Lakshman Abhilash, Arshad Kalliyil, and Vasu Sheeba

Even though the rhythm in adult emergence and rhythm in locomotor activity are two different rhythmic phenomena that occur at distinct life-stages of the fly life cycle, previous studies have hinted at similarities in certain aspects of the organisation of the circadian clock driving these two rhythms. For instance, the period gene plays an important regulatory role in both rhythms. In an earlier study, we have shown that selection on timing of adult emergence behaviour in populations of Drosophila melanogaster leads to the co-evolution of temperature sensitivity of circadian clocks driving eclosion. In this study, we were interested in asking if temperature sensitivity of the locomotor activity rhythm has evolved in our populations with divergent timing of adult emergence rhythm, with the goal of understanding the extent of similarity (or lack of it) in circadian organisation between the two rhythms. We found that in response to simulated jetlag with temperature cycles, late chronotypes (populations selected for predominant emergence during dusk) indeed re-entrain faster than early chronotypes (populations selected for predominant emergence during dawn) to 6-h phase-delays, thereby indicating enhanced sensitivity of the activity/rest clock to temperature cues in these stocks (entrainment is the synchronisation of internal rhythms to cyclic environmental time-cues). Additionally, we found that late chronotypes show higher plasticity of phases across regimes, day-to-day stability in phases and amplitude of entrainment, all indicative of enhanced temperature sensitive activity/rest rhythms. Our results highlight remarkably similar organisation principles between emergence and activity/rest rhythms.

Jing Wen, Tingbei Bo, Xueying Zhang, Zuoxin Wang, and Dehua Wang

Ambient temperature and food composition can affect energy metabolism of the host. Thermal transient receptor potential (thermo-TRPs) ion channels can detect temperature signals and are involved in the regulation of thermogenesis and energy homeostasis. Further, the gut microbiota has also been implicated in thermogenesis and obesity. In the present study, we tested the hypothesis that thermo-TRPs and gut microbiota are involved in reducing diet-induced obesity (DIO) during low temperature exposure. C57BL/6J mice in obese (body mass gain >45%), lean (body mass gain <15%), and control (body mass gain<1%) groups were exposed to high (23±1°C) or low (4±1°C) ambient temperature for 28 days. Our data showed that low temperature exposure attenuated DIO, but enhanced brown adipose tissue (BAT) thermogenesis. Low temperature exposure also resulted in increased norepinephrine (NE) concentrations in the hypothalamus, decreased TRP melastatin 8 (TRPM8) expression in the small intestine, and altered composition and diversity of gut microbiota. In DIO mice, there was a decrease in overall energy intake along with a reduction in TRP ankyrin 1 (TRPA1) expression and an increase in NE concentration in the small intestine. DIO mice also showed increases in Oscillospira, [Ruminococcus], Lactococcus, and Christensenella and decreases in Prevotella, Odoribacter, and Lactobacillus at the genus level in fecal samples. Together, our data suggest that thermos-TRPs and gut microbiota are involved in thermogenesis and energy metabolism during low temperature exposure in DIO mice.

Itsumi Nakamura, Rui Matsumoto, and Katsufumi Sato

It is generally assumed that the body temperature of large animals is less likely to change due to their large body size, resulting in a high thermal inertia and a smaller surface area to volume ratio. The goal of this study was to investigate the stability of body temperature in large fish using data from field experiments. We measured the muscle temperatures of free-ranging whale sharks (Rhincodon typus), the largest extant fish globally, and investigated their ectothermic physiology and the stability of their body temperatures. The measured muscle temperature of the whale sharks changed substantially more slowly than the water temperature fluctuations associated with vertical movements, and the whole-body heat-transfer coefficients (HTC) of whale sharks estimated using heat-budget models were lower than those of any other fish species measured to date. The heat-budget models also showed that internal heat production does not contribute to changes in muscle temperature. A comparative analysis showed that the HTC at cooling in various fish species including both ectothermic and endothermic species ranging from 10^–4 to 10³ kg was proportional to body mass^–0.63. This allometry was present regardless of whether the fish were ectothermic or endothermic, and was an extension of the relationship observed in previous studies on small fish. Thus, large fish have the advantage of body temperature stability while moving in environments with large temperature variations. Our results suggest that the large body size of whale sharks aids in preventing a decrease in body temperature during deep excursions to more than 1000 m depths without high metabolic costs of producing heat.

Nadia Vicenzi, Alejandro Laspiur, Paola L. Sassi, Ruben Massarelli, John Krenz, and Nora R. Ibargüengoytia

In ectotherms, temperature exerts a strong influence on the performance of physiological and ecological traits. One approach to understand the impact of rising temperatures on animals and their ability to cope with climate change is to quantify variation in thermal-sensitive traits. Here, we examined the thermal biology, the temperature dependence and the thermal plasticity of bite force (endurance and magnitude) in Diplolaemus leopardinus, an aggressive and territorial lizard, endemic to Mendoza province, Argentina. Our results indicated that this lizard behaves like a moderate thermoregulator which uses the rocks of its environment as the main heat source. Bite endurance was not influenced by head morphometry and body temperature, whereas bite force was influenced by head length and jaw length, and exhibited thermal dependence. Before thermal acclimation treatments, the maximum bite force for D. leopardinus occured at the lowest body temperature and fell sharply with increasing body temperature. After acclimation treatments, lizards acclimated at higher temperatures exhibited greater bite force. Bite force showed phenotypic plasticity, which reveals that leopard iguanas are able to maintain (and even improve) their bite force under a rising-temperature scenario.

Jorge de Costa, Gustavo Barja, and Pedro F. Almaida-Pagan

Lipid composition of cell membranes is linked to metabolic rate and lifespan in mammals and birds but very little information is available for fishes. In this study, three fish species of the short-lived annual genus Nothobranchius with different maximum lifespan potentials (MLSP) and the longer-lived outgroup species Aphyosemion australe were studied to test whether they conform to the predictions of the longevity-homeoviscous adaptation (LHA) theory of aging. Lipid analyses were performed in whole fish samples and peroxidation indexes (PIn) for every PL class and for the whole membrane, were calculated. Total PL content was significantly lower in A. australe and N. korthausae, the two species with the highest MLSP, and a negative correlation between membrane total PIn and fish MLSP was found, this meaning that the longer-lived fish species have more saturated membranes and therefore, a lower susceptibility to oxidative damage, as the LHA theory posits.

