Plants require a high concentration of ascorbate as a redox buffer for survival under stress conditions, such as high light. Dehydroascorbate reductases (DHARs) are enzymes that catalyze the reduction of DHA to ascorbate using reduced glutathione (GSH) as an electron donor, allowing rapid ascorbate recycling. However, a recent study using an Arabidopsis (Arabidopsis thaliana) triple mutant lacking all three DHAR genes (herein called dhar) did not find evidence for their role in ascorbate recycling under oxidative stress. To further study the function of DHARs, we generated dhar Arabidopsis plants as well as a quadruple mutant line combining dhar with an additional vtc2 mutation that causes ascorbate deficiency. Measurements of ascorbate in these mutants under low- or high-light conditions indicated that DHARs have a nonnegligible impact on full ascorbate accumulation under high light, but that they are dispensable when ascorbate concentrations are low to moderate. Because GSH itself can reduce DHA nonenzymatically, we used the pad2 mutant that contains ~30% of the wild-type GSH level. The pad2 mutant accumulated ascorbate at a wild-type level under high light; however, when the pad2 mutation was combined with dhar, there was near-complete inhibition of high-light–dependent ascorbate accumulation. The lack of ascorbate accumulation was consistent with a marked increase in the ascorbate degradation product threonate. These findings indicate that ascorbate recycling capacity is limited in dhar pad2 plants, and that both DHAR activity and GSH content set a threshold for high-light–induced ascorbate accumulation.

Objective

To investigate the frequency of levodopa-induced dyskinesia in dementia with Lewy bodies (DLBs) and Parkinson disease with dementia (PDD) and compare these frequencies with patients with incident Parkinson disease (PD) through a population-based cohort study.

We appreciate the readers' comments on the prevalence and impact of apathy on quality of life among individuals with Parkinson disease. In constructing our survey instrument, we discussed the inclusion of apathy as a symptom. However, we ultimately opted against inclusion because of concerns about the specificity of terminology in our online survey. Patients and care partners may not be familiar with the term "apathy,"and near-synonyms such as "reduced motivation" have substantial overlap with other nonmotor features. Still, as the readers point out, apathy is extremely common and under-recognized. Similar to many of the nonmotor symptoms identified in our study,¹ we agree that clinicians should be screening for apathy among those with Parkinson disease.

We read with great interest the study by Tarolli et al.,¹ which explored the burden of disease in Parkinson disease (PD) by evaluating the prevalence of nonmotor symptoms and their association with quality of life. The authors selected nonmotor symptoms based on literature review, expert opinions, and patient interviews. We note that apathy, which has major consequences for patients and carers, was not included as a relevant nonmotor symptom in their study. We performed a subcohort analysis of 60 patients from a study of pain in PD in 110 outpatients (PaCoMo-study, registered trial number: NL6311402917 [toetsingonline.nl]). We retrospectively reviewed the medical records to check whether the clinician identified apathy in these patients in the previous year, which was the case in 15% of the patients (n = 9). Blind to those results, patients were examined with the Apathy Scale (AS).² In total, 63.3% (n = 38) of the patients scored positive on the AS. Only 18.4% of the patients who scored positive on the AS were also classified or mentioned with apathy in the medical records by clinicians.

With high sensitivity in detecting acute brain events such as seizures, FDG PET can be used as an important tool for neurocutaneous melanosis disease monitoring.

BACKGROUND:

Although autodialer centralized reminder and recall (C-R/R) from state immunization information systems (IISs) has been shown to raise childhood vaccination rates, its impact on human papillomavirus (HPV) vaccination rates is unclear.

OBJECTIVES:

To determine if in utero selective serotonin reuptake inhibitor (SSRI) or selective serotonin norepinephrine inhibitor (SNRI) exposure is associated with developmental vulnerability in kindergarten among children whose mothers were diagnosed with prenatal mood or anxiety disorder.

The vertebrate limb serves as an experimental paradigm to study mechanisms that regulate development of the stereotypical skeletal elements. In this study, we simultaneously inactivated Sall4 using Hoxb6Cre and Plzf in mouse embryos, and found that their combined function regulates development of the proximal-anterior skeletal elements in hindlimbs. The Sall4; Plzf double knockout exhibits severe defects in the femur, tibia, and anterior digits, distinct defects compared to other allelic series of Sall4; Plzf. We found that Sall4 regulates Plzf expression prior to hindlimb outgrowth. Further expression analysis indicated that Hox10 genes and GLI3 are severely downregulated in the Sall4; Plzf double knockout hindlimb bud. In contrast, PLZF expression is reduced but detectable in Sall4; Gli3 double knockout limb buds, and SALL4 is expressed in the Plzf; Gli3 double knockout limb buds. These results indicate that Plzf, Gli3, and Hox10 genes downstream of Sall4, regulate femur and tibia development. In the autopod, we show that Sall4 negatively regulates Hedgehog signaling, which allows for development of the most anterior digit. Collectively, our study illustrates genetic systems that regulate development of the proximal-anterior skeletal elements in hindlimbs.

