: gene organization and transcriptional regulation.

Growth experiments showed that adenine and hypoxanthine can be used as nitrogen sources by several strains of K. pneumoniae under aerobic conditions. The assimilation of all nitrogens from these purines indicates that the catabolic pathway is complete and proceeds past allantoin. Here we identify the genetic system responsible for the oxidation of hypoxanthine to allantoin in K. pneumoniae. The hpx cluster consists of seven genes, for which an organization in four transcriptional units, hpxDE, hpxR, hpxO, and hpxPQT, is proposed. The proteins involved in the oxidation of hypoxanthine (HpxDE) or uric acid (HpxO) did not display any similarity to other reported enzymes known to catalyze these reactions but instead are similar to oxygenases acting on aromatic compounds. Expression of the hpx system is activated by nitrogen limitation and by the presence of specific substrates, with hpxDE and hpxPQT controlled by both signals. Nitrogen control of hpxPQT transcription, which depends on sigma(54), is mediated by the Ntr system. In contrast, neither NtrC nor the nitrogen assimilation control protein is involved in the nitrogen control of hpxDE, which is dependent on sigma(70) for transcription. Activation of these operons by the specific substrates is also mediated by different effectors and regulatory proteins. Induction of hpxPQT requires uric acid formation, whereas expression of hpxDE is induced by the presence of hypoxanthine through the regulatory protein HpxR. This LysR-type regulator binds to a TCTGC-N(4)-GCAAA site in the intergenic hpxD-hpxR region. When bound to this site for hpxDE activation, HpxR negatively controls its own transcription.

Molecular Profiling of Patients with Pancreatic Cancer: Initial Results from the Know Your Tumor Initiative.

Purpose: To broaden access to and implementation of precision medicine in the care of patients with pancreatic cancer, the Know Your Tumor (KYT) program was initiated using a turn-key precision medicine system. Patients undergo commercially available multiomic profiling to determine molecularly rationalized clinical trials and off-label therapies.Experimental Design: Tumor samples were obtained for 640 patients from 287 academic and community practices covering 44 states. College of American Pathologists/Clinical Laboratory Improvement Amendments-accredited laboratories were used for genomic, proteomic, and phosphoprotein-based molecular profiling.Results: Tumor samples were adequate for next-generation sequencing in 96% and IHC in 91% of patients. A tumor board reviewed the results for every patient and found actionable genomic alterations in 50% of patients (with 27% highly actionable) and actionable proteomic alterations (excluding chemopredictive markers) in 5%. Actionable alterations commonly found were in DNA repair genes (BRCA1/2 or ATM mutations, 8.4%) and cell-cycle genes (CCND1/2/3 or CDK4/6 alterations, 8.1%). A subset of samples was assessed for actionable phosphoprotein markers. Among patients with highly actionable biomarkers, those who received matched therapy (n = 17) had a significantly longer median progression-free survival (PFS) than those who received unmatched therapy [n = 18; PFS = 4.1 vs. 1.9 months; HR, 0.47; 95% confidence interval (CI): 0.24-0.94; P adj = 0.03].Conclusions: A comprehensive precision medicine system can be implemented in community and academic settings, with highly actionable findings observed in over 25% of pancreatic cancers. Patients whose tumors have highly actionable alterations and receive matched therapy demonstrated significantly increased PFS. Our findings support further prospective evaluation of precision oncology in pancreatic cancer. Clin Cancer Res; 24(20); 5018-27. ©2018 AACR.

Impact of the Australian gender specific thresholds using the Abbott high sensitivity troponin I assay in clinical care.

