In recent years, there have been intensive debates on whether healthy adults acquire new word knowledge through fast mapping (FM) by a different mechanism from explicit encoding (EE). In this study, we focused on this issue and investigated to what extent retention interval, prior knowledge (PK), and lure type modulated memory after FM and EE. Healthy young participants were asked to learn novel word-picture associations through both FM and EE. Half of the pictures were from familiar categories (i.e., high PK) and the other half were from unfamiliar categories (i.e., low PK). After 10 min and 1 wk, the participants were tested by forced-choice (FC) tasks, with lures from different categories (Experiment 1) or from the same categories of the target pictures (Experiment 2). Pseudowords were used to denote names of the novel pictures and baseline performance was controlled for each task. The results showed that in both Experiments 1 and 2, memory performance remained stable after FM, while it declined after EE from 10 min to 1 wk. Moreover, the effect of PK appeared at 10 min after FM while at 1 wk after EE in Experiment 2. PK enhanced memory of word-picture associations when the lures were from the same categories (Experiment 2), rather than from different categories (Experiment 1). These results were largely confirmed in Experiment 3 when encoding condition was manipulated as a between-subjects factor, while lure type as a within-subjects factor. The findings suggest that different from EE, FM facilitates rapid acquisition and consolidation of word-picture knowledge, and highlight that PK plays an important role in this process by enhancing access to detailed information.

Asja Guzman, Rachel C. Avard, Alexander J. Devanny, Oh Sang Kweon, and Laura J. Kaufman

The study of cancer cell invasion in 3D environments in vitro has revealed a variety of invasive modes, including amoeboid migration, characterized by primarily round cells that invade in a protease- and adhesion-independent manner. Here, we delineate a contractility-dependent migratory mode of primarily round breast cancer cells that is associated with extensive integrin-mediated extracellular matrix (ECM) reorganization that occurs at membrane blebs, with bleb necks sites of integrin clustering and integrin-dependent ECM alignment. We show that the spatiotemporal distribution of blebs and their utilization for ECM reorganization is mediated by functional β1 integrin receptors and other components of focal adhesions. Taken together, the work presented here characterizes a migratory mode of primarily round cancer cells in complex 3D environments and reveals a fundamentally new function for membrane blebs in cancer cell invasion.

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.

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.

Neuroendocrinelike transdifferentiation of prostate cancer adenocarcinomas correlates with serum levels of chromogranin A (CgA) and drives treatment resistance. The aim of this work was to evaluate whether CgA can serve as a response predictor for ¹⁷⁷Lu-prostate-specific membrane antigen 617 (PSMA) radioligand therapy (RLT) in comparison with the established tumor markers. Methods: One hundred consecutive patients with metastasized castration-resistant prostate cancer scheduled for PSMA RLT were evaluated for prostate-specific antigen (PSA), lactate dehydrogenase (LDH), and CgA at baseline and in follow-up of PSMA RLT. Tumor uptake of PSMA ligand, a known predictive marker for response, was assessed as a control variable. Results: From the 100 evaluated patients, 35 had partial remission, 16 stable disease, 15 mixed response, and 36 progression of disease. Tumor uptake above salivary gland uptake translated into partial remission, with an odds ratio (OR) of 60.265 (95% confidence interval [CI], 5.038–720.922). Elevated LDH implied a reduced chance for partial remission, with an OR of 0.094 (95% CI, 0.017–0.518), but increased the frequency of progressive disease (OR, 2.717; 95% CI, 1.391–5.304). All patients who achieved partial remission had a normal baseline LDH. Factor-2 elevation of CgA increased the risk for progression, with an OR of 3.089 (95% CI, 1.302–7.332). Baseline PSA had no prognostic value for response prediction. Conclusion: In our cohort, baseline PSA had no prognostic value for response prediction. LDH was the marker with the strongest prognostic value, and elevated LDH increased the risk for progression of disease under PSMA RLT. Elevated CgA demonstrated a moderate impact as a negative prognostic marker in general but was explicitly related to the presence of liver metastases. Well in line with the literature, sufficient tumor uptake is a prerequisite to achieve tumor response.