Replication initiation in eukaryotic cells occurs asynchronously throughout S phase, yielding early- and late-replicating regions of the genome, a process known as replication timing (RT). RT changes during development to ensure accurate genome duplication and maintain genome stability. To understand the relative contributions that cell lineage, cell cycle, and replication initiation regulators have on RT, we utilized the powerful developmental systems available in Drosophila melanogaster. We generated and compared RT profiles from mitotic cells of different tissues and from mitotic and endocycling cells of the same tissue. Our results demonstrate that cell lineage has the largest effect on RT, whereas switching from a mitotic to an endoreplicative cell cycle has little to no effect on RT. Additionally, we demonstrate that the RT differences we observed in all cases are largely independent of transcriptional differences. We also employed a genetic approach in these same cell types to understand the relative contribution the eukaryotic RT control factor, Rif1, has on RT control. Our results demonstrate that Rif1 can function in a tissue-specific manner to control RT. Importantly, the Protein Phosphatase 1 (PP1) binding motif of Rif1 is essential for Rif1 to regulate RT. Together, our data support a model in which the RT program is primarily driven by cell lineage and is further refined by Rif1/PP1 to ultimately generate tissue-specific RT programs.

Key Points

Key Points

Key Points

The spirochete Borrelia burgdorferi sensu lato is the causative agent of Lyme disease (LD). The spirochetes produce the CspZ protein that binds to a complement regulator, factor H (FH). Such binding downregulates activation of host complement to facilitate spirochete evasion of complement killing. However, vaccination with CspZ does not protect against LD infection. In this study, we demonstrated that immunization with CspZ-YA, a CspZ mutant protein with no FH-binding activity, protected mice from infection by several spirochete genotypes introduced via tick feeding. We found that the sera from CspZ-YA-vaccinated mice more efficiently eliminated spirochetes and blocked CspZ FH-binding activity than sera from CspZ-immunized mice. We also found that vaccination with CspZ, but not CspZ-YA, triggered the production of anti-FH antibodies, justifying CspZ-YA as an LD vaccine candidate. The mechanistic and efficacy information derived from this study provides insights into the development of a CspZ-based LD vaccine.

Endothelial activation and microvascular dysfunction are key pathogenic processes in severe malaria. We evaluated the early role of these processes in experimentally induced Plasmodium falciparum and P. vivax infection. Participants were enrolled in induced blood-stage malaria clinical trials. Plasma osteoprotegerin, angiopoietin-2, and von Willebrand Factor (vWF) levels were measured as biomarkers of endothelial activation. Microvascular function was assessed using peripheral arterial tonometry and near-infrared spectroscopy, and the endothelial glycocalyx was assessed by sublingual videomicroscopy and measurement of biomarkers of degradation. Forty-five healthy, malaria-naive participants were recruited from 5 studies. Osteoprotegerin and vWF levels increased in participants following inoculation with P. vivax (n = 16) or P. falciparum (n = 15), with the angiopoietin-2 level also increasing in participants following inoculation with P. falciparum. For both species, the most pronounced increase was seen in osteoprotegerin. This was particularly marked in participants inoculated with P. vivax, where the osteoprotegerin level correlated with the levels of parasitemia and the malaria clinical score. There were no changes in measures of endothelial glycocalyx or microvascular function. Plasma biomarkers of endothelial activation increased in early P. falciparum and P. vivax infection and preceded changes in the endothelial glycocalyx or microvascular function. The more pronounced increase in osteoprotegerin suggests that this biomarker may play a role in disease pathogenesis.

Implanted medical device-associated infections pose significant health risks, as they are often the result of bacterial biofilm formation. Staphylococcus aureus is a leading cause of biofilm-associated infections which persist due to mechanisms of device surface adhesion, biofilm accumulation, and reprogramming of host innate immune responses. We found that the S. aureus fibronectin binding protein A (FnBPA) is required for normal biofilm development in mammalian serum and that the SaeRS two-component system is required for functional FnBPA activity in serum. Furthermore, serum-developed biofilms deficient in FnBPA were more susceptible to macrophage invasion, and in a model of biofilm-associated implant infection, we found that FnBPA is crucial for the establishment of infection. Together, these findings show that S. aureus FnBPA plays an important role in physical biofilm development and represents a potential therapeutic target for the prevention and treatment of device-associated infections.

The translocated actin recruiting phosphoprotein (Tarp) is a multidomain type III secreted effector used by Chlamydia trachomatis. In aggregate, existing data suggest a role of this effector in initiating new infections. As new genetic tools began to emerge to study chlamydial genes in vivo, we speculated as to what degree Tarp function contributes to Chlamydia’s ability to parasitize mammalian host cells. To address this question, we generated a complete tarP deletion mutant using the fluorescence-reported allelic exchange mutagenesis (FRAEM) technique and complemented the mutant in trans with wild-type tarP or mutant tarP alleles engineered to harbor in-frame domain deletions. We provide evidence for the significant role of Tarp in C. trachomatis invasion of host cells. Complementation studies indicate that the C-terminal filamentous actin (F-actin)-binding domains are responsible for Tarp-mediated invasion efficiency. Wild-type C. trachomatis entry into HeLa cells resulted in host cell shape changes, whereas the tarP mutant did not. Finally, using a novel cis complementation approach, C. trachomatis lacking tarP demonstrated significant attenuation in a murine genital tract infection model. Together, these data provide definitive genetic evidence for the critical role of the Tarp F-actin-binding domains in host cell invasion and for the Tarp effector as a bona fide C. trachomatis virulence factor.