The aim of this study was to ascertain the impact of gender specific hs-TnI thresholds in a clinical setting and determine the clinical characteristics and discharge diagnosis for individuals presenting to the Emergency Department (ED) with elevated troponin I with the Abbott high-sensitivity troponin I (hs-TnI) assay, but non-elevated troponin I on the previous generation assay (STAT TnI-II). Medical records of individuals presenting to the Royal Perth Hospital ED with elevated hs-TnI between 12 November 2013 and 24 December 2013 were retrospectively reviewed. The 99th percentile hs-TnI thresholds were ≥26 ng/L for males and ≥16 ng/L for females. TnI-II assays were performed concomitantly. In total, 1449 individuals [855 (59%) males] had 3580 troponin measurements. hs-TnI was elevated in 1569 (43.8%) measurements. Elevated hs-TnI with normal TnI-II was found in 120 (8.3%) individuals: 77 (64%) females and 43 (36%) males. Eight (6.7%) individuals were diagnosed with acute coronary syndrome (ACS): four (9.3%) males and four (5.2%) females. Other cardiac aetiologies were found in 33 (42%) females and 17 (40%) males. Individuals with elevated hs-TnI had high rates of hypertension (80%), diabetes mellitus (33%), cardiac failure (23%), aspirin use (53%) and lipid lowering therapy (52%). Significantly fewer females than males with discrepant troponin I results had previous ischaemic heart disease. The hsTnI assay identifies 8% more individuals with elevated troponin in an acute setting, with a female predominance (64%). However, only 6.7% of these individuals with multiple cardiovascular risk factors were diagnosed with ACS, a ∼0.5% increase overall. Outcome studies are required to determine if the Australian hs-TnI thresholds are clinically appropriate.

Post-tonsillectomy hemorrhage rates in children compared by surgical technique.

Despite the sheer number of pediatric tonsillectomies performed in the United States annually, there is no clear consensus as to which surgical technique is superior. One way to compare surgical techniques is to study the morbidity associated with each. We report postoperative hemorrhage rates, one of the frequently encountered major adverse events, as part of a retrospective chart review across four different surgical techniques. These surgeries involved either (1) Coblation, (2) Co-blation with partial suture closure of the tonsillar fossa, (3) diathermy, or (4) partial intracapsular tonsillectomy (PIT). Of the 7,024 children we evaluated, 99 (1.4%) experienced a postoperative hemorrhage that required a second surgery; hemorrhage occurred after 33 of the 3,177 Coblation-alone procedures (1.04%), 28 of the 1,633 Coblation with partial suture closure procedures (1.71%), 29 of the 1,850 diathermies (1.57%), and 9 of the 364 PIT procedures (2.47%). Statistical analysis of hemorrhage rates with each surgical technique yielded p values >0.05 in each case (Coblation alone and Coblation with partial suture closure: p = 0.29; diathermy: p = 0.47; PIT, p = 0.20). Based on these data, we conclude that none of these techniques is significantly superior in terms of decreasing the risk of post-tonsillectomy hemorrhage in children. Therefore, surgeons should continue to use the surgical procedure they are most familiar with to optimize recovery in the postoperative period.

Interpretative comments specifically suggesting specialist referral increase the detection of familial hypercholesterolaemia.

Familial hypercholesterolaemia (FH) is an under-diagnosed inherited condition characterised by elevated low density lipoprotein (LDL)-cholesterol and premature coronary artery disease. The requesting general practitioner of individuals with extremely elevated LDL-cholesterol measured by St John of God Pathology receives an interpretative comment on the lipid results highlighting possible FH. We sought to determine whether specifically recommending referral to the regional Lipid Disorders Clinic (LDC) increased referral and FH detection rates. A prospective case-control study of individuals with LDL-cholesterol ≥6.5 mmol/L was conducted. All individuals received an interpretative comment highlighting the possibility of FH. The cases comment also suggested LDC referral, and a subset of cases received the LDC's fax number (fax-cases) in addition. There were 231 individuals with an LDL-cholesterol ≥6.5 mmol/L; 96 (42%) controls and 135 (58%) cases, of which 99 were fax-cases. Twenty-four (18%) cases were referred to clinic compared with eight (8%) controls (p = 0.035). After specialist review and genetic testing, four probable and four definite FH individuals were detected amongst controls, compared with seven possible, eight probable and nine definite FH amongst cases. Genetic testing was performed in 31 (94%) individuals, 13 (42%) had a causative mutation identified. Interpretative commenting specifically recommending specialist review augments the detection of FH in the community.