Flemming Dahlke, Magnus Lucassen, Ulf Bickmeyer, Sylke Wohlrab, Velmurugu Puvanendran, Atle Mortensen, Melissa Chierici, Hans-Otto Pörtner, and Daniela Storch

The vulnerability of fish embryos and larvae to environmental factors is often attributed to a lack of adult-like organ systems (gills) and thus insufficient homeostatic capacity. However, experimental data supporting this hypothesis are scarce. Here, by using Atlantic cod (Gadus morhua) as a model, the relationship between embryo vulnerability (to projected ocean acidification and warming) and homeostatic capacity was explored through parallel analyses of stage-specific mortality and in vitro activity and expression of major ion pumps (ATP-Synthase, Na⁺/K⁺-ATPase, H⁺-ATPase) and co-transporters (NBC1, NKCC1). Immunolocalization of these transporters was used to study ionocyte morphology in newly-hatched larvae. Treatment-related embryo mortality until hatch (+20% due to acidification and warming) occurred primarily during an early period (gastrulation) characterized by extremely low ion transport capacities. Thereafter, embryo mortality decreased in parallel with an exponential increase in activity and expression of all investigated ion transporters. Significant changes in transporter activity and expression in response to acidification (+15% activity) and warming (-30% expression) indicate some potential for short-term acclimatization, although likely associated with energetic trade-offs. Interestingly, whole-larvae enzyme capacities (supported by abundant epidermal ionocytes) reached levels similar to those previously measured in gill tissue of adult cod, suggesting that early-life stages without functional gills are better equipped in terms of ion homeostasis than previously thought. This study implies that the gastrulation period represents a critical transition from inherited (maternal) defenses to active homeostatic regulation, which facilitates enhanced resilience of later stages to environmental factors.

Brad A. Seibel and Curtis Deutsch

The capacity to extract oxygen from the environment and transport it to respiring tissues in support of metabolic demand reportedly has implications for species’ thermal tolerance, body-size, diversity and biogeography. Here we derive a quantifiable linkage between maximum and basal metabolic rate and their oxygen, temperature and size dependencies. We show that, regardless of size or temperature, the physiological capacity for oxygen supply precisely matches the maximum evolved demand at the highest persistently available oxygen pressure and this is the critical PO₂ for the maximum metabolic rate. For most terrestrial and shallow-living marine species, this "P_crit-max" is the current atmospheric pressure, 21 kPa. Any reduction in oxygen partial pressure from current values will result in a calculable decrement in maximum metabolic performance. However, oxygen supply capacity has evolved to match demand across temperatures and body sizes and so does not constrain thermal tolerance or cause the well-known reduction in mass-specific metabolic rate with increasing body mass. The critical oxygen pressure for resting metabolic rate, typically viewed as an indicator of hypoxia tolerance, is, instead, simply a rate-specific reflection of the oxygen supply capacity. A compensatory reduction in maintenance metabolic costs in warm-adapted species constrains factorial aerobic scope and the critical PO₂ to a similar range, between ~2 and 6, across each species’ natural temperature range. The simple new relationship described here redefines many important physiological concepts and alters their ecological interpretation.

Maria Stager, Nathan R. Senner, Bret W. Tobalske, and Zachary A. Cheviron

Flexibility in heat generation and dissipation mechanisms provides endotherms the ability to match their thermoregulatory strategy with external demands. However, the degree to which these two mechanisms account for seasonal changes in body temperature regulation is little explored. Here we present novel data on the regulation of avian body temperature to investigate how birds alter mechanisms of heat production and heat conservation to deal with variation in ambient conditions. We subjected Dark-eyed Juncos (Junco hyemalis) to chronic cold acclimations of varying duration and subsequently quantified their metabolic rates, thermal conductance, and ability to maintain normothermia. Cold-acclimated birds adjusted traits related to both heat generation (increased summit metabolic rate) and heat conservation (decreased conductance) to improve their body temperature regulation. Increases in summit metabolic rate occurred rapidly, but plateaued after one week of cold exposure. In contrast, changes to conductance occurred only after nine weeks of cold exposure. Thus, the ability to maintain body temperature continued to improve throughout the experiment, but the mechanisms underlying this improvement changed through time. Our results demonstrate the ability of birds to adjust thermoregulatory strategies in response to thermal cues and reveal that birds may combine multiple responses to meet the specific demands of their environments.

Background

Experimental and observational studies have raised concerns that giving intravenous (IV) iron to patients, such as individuals receiving maintenance hemodialysis, might increase the risk of infections. The Proactive IV Iron Therapy in Haemodialysis Patients (PIVOTAL) trial randomized 2141 patients undergoing maintenance hemodialysis for ESKD to a high-dose or a low-dose IV iron regimen, with a primary composite outcome of all-cause death, heart attack, stroke, or hospitalization for heart failure. Comparison of infection rates between the two groups was a prespecified secondary analysis.

Background

Fluid overload in patients undergoing hemodialysis contributes to cardiovascular morbidity and mortality. There is a global trend to lower dialysate sodium with the goal of reducing fluid overload.

Background

Studies have linked mutations in genes encoding the eight-protein exocyst protein complex to kidney disease, but the underlying mechanism is unclear. Because Drosophila nephrocytes share molecular and structural features with mammalian podocytes, they provide an efficient model for studying this issue.

Background

Mutations in CTNS—a gene encoding the cystine transporter cystinosin—cause the rare, autosomal, recessive, lysosomal-storage disease cystinosis. Research has also implicated cystinosin in modulating the mTORC1 pathway, which serves as a core regulator of cellular metabolism, proliferation, survival, and autophagy. In its severest form, cystinosis is characterized by cystine accumulation, renal proximal tubule dysfunction, and kidney failure. Because treatment with the cystine-depleting drug cysteamine only slows disease progression, there is an urgent need for better treatments.

Background

The utility of kidney organoids in regenerative medicine will rely on the functionality of the glomerular and tubular structures in these tissues. Recent studies have demonstrated the vascularization and subsequent maturation of human pluripotent stem cell–derived kidney organoids after renal subcapsular transplantation. This raises the question of whether the glomeruli also become functional upon transplantation.

Glycine riboswitches utilize both single- and tandem-aptamer architectures. In the tandem system, the relative contribution of each aptamer toward gene regulation is not well understood. To dissect these contributions, the effects of 684 single mutants of a tandem ON switch from Bacillus subtilis were characterized for the wild-type construct and binding site mutations that selectively restrict ligand binding to either the first or second aptamer. Despite the structural symmetry of tandem aptamers, the response to these mutations was frequently asymmetrical. Mutations in the first aptamer often significantly weakened the K_1/2, while several mutations in the second aptamer improved the amplitude. These results demonstrate that this ON switch favors ligand binding to the first aptamer. This is in contrast to the tandem OFF switch variant from Vibrio cholerae, which was previously shown to have preferential binding to its second aptamer. A bioinformatic analysis of tandem glycine riboswitches revealed that the two binding pockets are differentially conserved between ON and OFF switches. Altogether, this indicates that tandem ON switch variants preferentially utilize binding to the first aptamer to promote helical switching, while OFF switch variants favor binding to the second aptamer. The data set also revealed a cooperative glycine response when both binding pockets were maximally stabilized with three GC base pairs. This indicates a cooperative response may sometimes be obfuscated by a difference in the affinities of the two aptamers. This conditional cooperativity provides an additional layer of tunability to tandem glycine riboswitches that adds to their versatility as genetic switches.

