The nuclear factor kappa B (NF-B) is a potent transcription factor, activation of which typically results in robust proinflammatory signaling and triggering of fast negative feedback modulators to avoid excessive inflammatory responses. Here, we report that infection of epithelial cells, including primary porcine respiratory epithelial cells, with the porcine alphaherpesvirus pseudorabies virus (PRV) results in the gradual and persistent activation of NF-B, illustrated by proteasome-dependent degradation of the inhibitory NF-B regulator IB and nuclear translocation and phosphorylation of the NF-B subunit p65. PRV-induced persistent activation of NF-B does not result in expression of negative feedback loop genes, like the gene for IBα or A20, and does not trigger expression of prototypical proinflammatory genes, like the gene for tumor necrosis factor alpha (TNF-α) or interleukin-6 (IL-6). In addition, PRV infection inhibits TNF-α-induced canonical NF-B activation. Hence, PRV infection triggers persistent NF-B activation in an unorthodox way and dramatically modulates the NF-B signaling axis, preventing typical proinflammatory gene expression and the responsiveness of cells to canonical NF-B signaling, which may aid the virus in modulating early proinflammatory responses in the infected host.

IMPORTANCE The NF-B transcription factor is activated via different key inflammatory pathways and typically results in the fast expression of several proinflammatory genes as well as negative feedback loop genes to prevent excessive inflammation. In the current report, we describe that infection of cells with the porcine alphaherpesvirus pseudorabies virus (PRV) triggers a gradual and persistent aberrant activation of NF-B, which does not result in expression of hallmark proinflammatory or negative feedback loop genes. In addition, although PRV-induced NF-B activation shares some mechanistic features with canonical NF-B activation, it also shows remarkable differences; e.g., it is largely independent of the canonical IB kinase (IKK) and even renders infected cells resistant to canonical NF-B activation by the inflammatory cytokine TNF-α. Aberrant PRV-induced NF-B activation may therefore paradoxically serve as a viral immune evasion strategy and may represent an important tool to unravel currently unknown mechanisms and consequences of NF-B activation.

IMMUNOLOGY AND INFLAMMATION Correction for “Treg-mediated prolonged survival of skin allografts without immunosuppression,” by Nina Pilat, Mario Wiletel, Anna M. Weijler, Romy Steiner, Benedikt Mahr, Joanna Warren, Theresa M. Corpuz, Thomas Wekerle, Kylie E. Webster, and Jonathan Sprent, which was first published June 13, 2019; 10.1073/pnas.1903165116 (Proc. Natl. Acad. Sci....

Microtubule-associated serine/threonine kinase like (MASTL), also known as Greatwall (Gwl) kinase, has an important role in the regulation of mitosis. By inhibiting protein phosphatase 2A (PP2A), it plays a crucial role in activating one of the most important mitotic kinases, known as cyclin-dependent kinase 1 (CDK1). MASTL has been seen to be upregulated in various types of cancers and is also involved in tumor recurrence. It is activated by CDK1 through phosphorylations in the activation/T-loop, but the complete mechanism of its activation is still unclear. Here, we report that AKT phosphorylates MASTL at residue T299, which plays a critical role in its activation. Our results suggest that AKT increases CDK1-mediated phosphorylation and hence the activity of MASTL, which, in turn, promotes mitotic progression through PP2A inhibition. We also show that the oncogenic potential of AKT is augmented by MASTL activation, since AKT-mediated proliferation in colorectal cell lines can be attenuated by inhibiting and/or silencing MASTL. In brief, we report that AKT plays an important role in the progression of mitosis in mammalian cells and that it does so through the phosphorylation and activation of MASTL.

Growing evidence indicates that oxidative and endoplasmic reticular stress, which trigger changes in ion channels and inflammatory pathways that may undermine cellular homeostasis and survival, are critical determinants of injury in the diabetic kidney. Cells are normally able to mitigate these cellular stresses by maintaining high levels of autophagy, an intracellular lysosome-dependent degradative pathway that clears the cytoplasm of dysfunctional organelles. However, the capacity for autophagy in both podocytes and renal tubular cells is markedly impaired in type 2 diabetes, and this deficiency contributes importantly to the intensity of renal injury. The primary drivers of autophagy in states of nutrient and oxygen deprivation—sirtuin-1 (SIRT1), AMP-activated protein kinase (AMPK), and hypoxia-inducible factors (HIF-1α and HIF-2α)—can exert renoprotective effects by promoting autophagic flux and by exerting direct effects on sodium transport and inflammasome activation. Type 2 diabetes is characterized by marked suppression of SIRT1 and AMPK, leading to a diminution in autophagic flux in glomerular podocytes and renal tubules and markedly increasing their susceptibility to renal injury. Importantly, because insulin acts to depress autophagic flux, these derangements in nutrient deprivation signaling are not ameliorated by antihyperglycemic drugs that enhance insulin secretion or signaling. Metformin is an established AMPK agonist that can promote autophagy, but its effects on the course of CKD have been demonstrated only in the experimental setting. In contrast, the effects of sodium-glucose cotransporter–2 (SGLT2) inhibitors may be related primarily to enhanced SIRT1 and HIF-2α signaling; this can explain the effects of SGLT2 inhibitors to promote ketonemia and erythrocytosis and potentially underlies their actions to increase autophagy and mute inflammation in the diabetic kidney. These distinctions may contribute importantly to the consistent benefit of SGLT2 inhibitors to slow the deterioration in glomerular function and reduce the risk of ESKD in large-scale randomized clinical trials of patients with type 2 diabetes.

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.

Optic atrophy 1 (OPA1) is a dynamin protein that mediates mitochondrial fusion at the inner membrane. OPA1 is also necessary for maintaining the cristae and thus essential for supporting cellular energetics. OPA1 exists as membrane-anchored long form (L-OPA1) and short form (S-OPA1) that lacks the transmembrane region and is generated by cleavage of L-OPA1. Mitochondrial dysfunction and cellular stresses activate the inner membrane–associated zinc metallopeptidase OMA1 that cleaves L-OPA1, causing S-OPA1 accumulation. The prevailing notion has been that L-OPA1 is the functional form, whereas S-OPA1 is an inactive cleavage product in mammals, and that stress-induced OPA1 cleavage causes mitochondrial fragmentation and sensitizes cells to death. However, S-OPA1 contains all functional domains of dynamin proteins, suggesting that it has a physiological role. Indeed, we recently demonstrated that S-OPA1 can maintain cristae and energetics through its GTPase activity, despite lacking fusion activity. Here, applying oxidant insult that induces OPA1 cleavage, we show that cells unable to generate S-OPA1 are more sensitive to this stress under obligatory respiratory conditions, leading to necrotic death. These findings indicate that L-OPA1 and S-OPA1 differ in maintaining mitochondrial function. Mechanistically, we found that cells that exclusively express L-OPA1 generate more superoxide and are more sensitive to Ca2+-induced mitochondrial permeability transition, suggesting that S-OPA1, and not L-OPA1, protects against cellular stress. Importantly, silencing of OMA1 expression increased oxidant-induced cell death, indicating that stress-induced OPA1 cleavage supports cell survival. Our findings suggest that S-OPA1 generation by OPA1 cleavage is a survival mechanism in stressed cells.

Glial cell line–derived neurotrophic factor (GDNF) is a growth factor that regulates the health and function of neurons and other cells. GDNF binds to GDNF family receptor α1 (GFRa1), and the resulting complex activates the RET receptor tyrosine kinase and subsequent downstream signals. This feature restricts GDNF activity to systems in which GFRa1 and RET are both present, a scenario that may constrain GDNF breadth of action. Furthermore, this co-dependence precludes the use of GDNF as a tool to study a putative functional cross-talk between GFRa1 and RET. Here, using biochemical techniques, terminal deoxynucleotidyl transferase dUTP nick end labeling staining, and immunohistochemistry in murine cells, tissues, or retinal organotypic cultures, we report that a naphthoquinone/quinolinedione family of small molecules (Q compounds) acts as RET agonists. We found that, like GDNF, signaling through the parental compound Q121 is GFRa1-dependent. Structural modifications of Q121 generated analogs that activated RET irrespective of GFRa1 expression. We used these analogs to examine RET–GFRa1 interactions and show that GFRa1 can influence RET-mediated signaling and enhance or diminish AKT Ser/Thr kinase or extracellular signal-regulated kinase signaling in a biased manner. In a genetic mutant model of retinitis pigmentosa, a lead compound, Q525, afforded sustained RET activation and prevented photoreceptor neuron loss in the retina. This work uncovers key components of the dynamic relationships between RET and its GFRa co-receptor and provides RET agonist scaffolds for drug development.