Pediatric recurrent laryngeal nerve reinnervation: A case series and analysis of post-operative outcomes.

OBJECTIVE: To provide detailed information about recurrent laryngeal nerve (RLN) reinnervation outcomes in children using objective measures. METHODS: The records of three pediatric patients with unilateral vocal cord paralysis that underwent RLN reinnervation were retrospectively reviewed. Fundamental frequency (F0), jitter, shimmer, noise-to-harmonic ratio (NHR), and voice phonation (sustained /s/, /z/, /a/) were measured preoperatively and post-operatively at 13, 9, and 33 months (each time period corresponding to one of the three patients). RESULTS: Mean preoperative and post-operative variables were as follows: shimmer, 9.65±1.02% vs. 4.46±0.71% (p=0.01); NHR, 0.296±0.063 vs. 0.127±0.011 (p=0.04); jitter, 3.57±0.89% vs. 1.46±0.54% (p=0.08); F0, 274.6±35.4Hz vs. 282.2±70.6Hz (p=0.44); maximum phonation time, 7.46±1.40s vs. 9.79±1.84s (p=0.22); /s:z/ ratio, 1.28±0.22 vs.1.07±0.09 (p=0.26). CONCLUSIONS: There was statistically significant improvement in shimmer and NHR. Jitter improvement approached statistical significance. All other variables failed to show significant improvement among this small sample size. RLN reinnervation for pediatric patients is an option for the treatment of vocal cord paralysis. Further studies with larger cohorts are needed to show the full benefits.

Synthetic, Structural, Spectroscopic, and Theoretical Studies of Structural Isomers of the Cluster Pt(3)(&mgr;-PPh(2))(3)Ph(PPh(3))(2). A Unique Example of Core Isomerism in Phosphine Phosphido-Rich Clusters.

Two isomers of the phosphido-bridged platinum cluster Pt(3)(&mgr;-PPh(2))(3)Ph(PPh(3))(2) (2 and 3) have been isolated, and their structures have been solved by single-crystal X-ray diffraction. Compound 2 crystallizes in the orthorhombic space group Cmc2(1) with a = 22.192(10) Å, b = 17.650(9) Å, c = 18.182(8) Å, and Z = 4. Compound 3 crystallizes with 2 molecules of dichloromethane in the monoclinic space group C2/c with a = 21.390(10) Å, b = 18.471(9) Å, c = 19.021(11) Å, beta = 105.27(5) degrees, and Z = 4. The two isomers differ essentially in their metal-metal distances and Pt-(&mgr;-PPh(2))-Pt angles. Thus 2, having an imposed C(s) symmetry, contains a bent chain of metal atoms with two short Pt-Pt distances of 2.758(3) Å and a long separation of 3.586(2) Å. In 3, which has an imposed C(2) symmetry, the metal atoms form an isosceles triangle with two Pt-Pt distances of 2.956(3) Å and one of 3.074(4) Å. These isomers can be smoothly interconverted by changing the crystallization solvents. Solution and solid-state (31)P NMR studies have been performed in order to assign the resonances of the different P nuclei and relate their chemical shifts with their structural environments. Raman spectroscopy was used to assign the nu(Pt-Pt) modes of the two structural isomers. Theoretical studies based on extended Hückel calculations and using the fragment molecular orbital approach show that the isomer with the three medium Pt-Pt distances is slightly more stable, in agreement with earlier theoretical predictions. Cluster core isomerism remains a rare phenomenon, and the present example emphasizes the role and the importance of flexible phosphido bridges in stabilizing clusters as well as the unprecedented features which can be observed in phosphine phosphido-rich metal clusters.