Transposable elements (TEs) can damage genomes, thus organisms use a variety of mechanisms to repress TE expression. The PIWI–piRNA pathway is a small RNA pathway that represses TE expression in the germline of animals. Here we explore the function of the pathway in the somatic stem cells of Hydra, a long-lived freshwater cnidarian. Hydra have three stem cell populations, all of which express PIWI proteins; endodermal and ectodermal epithelial stem cells (ESCs) are somatic, whereas the interstitial stem cells have germline competence. To study somatic function of the pathway, we isolated piRNAs from Hydra that lack the interstitial lineage and found that these somatic piRNAs map predominantly to TE transcripts and display the conserved sequence signatures typical of germline piRNAs. Three lines of evidence suggest that the PIWI–piRNA pathway represses TEs in Hydra ESCs. First, epithelial knockdown of the Hydra piwi gene hywi resulted in up-regulation of TE expression. Second, degradome sequencing revealed evidence of PIWI-mediated cleavage of TE RNAs in epithelial cells using the ping-pong mechanism. Finally, we demonstrated a direct association between Hywi protein and TE transcripts in epithelial cells using RNA immunoprecipitation. Altogether, our data reveal that the PIWI–piRNA pathway represses TE expression in the somatic cell lineages of Hydra, which we propose contributes to the extreme longevity of the organism. Furthermore, our results, in combination with others, suggest that somatic TE repression is an ancestral function of the PIWI–piRNA pathway.

VOLUME 287 (2012) PAGES 44508–44517In Fig. 1A, the wrong image for the control group was presented. The authors inadvertently cropped the control images in Fig. 1, A and E, from the same raw image. Fig. 1A has now been corrected and does not affect the results or conclusions of the work. The authors sincerely apologize for their mistake during figure preparation and for any inconvenience this may have caused readers.jbc;295/19/6781/F1F1F1Figure 1A.

The atypical trichromatic cyanobacterial phytochrome NpTP1 from Nostoc punctiforme ATCC 29133 is a linear tetrapyrrole (bilin)-binding photoreceptor protein that possesses tandem-cysteine residues responsible for shifting its light-sensing maximum to the violet spectral region. Using bioinformatics and phylogenetic analyses, here we established that tandem-cysteine cyanobacterial phytochromes (TCCPs) compose a well-supported monophyletic phytochrome lineage distinct from prototypical red/far-red cyanobacterial phytochromes. To investigate the light-sensing diversity of this family, we compared the spectroscopic properties of NpTP1 (here renamed NpTCCP) with those of three phylogenetically diverged TCCPs identified in the draft genomes of Tolypothrix sp. PCC7910, Scytonema sp. PCC10023, and Gloeocapsa sp. PCC7513. Recombinant photosensory core modules of ToTCCP, ScTCCP, and GlTCCP exhibited violet-blue–absorbing dark-states consistent with dual thioether-linked phycocyanobilin (PCB) chromophores. Photoexcitation generated singly-linked photoproduct mixtures with variable ratios of yellow-orange and red-absorbing species. The photoproduct ratio was strongly influenced by pH and by mutagenesis of TCCP- and phytochrome-specific signature residues. Our experiments support the conclusion that both photoproduct species possess protonated 15E bilin chromophores, but differ in the ionization state of the noncanonical “second” cysteine sulfhydryl group. We found that the ionization state of this and other residues influences subsequent conformational change and downstream signal transmission. We also show that tandem-cysteine phytochromes present in eukaryotes possess similar amino acid substitutions within their chromophore-binding pocket, which tune their spectral properties in an analogous fashion. Taken together, our findings provide a roadmap for tailoring the wavelength specificity of plant phytochromes to optimize plant performance in diverse natural and artificial light environments.

Protease-activated receptor 2 (PAR-2) is activated by secreted proteases from immune cells or fungi. PAR-2 is normally expressed basolaterally in differentiated nasal ciliated cells. We hypothesized that epithelial remodeling during diseases characterized by cilial loss and squamous metaplasia may alter PAR-2 polarization. Here, using a fluorescent arrestin assay, we confirmed that the common fungal airway pathogen Aspergillus fumigatus activates heterologously-expressed PAR-2. Endogenous PAR-2 activation in submerged airway RPMI 2650 or NCI–H520 squamous cells increased intracellular calcium levels and granulocyte macrophage–colony-stimulating factor, tumor necrosis factor α, and interleukin (IL)-6 secretion. RPMI 2650 cells cultured at an air–liquid interface (ALI) responded to apically or basolaterally applied PAR-2 agonists. However, well-differentiated primary nasal epithelial ALIs responded only to basolateral PAR-2 stimulation, indicated by calcium elevation, increased cilia beat frequency, and increased fluid and cytokine secretion. We exposed primary cells to disease-related modifiers that alter epithelial morphology, including IL-13, cigarette smoke condensate, and retinoic acid deficiency, at concentrations and times that altered epithelial morphology without causing breakdown of the epithelial barrier to model early disease states. These altered primary cultures responded to both apical and basolateral PAR-2 stimulation. Imaging nasal polyps and control middle turbinate explants, we found that nasal polyps, but not turbinates, exhibit apical calcium responses to PAR-2 stimulation. However, isolated ciliated cells from both polyps and turbinates maintained basolateral PAR-2 polarization, suggesting that the calcium responses originated from nonciliated cells. Altered PAR-2 polarization in disease-remodeled epithelia may enhance apical responses and increase sensitivity to inhaled proteases.

Contact between inflammatory cells and endothelial cells (ECs) is a crucial step in vascular inflammation. Recently, we demonstrated that the cell-surface level of endomucin (EMCN), a heavily O-glycosylated single-transmembrane sialomucin, interferes with the interactions between inflammatory cells and ECs. We have also shown that, in response to an inflammatory stimulus, EMCN is cleared from the cell surface by an unknown mechanism. In this study, using adenovirus-mediated overexpression of a tagged EMCN in human umbilical vein ECs, we found that treatment with tumor necrosis factor α (TNF-α) or the strong oxidant pervanadate leads to loss of cell-surface EMCN and increases the levels of the C-terminal fragment of EMCN 3- to 4-fold. Furthermore, treatment with the broad-spectrum matrix metalloproteinase inhibitor batimastat (BB94) or inhibition of ADAM metallopeptidase domain 10 (ADAM10) and ADAM17 with two small-molecule inhibitors, GW280264X and GI254023X, or with siRNA significantly reduced basal and TNFα-induced cell-surface EMCN cleavage. Release of the C-terminal fragment of EMCN by TNF-α treatment was blocked by chemical inhibition of ADAM10 alone or in combination with ADAM17. These results indicate that cell-surface EMCN undergoes constitutive cleavage and that TNF-α treatment dramatically increases this cleavage, which is mediated predominantly by ADAM10 and ADAM17. As endothelial cell-surface EMCN attenuates leukocyte–EC interactions during inflammation, we propose that EMCN is a potential therapeutic target to manage vascular inflammation.

