Simple nutrients bypass the requirement for HLH-30 in coupling lysosomal nutrient sensing to survival.

Lysosomes are ubiquitous acidified organelles that degrade intracellular and extracellular material trafficked via multiple pathways. Lysosomes also sense cellular nutrient levels to regulate target of rapamycin (TOR) kinase, a signaling enzyme that drives growth and suppresses activity of the MiT/TFE family of transcription factors that control biogenesis of lysosomes. In this study, we subjected worms lacking basic helix-loop-helix transcription factor 30 (hlh-30), the Caenorhabditis elegans MiT/TFE ortholog, to starvation followed by refeeding to understand how this pathway regulates survival with variable nutrient supply. Loss of HLH-30 markedly impaired survival in starved larval worms and recovery upon refeeding bacteria. Remarkably, provision of simple nutrients in a completely defined medium (C. elegans maintenance medium [CeMM]), specifically glucose and linoleic acid, restored lysosomal acidification, TOR activation, and survival with refeeding despite the absence of HLH-30. Worms deficient in lysosomal lipase 2 (lipl-2), a lysosomal enzyme that is transcriptionally up-regulated in starvation in an HLH-30-dependent manner, also demonstrated increased mortality with starvation-refeeding that was partially rescued with glucose, suggesting a critical role for LIPL-2 in lipid metabolism under starvation. CeMM induced transcription of vacuolar proton pump subunits in hlh-30 mutant worms, and knockdown of vacuolar H+-ATPase 12 (vha-12) and its upstream regulator, nuclear hormone receptor 31 (nhr-31), abolished the rescue with CeMM. Loss of Ras-related GTP binding protein C homolog 1 RAGC-1, the ortholog for mammalian RagC/D GTPases, conferred starvation-refeeding lethality, and RAGC-1 overexpression was sufficient to rescue starved hlh-30 mutant worms, demonstrating a critical need for TOR activation with refeeding. These results show that HLH-30 activation is critical for sustaining survival during starvation-refeeding stress via regulating TOR. Glucose and linoleic acid bypass the requirement for HLH-30 in coupling lysosome nutrient sensing to survival.

Ureteral stenting after routine ureteroscopy: Is earlier stent removal feasible?

INTRODUCTION: Ureteroscopy and laser lithotripsy is a common treatment option for upper urinary tract calculi. Currently, ureteral stents are placed after uncomplicated ureteroscopy for up to 1 week, but the optimal length of placement is not well defined. Ureteral stents are associated with significant morbidity, particularly stent discomfort. This study aims to determine differences in postoperative unplanned clinic or ED visits based on duration of stent placement. MATERIALS AND METHODS: This is a single-institution, IRB-approved, retrospective cohort study of 559 ureteroscopy cases with laser lithotripsy for urinary tract calculi performed from 2016 to 2018. The primary outcome was unplanned ED or clinic visits within 30 days following surgery and there. The patients were separated into three groups based on stent duration: 1 (0-3 days), 2 (4-6 days), and 3 (> 6 days). RESULTS: Fifty-eight (10.31%) patients experienced an unplanned visit within 30 days of the procedure. There was no significant difference in unplanned visits among groups for stent duration (p = 0.45). A Clavien grade analysis showed no difference in grades between groups (p = 0.59). A Cox regression model showed no difference in risk of unplanned visit comparing those in groups 2 and 3 to group 1 (p = 0.157 and 0.374, respectively). This also remains to be the case after adjusting for age, sex, and surgeon (p = 0.166 and 0.376, respectively). CONCLUSIONS: We found no difference in unplanned visits in patients based on the duration of stent placement following routine ureteroscopy. Stent removal within 3 days of surgery appears to be sufficient to minimize morbidity after uncomplicated ureteroscopy.

Analysis of common oral and topical dermatologic medication price variation by source.

Online coupon retailers and pharmacies are popular sites that patients can access discounted medications when compared to cash prices. These sources are especially important for those patients without insurance. In our study, we analyzed commonly prescribed topical and oral medications and compared the cash prices to the discounted medications based on a typical month of usage. We found savings in every one of the medications that we analyzed, some with savings up to hundreds of dollars. Savings were present in all the sources analyzed, with the coupon-based programs often having the lowest price. We suggest certain alternative prescribing guidelines when considering patients who may not be able to afford cash prices of medications. Our hopes with this study are to quantify savings for discounted medications as well as to help physicians target more affordable medications for their patients.

A solution-phase parallel synthesis of alkylated guanidines from thioisocyanates and amines.