A Yersinia pestis mutant synthesizing an adjuvant form of lipid A (monophosphoryl lipid A, MPLA) displayed increased biogenesis of bacterial outer membrane vesicles (OMVs). To enhance the immunogenicity of the OMVs, we constructed an Asd-based balanced-lethal host-vector system that oversynthesized the LcrV antigen of Y. pestis, raised the amounts of LcrV enclosed in OMVs by the type II secretion system, and eliminated harmful factors like plasminogen activator (Pla) and murine toxin from the OMVs. Vaccination with OMVs containing MPLA and increased amounts of LcrV with diminished toxicity afforded complete protection in mice against subcutaneous challenge with 8 x 10⁵ CFU (80,000 50% lethal dose [LD₅₀]) and intranasal challenge with 5 x 10³ CFU (50 LD₅₀) of virulent Y. pestis. This protection was significantly superior to that resulting from vaccination with LcrV/alhydrogel or rF1-V/alhydrogel. At week 4 postimmunization, the OMV-immunized mice showed more robust titers of antibodies against LcrV, Y. pestis whole-cell lysate (YPL), and F1 antigen and more balanced IgG1:IgG2a/IgG2b-derived Th1 and Th2 responses than LcrV-immunized mice. Moreover, potent adaptive and innate immune responses were stimulated in the OMV-immunized mice. Our findings demonstrate that self-adjuvanting Y. pestis OMVs provide a novel plague vaccine candidate and that the rational design of OMVs could serve as a robust approach for vaccine development.

Programmed death-ligand 1 (PD-L1/B7-H1) serves as a cosignaling molecule in cell-mediated immune responses and contributes to chronicity of inflammation and the escape of tumor cells from immunosurveillance. Here, we investigated the molecular mechanisms leading to PD-L1 upregulation in human oral carcinoma cells and in primary human gingival keratinocytes in response to infection with Porphyromonas gingivalis (P. gingivalis), a keystone pathogen for the development of periodontitis. The bacterial cell wall component peptidoglycan uses bacterial outer membrane vesicles to be taken up by cells. Internalized peptidoglycan triggers cytosolic receptors to induce PD-L1 expression in a myeloid differentiation primary response 88 (Myd88)-independent and receptor-interacting serine/threonine-protein kinase 2 (RIP2)-dependent fashion. Interference with the kinase activity of RIP2 or mitogen-activated protein (MAP) kinases interferes with inducible PD-L1 expression.

Phagocytosis is the key mechanism for host control of Pseudomonas aeruginosa, a motile Gram-negative, opportunistic bacterial pathogen which frequently undergoes adaptation and selection for traits that are advantageous for survival. One such clinically relevant adaptation is the loss of bacterial motility, observed within chronic infections, that is associated with increased antibiotic tolerance and phagocytic resistance. Previous studies using phagocytes from a leukocyte adhesion deficiency type 1 (LAD-I) patient identified CD18 as a putative cell surface receptor for uptake of live P. aeruginosa. However, how bacterial motility alters direct engagement with CD18-containing integrins remains unknown. Here we demonstrate, with the use of motile and isogenic nonmotile deletion mutants of two independent strains of P. aeruginosa and with CRISPR-generated CD18-deficient cell lines in human monocytes and murine neutrophils, that CD18 expression facilitates the uptake of both motile and nonmotile P. aeruginosa. However, unexpectedly, mechanistic studies revealed that CD18 expression was dispensable for the initial attachment of the bacteria to the host cells, which was validated with ectopic expression of complement receptor 3 (CR3) by CHO cells. Our data support that surface N-linked glycan chains (N-glycans) likely facilitate the initial interaction of bacteria with monocytes and cooperate with CD18 integrins in trans to promote internalization of bacteria. Moreover, talin-1 and kindlin-3 proteins promote uptake, but not binding, of P. aeruginosa by murine neutrophils, which supports a role for CD18 integrin signaling in this process. These findings provide novel insights into the cellular determinants for phagocytic recognition and uptake of P. aeruginosa.

Kaposi sarcoma-associated herpesvirus (KSHV) is necessary but not sufficient for primary effusion lymphoma (PEL) development. Alterations in cellular signaling pathways are also a characteristic of PEL. Other B cell lymphomas have acquired an oncogenic mutation in the myeloid differentiation primary response 88 (MYD88) gene. The MYD88 L265P mutant results in the activation of interleukin-1 receptor associated kinase (IRAK). To probe IRAK/MYD88 signaling in PEL, we employed CRISPR/Cas9 technology to generate stable deletion clones in BCBL-1Cas9 and BC-1Cas9 cells. To look for off-target effects, we determined the complete exome of the BCBL-1Cas9 and BC-1Cas9 cells. Deletion of either MYD88, IRAK4, or IRAK1 abolished interleukin-1 beta (IL-1β) signaling; however, we were able to grow stable subclones from each population. Transcriptome sequencing (RNA-seq) analysis of IRAK4 knockout cell lines (IRAK4 KOs) showed that the IRAK pathway induced cellular signals constitutively, independent of IL-1β stimulation, which was abrogated by deletion of IRAK4. Transient complementation with IRAK1 increased NF-B activity in MYD88 KO, IRAK1 KO, and IRAK4 KO cells even in the absence of IL-1β. IL-10, a hallmark of PEL, was dependent on the IRAK pathway, as IRAK4 KOs showed reduced IL-10 levels. We surmise that, unlike B cell receptor (BCR) signaling, MYD88/IRAK signaling is constitutively active in PEL, but that under cell culture conditions, PEL rapidly became independent of this pathway.