Much of our current knowledge of biological chemistry is founded in the structure-function relationship, whereby sequence determines structure that determines function. Thus, the discovery that a large fraction of the proteome is intrinsically disordered, while being functional, has revolutionized our understanding of proteins and raised new and interesting questions. Many intrinsically disordered proteins (IDPs) have been determined to undergo a disorder-to-order transition when recognizing their physiological partners, suggesting that their mechanisms of folding are intrinsically different from those observed in globular proteins. However, IDPs also follow some of the classic paradigms established for globular proteins, pointing to important similarities in their behavior. In this review, we compare and contrast the folding mechanisms of globular proteins with the emerging features of binding-induced folding of intrinsically disordered proteins. Specifically, whereas disorder-to-order transitions of intrinsically disordered proteins appear to follow rules of globular protein folding, such as the cooperative nature of the reaction, their folding pathways are remarkably more malleable, due to the heterogeneous nature of their folding nuclei, as probed by analysis of linear free-energy relationship plots. These insights have led to a new model for the disorder-to-order transition in IDPs termed “templated folding,” whereby the binding partner dictates distinct structural transitions en route to product, while ensuring a cooperative folding.

Leukocyte recruitment is a universal feature of tissue inflammation and regulated by the interactions of chemokines with their G protein–coupled receptors. Activation of CC chemokine receptor 2 (CCR2) by its cognate chemokine ligands, including CC chemokine ligand 2 (CCL2), plays a central role in recruitment of monocytes in several inflammatory diseases. In this study, we used phosphoproteomics to conduct an unbiased characterization of the signaling network resulting from CCL2 activation of CCR2. Using data-independent acquisition MS analysis, we quantified both the proteome and phosphoproteome in FlpIn-HEK293T cells stably expressing CCR2 at six time points after activation with CCL2. Differential expression analysis identified 699 significantly regulated phosphorylation sites on 441 proteins. As expected, many of these proteins are known to participate in canonical signal transduction pathways and in the regulation of actin cytoskeleton dynamics, including numerous guanine nucleotide exchange factors and GTPase-activating proteins. Moreover, we identified regulated phosphorylation sites in numerous proteins that function in the nucleus, including several constituents of the nuclear pore complex. The results of this study provide an unprecedented level of detail of CCR2 signaling and identify potential targets for regulation of CCR2 function.

Multidrug resistance (MDR) in cancer arises from cross-resistance to structurally- and functionally-divergent chemotherapeutic drugs. In particular, MDR is characterized by increased expression and activity of ATP-binding cassette (ABC) superfamily transporters. Sphingolipids are substrates of ABC proteins in cell signaling, membrane biosynthesis, and inflammation, for example, and their products can favor cancer progression. Glucosylceramide (GlcCer) is a ubiquitous glycosphingolipid (GSL) generated by glucosylceramide synthase, a key regulatory enzyme encoded by the UDP-glucose ceramide glucosyltransferase (UGCG) gene. Stressed cells increase de novo biosynthesis of ceramides, which return to sub-toxic levels after UGCG mediates incorporation into GlcCer. Given that cancer cells seem to mobilize UGCG and have increased GSL content for ceramide clearance, which ultimately contributes to chemotherapy failure, here we investigated how inhibition of GSL biosynthesis affects the MDR phenotype of chronic myeloid leukemias. We found that MDR is associated with higher UGCG expression and with a complex GSL profile. UGCG inhibition with the ceramide analog d-threo-1-(3,4,-ethylenedioxy)phenyl-2-palmitoylamino-3-pyrrolidino-1-propanol (EtDO-P4) greatly reduced GSL and monosialotetrahexosylganglioside levels, and co-treatment with standard chemotherapeutics sensitized cells to mitochondrial membrane potential loss and apoptosis. ABC subfamily B member 1 (ABCB1) expression was reduced, and ABCC-mediated efflux activity was modulated by competition with nonglycosylated ceramides. Consistently, inhibition of ABCC-mediated transport reduced the efflux of exogenous C6-ceramide. Overall, UGCG inhibition impaired the malignant glycophenotype of MDR leukemias, which typically overcomes drug resistance through distinct mechanisms. This work sheds light on the involvement of GSL in chemotherapy failure, and its findings suggest that targeted GSL modulation could help manage MDR leukemias.

Site-specific arrest of RNA polymerases (RNAPs) is fundamental to several technologies that assess RNA structure and function. Current in vitro transcription “roadblocking” approaches inhibit transcription elongation by blocking RNAP with a protein bound to the DNA template. One limitation of protein-mediated transcription roadblocking is that it requires inclusion of a protein factor extrinsic to the minimal in vitro transcription reaction. In this work, we developed a chemical approach for halting transcription by Escherichia coli RNAP. We first established a sequence-independent method for site-specific incorporation of chemical lesions into dsDNA templates by sequential PCR and translesion synthesis. We then show that interrupting the transcribed DNA strand with an internal desthiobiotin-triethylene glycol modification or 1,N6-etheno-2'-deoxyadenosine base efficiently and stably halts Escherichia coli RNAP transcription. By encoding an intrinsic stall site within the template DNA, our chemical transcription roadblocking approach enables display of nascent RNA molecules from RNAP in a minimal in vitro transcription reaction.

Triple-negative breast cancer (TNBC) is characterized by its aggressive biology, early metastatic spread, and poor survival outcomes. TNBC lacks expression of the targetable receptors found in other breast cancer subtypes, mandating use of cytotoxic chemotherapy. However, resistance to chemotherapy is a significant problem, encountered in about two-thirds of TNBC patients, and new strategies are needed to mitigate resistance. In this issue of the Journal of Biological Chemistry, Geck et al. report that TNBC cells are highly sensitive to inhibition of the de novo polyamine synthesis pathway and that inhibition of this pathway sensitizes cells to TNBC-relevant chemotherapy, uncovering new opportunities for addressing chemoresistance.