Reactions of trinuclear platinum clusters with electrophiles: ionisation isomerism with [Pt3(µ2-I)(µ-PPh2)2(PPh3)3]I and [Pt3(µ-PPh2)2I2(PPh3)3]. Structures of [Pt3(µ2-Cl)(µ-PPh2)2(PPh3)3]PF6, [Pt3(µ-PPh2)2I2(PPh3)3] and of the Pt-Ag cluster [Pt3{µ3-AgBF4}(µ2-I)(µ-PPh2)2(PPh3)3]BF4.

A reaction of the 44 electron cluster [Pt3(µ-PPh2)3Ph(PPh3)2] (1) with wet AgBF4 afforded the cationic cluster [Pt3(µ2-OH)(µ-PPh2)2(PPh3)3]BF4 (3(BF4)) which slowly transformed into [Pt3(µ2-Cl)(µ-PPh2)2(PPh3)3]BF4 (4(BF4)) upon recrystallization from CH2Cl2. These 44 electron clusters have been characterized by (31)P{(1)H} NMR, and the crystal structure of 4(PF6) has been determined by X-ray diffraction, as well as that of [Pt3(µ-PPh2)2I2(PPh3)3] (5), which was obtained by recrystallization of the known cluster [Pt3(µ2-I)(µ-PPh2)2(PPh3)3]I (2(I)) from toluene and represents a neutral formula isomer of the latter. In addition, we have prepared the adducts of cluster 1 containing the moieties [Cu(NCMe)2](+) and [Au(PPh3)](+) in 6 and 7, respectively, and on the basis of their spectroscopic data, it was concluded that these complexes have similar structures to that previously established for the adduct of 1 with Ag(TFA) (TFA = OC(O)CF3), [Pt3{µ3-Ag(TFA)}(µ-PPh2)3Ph(PPh3)2] (8). The cationic clusters in 3(BF4) and 4(BF4) react with Ag(TFA) to afford cationic adducts in [Pt3{µ3-Ag(TFA)}(µ2-X)(µ-PPh2)2(PPh3)3]BF4 (9(BF4), X = OH; 10(BF4), X = Cl). The structure of the mixed-metal cluster [Pt3(µ3-AgBF4)(µ2-I)(µ-PPh2)2(PPh3)3]BF4 (11(BF4)), obtained by reaction of the complex 2(I) with AgBF4, was determined by X-ray diffraction.

Impact of interpretative commenting on lipid profiles in people at high risk of familial hypercholesterolaemia.

BACKGROUND: Familial hypercholesterolaemia (FH) is an autosomal dominant condition characterised by increased low density lipoprotein cholesterol (LDL-c), xanthomata and premature cardiovascular disease. However, it is currently underdiagnosed and undertreated in Australasia. We sought to investigate whether interpretative commenting on lipid profiles could improve FH detection and treatment. METHODS: A case-historical control study of individuals with serum LDL-c concentrations ≥6.5 mmol/L; 96 cases receiving an interpretative comment suggesting FH compared with 100 controls not receiving a comment. RESULTS: Serum LDL-c was repeated in 63 (66%) cases and 70 (70%) controls within 12 months. LDL-c decreased in 59 (94%) cases and in 61 (87%) controls. In individuals with a repeat LDL-c, a mean LDL-c reduction of 2.3 mmol/L (32%; p<0.0001) was demonstrated in controls, compared with 3.0 mmol/L (42%; p<0.0001) in cases; significantly greater than that of controls (p<0.005). Interpretative comments suggesting specialist review were associated with a higher referral rate compared with controls (11.5% vs 1%, p<0.05). CONCLUSION: Interpretative commenting was associated with a significant additional LDL-c reduction and increased specialist referrals compared with controls. However, only a minority of individuals received a specialist referral. Interpretative commenting may play an important role in the detection and management of FH.