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.

Arrestin-1 is the arrestin family member responsible for inactivation of the G protein–coupled receptor rhodopsin in photoreceptors. Arrestin-1 is also well-known to interact with additional protein partners and to affect other signaling cascades beyond phototransduction. In this study, we investigated one of these alternative arrestin-1 binding partners, the glycolysis enzyme enolase-1, to map the molecular contact sites between these two proteins and investigate how the binding of arrestin-1 affects the catalytic activity of enolase-1. Using fluorescence quench protection of strategically placed fluorophores on the arrestin-1 surface, we observed that arrestin-1 primarily engages enolase-1 along a surface that is opposite of the side of arrestin-1 that binds photoactivated rhodopsin. Using this information, we developed a molecular model of the arrestin-1–enolase-1 complex, which was validated by targeted substitutions of charge-pair interactions. Finally, we identified the likely source of arrestin's modulation of enolase-1 catalysis, showing that selective substitution of two amino acids in arrestin-1 can completely remove its effect on enolase-1 activity while still remaining bound to enolase-1. These findings open up opportunities for examining the functional effects of arrestin-1 on enolase-1 activity in photoreceptors and their surrounding cells.

Reactive immunization with a single-dose cholera vaccine that could rapidly (within days) protect immunologically naive individuals during virgin soil epidemics, when cholera reaches immunologically naive populations that have not experienced cholera for decades, would facilitate cholera control. One dose of attenuated Vibrio cholerae O1 classical Inaba vaccine CVD 103-HgR (Vaxchora) containing ≥2 x 10⁸ CFU induces vibriocidal antibody seroconversion (a correlate of protection) in >90% of U.S. adults. A previous CVD 103-HgR commercial formulation required ≥2 x 10⁹ CFU to elicit high levels of seroconversion in populations in developing countries. We compared the vibriocidal responses of Malians (individuals 18 to 45 years old) randomized to ingest a single ≥2 x 10⁸-CFU standard dose (n = 50) or a ≥2 x 10⁹-CFU high dose (n = 50) of PaxVax CVD 103-HgR with buffer or two doses (n = 50) of Shanchol inactivated cholera vaccine (the immunologic comparator). To maintain blinding, participants were dosed twice 2 weeks apart; CVD 103-HgR recipients ingested placebo 2 weeks before or after ingesting vaccine. Seroconversion (a ≥4-fold vibriocidal titer rise) between the baseline and 14 days after CVD 103-HgR ingestion and following the first and second doses of Shanchol were the main outcomes measured. By day 14 postvaccination, the rates of seroconversion after ingestion of a single standard dose and a high dose of CVD 103-HgR were 71.7% (33/46 participants) and 83.3% (40/48 participants), respectively. The rate of seroconversion following the first dose of Shanchol, 56.0% (28/50 participants), was significantly lower than that following the high dose of CVD 103-HgR (P = 0.003). The vibriocidal geometric mean titer (GMT) of the high dose of CVD 103-HgR exceeded the GMT of the standard dose at day 14 (214 versus 95, P = 0.045) and was ~2-fold higher than the GMT on day 7 and day 14 following the first Shanchol dose (P > 0.05). High-dose CVD 103-HgR is recommended for accelerated evaluation in developing countries to assess its efficacy and practicality in field situations. (This study has been registered at ClinicalTrials.gov under registration no. NCT02145377.)

RiVax is a promising recombinant ricin toxin A subunit (RTA) vaccine antigen that has been shown to be safe and immunogenic in humans and effective at protecting rhesus macaques against lethal-dose aerosolized toxin exposure. We previously used a panel of RTA-specific monoclonal antibodies (MAbs) to demonstrate, by competition enzyme-linked immunosorbent assay (ELISA), that RiVax elicits similar serum antibody profiles in humans and macaques. However, the MAb binding sites on RiVax have yet to be defined. In this study, we employed hydrogen exchange-mass spectrometry (HX-MS) to localize the epitopes on RiVax recognized by nine toxin-neutralizing MAbs and one nonneutralizing MAb. Based on strong protection from hydrogen exchange, the nine MAbs grouped into four spatially distinct epitope clusters (namely, clusters I to IV). Cluster I MAbs protected RiVax's α-helix B (residues 94 to 107), a protruding immunodominant secondary structure element known to be a target of potent toxin-neutralizing antibodies. Cluster II consisted of two subclusters located on the "back side" (relative to the active site pocket) of RiVax. One subcluster involved α-helix A (residues 14 to 24) and α-helices F-G (residues 184 to 207); the other encompassed β-strand d (residues 62 to 69) and parts of α-helices D-E (154 to 164) and the intervening loop. Cluster III involved α-helices C and G on the front side of RiVax, while cluster IV formed a sash from the front to back of RiVax, spanning strands b, c, and d (residues 35 to 59). Having a high-resolution B cell epitope map of RiVax will enable the development and optimization of competitive serum profiling assays to examine vaccine-induced antibody responses across species.

The presence of large palaeo-landslides can hinder the construction of railway lines if they cause an alteration of the natural balance of the slope, significantly increasing the cost of the project. During the construction works in a section of the Madrid–Asturias high-speed railway line (Spain), a large-scale hillside instability affected 460 m of the section. The ground movement began 10 months after the start of the excavations and remained active throughout the observation period (2008–2010). Data provided by fieldwork, boreholes and instrumental monitoring have allowed the investigation of the geological units involved, together with the geometry and the kinematics of the mass movement. This landslide involves a Paleozoic basement with an estimated volume of 4 400 000 m³. It shows low displacement rates (<45 mm a^–1) and was accommodated on a single surface of rupture, which reaches more than 60 m depth. The kinematics is favoured by structural and lithological conditions, there being a strong rheological contrast in the lower part of the mobilized ground. This movement is linked to a large palaeo-landslide that was partially reactivated when excavations undermined the lower slopes of the hillside. Technical solutions for the stabilization have already caused an additional cost of 17% over the initial budget.

A logistic regression model and a bivariate statistical analysis were used in this paper to evaluate the groundwater recharge susceptibility. The approach is based on the assessment of the relationship involving groundwater recharge and parameters that influence this hydrological process. Surface parameters and aquifer-related parameters were evaluated as thematic map layers using ArcGIS. Then, a weighted-rating method was adopted to categorize each parameter's map. To assess the role of each parameter in the aquifer recharge, a logistic regression model and a bivariate statistical analysis were applied to the Guenniche phreatic aquifer (Tunisia). Models are explored to establish a map showing the aquifer recharge susceptibility. The code Modflow was used to simulate the consequence of the recharge. The recharge amount was introduced in the model and was tested to verify the recharge effect on the hydraulic head for the two models. The obtained results reveal that the recharge as mapped in the bivariate statistical model has a minor impact on the hydraulic head. Results of the logistic regression model are more significant as the hydraulic head is widely affected. This model provides good results in mapping the spatial distribution of the aquifer recharge susceptibility.