Treatment of patients with triple-negative breast cancer (TNBC) is limited by a lack of effective molecular therapies targeting this disease. Recent studies have identified metabolic alterations in cancer cells that can be targeted to improve responses to standard-of-care chemotherapy regimens. Using MDA-MB-468 and SUM-159PT TNBC cells, along with LC-MS/MS and HPLC metabolomics profiling, we found here that exposure of TNBC cells to the cytotoxic chemotherapy drugs cisplatin and doxorubicin alter arginine and polyamine metabolites. This alteration was because of a reduction in the levels and activity of a rate-limiting polyamine biosynthetic enzyme, ornithine decarboxylase (ODC). Using gene silencing and inhibitor treatments, we determined that the reduction in ODC was mediated by its negative regulator antizyme, targeting ODC to the proteasome for degradation. Treatment with the ODC inhibitor difluoromethylornithine (DFMO) sensitized TNBC cells to chemotherapy, but this was not observed in receptor-positive breast cancer cells. Moreover, TNBC cell lines had greater sensitivity to single-agent DFMO, and ODC levels were elevated in TNBC patient samples. The alterations in polyamine metabolism in response to chemotherapy, as well as DFMO-induced preferential sensitization of TNBC cells to chemotherapy, reported here suggest that ODC may be a targetable metabolic vulnerability in TNBC.

Premature infants have a higher incidence of indirect hyperbilirubinemia than term infants. Management of neonatal indirect hyperbilirubinemia in late preterm and term neonates has been well addressed by recognized, consensus-based guidelines. However, the extension of these guidelines to the preterm population has been an area of uncertainty because of limited evidence. This leads to variation in clinical practice and lack of recognition of the spectrum of bilirubin-induced neurologic dysfunction (BIND) in this population. Preterm infants are metabolically immature and at higher risk for BIND at lower bilirubin levels than their term counterparts. Early use of phototherapy to eliminate BIND and minimize the need for exchange transfusion is the goal of treatment in premature neonates. Although considered relatively safe, phototherapy does have side effects, and some NICUs tend to overuse phototherapy. In this review, we describe the epidemiology and pathophysiology of BIND in preterm neonates, and discuss our approach to standardized management of indirect hyperbilirubinemia in the vulnerable preterm population. The proposed treatment charts suggest early use of phototherapy in preterm neonates with the aim of reducing exposure to high irradiance levels, minimizing the need for exchange transfusions, and preventing BIND. The charts are pragmatic and have additional curves for stopping phototherapy and escalating its intensity. Having a standardized approach would support future research and quality improvement initiatives that examine dose and duration of phototherapy exposure with relation to outcomes.

We present a review of the Chang 7 Member oil shale, which occurs in the middle–late Triassic Yanchang Formation of the Ordos Basin in central north China. The oil shale has a thickness of 28 m (average), an area of around 30 000 km² and a Ladinian age. It is mainly brown-black to black in colour with a laminar structure. It is characterized by average values of 18 wt% TOC (total organic carbon), 8 wt% oil yield, a 8.35 MJ kg^–1 calorific value, 400 kg t^–1 hydrocarbon productivity and kerogen of type I–II₁, showing a medium quality. On average, it comprises 49% clay minerals, 29% quartz, 16% feldspar and some iron oxides, which is close to the average mineral composition of global shale. The total SiO₂ and Al₂O₃ comprise 63.69 wt% of the whole rock, indicating a medium ash type. The Sr/Ba is 0.33, the V/Ni is 7.8, the U/Th is 4.8 and the FeO/Fe₂O₃ is 0.5, indicating formation in a strongly reducing, freshwater or low-salinity sedimentary environment. Multilayered intermediate-acid tuff is developed in the basin, which may have promoted the formation of the oil shale. The Ordos Basin was formed during the northwards subduction of the Qinling oceanic plate during the Ladinian–Norian in a back-arc basin context. The oil shale of the Ordos Basin has a large potential for hydrocarbon generation.

Supplementary material: Tables of oil-shale geochemical composition, proximate and organic matter analyses from the Chang 7 Member oil shale, the Ordos Basin, Central north China are available at https://doi.org/10.6084/m9.figshare.c.4411703

The Carboniferous Bowland Shale Formation of the UK is a proven hydrocarbon source rock and currently a target for shale gas exploration. Most existing analysis details lithofacies and geochemical assessment of a small number of boreholes. Given a paucity of relevant borehole cores, surface samples provide a valuable contribution to the assessment of this unconventional gas source. This study reviews existing literature on the formation's hydrocarbon geochemistry and provides new lithological descriptions of seven lithofacies, XRD mineralogy and hydrocarbon-specific geochemical data for 32 outcrop localities within the Craven and Edale basins, respectively in the northern and southern parts of the resource area. Low oxygen indices suggest that the majority of samples are relatively unaltered (in terms of hydrocarbon geochemistry), and therefore suitable for the characterization of the shale organic character. Total organic carbon (TOC) ranges from 0.7 to 6.5 wt%, with highest values associated with maximum flooding surfaces. Mean T_max values of 447 and 441°C for the Edale and Craven basins, respectively, suggest that nearly all the samples were too immature to have generated appreciable amounts of dry gas. The oil saturation index is consistently below the >100 mg g^–1 TOC benchmark, suggesting that they are not prospective for shale oil.

Supplementary material: A table summarizing the location, geological description and age of all of the samples in this paper is available at https://doi.org/10.6084/m9.figshare.c.4444589

The Northern Paraguai Belt, at the SE border of the Amazonian Craton, central Brazil, has been interpreted as a Brasiliano–Pan-African (c. 650–600 Ma) belt with a foreland basin, recording collisional polyphase tectonism and greenschist-facies metamorphism extending from the late Precambrian to the Cambrian–Ordovician. New structural investigations indicate that the older metasedimentary rocks of the Cuiabá Group represent a Tonian–Cryogenian basement assemblage deformed in two contemporaneous fault-bounded structural sub-domains of wrench-dominated (rake <10°) and contraction-dominated (rake ~30–40°) sinistral transpression, with tectonic vergence towards the SE. The younger late Cryogenian to early Cambrian sedimentary rocks lying to the NW of the Cuiabá Group are non-metamorphic and display only pervasive brittle transtension characterized by normal-oblique faults, fractures and forced drag folds with no consistent vergence pattern. Our analyses suggest that an unconformity separates the metasedimentary Cuiabá Group basement of the Northern Paraguai Belt from the unmetamorphosed sedimentary cover. It is proposed that the latter units were deposited during a post-glacial marine transgression (after c. 635 Ma) in a post-collisional basin. The Paraguai Belt basement and its post-collisional sedimentary cover share a number of significant geological similarities with sequences in the Bassarides Belt and Taoudéni Basin in the SW portion of the West African Craton.

