Mitochondrial Oxidative Phosphorylation Complex Regulates NLRP3 Inflammasome Activation and Predicts Patient Survival in Nasopharyngeal Carcinoma

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Highlights

• •Flow cytometry analysis is used to isolate ASC speck⁽⁺⁾ NPC cells.
• •Proteome analysis of ASC speck⁽⁺⁾ NPC cells reveals enriched mitochondrial OxPhos proteins.
• •OxPhos proteins mediate NLRP3 inflammasome activation through mtROS.
• •OxPhos proteins, NDUFB8 and ATP5B are correlated with NPC local recurrence.

     

Characterizing Patients with Recurrent Urinary Tract Infections in Vesicoureteral Reflux: A Pilot Study of the Urinary Proteome

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Highlights

• •Urinary proteomes of patients with recurrent UTI, renal scarring, and VUR.
• •80 proteins differentially expressed, compared to healthy controls.
• •62 proteins may be indicative of susceptibility for UTI.
• •Altered acute phase response, extracellular matrix and carbohydrate metabolism.

     

The impact of global climate change on genetic diversity within populations and species

Genetic diversity provides the basic substrate for evolution, yet few studies assess the impacts of global climate change (GCC) on intraspecific genetic variation. In this review, we highlight the importance of incorporating neutral and non‐neutral genetic diversity when assessing the impacts of GCC, for example, in studies that aim to predict the future distribution and fate of a species or ecological community. Specifically, we address the following questions: Why study the effects of GCC on intraspecific genetic diversity? How does GCC affect genetic diversity? How is the effect of GCC on genetic diversity currently studied? Where is potential for future research? For each of these questions, we provide a general background and highlight case studies across the animal, plant and microbial kingdoms. We further discuss how cryptic diversity can affect GCC assessments, how genetic diversity can be integrated into studies that aim to predict species' responses on GCC and how conservation efforts related to GCC can incorporate and profit from inclusion of genetic diversity assessments. We argue that studying the fate of intraspecifc genetic diversity is an indispensable and logical venture if we are to fully understand the consequences of GCC on biodiversity on all levels.     

Metabolic Labeling of Leucine Rich Repeat Kinases 1 and 2 with Radioactive Phosphate

Leucine rich repeat kinases 1 and 2 (LRRK1 and LRRK2) are paralogs which share a similar domain organization, including a serine-threonine kinase domain, a Ras of complex proteins domain (ROC), a C-terminal of ROC domain (COR), and leucine-rich and ankyrin-like repeats at the N-terminus. The precise cellular roles of LRRK1 and LRRK2 have yet to be elucidated, however LRRK1 has been implicated in tyrosine kinase receptor signaling^1,2, while LRRK2 is implicated in the pathogenesis of Parkinson''s disease^3,4. In this report, we present a protocol to label the LRRK1 and LRRK2 proteins in cells with ³²P orthophosphate, thereby providing a means to measure the overall phosphorylation levels of these 2 proteins in cells. In brief, affinity tagged LRRK proteins are expressed in HEK293T cells which are exposed to medium containing ³²P-orthophosphate. The ³²P-orthophosphate is assimilated by the cells after only a few hours of incubation and all molecules in the cell containing phosphates are thereby radioactively labeled. Via the affinity tag (3xflag) the LRRK proteins are isolated from other cellular components by immunoprecipitation. Immunoprecipitates are then separated via SDS-PAGE, blotted to PVDF membranes and analysis of the incorporated phosphates is performed by autoradiography (³²P signal) and western detection (protein signal) of the proteins on the blots. The protocol can readily be adapted to monitor phosphorylation of any other protein that can be expressed in cells and isolated by immunoprecipitation.     

Structural anatomy of telomere OB proteins

Telomere DNA-binding proteins protect the ends of chromosomes in eukaryotes. A subset of these proteins are constructed with one or more OB folds and bind with G+T-rich single-stranded DNA found at the extreme termini. The resulting DNA-OB protein complex interacts with other telomere components to coordinate critical telomere functions of DNA protection and DNA synthesis. While the first crystal and NMR structures readily explained protection of telomere ends, the picture of how single-stranded DNA becomes available to serve as primer and template for synthesis of new telomere DNA is only recently coming into focus. New structures of telomere OB fold proteins alongside insights from genetic and biochemical experiments have made significant contributions towards understanding how protein-binding OB proteins collaborate with DNA-binding OB proteins to recruit telomerase and DNA polymerase for telomere homeostasis. This review surveys telomere OB protein structures alongside highly comparable structures derived from replication protein A (RPA) components, with the goal of providing a molecular context for understanding telomere OB protein evolution and mechanism of action in protection and synthesis of telomere DNA.     