In Arabidopsis, LTR retrotransposons are activated by mutations in the chromatin gene DECREASE in DNA METHYLATION 1 (DDM1), giving rise to 21- to 22-nt epigenetically activated siRNA (easiRNA) that depend on RNA DEPENDENT RNA POLYMERASE 6 (RDR6). We purified virus-like particles (VLPs) from ddm1 and ddm1rdr6 mutants in which genomic RNA is reverse transcribed into complementary DNA. High-throughput short-read and long-read sequencing of VLP DNA (VLP DNA-seq) revealed a comprehensive catalog of active LTR retrotransposons without the need for mapping transposition, as well as independent of genomic copy number. Linear replication intermediates of the functionally intact COPIA element EVADE revealed multiple central polypurine tracts (cPPTs), a feature shared with HIV in which cPPTs promote nuclear localization. For one member of the ATCOPIA52 subfamily (SISYPHUS), cPPT intermediates were not observed, but abundant circular DNA indicated transposon "suicide" by auto-integration within the VLP. easiRNA targeted EVADE genomic RNA, polysome association of GYPSY (ATHILA) subgenomic RNA, and transcription via histone H3 lysine-9 dimethylation. VLP DNA-seq provides a comprehensive landscape of LTR retrotransposons and their control at transcriptional, post-transcriptional, and reverse transcriptional levels.

Early sorting endosomes are responsible for the trafficking and function of transferrin receptor (TfR) and EGFR. These receptors play important roles in iron uptake and signaling and are critical for breast cancer development. However, the role of morphology, receptor composition, and signaling of early endosomes in breast cancer remains poorly understood. A novel population of enlarged early endosomes was identified in breast cancer cells and tumor xenografts but not in noncancerous MCF10A cells. Quantitative analysis of endosomal morphology, cargo sorting, EGFR activation, and Rab GTPase regulation was performed using super-resolution and confocal microscopy followed by 3D rendering. MDA-MB-231 breast cancer cells have fewer, but larger EEA1-positive early endosomes compared with MCF10A cells. Live-cell imaging indicated dysregulated cargo sorting, because EGF and Tf traffic together via enlarged endosomes in MDA-MB-231, but not in MCF10A. Large EEA1-positive MDA-MB-231 endosomes exhibited prolonged and increased EGF-induced activation of EGFR upon phosphorylation at tyrosine-1068 (EGFR-p1068). Rab4A overexpression in MCF10A cells produced EEA1-positive enlarged endosomes that displayed prolonged and amplified EGF-induced EGFR-p1068 activation. Knockdown of Rab4A lead to increased endosomal size in MCF10A, but not in MDA-MB-231 cells. Nevertheless, Rab4A knockdown resulted in enhanced EGF-induced activation of EGFR-p1068 in MDA-MB-231 as well as downstream signaling in MCF10A cells. Altogether, this extensive characterization of early endosomes in breast cancer cells has identified a Rab4-modulated enlarged early endosomal compartment as the site of prolonged and increased EGFR activation.

Implications:

Enlarged early endosomes play a Rab4-modulated role in regulation of EGFR activation in breast cancer cells.

High-constitutive activity of the DNA damage response protein checkpoint kinase 1 (CHK1) has been shown in glioblastoma (GBM) cell lines and in tissue sections. However, whether constitutive activation and overexpression of CHK1 in GBM plays a functional role in tumorigenesis or has prognostic significance is not known. We interrogated multiple glioma patient cohorts for expression levels of CHK1 and the oncogene cancerous inhibitor of protein phosphatase 2A (CIP2A), a known target of high-CHK1 activity, and examined the relationship between these two proteins in GBM. Expression levels of CHK1 and CIP2A were independent predictors for reduced overall survival across multiple glioma patient cohorts. Using siRNA and pharmacologic inhibitors we evaluated the impact of their depletion using both in vitro and in vivo models and sought a mechanistic explanation for high CIP2A in the presence of high-CHK1 levels in GBM and show that; (i) CHK1 and pSTAT3 positively regulate CIP2A gene expression; (ii) pSTAT3 and CIP2A form a recursively wired transcriptional circuit; and (iii) perturbing CIP2A expression induces GBM cell senescence and retards tumor growth in vitro and in vivo. Taken together, we have identified an oncogenic transcriptional circuit in GBM that can be destabilized by targeting CIP2A.

Implications:

High expression of CIP2A in gliomas is maintained by a CHK1-dependent pSTAT3–CIP2A recursive loop; interrupting CIP2A induces cell senescence and slows GBM growth adding impetus to the development of CIP2A as an anticancer drug target.

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.

Implications:

Our studies identify that JMJD1A promotes the proliferation and progression of prostate cancer cells through enabling Snail transcriptional activation, and thus highlight JMJD1A as potential therapeutic target for advanced prostate cancer.

Decreased release of palmitic acid methyl ester (PAME), a vasodilator, from perivascular adipose tissue (PVAT) might contribute to hypertension pathogenesis. However, the PAME biosynthetic pathway remains unclear. In this study, we hypothesized that PAME is biosynthesized from palmitic acid (PA) via human catechol-O-methyltransferase (COMT) catalysis and that decreased PAME biosynthesis plays a role in hypertension pathogenesis. We compared PAME biosynthesis between age-matched normotensive Wistar Kyoto (WKY) rats and hypertensive spontaneously hypertensive rats (SHRs) and investigated the effects of losartan treatment on PAME biosynthesis. Computational molecular modeling indicated that PA binds well at the active site of COMT. Furthermore, in in vitro enzymatic assays in the presence of COMT and S-5'-adenosyl-L-methionine (AdoMet), the stable isotope [¹³C₁₆]-PA was methylated to form [¹³C₁₆]-PAME in incubation medium or the Krebs–Henseleit solution containing 3T3-L1 adipocytes or rat PVAT. The adipocytes and PVATs expressed membrane-bound (MB)-COMT and soluble (S)-COMT proteins. [¹³C₁₆]-PA methylation to form [¹³C₁₆]-PAME in 3T3-L1 adipocytes and rat PVAT was blocked by various COMT inhibitors, such as S-(5'-adenosyl)-L-homocysteine, adenosine-2',3'-dialdehyde, and tolcapone. MB- and S-COMT levels in PVATs of established SHRs were significantly lower than those in PVATs of age-matched normotensive WKY rats, with decreased [¹³C₁₆]-PA methylation to form [¹³C₁₆]-PAME. This decrease was reversed by losartan, an angiotensin II (Ang II) type 1 receptor antagonist. Therefore, PAME biosynthesis in rat PVAT is dependent on AdoMet, catalyzed by COMT, and decreased in SHRs, further supporting the role of PVAT/PAME in hypertension pathogenesis. Moreover, the antihypertensive effect of losartan might be due partly to its increased PAME biosynthesis.

SIGNIFICANCE STATEMENT

PAME is a key PVAT-derived relaxing factor. We for the first time demonstrate that PAME is synthesized through PA methylation via the S-5'-adenosyl-L-methionine–dependent COMT catalyzation pathway. Moreover, we confirmed PVAT dysfunction in the hypertensive state. COMT-dependent PAME biosynthesis is involved in Ang II receptor type 1–mediated blood pressure regulation, as evidenced by the reversal of decreased PAME biosynthesis in PVAT by losartan in hypertensive rats. This finding might help in developing novel therapeutic or preventive strategies against hypertension.

The 78-kDa glucose-regulated protein (GRP78), an endoplasmic reticulum (ER) chaperone, is a master regulator of the ER stress. A number of studies revealed that high levels of GRP78 protein in cancer cells confer multidrug resistance (MDR) to therapeutic treatment. Therefore, drug candidate that reduces GRP78 may represent a novel approach to eliminate MDR cancer cells. Our earlier studies showed that a set of 4H-chromene derivatives induced selective cytotoxicity in MDR cancer cells. In the present study, we elucidated its selective mechanism in four MDR cancer cell lines with one lead candidate (CXL146). Cytotoxicity results confirmed the selective cytotoxicity of CXL146 toward the MDR cancer cell lines. We noted significant overexpression of GRP78 in all four MDR cell lines compared with the parental cell lines. Unexpectedly, CXL146 treatment rapidly and dose-dependently reduced GRP78 protein in MDR cancer cell lines. Using human leukemia (HL) 60/mitoxantrone (MX) 2 cell line as the model, we demonstrated that CXL146 treatment activated the unfolded protein response (UPR); as evidenced by the activation of inositol-requiring enzyme 1α, protein kinase R–like ER kinase, and activating transcription factor 6. CXL146-induced UPR activation led to a series of downstream events, including extracellular signal-regulated kinase 1/2 and c-Jun N-terminal kinase activation, which contributed to CXL146-induced apoptosis. Targeted reduction in GRP78 resulted in reduced sensitivity of HL60/MX2 toward CXL146. Long-term sublethal CXL146 exposure also led to reduction in GRP78 in HL60/MX2. These data collectively support GRP78 as the target of CXL146 in MDR treatment. Interestingly, HL60/MX2 upon long-term sublethal CXL146 exposure regained sensitivity to mitoxantrone treatment. Therefore, further exploration of CXL146 as a novel therapy in treating MDR cancer cells is warranted.