IMPORTANCE One hundred percent of primary effusion lymphoma (PEL) cases are associated with Kaposi sarcoma-associated herpesvirus (KSHV). PEL cell lines, such as BCBL-1, are the workhorse for understanding this human oncovirus and the host pathways that KSHV dysregulates. Understanding their function is important for developing new therapies as well as identifying high-risk patient groups. The myeloid differentiation primary response 88 (MYD88)/interleukin-1 receptor associated kinase (IRAK) pathway, which has progrowth functions in other B cell lymphomas, has not been fully explored in PEL. By performing CRISPR/Cas9 knockout (KO) studies targeting the IRAK pathway in PEL, we were able to determine that established PEL cell lines can circumvent the loss of IRAK1, IRAK4, and MYD88; however, the deletion clones are deficient in interleukin-10 (IL-10) production. Since IL-10 suppresses T cell function, this suggests that the IRAK pathway may serve a function in vivo and during early-stage development of PEL.

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.

Members of the flavivirus genus share a high level of sequence similarity and often circulate in the same geographical regions. However, whether T cells induced by one viral species cross-react with other related flaviviruses has not been globally addressed. In this study, we tested pools of epitopes derived from dengue (DENV), Zika (ZIKV), Japanese encephalitis (JEV), West Nile (WNV), and yellow fever (YFV) viruses by intracellular cytokine staining (ICS) using peripheral blood mononuclear cells (PBMCs) of individuals naturally exposed to DENV or immunized with DENV (TV005) or YF17D vaccine. CD8 T cell responses recognized epitopes from multiple flaviviruses; however, the magnitude of cross-reactive responses was consistently severalfold lower than those to the autologous epitope pools and was associated with lower expression of activation markers such as CD40L, CD69, and CD137. Next, we characterized the antigen sensitivity of short-term T cell lines (TCL) representing 29 different individual epitope/donor combinations. TCL derived from DENV monovalent vaccinees induced CD8 and CD4 T cells that cross-reacted within the DENV serocomplex but were consistently associated with >100-fold-lower antigen sensitivity for most other flaviviruses, with no cross-recognition of YFV-derived peptides. CD8 and CD4 TCL from YF17D vaccinees were associated with very limited cross-reactivity with any other flaviviruses and in five out of eight cases >1,000-fold-lower antigen sensitivity. Overall, our data suggest limited cross-reactivity for both CD4 and CD8 T cell responses between flaviviruses and have implications for understanding immunity elicited by natural infection and strategies to develop live attenuated vaccines against flaviviral species.

IMPORTANCE The envelope (E) protein is the dominant target of neutralizing antibodies for dengue virus (DENV) and yellow fever virus (YFV). Accordingly, several DENV vaccine constructs use the E protein in a live attenuated vaccine format, utilizing a backbone derived from a heterologous flavivirus (such as YF) as a delivery vector. This backbone comprises the nonstructural (NS) and capsid (C) antigens, which are dominant targets of T cell responses. Here, we demonstrate that cross-reactivity at the level of T cell responses among different flaviviruses is very limited, despite high levels of sequence homology. Thus, the use of heterologous flavivirus species as a live attenuated vaccine vector is not likely to generate optimal T cell responses and might thus impair vaccine performance.

Genetic variants within complement factor H (CFH), a major alternative complement pathway regulator, are associated with the development of age-related macular degeneration (AMD) and other complementopathies. This is explained with the reduced binding of CFH or its splice variant factor H-like protein 1 (FHL-1) to self-ligands or altered self-ligands (e.g.,...

At the pinnacle of the 17th century scientific revolution, René Descartes, the father of modern philosophy, published his monumental Meditations on First Philosophy (1), in which he proposed a division between soul and body—mind and brain—with the former in charge of our thoughts and conscious decisions (res cogitans) and the...

Proteasomes are essential protease complexes that maintain cellular homeostasis, and aberrant proteasomal activity supports cancer development. The regulatory mechanisms and biological function of the ubiquitin-26S proteasome have been studied extensively, while those of the ubiquitin-independent 20S proteasome system remain obscure. Here, we show that the cap ’n’ collar (CNC) family transcription factor NRF3 specifically enhances 20S proteasome assembly in cancer cells and that 20S proteasomes contribute to colorectal cancer development through ubiquitin-independent proteolysis of the tumor suppressor p53 and retinoblastoma (Rb) proteins. The NRF3 gene is highly expressed in many cancer tissues and cell lines and is important for cancer cell growth. In cancer cells, NRF3 upregulates the assembly of the 20S proteasome by directly inducing the gene expression of the 20S proteasome maturation protein POMP. Interestingly, NRF3 knockdown not only increases p53 and Rb protein levels but also increases p53 activities for tumor suppression, including cell cycle arrest and induction of apoptosis. Furthermore, protein stability and cell viability assays using two distinct proteasome inhibitor anticancer drugs, the 20S proteasome inhibitor bortezomib and the ubiquitin-activating enzyme E1 inhibitor TAK-243, show that the upregulation of the NRF3-POMP axis leads to ubiquitin-independent proteolysis of p53 and Rb and to impaired sensitivity to bortezomib but not TAK-243. More importantly, the NRF3-POMP axis supports tumorigenesis and metastasis, with higher NRF3/POMP expression levels correlating with poor prognoses in patients with colorectal or rectal adenocarcinoma. These results suggest that the NRF3-POMP-20S proteasome assembly axis is significant for cancer development via ubiquitin-independent proteolysis of tumor suppressor proteins.