Parallel to the application of new PET radiopharmaceuticals for pheochromocytoma and paraganglioma (collectively named PPGLs) imaging, several studies have increased our understanding on their biology, genetics, metabolomics, and embryologic origin. In this review, we highlight the current relationship between genotypes and molecular imaging phenotypes. Additionally, we summarize the referral guidelines for imaging of PPGL patients with or without knowledge of their genetic background.

Chronic cough is a common complaint in the general population but there are no precise data on the incidence of, and prospectively examined risk factors for chronic cough in a population-based setting. Therefore, we investigated the period prevalence, incidence and risk factors for chronic cough in adult subjects.

In a prospective population-based cohort study among subjects aged ≥45 years, data on chronic cough were collected on two separate occasions using a standardised questionnaire. Chronic cough was defined as daily coughing for at least 3 months duration during the preceding 2 years. Potential risk factors were gathered by interview, physical examination and several investigations.

Of the 9824 participants in this study, 1073 (10.9%) subjects had chronic cough at baseline. The prevalence of chronic cough increased with age and peaked in the eighth decade. In subjects aged <70 years, chronic cough was more common in women. During an average follow-up of 6 years, 439 incident cases of chronic cough occurred with an overall incidence rate of 11.6 per 1000 person-years (95% CI 10.6–12.8). In current smokers, the incidence of chronic cough was higher in men. In the multivariable analysis, current smoking, gastro-oesophageal reflux disease (GORD), asthma and COPD were identified as risk factors for chronic cough.

Chronic cough is common among adults and highly prevalent in the older population. Current smoking, GORD, asthma and COPD are independent risk factors for chronic cough. Individuals at risk of developing chronic cough may benefit from smoking cessation and control of the underlying disease.

OBJECTIVE

We investigated the association between changes in weight status from childhood through adulthood and subsequent type 2 diabetes risks and whether educational attainment, smoking, and leisure time physical activity (LTPA) modify this association.

OBJECTIVE

To determine the incidence of and factors associated with an estimated glomerular filtration rate (eGFR) <60 mL/min/1.73 m² in people with diabetes.

In patients with cancer-associated venous thromboembolism, knowledge of the estimated rate of recurrent events is important for clinical decision-making regarding anticoagulant therapy. The Ottawa score is a clinical prediction rule designed for this purpose, stratifying patients according to their risk of recurrent venous thromboembolism during the first six months of anticoagulation. We conducted a systematic review and meta-analysis of studies validating either the Ottawa score in its original or modified versions. Two investigators independently reviewed the relevant articles published from 1st June 2012 to 15th December 2018 and indexed in MEDLINE and EMBASE. Nine eligible studies were identified; these included a total of 14,963 patients. The original score classified 49.3% of the patients as high-risk, with a sensitivity of 0.7 [95% confidence interval (CI): 0.6-0.8], a 6-month pooled rate of recurrent venous thromboembolism of 18.6% (95%CI: 13.9-23.9). In the low-risk group, the recurrence rate was 7.4% (95%CI: 3.4-12.5). The modified score classified 19.8% of the patients as low-risk, with a sensitivity of 0.9 (95%CI: 0.4-1.0) and a 6-month pooled rate of recurrent venous thromboembolism of 2.2% (95%CI: 1.6-2.9). In the high-risk group, recurrence rate was 10.2% (95%CI: 6.4-14.6). Limitations of our analysis included type and dosing of anticoagulant therapy. We conclude that new therapeutic strategies are needed in patients at high risk for recurrent cancer-associated venous thromboembolism. Low-risk patients, as per the modified score, could be good candidates for oral anticoagulation. (This systematic review was registered with the International Prospective Registry of Systematic Reviews as: PROSPERO CRD42018099506).