SIGNIFICANCE STATEMENT

Multidrug resistance is one major challenge to cancer treatment. This study provides evidence that cancer cells overexpress 78-kDa glucose-regulated protein (GRP78) as a mechanism to acquire resistance to standard cancer therapies. A chromene-based small molecule, CXL146, selectively eliminates cancer cells with GRP78 overexpression via activating unfolded protein response–mediated apoptosis. Further characterization indicates that CXL146 and standard therapies complementarily target different populations of cancer cells, supporting the potential of CXL146 to overcome multidrug resistance in cancer treatment.

Proteinase-activated receptors (PARs) are a four-member family of G-protein–coupled receptors that are activated via proteolysis. PAR4 is a member of this family that is cleaved and activated by serine proteinases such as thrombin, trypsin, and cathepsin-G. PAR4 is expressed in a variety of tissues and cell types, including platelets, vascular smooth muscle cells, and neuronal cells. In studying PAR4 signaling and trafficking, we observed dynamic changes in the cell membrane, with spherical membrane protrusions that resemble plasma membrane blebbing. Since nonapoptotic membrane blebbing is now recognized as an important regulator of cell migration, cancer cell invasion, and vesicular content release, we sought to elucidate the signaling pathway downstream of PAR4 activation that leads to such events. Using a combination of pharmacological inhibition and CRISPR/CRISPR-associated protein 9 (Cas9)–mediated gene editing approaches, we establish that PAR4-dependent membrane blebbing occurs independently of the Gα_q/11- and Gα_i-signaling pathways and is dependent on signaling via the β-arrestin-1/2 and Ras homolog family member A (RhoA) signaling pathways. Together these studies provide further mechanistic insight into PAR4 regulation of cellular function.

SIGNIFICANCE STATEMENT

We find that the thrombin receptor PAR4 triggers cell membrane blebbing in a RhoA–and β-arrestin–dependent manner. In addition to identifying novel cellular responses mediated by PAR4, these data provide further evidence for biased signaling in PAR4 since membrane blebbing was dependent on some, but not all, signaling pathways activated by PAR4.

Before it was molecularly cloned in 1994, acute-phase response factor or signal transducer and activator of transcription (STAT)3 was the focus of intense research into understanding the mammalian response to injury, particularly the acute-phase response. Although known to be essential for liver production of acute-phase reactant proteins, many of which augment innate immune responses, molecular cloning of acute-phase response factor or STAT3 and the research this enabled helped establish the central function of Janus kinase (JAK) family members in cytokine signaling and identified a multitude of cytokines and peptide hormones, beyond interleukin-6 and its family members, that activate JAKs and STAT3, as well as numerous new programs that their activation drives. Many, like the acute-phase response, are adaptive, whereas several are maladaptive and lead to chronic inflammation and adverse consequences, such as cachexia, fibrosis, organ dysfunction, and cancer. Molecular cloning of STAT3 also enabled the identification of other noncanonical roles for STAT3 in normal physiology, including its contribution to the function of the electron transport chain and oxidative phosphorylation, its basal and stress-related adaptive functions in mitochondria, its function as a scaffold in inflammation-enhanced platelet activation, and its contributions to endothelial permeability and calcium efflux from endoplasmic reticulum. In this review, we will summarize the molecular and cellular biology of JAK/STAT3 signaling and its functions under basal and stress conditions, which are adaptive, and then review maladaptive JAK/STAT3 signaling in animals and humans that lead to disease, as well as recent attempts to modulate them to treat these diseases. In addition, we will discuss how consideration of the noncanonical and stress-related functions of STAT3 cannot be ignored in efforts to target the canonical functions of STAT3, if the goal is to develop drugs that are not only effective but safe.

Significance Statement

Key biological functions of Janus kinase (JAK)/signal transducer and activator of transcription (STAT)3 signaling can be delineated into two broad categories: those essential for normal cell and organ development and those activated in response to stress that are adaptive. Persistent or dysregulated JAK/STAT3 signaling, however, is maladaptive and contributes to many diseases, including diseases characterized by chronic inflammation and fibrosis, and cancer. A comprehensive understanding of JAK/STAT3 signaling in normal development, and in adaptive and maladaptive responses to stress, is essential for the continued development of safe and effective therapies that target this signaling pathway.

Monoclonal antibodies (mAbs) against programmed cell death 1 (PD-1), such as nivolumab and pembrolizumab, are associated with high response rates in patients with relapsed or refractory classic Hodgkin lymphoma (HL). To date, no prognostic factor for overall survival (OS) has been established with these agents in HL. We examined whether the first early response assessment evaluated using ¹⁸F-FDG PET/CT may be associated with OS in this setting. Methods: This retrospective study included 45 patients from 34 institutions. In a masked, centralized review, 3 independent radiologists classified PET/CT scans obtained at a median of 2.0 mo (interquartile range, 1.7–3.7 mo) after nivolumab initiation using existing criteria (i.e., 2014 Lugano classification and 2016 LYRIC). Patients were classified according to 4 possible response categories: complete metabolic response (CMR), partial metabolic response (PMR), no metabolic response (NMR), or progressive metabolic disease (PMD). Because the OS of patients with NMR and PMR was similar, they were grouped together. OS was estimated using the Kaplan–Meier method and compared between groups using log-rank testing. Results: Eleven patients (24%) died after a median follow-up of 21.2 mo. The classification was identical between Lugano and LYRIC because all 16 progression events classified as indeterminate response per LYRIC were confirmed on subsequent evaluations. Both Lugano and LYRIC classified patients as CMR in 13 cases (29%), PMD in 16 (36%), NMR in 4 (9%), and PMR in 12 (27%). The 2-y OS probability was significantly different in patients with PMD (0.53; 95% confidence interval [95%CI], 0.32–0.87), NMR or PMR (0.80; 95%CI, 0.63–1.00), and CMR (1.00; 95%CI, 1.00–1.00) in the overall population (P = 0.02, 45 patients), as well as according to a landmark analysis at 3 mo (P = 0.05, 32 patients). Conclusion: In relapsed or refractory HL patients treated with anti-PD-1 mAbs, the first early PET/CT assessment using either Lugano or LYRIC predicted OS and allowed early risk stratification, suggesting that PET/CT might be used to develop risk-adapted strategies.

Autophagy captures intracellular components and delivers them to lysosomes for degradation and recycling. Conditional autophagy deficiency in adult mice causes liver damage, shortens life span to 3 mo due to neurodegeneration, and is lethal upon fasting. As autophagy deficiency causes p53 induction and cell death in neurons, we sought to test whether p53 mediates the lethal consequences of autophagy deficiency. Here, we conditionally deleted Trp53 (p53 hereafter) and/or the essential autophagy gene Atg7 throughout adult mice. Compared with Atg7^/ mice, the life span of Atg7^/p53^/ mice was extended due to delayed neurodegeneration and resistance to death upon fasting. Atg7 also suppressed apoptosis induced by p53 activator Nutlin-3, suggesting that autophagy inhibited p53 activation. To test whether increased oxidative stress in Atg7^/ mice was responsible for p53 activation, Atg7 was deleted in the presence or absence of the master regulator of antioxidant defense nuclear factor erythroid 2-related factor 2 (Nrf2). Nrf2^–/–Atg7^/ mice died rapidly due to small intestine damage, which was not rescued by p53 codeletion. Thus, Atg7 limits p53 activation and p53-mediated neurodegeneration. In turn, NRF2 mitigates lethal intestine degeneration upon autophagy loss. These findings illustrate the tissue-specific roles for autophagy and functional dependencies on the p53 and NRF2 stress response mechanisms.