The Tabaquito batholith (Frontal Cordillera, western Argentina), is mainly composed of shallowly emplaced granodiorite to minor monzogranite with abundant mafic microgranular enclaves. New sensitive high-resolution ion microprobe U–Pb zircon ages of c. 337 Ma (biotite granodiorite) and c. 284 Ma (mafic dyke) along with previously published geochronological data suggest that a long-lived magmatic system formed through at least two magmatic pulses at c. 337 and c. 322 Ma with later superimposition of Permian magmatism. The Tabaquito granitoids are metaluminous, calc-alkalic and magnesian with I-type affinity. Elevated Th/Nb, Y/Nb and La/Nb ratios along with negative Nb–Ta and positive Pb anomalies are consistent with a continental arc setting. Hf, Nd and Sr isotopic composition of the Tabaquito granitoids suggests that their source could result from mixing of an old felsic crustal component and a juvenile mafic to intermediate component. New geochronological and geochemical data together with published data reveal a continuous arc setting from the Carboniferous to the Permian in Argentina, and important magmatic compositional variations through time and space controlled by episodic fluctuations in the subduction angle of the oceanic plate. Reported and compiled data allow us to infer the continuity of the Carboniferous magmatic arc along the west margin of Gondwana.

Supplementary material: Detailed petrography, analytical methods and data, zircon cathodoluminescence images and supplementary figures are available at https://doi.org/10.6084/m9.figshare.c.4763993

Alemtuzumab is approved for the treatment of relapsing-remitting MS and is used off-label for patients with chronic lymphocytic leukemia and as induction and antirejection therapy in kidney transplant recipients.¹ Guillain-Barré syndrome (GBS) or chronic inflammatory demyelinating polyradiculoneuropathy (CIDP) complicating alemtuzumab treatment was reported in 9 patients with hematologic malignancy or MS.^1–3 The risk of GBS or CIDP in solid organ transplant recipients treated with alemtuzumab is unknown.

Most British railway embankments were constructed between 120 and 180 years ago without the benefit of modern design and construction methods. This can result in undesirable load-deformation characteristics and consequent disruption to present-day railway operations, for which there is unprecedented demand. Annual rail passenger kilometres have approximately doubled in the last 20 years and freight has increased by 60% over the same period. Whereas elements such as rails or bridges can be refurbished or replaced to meet increasing demand, the same is not usually feasible for embankments. Development of techniques to assess embankment performance risks posed by operational capacity enhancements is therefore of increasing significance to railway geotechnical asset management. The two case studies presented in this paper demonstrate how geospatial analysis and data management techniques may be applied to this challenge at both strategic (regional or national) and tactical (site-specific) scales for embankments incorporating plastic clay fill. The case studies also demonstrate, in a world of ever more abundant data, the growing need for engineering geologists and geotechnical engineers to augment their traditional knowledge with comprehensive data management and geospatial analysis skills, these being essential for modern infrastructure asset management.

Thematic collection: This article is part of the ‘Ground-related risk to transportation infrastructure’ collection available at: https://www.lyellcollection.org/cc/Ground-related-risk-to-transportation-infrastructure

Strategic geotechnical asset management considers the whole of an organization's earthworks portfolio and is concerned with setting an overall earthworks asset management policy with long-term objectives related to asset performance, safety and condition, and identifying how those objectives can best be met, now and into the future. A risk-based approach is adopted that requires an understanding of the likelihood that any of the earthworks may fail, combined with a knowledge of the consequences should they fail. Procedures are required to identify those earthworks that are most vulnerable to failure under the influence of triggering events, such as extreme weather. The risks are managed through a mix of interventions to reduce the likelihood of failure and mitigations to reduce the impact of failure. Many of the challenges of implementing a strategic earthworks policy have, or are, being met by the main UK transportation infrastructure organizations.

Thematic collection: This article is part of the Ground-related risk to transportation infrastructure collection available at https://www.lyellcollection.org/cc/Ground-related-risk-to-transportation-infrastructure

Groundwater recharge and runoff conditions are ascertained in the Suixiao coal-mining district using the hydrogen and oxygen isotopes and the trace elements in the unconsolidated pore aquifer of the Cenozoic group, the fissured sandstone aquifer of the Permian system, and the karst fissured limestone aquifer of the Carboniferous Taiyuan Formation and the Ordovician system, which are the main recharge aquifers during coal mining. The main water–rock interactions are pyrite oxidation, cation exchange and adsorption, and carbonate acidification, which are educed by principal component analysis of conventional ions. These results combined with geological conditions prove that hydraulic connection exists generally between the main recharge aquifers, and the groundwater circulation is controlled by faults in the sandstone and limestone aquifers. The water–rock interaction is very weak in the east of the district, which is proved to be a recharge area by Fisher discriminant analysis. This study provides the theoretical basis for the hydrochemistry exploration and the establishment of a water-inrush warning system in a concealed coalfield.

The need to characterize and distinguish geographically adjacent aggregate quarry sources prompted the SEM-EDS analysis of pyrite (FeS₂) within fill material taken from eight different quarry sources. This experiment was undertaken to investigate the possibility of geochemically separating these quarry sources based on the major element concentration of their pyrite. The results show that median values for Fe and S vary by up to 7.6 and 8.55 wt% respectively. By implementing statistical methods, including k-means clustering and principal component analysis, it is possible to geochemically discriminate three of the eight sources.

In this paper, a decision tree is presented, constructed on the basis of hydrogeological characteristics (water table depth, freshwater thickness, surface area required and distance between wells), to choose the optimal groundwater extraction method in the case of a coastal unconfined aquifer. A comparison is made of the groundwater extraction methods in a freshwater aquifer of limited thickness occurring in coastal dunes in the eastern region of the Province of Buenos Aires (Argentina). The negative effects brought about by the wrong use of the groundwater extraction methods are analysed, because, as a result of excessive extraction, such methods lead to the dramatic decrease of the freshwater reserves. The decision tree is a useful tool to assist decision-makers as it suggests the most suitable groundwater extraction method options (vertical wells or wellpoints), as well as identifying areas that are unsuitable for sustainable groundwater extraction.

In 2015, the M_w 7.8 Gorkha earthquake struck Nepal, triggering thousands of landslides across the central and eastern Himalayas. These landslides had many adverse effects, including causing widespread damage to low-grade transport routes (e.g. tracks, footpaths) in rural regions that depend on tourism for survival. Langtang Valley is a glacial–periglacial landscape located 60 km north of Kathmandu. It is one of the most popular trekking regions in Nepal and has been severely affected by Gorkha earthquake-triggered and monsoon-triggered landsliding. Here, qualitative and quantitative observations from fieldwork and remote sensing are used to describe the materials and geomorphology of the landslides across Langtang Valley, and to quantify the extent to which coseismic and monsoon-triggered landslides have affected Langtang's trekking infrastructure. The dominant bedrock materials involved within Langtang landslides are found to be a range of gneisses and intruded leucogranites. In total, 64 landslides are found to have intersected trekking paths across Langtang, with coseismic and monsoon-triggered landslides having an impact on c. 3 km and 0.8 km of path respectively. It is observed that the practice of reconstructing paths through unstable landslide deposits is leaving the trekking infrastructure across Langtang increasingly vulnerable to future failure.