Background:

Patient identification is an important step for advance care planning (ACP) discussions.

Erin Gorrell, Ashley Shemery, Jesse Kowalski, Miranda Bodziony, Nhlalala Mavundza, Amber R. Titus, Mark Yoder, Sarah Mull, Lydia A. Heemstra, Jacob G. Wagner, Megan Gibson, Olivia Carey, Diamond Daniel, Nicholas Harvey, Meredith Zendlo, Megan Rich, Scott Everett, Chaitanya K. Gavini, Tariq I. Almundarij, Diane Lorton, and Colleen M. Novak

Non-shivering thermogenesis can promote negative energy balance and weight loss. In this study, we identified a contextual stimulus that induces rapid and robust thermogenesis in skeletal muscle. Rats exposed to the odor of a natural predator (ferret) showed elevated skeletal muscle temperatures detectable as quickly as 2 min after exposure, reaching maximum thermogenesis of >1.5°C at 10–15 min. Mice exhibited a similar thermogenic response to the same odor. Ferret odor induced a significantly larger and qualitatively different response from that of novel or aversive odors, fox odor or moderate restraint stress. Exposure to predator odor increased energy expenditure, and both the thermogenic and energetic effects persisted when physical activity levels were controlled. Predator odor-induced muscle thermogenesis is subject to associative learning as exposure to a conditioned stimulus provoked a rise in muscle temperature in the absence of the odor. The ability of predator odor to induce thermogenesis is predominantly controlled by sympathetic nervous system activation of β-adrenergic receptors, as unilateral sympathetic lumbar denervation and a peripherally acting β-adrenergic antagonist significantly inhibited predator odor-induced muscle thermogenesis. The potential survival value of predator odor-induced changes in muscle physiology is reflected in an enhanced resistance to running fatigue. Lastly, predator odor-induced muscle thermogenesis imparts a meaningful impact on energy expenditure as daily predator odor exposure significantly enhanced weight loss with mild calorie restriction. This evidence signifies contextually provoked, centrally mediated muscle thermogenesis that meaningfully impacts energy balance.

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.

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 serine/threonine kinases MST1 and MST2 are core components of the Hippo pathway, which has been found to be critically involved in embryonic kidney development. Yes-associated protein (YAP) and transcriptional coactivator with PDZ-binding motif (TAZ) are the pathway’s main effectors. However, the biologic functions of the Hippo/YAP pathway in adult kidneys are not well understood, and the functional role of MST1 and MST2 in the kidney has not been studied.

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.

Hypoxia is a hallmark of solid cancers, supporting proliferation, angiogenesis, and escape from apoptosis. There is still limited understanding of how cancer cells adapt to hypoxic conditions and survive. We analyzed transcriptome changes of human lung and breast cancer cells under chronic hypoxia. Hypoxia induced highly concordant changes in transcript abundance, but divergent splicing responses, underlining the cell type-specificity of alternative splicing programs. While RNA-binding proteins were predominantly reduced, hypoxia specifically induced muscleblind-like protein 2 (MBNL2). Strikingly, MBNL2 induction was critical for hypoxia adaptation by controlling the transcript abundance of hypoxia response genes, such as vascular endothelial growth factor A (VEGFA). MBNL2 depletion reduced the proliferation and migration of cancer cells, demonstrating an important role of MBNL2 as cancer driver. Hypoxia control is specific for MBNL2 and not shared by its paralog MBNL1. Thus, our study revealed MBNL2 as central mediator of cancer cell responses to hypoxia, regulating the expression and alternative splicing of hypoxia-induced genes.

Functions of eukaryotic mRNAs are characterized by intramolecular interactions between their ends. We have addressed the question whether 5' and 3' ends meet by diffusion-controlled encounter "through solution" or by a mechanism involving the RNA backbone. For this purpose, we used a translation system derived from Drosophila embryos that displays two types of 5'–3' interactions: Cap-dependent translation initiation is stimulated by the poly(A) tail and inhibited by Smaug recognition elements (SREs) in the 3' UTR. Chimeric RNAs were made consisting of one RNA molecule carrying a luciferase coding sequence and a second molecule containing SREs and a poly(A) tail; the two were connected via a protein linker. The poly(A) tail stimulated translation of such chimeras even when disruption of the RNA backbone was combined with an inversion of the 5'–3' polarity between the open reading frame and poly(A) segment. Stimulation by the poly(A) tail also decreased with increasing RNA length. Both observations suggest that contacts between the poly(A) tail and the 5' end are established through solution, independently of the RNA backbone. In the same chimeric constructs, SRE-dependent inhibition of translation was also insensitive to disruption of the RNA backbone. Thus, tracking of the backbone is not involved in the repression of cap-dependent initiation. However, SRE-dependent repression was insensitive to mRNA length, suggesting that the contact between the SREs in the 3' UTR and the 5' end of the RNA might be established in a manner that differs from the contact between the poly(A) tail and the cap.