Management of cystic fibrosis has been revolutionised by the introduction of cystic fibrosis transmembrane conductance regulator (CFTR) modulators. These compounds treat the underlying molecular basis of the disease by increasing activity of defective CFTR channels, which improves many clinical parameters and enhances patient quality of life [1]. Next-generation modulators, also known as triple combination therapy, promise to be highly efficacious in up to 90% of patients [2] and will likely dramatically change the landscape of cystic fibrosis disease. Studies examining individuals before and after initiation of CFTR modulators have revealed novel functions of CFTR and shown that CFTR modulators do not reverse all disease manifestations [3–5]. Thus, knowledge of the post-modulator cystic fibrosis disease state is crucial for understanding what continued therapies will be needed for people with cystic fibrosis and what new challenges may arise.

Background

COPD patients account for a large proportion of lung transplants; lung transplantation survival benefit for COPD patients is not well established.

Methods

We identified 4521 COPD patients in the United Network for Organ Sharing (UNOS) dataset transplanted from May 2005 to August 2016, and 604 patients assigned to receive pulmonary rehabilitation and medical management in the National Emphysema Treatment Trial (NETT). After trimming the populations for NETT eligibility criteria and data completeness, 1337 UNOS and 596 NETT patients remained. Kaplan–Meier estimates of transplant-free survival from transplantation for UNOS, and NETT randomisation, were compared between propensity score-matched UNOS (n=401) and NETT (n=262) patients.

Results

In propensity-matched analyses, transplanted patients had better survival compared to medically managed patients in NETT (p=0.003). Stratifying on 6 min walk distance (6 MWD) and FEV₁, UNOS patients with 6 MWD <1000 ft (~300 m) or FEV₁ <20% of predicted had better survival than NETT counterparts (median survival 5.0 years UNOS versus 3.4 years NETT; log-rank p<0.0001), while UNOS patients with 6 MWD ≥1000 ft (~300 m) and FEV₁ ≥20% had similar survival to NETT counterparts (median survival, 5.4 years UNOS versus 4.9 years NETT; log-rank p=0.73), interaction p=0.01.

Conclusions

Overall survival is better for matched lung transplant patients compared with medical management alone. Patients who derive maximum benefit are those with 6 MWD <1000 ft (~300 m) or FEV₁ <20% of predicted, compared with pulmonary rehabilitation and medical management.

Streptococcus pyogenes (Lancefield group A Streptococcus [GAS]) is a β-hemolytic human-selective pathogen that is responsible for a large number of morbid and mortal infections in humans. For efficient infection, GAS requires different types of surface proteins that provide various mechanisms for evading human innate immune responses, thus enhancing pathogenicity of the bacteria. Many such virulence-promoting proteins, including the major surface signature M protein, are translocated after biosynthesis through the cytoplasmic membrane and temporarily tethered to this membrane via a type 1 transmembrane domain (TMD) positioned near the COOH terminus. In these proteins, a sorting signal, LPXTG, is positioned immediately upstream of the TMD, which is cleaved by the membrane-associated transpeptidase, sortase A (SrtA), leading to the covalent anchoring of these proteins to newly emerging l-Ala–l-Ala cross-bridges of the growing peptidoglycan cell wall. Herein, we show that inactivation of the srtA gene in a skin-tropic pattern D GAS strain (AP53) results in retention of the M protein in the cell membrane. However, while the isogenic AP53 srtA strain is attenuated in overall pathogenic properties due to effects on the integrity of the cell membrane, our data show that the M protein nonetheless can extend from the cytoplasmic membrane through the cell wall and then to the surface of the bacteria and thereby retain its important properties of productively binding and activating fluid-phase host plasminogen (hPg). The studies presented herein demonstrate an underappreciated additional mechanism of cell surface display of bacterial virulence proteins via their retention in the cell membrane and extension to the GAS surface.

IMPORTANCE Group A Streptococcus pyogenes (GAS) is a human-specific pathogen that produces many surface factors, including its signature M protein, that contribute to its pathogenicity. M proteins undergo specific membrane localization and anchoring to the cell wall via the transpeptidase sortase A. Herein, we explored the role of sortase A function on M protein localization, architecture, and function, employing, a skin-tropic GAS isolate, AP53, which expresses a human plasminogen (hPg)-binding M (PAM) Protein. We showed that PAM anchored in the cell membrane, due to the targeted inactivation of sortase A, was nonetheless exposed on the cell surface and functionally interacted with host hPg. We demonstrate that M proteins, and possibly other sortase A-processed proteins that are retained in the cell membrane, can still function to initiate pathogenic processes by this underappreciated mechanism.

Shigella species, the causal agents of bacillary dysentery, use a type III secretion system (T3SS) to inject two waves of virulence proteins, known as effectors, into the colonic epithelium to subvert host cell machinery. Prior to host cell contact and secretion of the first wave of T3SS effectors, OspD1, an effector and antiactivator protein, prevents premature production of the second wave of effectors. Despite this important role, regulation of the ospD1 gene is not well understood. While ospD1 belongs to the large regulon of VirB, a transcriptional antisilencing protein that counters silencing mediated by the histone-like nucleoid structuring protein H-NS, it remains unclear if VirB directly or indirectly regulates ospD1. Additionally, it is not known if ospD1 is regulated by H-NS. Here, we identify the primary ospD1 transcription start site (+1) and show that the ospD1 promoter is remotely regulated by both VirB and H-NS. Our findings demonstrate that VirB regulation of ospD1 requires at least one of the two newly identified VirB regulatory sites, centered at –978 and –1270 relative to the ospD1 +1. Intriguingly, one of these sites lies on a 193-bp sequence found in three conserved locations on the large virulence plasmids of Shigella. The region required for H-NS-dependent silencing of ospD1 lies between –1120 and –820 relative to the ospD1 +1. Thus, our study provides further evidence that cis-acting regulatory sequences for transcriptional antisilencers and silencers, such as VirB and H-NS, can lie far upstream of the canonical bacterial promoter region (i.e., –250 to +1).

IMPORTANCE Transcriptional silencing and antisilencing mechanisms regulate virulence gene expression in many important bacterial pathogens. In Shigella species, plasmid-borne virulence genes, such as those encoding the type III secretion system (T3SS), are silenced by the histone-like nucleoid structuring protein H-NS and antisilenced by VirB. Previous work at the plasmid-borne icsP locus revealed that VirB binds to a remotely located cis-acting regulatory site to relieve transcriptional silencing mediated by H-NS. Here, we characterize a second example of remote VirB antisilencing at ospD1, which encodes a T3SS antiactivator and effector. Our study highlights that remote transcriptional silencing and antisilencing occur more frequently in Shigella than previously thought, and it raises the possibility that long-range transcriptional regulation in bacteria is commonplace.

Mutation and translocation of fibroblast growth factor receptors often lead to aberrant signaling and cancer. This work focuses on the t(8;22)(p11;q11) chromosomal translocation which creates the breakpoint cluster region (BCR) fibroblast growth factor receptor1 (FGFR1) (BCR-FGFR1) fusion protein. This fusion occurs in stem cell leukemia/lymphoma, which can progress to atypical chronic myeloid leukemia, acute myeloid leukemia, or B-cell lymphoma. This work focuses on the biochemical characterization of BCR-FGFR1 and identification of novel therapeutic targets. The tyrosine kinase activity of FGFR1 is required for biological activity as shown using transformation assays, interleukin-3 independent cell proliferation, and liquid chromatography/mass spectroscopy analyses. Furthermore, BCR contributes a coiled-coil oligomerization domain, also essential for oncogenic transformation by BCR-FGFR1. The importance of salt bridge formation within the coiled-coil domain is demonstrated, as disruption of three salt bridges abrogates cellular transforming ability. Lastly, BCR-FGFR1 acts as a client of the chaperonin heat shock protein 90 (Hsp90), suggesting that BCR-FGFR1 relies on Hsp90 complex to evade proteasomal degradation. Transformed cells expressing BCR-FGFR1 are sensitive to the Hsp90 inhibitor Ganetespib, and also respond to combined treatment with Ganetespib plus the FGFR inhibitor BGJ398. Collectively, these data suggest novel therapeutic approaches for future stem cell leukemia/lymphoma treatment: inhibition of BCR oligomerization by disruption of required salt bridges; and inhibition of the chaperonin Hsp90 complex.