Routine identification of fungal pathogens from positive blood cultures by culture-based methods can be time-consuming, delaying treatment with appropriate antifungal agents. The GenMark Dx ePlex investigational use only blood culture identification fungal pathogen panel (BCID-FP) rapidly detects 15 fungal targets simultaneously in blood culture samples positive for fungi by Gram staining. We aimed to determine the performance of the BCID-FP in a multicenter clinical study. Blood culture samples collected at 10 United States sites and tested with BCID-FP at 4 sites were compared to the standard-of-care microbiological and biochemical techniques, fluorescence in situ hybridization using peptide nucleic acid probes (PNA-FISH) and matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS). Discrepant results were analyzed by bi-directional PCR/sequencing of residual blood culture samples. A total of 866 clinical samples, 120 retrospectively and 21 prospectively collected, along with 725 contrived samples were evaluated. Sensitivity and specificity of detection of Candida species (C. albicans, C. auris, C. dubliniensis, C. famata, C. glabrata, C. guilliermondii, C. kefyr, C. krusei, C. lusitaniae, C. parapsilosis, and C. tropicalis) ranged from 97.1 to 100% and 99.8 to 100%, respectively. For the other organism targets, sensitivity and specificity were as follows: 100% each for Cryptococcus neoformans and C. gattii, 98.6% and 100% for Fusarium spp., and 96.2% and 99.9% for Rhodotorula spp., respectively. In 4 of the 141 clinical samples, the BCID-FP panel correctly identified an additional Candida species, undetected by standard-of-care methods. The BCID-FP panel offers a faster turnaround time for identification of fungal pathogens in positive blood cultures that may allow for earlier antifungal interventions and includes C. auris, a highly multidrug-resistant fungus.

Q fever, caused by Coxiella burnetii, is a worldwide zoonotic disease that may cause severe forms in humans and requires a specific and prolonged antibiotic treatment. Although current serological and molecular detection tools allow a reliable diagnosis of the disease, culture of C. burnetii strains is mandatory to assess their susceptibility to antibiotics and sequence their genome in order to optimize patient management and epidemiological studies. However, cultivating this fastidious microorganism is difficult and restricted to reference centers, as it requires biosafety level 3 laboratories and relies on cell culture performed by experienced technicians. In addition, the culture yield is low, which results in a small number of isolates being available. In this work, we developed a novel high-content screening (HCS) isolation strategy based on optimized high-throughput cell culture and automated microscopic detection of infected cells with specifically designed algorithms targeting cytopathic effects. This method was more efficient than the shell vial assay, at the level of time dependency, when applied to both frozen specimens (7 isolates recovered by HCS only, sensitivity 91% versus 78% for shell vial) and fresh samples (1 additional isolate using HCS, sensitivity 7% versus 5% for shell vial), for which most strains were recovered more rapidly with the new technique. In addition, detecting positive cultures by an automated microscope reduced the need for expertise and saved 24% of technician working time. Application of HCS to antibiotic susceptibility testing of 12 strains demonstrated that it was as efficient as the standard procedure that combines shell vial culture and quantitative PCR.

Limited treatment options contribute to high morbidity/mortality rates with carbapenem-resistant, Gram-negative bacterial infections. New approaches for carbapenemase-producing organism (CPO) detection may help inform clinician decision-making on patient treatment and infection control. BD Phoenix CPO detect (CPO detect) detects and classifies carbapenemases in Enterobacterales, Acinetobacter baumannii, and Pseudomonas aeruginosa during susceptibility testing. The clinical performance of CPO detect is reported here. Enterobacterales, Acinetobacter baumannii, and Pseudomonas aeruginosa isolates were evaluated across three sites using CPO detect and a composite reference method (RM); the latter was comprised of the modified carbapenem inactivation method and a MIC screen for ertapenem, imipenem, and meropenem. Multiplex PCR testing was also utilized for Ambler class determination. Positive and negative percentages of agreement (PPA and NPA, respectively) between CPO detect and the RM were determined. The PPA and NPA for Enterobacterales were 98.5% (confidence intervals, 96.6%, 99.4%) and 97.2% (95.8%, 98.2%), respectively. The A. baumannii PPA and NPA, respectively, were 97.1% (90.2%, 99.2%) and 97.1% (89.9%, 99.2%). The P. aeruginosa PPA and NPA, respectively, were 95.9% (88.6%, 98.6%) and 92.3% (86.7%, 95.6%). The PPA values for carbapenemase class designations for all organisms combined and Enterobacterales alone, respectively, were 95.3% (90.2%, 97.8%) and 94.6% (88.8%, 97.5%) for class A, 94.0% (88.7%, 96.6%) and 96.4% (90.0%, 98.8%) for class B, and 95.0% (90.1%, 97.6%) and 99.0% (94.4%, 99.8%) for class D carbapenemases. NPA values for all organisms and Enterobacterales alone ranged from 98.5% to 100%. CPO detect provided accurate detection and classification of CPOs for the majority of isolates of Enterobacterales, Acinetobacter baumannii, and Pseudomonas aeruginosa tested.

The number of onsite clinical microbiology laboratories in hospitals is decreasing, likely related to the business model for laboratory consolidation and labor shortages, and this impacts a variety of clinical practices, including that of banking isolates for clinical or epidemiologic purposes. To determine the impact of these trends, infectious disease (ID) physicians were surveyed regarding their perceptions of offsite services. Clinical microbiology practices for retention of clinical isolates for future use were also determined. Surveys were sent to members of the Infectious Diseases Society of America’s (IDSA) Emerging Infections Network (EIN). The EIN is a sentinel network of ID physicians who care for adult and/or pediatric patients in North America and who are members of IDSA. The response rate was 763 (45%) of 1,680 potential respondents. Five hundred forty (81%) respondents reported interacting with the clinical microbiology laboratory. Eighty-six percent of respondents thought an onsite laboratory very important for timely diagnostic reporting and ongoing communication with the clinical microbiologist. Thirty-five percent practiced in institutions where the core microbiology laboratory has been moved offsite, and an additional 7% (n = 38) reported that movement of core laboratory functions offsite was being considered. The respondents reported that only 24% of laboratories banked all isolates, with the majority saving isolates for less than 30 days. Based on these results, the trend toward centralized core laboratories negatively impacts the practice of ID physicians, potentially delays effective implementation of prompt and targeted care for patients with serious infections, and similarly adversely impacts infection control epidemiologic investigations.