A systems level engineered E. coli capable of efficiently producing L-phenylalanine

The biosynthesis of L-phenylalanine (Phe) is one of the most complicated amino acid synthesis pathways. In this study, the engineering of Phe producer was carried out to illustrate the effectiveness of systems level engineering: (1) inactivated glucose specific phosphoenolpyruvate-carbohydrate phosphotransferase (PTS) system by inactivation of crr to moderate the glucose uptake rate to reduce overflow metabolism; (2) genetic switch on or off the expression of phe^fbr, aroG15, ydiB, aroK, and tyrB to increase the supply of precursors; (3) employed a tyrA mutant strain to reduce carbon diversion and to result in non-growing cells; (4) enhanced the efflux of Phe by overexpressing yddG to shift equilibrium towards Phe synthesis and to release the feedback regulation in Phe synthesis. The mutants in PTS were firstly compared and a crr⁻ mutant was firstly screened. The mutant AroG15 was demonstrated to a thermostable mutant. The strains expressing yddG excreted Phe into the medium at higher rate and less intracellular Phe accumulated. By systems level engineering, an engineered Phe producer achieved 47.0 g/L Phe with a yield of 0.252 g/g which was the highest under the non-optimized fermentation condition.     

In-vitro circadian rhythm of murine bone marrow progenitor production

Hematopoietic processes display 24h rhythms both in rodents and in human beings. We hypothesized these rhythms to be in part generated by a circadian oscillator within the bone marrow. The ability of murine bone marrow granulo-monocytic (GM) precursors to form colonies following colony-stimulating factor (rm GM-CSF) exposure was investigated in liquid culture samples obtained every 3 h for a span of up to 198 h. The CFU-GM count varied rhythmically over the first 4 d of culture, with a reproducible maximum in the early morning hours, similar to that observed in vivo. These experiments provide the first evidence that bone marrow progenitors sustain in vitro circadian rhythmicity, and they demonstrate the presence of a circadian time-keeping system within these cells. The results support the potential usefulness of bone marrow cultures for investigating chronopharmacologic effects of anticancer drugs and cytokines on this target system.     

An Ex vivo Culture System to Study Thyroid Development

The thyroid is a bilobated endocrine gland localized at the base of the neck, producing the thyroid hormones T3, T4, and calcitonin. T3 and T4 are produced by differentiated thyrocytes, organized in closed spheres called follicles, while calcitonin is synthesized by C-cells, interspersed in between the follicles and a dense network of blood capillaries. Although adult thyroid architecture and functions have been extensively described and studied, the formation of the “angio-follicular” units, the distribution of C-cells in the parenchyma and the paracrine communications between epithelial and endothelial cells is far from being understood.This method describes the sequential steps of mouse embryonic thyroid anlagen dissection and its culture on semiporous filters or on microscopy plastic slides. Within a period of four days, this culture system faithfully recapitulates in vivo thyroid development. Indeed, (i) bilobation of the organ occurs (for e12.5 explants), (ii) thyrocytes precursors organize into follicles and polarize, (iii) thyrocytes and C-cells differentiate, and (iv) endothelial cells present in the microdissected tissue proliferate, migrate into the thyroid lobes, and closely associate with the epithelial cells, as they do in vivo.Thyroid tissues can be obtained from wild type, knockout or fluorescent transgenic embryos. Moreover, explants culture can be manipulated by addition of inhibitors, blocking antibodies, growth factors, or even cells or conditioned medium. Ex vivo development can be analyzed in real-time, or at any time of the culture by immunostaining and RT-qPCR.In conclusion, thyroid explant culture combined with downstream whole-mount or on sections imaging and gene expression profiling provides a powerful system for manipulating and studying morphogenetic and differentiation events of thyroid organogenesis.