Alignment of multiple chromosomes along helical ParA scaffolding in sporulating Streptomyces hyphae

The dynamic, mitosis-like segregation of bacterial chromosomes and plasmids often involves proteins of the ParA (ATPase) and ParB (DNA-binding protein) families. The conversion of multigenomic aerial hyphae of the mycelial organism Streptomyces coelicolor into chains of unigenomic spores requires the synchronous segregation of multiple chromosomes, providing an unusual context for chromosome segregation. Correct spatial organization of the oriC-proximal region prior to septum formation is achieved by the assembly of ParB into segregation complexes (Jakimowicz et al., 2005; J Bacteriol 187: 3572-3580). Here, we focus on the contribution of ParA to sporulation-associated chromosome segregation. Elimination of ParA strongly affects not only chromosome segregation but also septation. In wild type hyphae about to undergo sporulation, immunostained ParA was observed as a stretched double-helical filament, which accompanies the formation of ParB foci. We show that ParA mediates efficient assembly of ParB complexes in vivo and in vitro, and that ATP binding is crucial for ParA dimerization and interaction with ParB but not for ParA localization in vivo. We suggest that S. coelicolor ParA provides scaffolding for proper distribution of ParB complexes and consequently controls synchronized segregation of several dozens of chromosomes, possibly mediating a segregation and septation checkpoint.     

The Concentration of Vanadium in Pathologically Altered Human Kidneys

This study evaluated serum and hair mineral and trace element levels as well as levels of other nutritional factors affecting growth and appetite in young children with non-organic failure to thrive (NOFTT) based on the presence or types of feeding difficulty (FD). Between August 2012 and July 2015, 136 children less than 6 years of age with NOFTT were included. FD was diagnosed based on Wolfson criteria and divided into subtypes according to Chatoor’s classification. Clinical data were reviewed, and serum and hair mineral levels were measured. Of all assessed serum and hair minerals, only hair sulfur contents differed significantly between subjects with and without infantile anorexia (39,392 ± 2211 vs. 40,332 ± 2551 μg/g, P = 0.034). There were no differences in serum and hair mineral levels between children with and without sensory food aversion. Hair copper contents were significantly lower in children with FD of reciprocity (12.3 ± 6.0 vs. 22.4 ± 25.1 μg/g, P = 0.049). While hair zinc contents were also lower, the difference was not statistically significant (49.2 ± 26.8 vs. 70.6 ± 41.0 μg/g, P = 0.055). Only hair manganese contents were significantly lower in children with posttraumatic FD (0.12 ± 0.04 vs. 0.26 ± 0.73 μg/g, P = 0.037). In conclusion, there were no differences in most serum and hair mineral levels in children with NOFTT, except for relatively lower hair levels of sulfur, copper and possibly zinc, and manganese in infantile anorexia, reciprocity, and posttraumatic types of FD, respectively.     

MLK4 regulates DNA damage response and promotes triple-negative breast cancer chemoresistance

Chemoresistance constitutes a major challenge in the treatment of triple-negative breast cancer (TNBC). Mixed-Lineage Kinase 4 (MLK4) is frequently amplified or overexpressed in TNBC where it facilitates the aggressive growth and migratory potential of breast cancer cells. However, the functional role of MLK4 in resistance to chemotherapy has not been investigated so far. Here, we demonstrate that MLK4 promotes TNBC chemoresistance by regulating the pro-survival response to DNA-damaging therapies. We observed that MLK4 knock-down or inhibition sensitized TNBC cell lines to chemotherapeutic agents in vitro. Similarly, MLK4-deficient cells displayed enhanced sensitivity towards doxorubicin treatment in vivo. MLK4 silencing induced persistent DNA damage accumulation and apoptosis in TNBC cells upon treatment with chemotherapeutics. Using phosphoproteomic profiling and reporter assays, we demonstrated that loss of MLK4 reduced phosphorylation of key DNA damage response factors, including ATM and CHK2, and compromised DNA repair via non-homologous end-joining pathway. Moreover, our mRNA-seq analysis revealed that MLK4 is required for DNA damage-induced expression of several NF-кB-associated cytokines, which facilitate TNBC cells survival. Lastly, we found that high MLK4 expression is associated with worse overall survival of TNBC patients receiving anthracycline-based neoadjuvant chemotherapy. Collectively, these results identify a novel function of MLK4 in the regulation of DNA damage response signaling and indicate that inhibition of this kinase could be an effective strategy to overcome TNBC chemoresistance.Subject terms: Chemotherapy, Oncogenes, Cell signalling, Breast cancer     