BACKGROUND:Patients with cystic fibrosis develop decreased exercise capacity. However, the main factors responsible for this decline are still unclear. Thus, the objective of this study was to evaluate the factors influencing exercise capacity assessed with the modified shuttle test (MST) in individuals with cystic fibrosis.METHODS:A cross-sectional study was carried out in subjects with a diagnosis of cystic fibrosis who were 6–26 y old and were regularly monitored at 2 cystic fibrosis reference centers in Brazil. Individuals who were unable to perform the tests or who exhibited hemodynamic instability and exacerbation of respiratory symptoms were excluded. Anthropometric, clinical, and genotype data were collected. In addition, lung function and exercise capacity were evaluated with the MST.RESULTS:73 subjects (mean age 12.2 ± 4.9 y and FEV1 76.8 ± 23.3%) were included. The mean distance achieved in the MST was 765 ± 258 m (71.6% of predicted). The distance achieved on the MST correlated significantly with age (r = 0.49, P < .001), body mass index (r = 0.41, P < .001), resting heart rate (r = −0.51, P < .001), and FEV1 (r = 0.24, P = .042). Subjects with FEV1 > 67% of predicted (P = .02) and those with resting heart rate < 100 beats/min (P = .01) had a greater exercise capacity. Resting heart rate, age, and FEV1 (%) were found as significant variables to explain the distance achieved on the MST (R2 = 0.48, standard error = 191.0 m).CONCLUSIONS:The main determinants of exercise capacity assessed with the MST in individuals with cystic fibrosis were resting heart rate, age, and lung function.

BACKGROUND:Prolonged ventilatory weaning may expose patients to unnecessary discomfort, increase the risk of complications, and raise the costs of hospital treatment. In this scenario, indexes that reliably predict successful liberation can be helpful.OBJECTIVE:To evaluate the intra- and interobserver reproducibility of the timed inspiratory effort index as a weaning predictor.METHODS:This prospective observational study included subjects judged as able to start liberation from mechanical ventilation. For the intra-observer analysis, the same investigator performed 2 measurements in each selected patient with an interval of 30 min a rest. For interobserver analysis, 2 measurements were obtained in another sample of subjects, also with an interval of 30 min rest, but each of one performed by a different investigator. The Bland-Altman diagram, the coefficient concordance of kappa, and the Pearson correlation coefficient were used to compare the measurements. The performance of the timed inspiratory effort index was assessed by receiver operating characteristic curves. Values of P < .05 were considered significant.RESULTS:We selected 113 subjects (43 males; mean ± SD age, 77 ± 14 y). Fifty-six (49.6%) achieved successful liberation, and 33 (29%) died in the ICU. The mean ± SD duration of mechanical ventilation was 14.4 ± 6.7 d. The Bland-Altman diagrams that addressed intra- and interobservers agreement showed low variability between measurements. Values of the concordance coefficients of kappa were 0.82 (0.68–0.95) and 0.80 (0.65–0.94), and of the linear correlation coefficients, 0.86 (0.77–0.91) and 0.89 (0.82–0.93) for the intra- and interobservers measurements, respectively. The mean ± SD values for the area under the curve for each pair of the intra- and interobserver measurements were 0.96 ± 0.07 versus 0.94 ± 0.07 (P = .41) and 0.94 ± 0.05 versus 0.90 ± 0.07 (P = .14), respectively.CONCLUSIONS:The variability of the measurement of the timed inspiratory effort index by intra- and interobservers showed very high reproducibility, which reinforced the index as a sensible, accurate, and reliable outcome predictor of liberation from mechanical ventilation.

Drug-induced liver injury (DILI) is a global medical problem. The risk of DILI is often related to expression and activities of drug-metabolizing enzymes, especially cytochrome P450s (P450s). However, changes on expression and activities of P450s after DILI have not been determined. The aim of this study is to fill this knowledge gap. Acetaminophen (APAP) was used as a model drug to induce DILI in C57BL/6J mice at different ages of days 10 (infant), 22 (child), and 60 (adult). DILI was assessed by levels of alanine aminotransferase and aspartate aminotransferase in plasma with a confirmation by H&E staining on liver tissue sections. The expression of selected P450s at mRNA and protein levels was measured by real-time polymerase chain reaction and liquid chromatography–tandem mass spectrometry, respectively. The activities of these P450s were determined by the formation of metabolites from probe drugs for each P450 using ultraperformance liquid chromatography–quadrupole time of flight mass spectrometry. DILI was induced at mild to severe levels in a dose-dependent manner in 200, 300, and 400 mg/kg APAP-treated groups at child and adult ages, but not at the infant age. Significantly decreased expression at mRNA and protein levels as well as enzymatic activities of CYP2E1, 3A11, 1A2, and 2C29 were found at child and adult ages. Adult male mice were more susceptible to APAP-induced liver injury than female mice with more decreased expression of P450s. These results suggest that altered levels of P450s in livers severely injured by drugs may affect the therapeutic efficacy of drugs, which are metabolized by P450s, more particularly for males.

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.