To identify genomic alterations contributing to the pathogenesis of high-risk chronic lymphocytic leukemia (CLL) beyond the well-established role of TP53 aberrations, we comprehensively analyzed 75 relapsed/refractory and 71 treatment-naïve high-risk cases from prospective clinical trials by single nucleotide polymorphism arrays and targeted next-generation sequencing. Increased genomic complexity was a hallmark of relapsed/refractory and treatment-naïve high-risk CLL. In relapsed/refractory cases previously exposed to the selective pressure of chemo(immuno)therapy, gain(8)(q24.21) and del(9)(p21.3) were particularly enriched. Both alterations affect key regulators of cell-cycle progression, namely MYC and CDKN2A/B. While homozygous CDKN2A/B loss has been directly associated with Richter transformation, we did not find this association for heterozygous loss of CDKN2A/B. Gains in 8q24.21 were either focal gains in a MYC enhancer region or large gains affecting the MYC locus, but only the latter type was highly enriched in relapsed/refractory CLL (17%). In addition to a high frequency of NOTCH1 mutations (23%), we found recurrent genetic alterations in SPEN (4% mutated), RBPJ (8% deleted) and SNW1 (8% deleted), all affecting a protein complex that represses transcription of NOTCH1 target genes. We investigated the functional impact of these alterations on HES1, DTX1 and MYC gene transcription and found derepression of these NOTCH1 target genes particularly with SPEN mutations. In summary, we provide new insights into the genomic architecture of high-risk CLL, define novel recurrent DNA copy number alterations and refine knowledge on del(9p), gain(8q) and alterations affecting NOTCH1 signaling. This study was registered at ClinicalTrials.gov with number NCT01392079.

Autophagy is a genetically regulated process of adaptation to metabolic stress and was recently shown to be involved in the treatment response of chronic myeloid leukemia (CML). However, in vivo data are limited and the molecular mechanism of autophagy regulators in the process of leukemogenesis is not completely understood. Here we show that Beclin-1 knockdown, but not Atg5 deletion in a murine CML model leads to a reduced leukemic burden and results in a significantly prolonged median survival of targeted mice. Further analyses of murine cell lines and primary patient material indicate that active BCR-ABL directly interacts with BECLIN-1 and phosphorylates its tyrosine residues 233 and 352, resulting in autophagy suppression. By using phosphorylation-deficient and phosphorylation-mimic mutants, we identify BCR-ABL induced BECLIN-1 phosphorylation as a crucial mechanism for BECLIN-1 complex formation: interaction analyses exhibit diminished binding of the positive autophagy regulators UVRAG, VPS15, ATG14 and VPS34 and enhanced binding of the negative regulator Rubicon to BCR-ABL-phosphorylated BECLIN-1. Taken together, our findings show interaction of BCR-ABL and BECLIN-1 thereby highlighting the importance of BECLIN-1-mediated autophagy in BCR-ABL⁺ cells.

Although highly effective, BCR-ABL1 tyrosine kinase inhibitors do not target chronic myeloid leukemia (CML) stem cells. Most patients relapse upon tyrosine kinase inhibitor therapy cessation. We reported previously that combined BCR-ABL1 and BCL-2 inhibition synergistically targets CML stem/progenitor cells. p53 induces apoptosis mainly by modulating BCL-2 family proteins. Although infrequently mutated in CML, p53 is antagonized by MDM2, which is regulated by BCR-ABL1 signaling. We hypothesized that MDM2 inhibition could sensitize CML cells to tyrosine kinase inhibitors. Using an inducible transgenic Scl-tTa-BCR-ABL1 murine CML model, we found, by RT-PCR and CyTOF proteomics increased p53 signaling in CML bone marrow (BM) cells compared with controls in CD45⁺ and linage-SCA-1⁺C-KIT⁺ populations. CML BM cells were more sensitive to exogenous BH3 peptides than controls. Combined inhibition of BCR-ABL1 with imatinib and MDM2 with DS-5272 increased NOXA level, markedly reduced leukemic linage-SCA-1⁺C-KIT⁺ cells and hematopoiesis, decreased leukemia burden, significantly prolonged the survival of mice engrafted with BM cells from Scl-tTa-BCR-ABL1 mice, and significantly decreased CML stem cell frequency in secondary transplantations. Our results suggest that CML stem/progenitor cells have increased p53 signaling and a propensity for apoptosis. Combined MDM2 and BCR-ABL1 inhibition targets CML stem/progenitor cells and has the potential to improve cure rates for CML.