An efficient solution-phase parallel synthesis of alkylated guanidines from commercial thioisocyanates and amines is described. In the first step, a thioisocyanate reacts with one equivalent of ammonia or a primary or secondary amine to give a thiourea intermediate. The latter is S-alkylated with n-dodecyl bromide resulting in the corresponding thiouronium bromide. Finally, the reaction of the thiouronium salt with a second equivalent of ammonia or a primary amine yields an alkylated guanidine. All three synthetic steps are easily combined in a one-pot high-yielding procedure with a simple work-up. Ca. 250 guanidine derivatives with high structural and functional diversity were synthesized by the developed method. 35 representatives reported in this study were fully characterized.

Predictors of Breast Cancer Outcome in a Cohort of Women Seeking Care at a Safety Net Hospital.

RATIONALE AND OBJECTIVES: This study aimed to identify predictors associated with lower mortality in a population of women diagnosed and treated for breast cancer at a safety net hospital. METHODS: From 2008 to 2014, 1115 patients were treated for breast cancer at our academic safety net hospital. 208 were excluded due to diagnosis at an outside facility, and the remaining 907 (81%) formed the study cohort. Retrospective charts and imaging reviews looked at race, ethnicity, insurance status, social determinants of health, screening utilization, treatment regimen, and 7-13-year follow-up care, including the cause of death. Multivariable logistic regression modeling assessed mortality, and adjusted odds ratios (aOR) with 95% confidence intervals (CI) were computed. RESULTS: Of the 907 women, the mean age was 59 years (inter-quartile range 50-68 years), with 40% White, 46% Black, 4% Asian, and 10% Other. Increasing age (aOR=1.03, p = 0.001) and more advanced stage at diagnosis (aOR=6.37, p < 0.0001) were associated with increased mortality. There was no significant difference in mortality based on race or ethnicity (p > 0.05). Of 494 with screening prior to diagnosis, longer screening time was observed for patients with advanced stage (median 521 days) vs. early stage (median 404 days), p = 0.0004. Patients with Medicaid, insurance not specified, and no insurance were less likely to undergo screening before diagnosis than privately insured (all p < 0.05). Shorter screening time was associated with lower all-cause mortality (aOR=0.57, 95% CI=0.36-0.89, p = 0.013). DISCUSSION: In a safety net population, a more advanced stage at diagnosis was associated with higher mortality and lower odds of undergoing screening mammography in the two years prior to a breast cancer diagnosis. Early screening was associated with lower mortality. Finally, given no racial or ethnic differences in mortality, the safety net infrastructure at our institution effectively provides equitable cancer care once a cancer is confirmed.

Collapsed Cu(II)-hydroxamate metallacrowns.

Degradation of a strained, thermodynamically destabilized pentanuclear copper(II) 12-metallacrown-4 complex based on a picoline hydroxamic acid resulted in the formation of the tetranuclear compounds which are the first examples of solely hydroxamate-based Cu(II) metallacrown complexes with a collapse of the metallamacrocyclic cavity.

Tris(2,2'-bipyridine-κ(2)N,N')cobalt(III) bis-[bis-(pyridine-2,6-dicarboxyl-ato-κ(3)O(2),N,O(6))cobaltate(III)] perchlorate dimethyl-formamide hemisolvate 1.3-hydrate.

In the title compound, [Co(C(10)H(8)N(2))(3)][Co(C(7)H(3)NO(4))(2)](2)(ClO(4))·0.5C(3)H(7)NO·1.3H(2)O, the Co(III) atom in the complex cation is pseudoocta-hedrally coordinated by six N atoms of three chelating bipyridine ligands. The Co(III) atom in the complex anion is coordinated by two pyridine N atoms and four carboxyl-ate O atoms of two doubly deprotonated pyridine-2,6-dicarboxyl-ate ligands in a distorted octa-hedral geometry. One dimethyl-formamide solvent mol-ecule and two water mol-ecules are half-occupied and one water mol-ecule is 0.3-occupied. O-H⋯O hydrogen bonds link the water mol-ecules, the perchlorate anions and the complex anions. π-π inter-actions between the pyridine rings of the complex anions are also observed [centroid-centroid distance = 3.804 (3) Å].

Poly[di-µ(2)-aqua-µ(5)-(pyridine-2,6-dicarboxyl-ato)-µ(3)-(pyridine-2,6-dicarboxyl-ato)-cobalt(II)disodium].

In the title compound, [CoNa(2)(C(7)H(3)NO(4))(2)(H(2)O)(2)](n), the Co(II) atom is coordinated by two pyridine N atoms and four carboxyl-ate O atoms from two doubly deprotonated pyridine-2,6-dicarboxyl-ate ligands in a distorted octa-hedral geometry. One Na(+) cation is coordinated by three carboxyl-ate O atoms and two water mol-ecules and the other is coordinated by five carboxyl-ate O atoms and two water mol-ecules in an irregular geometry. The bis-(pyridine-2,6-dicarboxyl-ato)cobalt complex units are connected by Na(+) cations and bridging water mol-ecules into a three-dimensional coordination network. O-H⋯O hydrogen bonds are formed between the water mol-ecules and the carboxyl-ate O atoms.