Progression from the initial vascular response upon hyperglycemia to a proliferative stage with neovacularizations is the hallmark of proliferative diabetic retinopathy. Here, we report on the novel diabetic pdx1^–/– zebrafish mutant as a model for diabetic retinopathy that lacks the transcription factor pdx1 through CRISPR-Cas9–mediated gene knockout leading to disturbed pancreatic development and hyperglycemia. Larval pdx1^–/– mutants prominently show vasodilation of blood vessels through increased vascular thickness in the hyaloid network as direct developmental precursor of the adult retinal vasculature in zebrafish. In adult pdx1^–/– mutants, impaired glucose homeostasis induces increased hyperbranching and hypersprouting with new vessel formation in the retina and aggravation of the vascular alterations from the larval to the adult stage. Both vascular aspects respond to antiangiogenic and antihyperglycemic pharmacological interventions in the larval stage and are accompanied by alterations in the nitric oxide metabolism. Thus, the pdx1^–/– mutant represents a novel model to study mechanisms of hyperglycemia-induced retinopathy wherein extensive proangiogenic alterations in blood vessel morphology and metabolic alterations underlie the vascular phenotype.

PURPOSE

The prognosis of older patients with dizziness in primary care is unknown. Our objective was to determine the prognosis and survival of patients with different subtypes and causes of dizziness.

METHODS

In a primary care prospective cohort study, 417 older adults with dizziness (mean age 79 years) received a full diagnostic workup in 2006-2008. A panel of physicians classified the subtype and primary cause of dizziness. Main outcome measures were mortality and dizziness-related impairment assessed at 10-year follow-up.

RESULTS

At 10-year follow-up 169 patients (40.5%) had died. Presyncope was the most common dizziness subtype (69.1%), followed by vertigo (41.0%), disequilibrium (39.8%), and other dizziness (1.7%). The most common primary causes of dizziness were cardiovascular disease (56.8%) and peripheral vestibular disease (14.4%). Multivariable adjusted Cox models showed a lower mortality rate for patients with the subtype vertigo compared with other subtypes (hazard ratio [HR] = 0.62; 95% CI, 0.40-0.96), and for peripheral vestibular disease vs cardiovascular disease as primary cause of dizziness (HR = 0.46; 95% CI, 0.25-0.84). After 10 years, 47.7% of patients who filled out the follow-up measurement experienced substantial dizziness-related impairment. No significant difference in substantial impairment was seen between different subtypes and primary causes of dizziness.

CONCLUSIONS

The 10-year mortality rate was lower for the dizziness subtype vertigo compared with other subtypes. Patients with dizziness primarily caused by peripheral vestibular disease had a lower mortality rate than patients with cardiovascular disease. Substantial dizziness-related impairment in older patients with dizziness 10 years later is high, and indicates that current treatment strategies by family physicians may be suboptimal.

Background and objectives

This study aimed to investigate the association between in-hospital trajectories of serum sodium and risk of in-hospital and 1-year mortality in patients in hospital.

Design, setting, participants, & measurements

This is a single-center cohort study. All adult patients who were hospitalized from years 2011 through 2013 who had available admission serum sodium and at least three serum sodium measurements during hospitalization were included. The trend of serum sodium during hospitalization was analyzed using group-based trajectory modeling; the five main trajectories were grouped as follows: (1) stable normonatremia, (2) uncorrected hyponatremia, (3) borderline high serum sodium, (4) corrected hyponatremia, and (5) fluctuating serum sodium. The outcome of interest was in-hospital mortality and 1-year mortality. Stable normonatremia was used as the reference group for outcome comparison.

Results

A total of 43,539 patients were analyzed. Of these, 47% had stable normonatremia, 15% had uncorrected hyponatremia, 31% had borderline high serum sodium, 3% had corrected hyponatremia, and 5% had fluctuating serum sodium trajectory. In adjusted analysis, there was a higher in-hospital mortality among those with uncorrected hyponatremia (odds ratio [OR], 1.33; 95% CI, 1.06 to 1.67), borderline high serum sodium (OR, 1.66; 95% CI, 1.38 to 2.00), corrected hyponatremia (OR, 1.50; 95% CI, 1.02 to 2.20), and fluctuating serum sodium (OR, 4.61; 95% CI, 3.61 to 5.88), compared with those with the normonatremia trajectory. One-year mortality was higher among those with uncorrected hyponatremia (hazard ratio [HR], 1.28; 95% CI, 1.19 to 1.38), borderline high serum sodium (HR, 1.18; 95% CI, 1.11 to 1.26), corrected hyponatremia (HR, 1.24; 95% CI, 1.08 to 1.42), and fluctuating serum sodium (HR, 2.10; 95% CI, 1.89 to 2.33) compared with those with the normonatremia trajectory.

Conclusions

More than half of patients who had been hospitalized had an abnormal serum sodium trajectory during hospitalization. This study demonstrated that not only the absolute serum sodium levels but also their in-hospital trajectories were significantly associated with in-hospital and 1-year mortality. The highest in-hospital and 1-year mortality risk was associated with the fluctuating serum sodium trajectory.

Podcast

This article contains a podcast at https://www.asn-online.org/media/podcast/CJASN/2020_03_25_CJN.12281019.mp3

The identification of biomarkers for patient stratification is fundamental to precision medicine efforts in oncology. Here, we identified two baseline, circulating immune cell subsets associated with overall survival in patients with metastatic pancreatic cancer who were enrolled in two phase II randomized studies of GVAX pancreas and CRS-207 immunotherapy. Single-cell mass cytometry was used to simultaneously measure 38 cell surface or intracellular markers in peripheral blood mononuclear cells obtained from a phase IIa patient subcohort (N = 38). CITRUS, an algorithm for identification of stratifying subpopulations in multidimensional cytometry datasets, was used to identify single-cell signatures associated with clinical outcome. Patients with a higher abundance of CD8⁺CD45RO^–CCR7^–CD57⁺ cells and a lower abundance of CD14⁺CD33⁺CD85j⁺ cells had improved overall survival [median overall survival, range (days) 271, 43–1,247] compared with patients with a lower abundance of CD8⁺CD45RO^–CCR7^–CD57⁺ cells and higher abundance of CD14⁺CD33⁺CD85j⁺ cells (77, 24–1,247 days; P = 0.0442). The results from this prospective–retrospective biomarker analysis were validated by flow cytometry in 200 patients with pancreatic cancer enrolled in a phase IIb study (P = 0.0047). The identified immune correlates provide potential prognostic or predictive signatures that could be employed for patient stratification.

Biofilm formation by Pseudomonas aeruginosa contributes to its survival on surfaces and represents a major clinical threat because of the increased tolerance of biofilms to disinfecting agents. This study aimed to investigate the efficacy of 405-nm light-emitting diode (LED) illumination in eliminating P. aeruginosa biofilms formed on stainless steel coupons under different temperatures. Time-dependent killing assays using planktonic and biofilm cells were used to determine the antimicrobial and antibiofilm activities of LED illumination. We also evaluated the effects of LED illumination on the disinfectant susceptibility, biofilm structure, extracellular polymeric substance (EPS) structure and composition, and biofilm-related gene expression of P. aeruginosa biofilm cells. Results showed that the abundance of planktonic P. aeruginosa cells was reduced by 0.88, 0.53, and 0.85 log CFU/ml following LED treatment for 2 h compared with untreated controls at 4, 10, and 25°C, respectively. For cells in biofilms, significant reductions (1.73, 1.59, and 1.68 log CFU/cm²) were observed following LED illumination for 2 h at 4, 10, and 25°C, respectively. Moreover, illuminated P. aeruginosa biofilm cells were more sensitive to benzalkonium chloride or chlorhexidine than untreated cells. Scanning electron microscopy and confocal laser scanning microscopic observation indicated that both the biofilm structure and EPS structure were disrupted by LED illumination. Further, reverse transcription-quantitative PCR revealed that LED illumination downregulated the transcription of several genes associated with biofilm formation. These findings suggest that LED illumination has the potential to be developed as an alternative method for prevention and control of P. aeruginosa biofilm contamination.