Although studies of mixed chimerism following hematopoietic stem cell transplantation in patients with sickle cell disease (SCD) may provide insights into the engraftment needed to correct the disease and into immunological reconstitution, an extensive multilineage analysis is lacking. We analyzed chimerism simultaneously in peripheral erythroid and granulomonocytic precursors/progenitors, highly purified B and T lymphocytes, monocytes, granulocytes and red blood cells (RBC). Thirty-four patients with mixed chimerism and ≥12 months of follow-up were included. A selective advantage of donor RBC and their progenitors/precursors led to full chimerism in mature RBC (despite partial engraftment of other lineages), and resulted in the clinical control of the disease. Six patients with donor chimerism <50% had hemolysis (reticulocytosis) and higher HbS than their donor. Four of them had donor chimerism <30%, including a patient with AA donor (hemoglobin >10 g/dL) and three with AS donors (hemoglobin <10 g/dL). However, only one vaso-occlusive crisis occurred with 68.7% HbS. Except in the patients with the lowest chimerism, the donor engraftment was lower for T cells than for the other lineages. In a context of mixed chimerism after hematopoietic stem cell transplantation for SCD, myeloid (rather than T cell) engraftment was the key efficacy criterion. Results show that myeloid chimerism as low as 30% was sufficient to prevent a vaso-occlusive crisis in transplants from an AA donor but not constantly from an AS donor. However, the correction of hemolysis requires higher donor chimerism levels (i.e. ≥50%) in both AA and AS recipients. In the future, this group of patients may need a different therapeutic approach.

Inadequate insulin secretion in response to glucose is an important factor for β-cell failure in type 2 diabetes (T2D). Although HMG-CoA reductase degradation 1 (HRD1), a subunit of the endoplasmic reticulum–associated degradation complex, plays a pivotal role in β-cell function, HRD1 elevation in a diabetic setting contributes to β-cell dysfunction. We report in this study the excessive HRD1 expression in islets from humans with T2D and T2D mice. Functional studies reveal that β-cell–specific HRD1 overexpression triggers impaired insulin secretion that will ultimately lead to severe hyperglycemia; by contrast, HRD1 knockdown improves glucose control and response in diabetic models. Proteomic analysis results reveal a large HRD1 interactome, which includes v-maf musculoaponeurotic fibrosarcoma oncogene homolog A (MafA), a master regulator of genes implicated in the maintenance of β-cell function. Furthermore, mechanistic assay results indicate that HRD1 is a novel E3 ubiquitin ligase that targets MafA for ubiquitination and degradation in diabetic β-cells, resulting in cytoplasmic accumulation of MafA and in the reduction of its biological function in the nucleus. Our results not only reveal the pathological importance of excessive HRD1 in β-cell dysfunction but also establish the therapeutic importance of targeting HRD1 in order to prevent MafA loss and suppress the development of T2D.

An aging global population combined with sedentary lifestyles and unhealthy diets has contributed to an increasing incidence of obesity and type 2 diabetes. These metabolic disorders are associated with perturbations to nitric oxide (NO) signaling and impaired glucose metabolism. Dietary inorganic nitrate, found in high concentration in green leafy vegetables, can be converted to NO in vivo and demonstrates antidiabetic and antiobesity properties in rodents. Alongside tissues including skeletal muscle and liver, white adipose tissue is also an important physiological site of glucose disposal. However, the distinct molecular mechanisms governing the effect of nitrate on adipose tissue glucose metabolism and the contribution of this tissue to the glucose-tolerant phenotype remain to be determined. Using a metabolomic and stable-isotope labeling approach, combined with transcriptional analysis, we found that nitrate increases glucose uptake and oxidative catabolism in primary adipocytes and white adipose tissue of nitrate-treated rats. Mechanistically, we determined that nitrate induces these phenotypic changes in primary adipocytes through the xanthine oxidoreductase–catalyzed reduction of nitrate to NO and independently of peroxisome proliferator–activated receptor-α. The nitrate-mediated enhancement of glucose uptake and catabolism in white adipose tissue may be a key contributor to the antidiabetic effects of this anion.