The quinoprotein glycine oxidase from the marine bacterium Pseudoalteromonas luteoviolacea (PlGoxA) uses a protein-derived cysteine tryptophylquinone (CTQ) cofactor to catalyze conversion of glycine to glyoxylate and ammonia. This homotetrameric enzyme exhibits strong cooperativity toward glycine binding. It is a good model for studying enzyme kinetics and cooperativity, specifically for being able to separate those aspects of protein function through directed mutagenesis. Variant proteins were generated with mutations in four active-site residues, Phe-316, His-583, Tyr-766, and His-767. Structures for glycine-soaked crystals were obtained for each. Different mutations had differential effects on kcat and K0.5 for catalysis, K0.5 for substrate binding, and the Hill coefficients describing the steady-state kinetics or substrate binding. Phe-316 and Tyr-766 variants retained catalytic activity, albeit with altered kinetics and cooperativity. Substitutions of His-583 revealed that it is essential for glycine binding, and the structure of H583C PlGoxA had no active-site glycine present in glycine-soaked crystals. The structure of H767A PlGoxA revealed a previously undetected reaction intermediate, a carbinolamine product-reduced CTQ adduct, and exhibited only negligible activity. The results of these experiments, as well as those with the native enzyme and previous variants, enabled construction of a detailed mechanism for the reductive half-reaction of glycine oxidation. This proposed mechanism includes three discrete reaction intermediates that are covalently bound to CTQ during the reaction, two of which have now been structurally characterized by X-ray crystallography.

Interferon-regulated myxovirus resistance protein B (MxB) is an interferon-induced GTPase belonging to the dynamin superfamily. It inhibits infection with a wide range of different viruses, including HIV-1, by impairing viral DNA entry into the nucleus. Unlike the related antiviral GTPase MxA, MxB possesses an N-terminal region that contains a nuclear localization signal and is crucial for inhibiting HIV-1. Because MxB previously has been shown to reside in both the nuclear envelope and the cytoplasm, here we used bioinformatics and biochemical approaches to identify a nuclear export signal (NES) responsible for MxB's cytoplasmic location. Using the online computational tool LocNES (Locating Nuclear Export Signals or NESs), we identified five putative NES candidates in MxB and investigated whether their deletion caused nuclear localization of MxB. Our results revealed that none of the five deletion variants relocates to the nucleus, suggesting that these five predicted NES sequences do not confer NES activity. Interestingly, deletion of one sequence, encompassing amino acids 505–527, abrogated the anti-HIV-1 activity of MxB. Further mutation experiments disclosed that amino acids 515–519, and Pro-515 in particular, regulate MxB oligomerization and its binding to HIV-1 capsid, thereby playing an important role in MxB-mediated restriction of HIV-1 infection. In summary, our results indicate that none of the five predicted NES sequences in MxB appears to be required for its nuclear export. Our findings also reveal several residues in MxB, including Pro-515, critical for its oligomerization and anti-HIV-1 function.

The heterodimeric cytokine interleukin-23 (IL-23 or IL23A/IL12B) is produced by dendritic cells and macrophages and promotes the proinflammatory and regenerative activities of T helper 17 (Th17) and innate lymphoid cells. A recent study has reported that IL-23 is also secreted by lung adenoma cells and generates an inflammatory and immune-suppressed stroma. Here, we observed that proinflammatory tumor necrosis factor (TNF)/NF-κB and mitogen-activated protein kinase (MAPK) signaling strongly induce IL23A expression in intestinal epithelial cells. Moreover, we identified a strong crosstalk between the NF-κB and MAPK/ERK kinase (MEK) pathways, involving the formation of a transcriptional enhancer complex consisting of proto-oncogene c-Jun (c-Jun), RELA proto-oncogene NF-κB subunit (RelA), RUNX family transcription factor 1 (RUNX1), and RUNX3. Collectively, these proteins induced IL23A secretion, confirmed by immunoprecipitation of endogenous IL23A from activated human colorectal cancer (CRC) cell culture supernatants. Interestingly, IL23A was likely secreted in a noncanonical form, as it was not detected by an ELISA specific for heterodimeric IL-23 likely because IL12B expression is absent in CRC cells. Given recent evidence that IL23A promotes tumor formation, we evaluated the efficacy of MAPK/NF-κB inhibitors in attenuating IL23A expression and found that the MEK inhibitor trametinib and BAY 11–7082 (an IKKα/IκB inhibitor) effectively inhibited IL23A in a subset of human CRC lines with mutant KRAS or BRAFV600E mutations. Together, these results indicate that proinflammatory and mitogenic signals dynamically regulate IL23A in epithelial cells. They further reveal its secretion in a noncanonical form independent of IL12B and that small-molecule inhibitors can attenuate IL23A secretion.

Carbon monoxide (CO) remains the most common cause of human poisoning. The consequences of CO poisoning include cardiac dysfunction, brain injury, and death. CO causes toxicity by binding to hemoglobin and by inhibiting mitochondrial cytochrome c oxidase (CcO), thereby decreasing oxygen delivery and inhibiting oxidative phosphorylation. We have recently developed a CO antidote based on human neuroglobin (Ngb-H64Q-CCC). This molecule enhances clearance of CO from red blood cells in vitro and in vivo. Herein, we tested whether Ngb-H64Q-CCC can also scavenge CO from CcO and attenuate CO-induced inhibition of mitochondrial respiration. Heart tissue from mice exposed to 3% CO exhibited a 42 ± 19% reduction in tissue respiration rate and a 33 ± 38% reduction in CcO activity compared with unexposed mice. Intravenous infusion of Ngb-H64Q-CCC restored respiration rates to that of control mice correlating with higher electron transport chain CcO activity in Ngb-H64Q-CCC–treated compared with PBS-treated, CO-poisoned mice. Further, using a Clark-type oxygen electrode, we measured isolated rat liver mitochondrial respiration in the presence and absence of saturating solutions of CO (160 μm) and nitric oxide (100 μm). Both CO and NO inhibited respiration, and treatment with Ngb-H64Q-CCC (100 and 50 μm, respectively) significantly reversed this inhibition. These results suggest that Ngb-H64Q-CCC mitigates CO toxicity by scavenging CO from carboxyhemoglobin, improving systemic oxygen delivery and reversing the inhibitory effects of CO on mitochondria. We conclude that Ngb-H64Q-CCC or other CO scavengers demonstrate potential as antidotes that reverse the clinical and molecular effects of CO poisoning.

