The complete genome sequence of Thiobacillus denitrificans ATCC 25259 is the first to become available for an obligately chemolithoautotrophic, sulfur-compound-oxidizing, β-proteobacterium. Analysis of the 2,909,809-bp genome will facilitate our molecular and biochemical understanding of the unusual metabolic repertoire of this bacterium, including its ability to couple denitrification to sulfur-compound oxidation, to catalyze anaerobic, nitrate-dependent oxidation of Fe(II) and U(IV), and to oxidize mineral electron donors. Notable genomic features include (i) genes encoding c -type cytochromes totaling 1 to 2 percent of the genome, which is a proportion greater than for almost all bacterial and archaeal species sequenced to date, (ii) genes encoding two [NiFe]hydrogenases, which is particularly significant because no information on hydrogenases has previously been reported for T. denitrificans and hydrogen oxidation appears to be critical for anaerobic U(IV) oxidation by this species, (iii) a diverse complement of more than 50 genes associated with sulfur-compound oxidation (including sox genes, dsr genes, and genes associated with the AMP-dependent oxidation of sulfite to sulfate), some of which occur in multiple (up to eight) copies, (iv) a relatively large number of genes associated with inorganic ion transport and heavy metal resistance, and (v) a paucity of genes encoding organic-compound transporters, commensurate with obligate chemolithoautotrophy. Ultimately, the genome sequence of T. denitrificans will enable elucidation of the mechanisms of aerobic and anaerobic sulfur-compound oxidation by β-proteobacteria and will help reveal the molecular basis of this organism's role in major biogeochemical cycles (i.e., those involving sulfur, nitrogen, and carbon) and groundwater restoration.

Cancer-associated fibroblasts (CAFs) are a prominent stromal cell type in solid tumors and molecules secreted by CAFs play an important role in tumor progression and metastasis. CAFs coexist as heterogeneous populations with potentially different biological functions. Although CAFs are a major component of the breast cancer stroma, molecular and phenotypic heterogeneity of CAFs in breast cancer is poorly understood. In this study, we investigated CAF heterogeneity in triple-negative breast cancer (TNBC) using a syngeneic mouse model, BALB/c-derived 4T1 mammary tumors. Using single-cell RNA sequencing (scRNA-seq), we identified six CAF subpopulations in 4T1 tumors including: 1) myofibroblastic CAFs, enriched for α-smooth muscle actin and several other contractile proteins; 2) ‘inflammatory’ CAFs with elevated expression of inflammatory cytokines; and 3) a CAF subpopulation expressing major histocompatibility complex (MHC) class II proteins that are generally expressed in antigen-presenting cells. Comparison of 4T1-derived CAFs to CAFs from pancreatic cancer revealed that these three CAF subpopulations exist in both tumor types. Interestingly, cells with inflammatory and MHC class II-expressing CAF profiles were also detected in normal breast/pancreas tissue, suggesting that these phenotypes are not tumor microenvironment-induced. This work enhances our understanding of CAF heterogeneity, and specifically targeting these CAF subpopulations could be an effective therapeutic approach for treating highly aggressive TNBCs.

We present results from experiments at the Linac Coherent Light Source (LCLS) demonstrating that serial femtosecond crystallography (SFX) can be performed to high resolution (~2.5 Å) using protein microcrystals deposited on an ultra-thin silicon nitride membrane and embedded in a preservation medium at room temperature. Data can be acquired at a high acquisition rate using x-ray free electron laser sources to overcome radiation damage, while sample consumption is dramatically reduced compared to flowing jet methods. We achieved a peak data acquisition rate of 10 Hz with a hit rate of ~38%, indicating that a complete data set could be acquired in about one 12-hour LCLS shift using the setup described here, or in even less time using hardware optimized for fixed target SFX. This demonstration opens the door to ultra low sample consumption SFX using the technique of diffraction-before-destruction on proteins that exist in only small quantities and/or do not produce the copious quantities of microcrystals required for flowing jet methods.

The new European X-ray Free-Electron Laser is the first X-ray free-electron laser capable of delivering X-ray pulses with a megahertz inter-pulse spacing, more than four orders of magnitude higher than previously possible. However, to date, it has been unclear whether it would indeed be possible to measure high-quality diffraction data at megahertz pulse repetition rates. Here, we show that high-quality structures can indeed be obtained using currently available operating conditions at the European XFEL. We present two complete data sets, one from the well-known model system lysozyme and the other from a so far unknown complex of a β-lactamase from K. pneumoniae involved in antibiotic resistance. This result opens up megahertz serial femtosecond crystallography (SFX) as a tool for reliable structure determination, substrate screening and the efficient measurement of the evolution and dynamics of molecular structures using megahertz repetition rate pulses available at this new class of X-ray laser source.

Purpose: To characterize the cellular functions associated with the altered transcript profiles of mouse brain exposed to low‐dose in vivo gamma‐irradiation. Materials and methods: Cerebral RNA was isolated at 30 min and 4 h after whole‐body irradiation at 0.1 or 2 Gy, hybridized to random oligonucleotide arrays, and evaluated for time and dose–response patterns by multifactorial analyses. Results: Brain irradiation modulated the expression patterns of 1574 genes, of which 855 showed more than 1.5‐fold variation. about 30% of genes showed dose‐dependent variations, including genes exclusively affected by 0.1 Gy. About 60% of genes showed time‐dependent variation with more genes affected at 30 min than at 4 h. Early changes involved signal transduction, ion regulation and synaptic signalling. Later changes involved metabolic functions including myelin and protein synthesis. Low‐dose radiation also modulated the expression of genes involved in stress response, cell‐cycle control and DNA synthesis/repair. Conclusions: Doses of 0.1 Gy induced changes in gene expression that were qualitatively different from those at 2 Gy. The findings suggest that low‐dose irradiation of the brain induces the expression of genes involved in protective and reparative functions, while down‐modulating genes involved in neural signalling activity.

Tumor cells, including cancer stem cells (CSCs) resistant to radio- and chemotherapy, must enhance metabolism to meet the extra energy demands to repair and survive such genotoxic conditions. However, such stress-induced adaptive metabolic alterations, especially in cancer cells that survive radiotherapy, remain unresolved. In this study, we found that CPT1 (Carnitine palmitoyl transferase I) and CPT2 (Carnitine palmitoyl transferase II), a pair of rate-limiting enzymes for mitochondrial fatty acid transportation, play a critical role in increasing fatty acid oxidation (FAO) required for the cellular fuel demands in radioresistant breast cancer cells (RBCs) and radiation-derived breast cancer stem cells (RD-BCSCs). Enhanced CPT1A/CPT2 expression was detected in the recurrent human breast cancers and associated with a worse prognosis in breast cancer patients. Blocking FAO via a FAO inhibitor or by CRISPR-mediated CPT1A/CPT2 gene deficiency inhibited radiation-induced ERK activation and aggressive growth and radioresistance of RBCs and RD-BCSCs. These results revealed that switching to FAO contributes to radiation-induced mitochondrial energy metabolism, and CPT1A/CPT2 is a potential metabolic target in cancer radiotherapy.