There are roughly 48,000 deaths caused by influenza annually and an estimated 200,000 people who have undiagnosed human immunodeficiency virus (HIV). These are examples of acute and chronic illnesses that can be identified by employing a CLIA-waived test. Pharmacies across the country have been incorporating CLIA-waived point-of-care tests (POCT) into disease screening and management programs offered in the pharmacy. The rationale behind these programs is discussed. Additionally, a summary of clinical data for some of these programs in the infectious disease arena is provided. Finally, we discuss the future potential for CLIA-waived POCT-based programs in community pharmacies.

The new decade of the 21^st century (2020) started with the emergence of a novel coronavirus known as SARS-CoV-2 that caused an epidemic of coronavirus disease (COVID-19) in Wuhan, China. It is the third highly pathogenic and transmissible coronavirus after severe acute respiratory syndrome coronavirus (SARS-CoV) and Middle East respiratory syndrome coronavirus (MERS-CoV) emerged in humans. The source of origin, transmission to humans, and mechanisms associated with the pathogenicity of SARS-CoV-2 are not yet clear, however, its resemblance to SARS-CoV and several other bat coronaviruses was recently confirmed through genome sequencing-related studies. The development of therapeutic strategies is necessary in order to prevent further epidemics and cure infections. In this review, we summarize current information about the emergence, origin, diversity, and epidemiology of three pathogenic coronaviruses with a specific focus on the current outbreak in Wuhan, China. Furthermore, we discuss the clinical features and potential therapeutic options that may be effective against SARS-CoV-2.

The IR Biotyper is a new automated typing system based on Fourier-transform infrared (FT-IR) spectroscopy that gives results within 4 h. We aimed (i) to use the IR Biotyper to retrospectively analyze an outbreak of extended-spectrum beta-lactamase-producing Klebsiella pneumoniae (ESBL-KP) in a neonatal intensive care unit and to compare results to BOX-PCR and whole-genome sequencing (WGS) results as the gold standard and (ii) to assess how the cutoff values used to define clusters affect the discriminatory power of the IR Biotyper. The sample consisted of 18 isolates from 14 patients. Specimens were analyzed in the IR Biotyper using the default analysis settings, and spectra were analyzed using OPUS 7.5 software. The software contains a feature that automatically proposes a cutoff value to define clusters; the cutoff value defines up to which distance the spectra are considered to be in the same cluster. Based on FT-IR, the outbreak represented 1 dominant clone, 1 secondary clone, and several unrelated clones. FT-IR results, using the cutoff value generated by the accompanying software after 4 replicates, were concordant with WGS for all but 1 isolate. BOX-PCR was underdiscriminatory compared to the other two methods. Using the cutoff value generated after 12 replicates, the results of FT-IR and WGS were completely concordant. The IR Biotyper can achieve the same typeability and discriminatory power as genome-based methods. However, to attain this high performance requires either previous, strain-dependent knowledge about the optimal technical parameters to be used or validation by a second method.

The regulation of transposable element (TE) activity by small RNAs is a ubiquitous feature of germlines. However, despite the obvious benefits to the host in terms of ensuring the production of viable gametes and maintaining the integrity of the genomes they carry, it remains controversial whether TE regulation evolves adaptively. We examined the emergence and evolutionary dynamics of repressor alleles after P-elements invaded the Drosophila melanogaster genome in the mid-twentieth century. In many animals including Drosophila, repressor alleles are produced by transpositional insertions into piRNA clusters, genomic regions encoding the Piwi-interacting RNAs (piRNAs) that regulate TEs. We discovered that ~94% of recently collected isofemale lines in the Drosophila melanogaster Genetic Reference Panel (DGRP) contain at least one P-element insertion in a piRNA cluster, indicating that repressor alleles are produced by de novo insertion at an exceptional rate. Furthermore, in our sample of approximately 200 genomes, we uncovered no fewer than 80 unique P-element insertion alleles in at least 15 different piRNA clusters. Finally, we observe no footprint of positive selection on P-element insertions in piRNA clusters, suggesting that the rapid evolution of piRNA-mediated repression in D. melanogaster was driven primarily by mutation. Our results reveal for the first time how the unique genetic architecture of piRNA production, in which numerous piRNA clusters can encode regulatory small RNAs upon transpositional insertion, facilitates the nonadaptive rapid evolution of repression.

Hepatocellular carcinomas (HCC) are adapted to survive extreme genomic stress conditions imposed by hyperactive DNA replication and genotoxic drug treatment. The underlying mechanisms remain unclear, but may involve intensified DNA damage response/repair programs. Here, we investigate a new role of nucleostemin (NS) in allowing HCC to survive its own malignancy, as NS was previously shown to promote liver regeneration via a damage repair mechanism. We first established that a higher NS transcript level correlates with high-HCC grades and poor prognostic signatures, and is an independent predictor of shorter overall and progression-free survival specifically for HCC and kidney cancer but not for others. Immunostaining confirmed that NS is most abundantly expressed in high-grade and metastatic HCCs. Genome-wide analyses revealed that NS is coenriched with MYC target and homologous recombination (HR) repair genes in human HCC samples and functionally intersects with those involved in replication stress response and HR repair in yeasts. In support, NS-high HCCs are more reliant on the replicative/oxidative stress response pathways, whereas NS-low HCCs depend more on the mTOR pathway. Perturbation studies showed NS function in protecting human HCC cells from replication- and drug-induced DNA damage. Notably, NS depletion in HCC cells increases the amounts of physical DNA damage and cytosolic double-stranded DNA, leading to a reactive increase of cytokines and PD-L1. This study shows that NS provides an essential mechanism for HCC to adapt to high genomic stress for oncogenic maintenance and propagation. NS deficiency sensitizes HCC cells to chemotherapy but also triggers tumor immune responses.

The histone demethylase JMJD1A plays a key functional role in spermatogenesis, sex determination, stem cell renewal, and cancer via removing mono- and di-methyl groups from H3K9 to epigenetically control gene expression. However, its role in prostate cancer progression remains unclear. Here, we found JMJD1A was significantly elevated in prostate cancer tissue compared with matched normal tissue. Ectopic JMJD1A expression in prostate cancer cells promoted proliferation, migration, and invasion in vitro, and tumorigenesis in vivo; JMJD1A knockdown exhibited the opposite effects. Mechanically, we revealed that JMJD1A directly interacted with the Snail gene promoter and regulated its transcriptional activity, promoting prostate cancer progression both in vitro and in vivo. Furthermore, we found that JMJD1A transcriptionally activated Snail expression via H3K9me1 and H3K9me2 demethylation at its special promoter region. In summary, our studies reveal JMJD1A plays an important role in regulating proliferation and progression of prostate cancer cells though Snail, and thus highlight JMJD1A as potential therapeutic target for advanced prostate cancer.