Diaqua-bis-(pyridine-2-carboxyl-ato-κN,O)manganese(II) dimethyl-formamide hemisolvate.

There are two crystallographically independent complex mol-ecules with very similar geometries in the unit cell of the title compound, [Mn(C(6)H(4)NO(2))(2)(H(2)O)(2)]·0.5C(3)H(7)NO. The central ion is situated in a distorted octa-hedral environment of two N- and four O-donor atoms from two pyridine-2-carboxyl-ate ligands and two cis-disposed water mol-ecules. The carboxyl-ate ligands are coordinated in a chelate fashion with the formation of two five-membered rings. In the crystal, the complex mol-ecules are connected by O-H⋯O hydrogen bonds between the coordinated water mol-ecules and the uncoordinated carboxyl-ate O atoms, thus forming hydrogen-bonded walls disposed perpendicularly to the bc plane.

fac-Tris(pyridine-2-carboxyl-ato-κN,O)cobalt(III).

In the title compound, [Co(C(6)H(4)NO(2))(3)], the Co(III) ion lies on a threefold rotation axis and is in a distorted octa-hedral environment defined by three N and three O donor atoms from three fac-disposed pyridine-2-carboxyl-ate ligands. The ligands are coordinated in a chelate fashion, forming three five-membered rings. In the crystal, translationally related complex molecules are organized into columns along [001] via C-H⋯O hydrogen bonds.

2-Hydroxy-amino-2-oxoacetohydrazide.

In the title compound, C(2)H(5)N(3)O(3), the hydroxamic group adopts an anti orientation with respect to the hydrazide group. In the crystal, mol-ecules are connected by N-H⋯O and O-H⋯N hydrogen bonds into zigzag chains along the c axis.

µ-Oxalato-bis-[(2,2'-bipyridyl)-copper(II)] bis(perchlorate) dimethyl-formamide disolvate monohydrate.

The title compound, [Cu(2)(C(2)O(4))(C(10)H(8)N(2))(4)](ClO(4))(2)·2C(3)H(7)NO·H(2)O, contains doubly charged centrosymmetric dinuclear oxalato-bridged copper(II) complex cations, perchlorate anions, and DMF and water solvate mol-ecules. In the complex cation, the oxalate ligand is coordinated in a bis-bidentate bridging mode to the Cu atoms. Each Cu atom has a distorted tetra-gonal-bipyramidal environment, being coordinated by two N atoms of the two chelating bipy ligands and two O atoms of the doubly deprotonated oxalate anion. Pairs of perchlorate anions and water mol-ecules are linked into recta-ngles by O-H⋯O bonds in which the perchlorate O atoms act as acceptors and the water mol-ecules as donors. Methyl groups of the DMF solvent molecule are disordered over two sites with occupancies of 0.453 (7):0.547 (7), and the water molecule is half-occupied.

Transgenerational changes in the genome stability and methylation in pathogen-infected plants: (virus-induced plant genome instability).

Previously, we reported the generation of a virus-induced systemic signal that increased the somatic and meiotic recombination rates in tobacco mosaic virus (TMV)-infected tobacco plants. Here, we analyzed the progeny of plants that received the signal and found that these plants also have a higher frequency of rearrangements in the loci carrying the homology to LRR region of the gene of resistance to TMV (N-gene). Analysis of the stability of repetitive elements from Nicotiana tabacum loci and 5.8S ribosomal RNA loci did not show any changes. Further analysis of the changes in the progeny of infected plants revealed that they had substantially hypermethylated genomes. At the same time, loci-specific methylation analysis showed: (1) profound hypomethylation in several LRR-containing loci; (2) substantial hypermethylation of actin loci and (3) no change in methylation in the loci of repetitive elements from N. tabacum or 5.8S ribosomal RNA. Global genome hypermethylation of the progeny is believed to be part of a general protection mechanism against stress, whereas locus-specific hypomethylation is associated with a higher frequency of rearrangements. Increased recombination events combined with the specific methylation pattern induced by pathogen attack could be a sign of an adaptive response by plants.

Homologous recombination in plants is organ specific.