IMPORTANCE Pseudomonas aeruginosa can form biofilms on medical implants, industrial equipment, and domestic surfaces, contributing to high morbidity and mortality rates. This study examined the antibiofilm activity of 405-nm light-emitting diode (LED) illumination against mature biofilms formed on stainless steel coupons. We found that the disinfectant susceptibility, biofilm structure, and extracellular polymeric substance structure and composition were disrupted by LED illumination. We then investigated the transcription of several critical P. aeruginosa biofilm-related genes and analyzed the effect of illumination temperature on the above characteristics. Our results confirmed that LED illumination could be developed into an effective and safe method to counter P. aeruginosa biofilm contamination. Further research will be focused on the efficacy and application of LED illumination for elimination of complicated biofilms in the environment.

Background:

Deaths from respiratory tract infections (RTIs) in children are preventable through timely access to public health and medical interventions. We aimed to assess whether socioeconomic disparities in mortality related to pediatric RTI persisted after accounting for health status at birth.

Methods:

We compared the prevalence of and risk factors for RTI-related death in singletons aged 28 days to 4 years across Ontario (Canada), Scotland and England (jurisdictions with universal health care) using linked administrative data for 2003–2013. We estimated rates of RTI-related mortality for children living in deprived areas and those born to teenage girls; we estimated both crude rates and those adjusted for health status at birth.

Results:

A total of 1 299 240 (Ontario), 547 556 (Scotland) and 3 910 401 (England) children were included in the study. Across all jurisdictions, children born in the most deprived areas experienced the highest rates of RTI-related mortality. After adjustment for high-risk chronic conditions and prematurity, we observed differences in mortality according to area-level deprivation in Ontario and England but not in Scotland. In Ontario, teenage motherhood was also an independent risk factor for RTI-related mortality.

Interpretation:

Socioeconomic disparities played a substantial role in child mortality related to RTI in all 3 jurisdictions. Context-specific investigations around the mechanisms of this increased risk and development of programs to address socioeconomic disparities are needed.

With increased interest in lipoprotein(a) (Lp[a]) concentration as a target for risk reduction and growing clinical evidence of its impact on cardiovascular disease (CVD) risk, rigorous analytical performance specifications (APS) and accuracy targets for Lp(a) are required. We investigated the biological variation (BV) of Lp(a), and 2 other major biomarkers of CVD, apolipoprotein A-I (apoA-I) and apolipoprotein B-100 (apoB), in the European Biological Variation Study population.Serum samples were drawn from 91 healthy individuals for 10 consecutive weeks at 6 European laboratories and analyzed in duplicate on a Roche Cobas 8000 c702. Outlier, homogeneity, and trend analysis were performed, followed by CV-ANOVA to determine BV estimates and their 95% CIs. These estimates were used to calculate APS and reference change values. For Lp(a), BV estimates were determined on normalized concentration quintiles.Within-subject BV estimates were significantly different between sexes for Lp(a) and between women aged <50 and >50 years for apoA-I and apoB. Lp(a) APS was constant across concentration quintiles and, overall, lower than APS based on currently published data, whereas results were similar for apoA-I and apoB.Using a fully Biological Variation Data Critical Appraisal Checklist (BIVAC)–compliant protocol, our study data confirm BV estimates of Lp(a) listed in the European Federation of Clinical Chemistry and Laboratory Medicine database and reinforce concerns expressed in recent articles regarding the suitability of older APS recommendations for Lp(a) measurements. Given the heterogeneity of Lp(a), more BIVAC-compliant studies on large numbers of individuals of different ethnic groups would be desirable.

Copy number variation (CNV) in the salivary amylase gene (AMY1) modulates salivary α-amylase levels and is associated with postprandial glycemic traits. Whether AMY1-CNV plays a role in age-mediated change in insulin resistance (IR) is uncertain.We measured AMY1-CNV using duplex quantitative real-time polymerase chain reaction in two studies, the Boston Puerto Rican Health Study (BPRHS, n = 749) and the Genetics of Lipid-Lowering Drug and Diet Network study (GOLDN, n = 980), and plasma metabolomic profiles in the BPRHS. We examined the interaction between AMY1-CNV and age by assessing the relationship between age with glycemic traits and type 2 diabetes (T2D) according to high or low copy numbers of the AMY1 gene. Furthermore, we investigated associations between metabolites and interacting effects of AMY1-CNV and age on T2D risk.We found positive associations of IR with age among subjects with low AMY1-copy-numbers in both studies. T2D was marginally correlated with age in participants with low AMY1-copy-numbers but not with high AMY1-copy-numbers in the BPRHS. Metabolic pathway enrichment analysis identified the pentose metabolic pathway based on metabolites that were associated with both IR and the interactions between AMY1-CNV and age. Moreover, in older participants, high AMY1-copy-numbers tended to be associated with lower levels of ribonic acid, erythronic acid, and arabinonic acid, all of which were positively associated with IR.We found evidence supporting a role of AMY1-CNV in modifying the relationship between age and IR. Individuals with low AMY1-copy-numbers tend to have increased IR with advancing age.

Purpose:

Unleashing the immune system by PD-1 and/or CTLA-4 blockade can cause severe immune-related toxicity necessitating immunosuppressive treatment. Whether immunosuppression for toxicity impacts survival is largely unknown.

Experimental Design:

Using data from the prospective nationwide Dutch Melanoma Treatment Registry (DMTR), we analyzed the association between severe toxicity and overall survival (OS) in 1,250 patients with advanced melanoma who were treated with immune checkpoint inhibitors (ICI) in first line between 2012 and 2017. Furthermore, we analyzed whether toxicity management affected survival in these patients.

Results:

A total of 1,250 patients were included, of whom 589 received anti-PD1 monotherapy, 576 ipilimumab, and 85 combination therapy. A total of 312 patients (25%) developed severe (grade ≥3) toxicity. Patients experiencing severe ICI toxicity had a significantly prolonged survival with a median OS of 23 months compared with 15 months for patients without severe toxicity [hazard ratio (HR_adj) = 0.77; 95% confidence interval (CI), 0.63–0.93]. Among patients experiencing severe toxicity, survival was significantly decreased in patients who received anti-TNF ± steroids for steroid-refractory toxicity compared with patients who were managed with steroids only (HR_adj = 1.61; 95% CI, 1.03–2.51), with a median OS of 17 and 27 months, respectively.

Conclusions:

Patients experiencing severe ICI toxicity have a prolonged OS. However, this survival advantage is abrogated when anti-TNF is administered for steroid-refractory toxicity. Further prospective studies are needed to assess the effect of different immunosuppressive regimens on checkpoint inhibitor efficacy.

See related commentary by Weber and Postow, p. 2085

Purpose:

To provide a better understanding of the interplay between the immune system and brain metastases to advance therapeutic options for this life-threatening disease.

Experimental Design:

Tumor-infiltrating lymphocytes (TIL) were quantified by semiautomated whole-slide analysis in brain metastases from 81 lung adenocarcinomas. Multi-color staining enabled phenotyping of TILs (CD3, CD8, and FOXP3) on a single-cell resolution. Molecular determinants of the extent of TILs in brain metastases were analyzed by transcriptomics in a subset of 63 patients. Findings in lung adenocarcinoma brain metastases were related to published multi-omic primary lung adenocarcinoma The Cancer Genome Atlas data (n = 230) and single-cell RNA-sequencing (scRNA-seq) data (n = 52,698).

Results:

TIL numbers within tumor islands was an independent prognostic marker in patients with lung adenocarcinoma brain metastases. Comparative transcriptomics revealed that expression of three surfactant metabolism-related genes (SFTPA1, SFTPB, and NAPSA) was closely associated with TIL numbers. Their expression was not only prognostic in brain metastasis but also in primary lung adenocarcinoma. Correlation with scRNA-seq data revealed that brain metastases with high expression of surfactant genes might originate from tumor cells resembling alveolar type 2 cells. Methylome-based estimation of immune cell fractions in primary lung adenocarcinoma confirmed a positive association between lymphocyte infiltration and surfactant expression. Tumors with a high surfactant expression displayed a transcriptomic profile of an inflammatory microenvironment.