The effect of the menstrual cycle on collagen metabolism,growth hormones and strength in young physically active women

This study aimed to investigate the effect of the menstrual cycle on strength, functioning of the GH/IGF-1 axis and collagen metabolism in physically active women. Twenty-four physically active and eumenorrheic women volunteered to participate in the study (body mass 60.3 ± 9.18 kg, age 21.8 ± 0.92 years). Blood samples were obtained between the 5th and 8th days (the follicular phase) and between the 19th and 22th days (the luteal phase) of the menstrual cycle to determine sex steroid concentrations (follicle-stimulating hormone (FSH), luteinizing hormone (LH), prolactin (PRL), oestradiol (E2) and progesterone (P)). Also insulin-like growth factor 1 (IGF-1) and collagen metabolism markers (synthesis (PICP) and breakdown (ICTP)) and maximum voluntary contraction (MVC) were measured. MVC was higher in the luteal phase 164.1 ± 34.77 [N m] (F(1.23) = 4.59; p = 0.043). The recorded collagen synthesis marker (PICP = 296.4 ± 35.61 [ng/ml]) was at the upper level of the reference range (30–300), with an insignificant decrease in the luteal phase (Z = 1.612; p = 0.107) and a significant increase in oestradiol concentration (Z = 4.286; p = 0.0001). The marker of collagen breakdown (ICTP = 4.16 ± 0.68 [μg/l]) was reduced by 6.8% in the same phase (Z = 1.764; p = 0.137). The variability of physical abilities (MVC) during the menstrual cycle showed that menstrual status should be taken into account in determination of the training loads. Increasing the load in the luteal phase seems to be favoured by a beneficial change in collagen metabolism (lower synthesis decrease, lower breakdown increase) observed in physically active women.     

Fission yeast Duf89 and Duf8901 are cobalt/nickel-dependent phosphatase–pyrophosphatases that act via a covalent aspartyl–phosphate intermediate

Domain of Unknown Function 89 (DUF89) proteins are metal-dependent phosphohydrolases. Exemplary DUF89 enzymes differ in their metal and phosphosubstrate preferences. Here, we interrogated the activities and structures of two DUF89 paralogs from fission yeast—Duf89 and Duf8901. We find that Duf89 and Duf8901 are cobalt/nickel-dependent phosphohydrolases adept at hydrolyzing p-nitrophenylphosphate and PP_i. Crystal structures of metal-free Duf89 and Co²⁺-bound Duf8901 disclosed two enzyme conformations that differed with respect to the position of a three-helix module, which is either oriented away from the active site in Duf89 or forms a lid over the active site in Duf8901. Lid closure results in a 16 Å movement of Duf8901 Asp195, vis-à-vis Asp199 in Duf89, that brings Asp195 into contact with an octahedrally coordinated cobalt. Reaction of Duf8901 with BeCl₂ and NaF in the presence of divalent cations Co²⁺, Ni²⁺, or Zn²⁺ generated covalent Duf8901-(Asp248)–beryllium trifluoride (BeF₃)•Co²⁺, Duf8901-(Asp248)–BeF₃•Ni²⁺, or Duf8901-(Asp248)–BeF₃•Zn²⁺ adducts, the structures of which suggest a two-step catalytic mechanism via formation and hydrolysis of an enzyme-(aspartyl)–phosphate intermediate. Alanine mutations of Duf8901 Asp248, Asn249, Lys401, Asp286, and Asp195 that interact with BeF₃•Co²⁺ squelched p-nitrophenylphosphatase activity. A 1.8 Å structure of a Duf8901-(Asp248)–AlF₄–OH₂•Co²⁺ transition-state mimetic suggests an associative mechanism in which Asp195 and Asp363 orient and activate the water nucleophile. Whereas deletion of the duf89 gene elicited a phenotype in which expression of phosphate homeostasis gene pho1 was derepressed, deleting duf8901 did not, thereby hinting that the DUF89 paralogs have distinct functional repertoires in vivo.