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.

The Dukla Nappe in the Skrzydlna area exposes two types of mélange reflecting two different phases of basinal and tectonic evolution of the Outer West Carpathian orogen in its Polish sector. The Oligocene-age sedimentary mélange (olistostrome) is related to growth of the accretionary wedge, whereas the Miocene-age diapiric mélange postdates the orogenic thrusting. Textural and structural features of the very coarse-grained sedimentary mélange suggest non-cohesive debris flows and high-density turbidity currents as predominant emplacement mechanisms. Growth strata, associated with progressive unconformities, and facies contrast between the underlying fine-grained unit and the overlying olistostrome reflect a considerable uplift of the source area and rotation of the adjacent part of the basin floor. The olistostrome and the overlying turbidite succession form a retrogressive sequence interpreted as a submarine canyon infill grading to a small submarine fan. The diapiric mélange, injected into the Oligocene-age succession of the Dukla Nappe, contains the Early and Late Cretaceous-age blocks and matrix derived from the underlying Silesian Nappe. The features reflecting diapiric emplacement include matrix proportion increase and block content decrease towards the mélange margins, scaly fabric and shear zones. Both mélanges, interpreted in the past as chaotic bodies, upon detailed examination reveal genesis-related subtle internal organization.

Laterites preserved on both sides of the Western Ghats Escarpment of Peninsular India have formed by long-term lateritic weathering essentially after India–Seychelles continental break-up following Deccan Traps emplacement (c. 63 myr ago). Supergene manganese ores of the Western Ghats were formed on Late Archean manganese protores. Among Mn oxides composing the ores, cryptomelane (K-rich Mn oxide) was characterized and dated by ⁴⁰Ar/³⁹Ar geochronology. Measured ages complement those previously obtained in other South Indian manganese ores from the hinterland plateau and further document three major weathering periods, c. 53–44, c. 39–22 and c. 14–10 Ma, the last being documented for the first time in India. These periods coincide with global palaeoclimatic proxies and date the lateritic weathering of three successive palaeolandscapes of the Western Ghats that evolved under slow denudation (c. 8 m Ma^–1) over the last 44 myr and were mostly incised during the Neogene (<22 Ma). This indicates that the Western Ghats are a relict of a South Indian plateau preserved at the headwaters of very long east-flowing river systems and above the Western Ghats Escarpment. Topography and denudation history of this landscape do not require Neogene tilt of the Peninsula as recently proposed.

Supplementary material: Full details of field and sample description, methods and analytical data including electron probe microanalyses of cryptomelane, and isotopic analyses and degassing spectra of irradiated cryptomelane grains are available at https://doi.org/10.6084/m9.figshare.c.4726661

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.)

We previously produced a heavy-chain-only antibody (Ab) VH domain (V_HH)-displayed phage library from two alpacas that had been immunized with ricin toxoid and nontoxic mixtures of the enzymatic ricin toxin A subunit (RTA) and binding ricin toxin B subunit (RTB) (D. J. Vance, J. M. Tremblay, N. J. Mantis, and C. B. Shoemaker, J Biol Chem 288:36538–36547, 2013, https://doi.org/10.1074/jbc.M113.519207). Initial and subsequent screens of that library by direct enzyme-linked immunosorbent assay (ELISA) yielded more than two dozen unique RTA- and RTB-specific V_HHs, including 10 whose structures were subsequently solved in complex with RTA. To generate a more complete antigenic map of ricin toxin and to define the epitopes associated with toxin-neutralizing activity, we subjected the V_HH-displayed phage library to additional "pannings" on both receptor-bound ricin and antibody-captured ricin. We now report the full-length DNA sequences, binding affinities, and neutralizing activities of 68 unique V_HHs: 31 against RTA, 33 against RTB, and 4 against ricin holotoxin. Epitope positioning was achieved through cross-competition ELISAs performed with a panel of monoclonal antibodies (MAbs) and verified, in some instances, with hydrogen-deuterium exchange mass spectrometry. The 68 V_HHs grouped into more than 20 different competition bins. The RTA-specific V_HHs with strong toxin-neutralizing activities were confined to bins that overlapped two previously identified neutralizing hot spots, termed clusters I and II. The four RTB-specific V_HHs with potent toxin-neutralizing activity grouped within three adjacent bins situated at the RTA-RTB interface near cluster II. These results provide important insights into epitope interrelationships on the surface of ricin and delineate regions of vulnerability that can be exploited for the purpose of vaccine and therapeutic development.