In this paper we analysed the genome stability of various Arabidopsis thaliana plant organs using a transgenic recombination system. The system was based on two copies of non-functional GUS (lines #651 and #11) or LUC (line #15D8) reporter genes serving as a recombination substrate. Both reporter assays showed that recombination in flowers or stems were rare events. Most of the recombination sectors were found in leaves and roots, with leaves having over 2-fold greater number of the recombination events per single cell genome as compared to roots. The recombination events per single genome were 9.7-fold more frequent on the lateral half of the leaves than on the medial halves. This correlated with a 2.5-fold higher metabolic activity in the energy source (lateral) versus energy sink (medial) of leaves. Higher metabolic activity was paralleled by a higher anthocyanin production in lateral halves. The level of double strand break (DSB) occurrence was also different among plant organs; the highest level was observed in roots and the lowest in leaves. High level of DSBs strongly positively correlated with the activity of the key repair enzymes, AtKU70 and AtRAD51. The ratio of AtRAD51 to AtKU70 expression was the highest in leaves, supporting the more active involvement of homologous recombination pathway in the repair of DSBs in this organ. Western blot analysis confirmed the real time PCR expression data for AtKU70 gene.

Homologous recombination in plants is temperature and day-length dependent.

Homologous recombination (HR) as a strand break repair mechanism was shown to be influenced by various factors. The balance of different vitamins, macro- and microelements, light spectrum, sodium chloride concentration as well as various environmental mutagens were shown to influence the frequency of HR. In this paper we analysed the influence of temperature (4, 22, and 32 degrees C) and day/night duration on the genome stability of plants. We analyzed the HR frequency in transgenic Arabidopsis thaliana plants carrying beta-glucuronidase based homologous recombination substrate. To find the recombination rate (RR), we related the HR frequency to the number of genomes present in plants grown under different conditions. The RR was also standardized to the transcription activity of the transgene. We found RR to be higher in plants grown at suboptimal temperatures (either 4 or 32 degrees C) as compared to plants grown at 22 degrees C. This negatively correlated with the plant metabolic rate and positively correlated with concentration of peroxide produced by plant. In contrast, the RR in plants grown at different day length (8-24 h) was the lowest in plants grown at the longest day (24 h) and highest in the plants grown at the shortest day (8 h). Over 15-fold difference in the RR between plants grown at the shortest and the longest day was observed. Such a difference in recombination rate was primarily due to the higher transgene activity and higher endoreduplication levels in plants grown at longer days. Our data suggests that even "moderate" changes of environmental conditions may have a significant effect on plant genome stability.

Luciferase-based transgenic recombination assay is more sensitive than beta-glucoronidase-based.

Study of the DNA repair and genome stability in plants is directly dependent on the availability of an easy, inexpensive, and reliable assay. Marker gene-based homologous recombination (HR) assays were introduced more than a decade ago and have been intensively used ever since. Here, we compared several transgenic Arabidopsis and tobacco lines that carried in their genome the luciferase (LUC) or the beta-glucoronidase (uidA or GUS) substrates for HR. The average recombination frequency detected with the luciferase transgene was nearly 9.0-fold higher in Arabidopsis and 12.4-fold higher in tobacco plants. Importantly, both transgenes were under the control of 35S promoter and had similar expression levels throughout the plants. Irradiation with UVC increased the HR frequency similarly in both transgenes. The actual difference in the frequency of HR in Arabidopsis and tobacco possibly results from differing sensitivity to detection of transgene activity. Thus, we could suggest that luciferase recombination assay, due to its higher sensitivity, should be the assay of choice when plant genome stability is studied.

Double-strand break repair in plants is developmentally regulated.

In this study, we analyzed double-strand break (DSB) repair in Arabidopsis (Arabidopsis thaliana) at various developmental stages. To analyze DSB repair, we used a homologous recombination (HR) and point mutation reversion assays based on nonfunctional beta-glucuronidase reporter genes. Activation of the reporter gene through HR or point mutation reversion resulted in the appearance of blue sectors after histochemical staining. Scoring of these sectors at 3-d intervals from 2 to 31 d post germination (dpg) revealed that, although there was a 100-fold increase in the number of genomes per plant, the recombination frequency only increased 30-fold. This translates to a recombination rate at 31 dpg (2.77 x 10(-8)) being only 30% of the recombination rate at 2 dpg (9.14 x 10(-8)). Conversely, the mutation frequency increased nearly 180-fold, resulting in a 1.8-fold increase in mutation rate from 2 to 31 dpg. Additional analysis of DSBs over the early developmental stages revealed a substantial increase in the number of strand breaks per unit of DNA. Furthermore, RNA analysis of Ku70 and Rad51, two key enzymes in two different DSB repair pathways, and further protein analysis of Ku70 revealed an increase in Ku70 levels and a decrease of Rad51 levels in the developing plants. These data suggest that DSB repair mechanisms are developmentally regulated in Arabidopsis, whereby the proportion of breaks repaired via HR substantially decreases as the plants mature.