Conclusions:

The expression of surfactant metabolism-related genes (SFTPA1, SFTPB, and NAPSA) defines an inflamed subtype of lung adenocarcinoma brain metastases characterized by high abundance of TILs in close vicinity to tumor cells, a prolonged survival, and a tumor microenvironment which might be more accessible to immunotherapeutic approaches.

Antiretroviral therapy (ART) suppresses HIV-1 replication but fails to cure the infection. The presence of an extremely stable viral latent reservoir, primarily in resting memory CD4⁺ T cells, remains a major obstacle to viral eradication. The "shock and kill" strategy targets these latently infected cells and boosts immune recognition and clearance, and thus, it is a promising approach for an HIV-1 functional cure. Although some latency-reversing agents (LRAs) have been reported, no apparent clinical progress has been made, so it is still vital to seek novel and effective LRAs. Here, we report that thiostrepton (TSR), a proteasome inhibitor, reactivates latent HIV-1 effectively in cellular models and in primary CD4⁺ T cells from ART-suppressed individuals ex vivo. TSR does not induce global T cell activation, severe cytotoxicity, or CD8⁺ T cell dysfunction, making it a prospective LRA candidate. We also observed a significant synergistic effect of reactivation when TSR was combined with JQ1, prostratin, or bryostatin-1. Interestingly, six TSR analogues also show reactivation abilities that are similar to or more effective than that of TSR. We further verified that TSR upregulated expression of heat shock proteins (HSPs) in CD4⁺ T cells, which subsequently activated positive transcriptional elongation factor b (p-TEFb) and NF-B signals, leading to viral reactivation. In summary, we identify TSR as a novel LRA which could have important significance for applications to an HIV-1 functional cure in the future.

Staphylococcus aureus biofilms are a significant problem in health care settings, partly due to the presence of a nondividing, antibiotic-tolerant subpopulation. Here we evaluated treatment of S. aureus UAMS-1 biofilms with HT61, a quinoline derivative shown to be effective against nondividing Staphylococcus spp. HT61 was effective at reducing biofilm viability and was associated with increased expression of cell wall stress and division proteins, confirming its potential as a treatment for S. aureus biofilm infections.

Plasmodium vivax relapse is one of the major causes of sustained global malaria transmission. Primaquine (PQ) is the only commercial drug available to prevent relapses, and its efficacy is dependent on metabolic activation by cytochrome P450 2D6 (CYP2D6). Impaired CYP2D6 function, caused by allelic polymorphisms, leads to the therapeutic failure of PQ as a radical cure for P. vivax malaria. Here, we hypothesized that the host immune response to malaria parasites modulates susceptibility to P. vivax recurrences in association with CYP2D6 activity. We performed a 10-year retrospective study by genotyping CYP2D6 polymorphisms in 261 malaria-exposed individuals from the Brazilian Amazon. The immune responses against a panel of P. vivax blood-stage antigens were evaluated by serological assays. We confirmed our previous findings, which indicated an association between impaired CYP2D6 activity and a higher risk of multiple episodes of P. vivax recurrence (risk ratio, 1.75; 95% confidence interval [CI], 1.2 to 2.6; P = 0.0035). An important finding was a reduction of 3% in the risk of recurrence (risk ratio, 0.97; 95% CI, 0.96 to 0.98; P < 0.0001) per year of malaria exposure, which was observed for individuals with both reduced and normal CYP2D6 activity. Accordingly, subjects with long-term malaria exposure and persistent antibody responses to various antigens showed fewer episodes of malaria recurrence. Our findings have direct implications for malaria control, since it was shown that nonimmune individuals who do not respond adequately to treatment due to reduced CYP2D6 activity may present a significant challenge for sustainable progress toward P. vivax malaria elimination.

Patients with HPV16⁺ cervical cancer and high T-cell responses to an HPV16 vaccine survived longer.

Background:

Asian Americans are at higher risk for noncardia gastric cancers (NCGC) relative to non-Hispanic Whites (NHW). Asian Americans are genetically, linguistically, and culturally heterogeneous, yet have mostly been treated as a single population in prior studies. This aggregation may obscure important subgroup-specific cancer patterns.

Methods:

We utilized data from 13 regional United States cancer registries from 1990 to 2014 to determine secular trends in incidence and survivorship from NCGC. Data were analyzed for NHWs and the six largest Asian American subgroups: Chinese, Japanese, Filipino, Korean, Vietnamese, and South Asian (Indian/Pakistani).

Results:

There exists substantial heterogeneity in NCGC incidence between Asian subgroups, with Koreans (48.6 per 100,000 person-years) having seven-fold higher age-adjusted incidence than South Asians (7.4 per 100,000 person-years). Asians had generally earlier stages of diagnosis and higher rates of surgical resection compared with NHWs. All Asian subgroups also demonstrated higher 5-year observed survival compared with NHWs, with Koreans (41.3%) and South Asians (42.8%) having survival double that of NHWs (20.1%, P < 0.001). In multivariable regression, differences in stage of diagnosis and rates of resection partially explained the difference in survivorship between Asian subgroups.

Conclusions:

We find substantial differences in incidence, staging, histology, treatment, and survivorship from NCGC between Asian subgroups, data which challenge our traditional perceptions about gastric cancer in Asians. Both biological heterogeneity and cultural/environmental differences may underlie these findings.

Impact:

These data are relevant to the national discourse regarding the appropriate role of gastric cancer screening, and identifies high-risk racial/ethnic subgroups who many benefit from customized risk attenuation programs.

BACKGROUND AND PURPOSE:

Diffuse intrinsic pontine glioma is a lethal childhood brain cancer with dismal prognosis and MR imaging is the primary methodology used for diagnosis and monitoring. Our aim was to determine whether advanced diffusion, perfusion, and permeability MR imaging metrics predict survival and pseudoprogression in children with newly diagnosed diffuse intrinsic pontine glioma.

MATERIALS AND METHODS:

A clinical trial using the poly (adenosine diphosphate ribose) polymerase (PARP) inhibitor veliparib concurrently with radiation therapy, followed by maintenance therapy with veliparib + temozolomide, in children with diffuse intrinsic pontine glioma was conducted by the Pediatric Brain Tumor Consortium. Standard MR imaging, DWI, dynamic contrast-enhanced perfusion, and DSC perfusion were performed at baseline and approximately every 2 months throughout treatment. ADC histogram metrics of T2-weighted FLAIR and enhancing tumor volume, dynamic contrast-enhanced permeability metrics for enhancing tumors, and tumor relative CBV from DSC perfusion MR imaging were calculated. Baseline values, post-radiation therapy changes, and longitudinal trends for all metrics were evaluated for associations with survival and pseudoprogression.

RESULTS:

Fifty children were evaluable for survival analyses. Higher baseline relative CBV was associated with shorter progression-free survival (P = .02, Q = 0.089) and overall survival (P = .006, Q = 0.055). Associations of higher baseline mean transfer constant from the blood plasma into the extravascular extracellular space with shorter progression-free survival (P = .03, Q = 0.105) and overall survival (P = .03, Q = 0.102) trended toward significance. An increase in relative CBV with time was associated with shorter progression-free survival (P < .001, Q < 0.001) and overall survival (P = .004, Q = 0.043). Associations of longitudinal mean extravascular extracellular volume fraction with progression-free survival (P = .03, Q = 0.104) and overall survival (P = .03, Q = 0.105) and maximum transfer constant from the blood plasma into the extravascular extracellular space with progression-free survival (P = .03, Q = 0.102) trended toward significance. Greater increases with time were associated with worse outcomes. True radiologic progression showed greater post-radiation therapy decreases in mode_ADC_FLAIR compared with pseudoprogression (means, –268.15 versus –26.11, P = .01.)

CONCLUSIONS:

ADC histogram, perfusion, and permeability MR imaging metrics in diffuse intrinsic pontine glioma are useful in predicting survival and pseudoprogression.

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