Yuta Sakae, Akira Oikawa, Yuki Sugiura, Masatoshi Mita, Shuhei Nakamura, Toshiya Nishimura, Makoto Suematsu, and Minoru Tanaka

The teleost fish, medaka (Oryzias latipes), employs the XX/XY genetic sex determination system. We show here that the phenotypic sex of medaka is affected by changes in lipid metabolism. Medaka larvae subjected to 5 days of starvation underwent female-to-male sex reversal. Metabolomic and RT-qPCR analyses indicated that pantothenate metabolism was suppressed by starvation. Consistently, inhibiting the pantothenate metabolic pathway caused sex reversal. The final metabolite in this pathway is coenzyme A, an essential factor for lipogenesis. Inhibiting fatty acid synthesis, the first step of lipogenesis, also caused sex reversal. The expression of dmrt1, a critical gene for male development, was suppressed by starvation, and a dmrt1 (13) mutant did not show sex reversal under starvation. Collectively, these results indicate that fatty acid synthesis is involved in female-to-male sex reversal through ectopic expression of male gene dmrt1 under starvation.

Background and objectives

Walking while talking is a dual cognitive-motor task that predicts frailty, falls, and cognitive decline in the general elderly population. Adults with CKD have gait abnormalities during usual walking. It is unknown whether they have greater gait abnormalities and cognitive-motor interference during walking while talking.

Background and objectives

Actionable genetic findings have implications for care of patients with kidney disease, and genetic testing is an emerging tool in nephrology practice. However, there are scarce data regarding best practices for return of results and clinical application of actionable genetic findings for kidney patients.

Although treatment with the glucocorticoid-induced tumor necrosis factor receptor–related protein (GITR) agonistic antibody (DTA-1) has shown antitumor activity in various tumor models, the underlying mechanism is not fully understood. Here, we demonstrate that interleukin (IL)-21–producing follicular helper T (Tfh) cells play a crucial role in DTA-1–induced tumor inhibition. The administration of DTA-1 increased IL21 expression by Tfh cells in an antigen-specific manner, and this activation led to enhanced antitumor cytotoxic T lymphocyte (CTL) activity. Mice treated with an antibody that neutralizes the IL21 receptor exhibited decreased antitumor activity when treated with DTA-1. Tumor growth inhibition by DTA-1 was abrogated in Bcl6^fl/flCd4^Cre mice, which are genetically deficient in Tfh cells. IL4 was required for optimal induction of IL21-expressing Tfh cells by GITR costimulation, and c-Maf mediated this pathway. Thus, our findings identify GITR costimulation as an inducer of IL21-expressing Tfh cells and provide a mechanism for the antitumor activity of GITR agonism.

T-cell receptor (TCR)–based therapeutic cells and agents have emerged as a new class of effective cancer therapies. These therapies work on cells that express intracellular cancer-associated proteins by targeting peptides displayed on MHC receptors. However, cross-reactivities of these agents to off-target cells and tissues have resulted in serious, sometimes fatal, adverse events. We have developed a high-throughput genetic platform (termed "PresentER") that encodes MHC-I peptide minigenes for functional immunologic assays and determines the reactivities of TCR-like therapeutic agents against large libraries of MHC-I ligands. In this article, we demonstrated that PresentER could be used to identify the on-and-off targets of T cells and TCR-mimic (TCRm) antibodies using in vitro coculture assays or binding assays. We found dozens of MHC-I ligands that were cross-reactive with two TCRm antibodies and two native TCRs and that were not easily predictable by other methods.

Dietary protein residue can result in microbial generation of various toxic metabolites in the gut, such as ammonia. A prebiotic is "a substrate that is selectively utilised by host microorganisms conferring a health benefit" (G. R. Gibson, R. Hutkins, M. E. Sanders, S. L. Prescott, et al., Nat Rev Gastroenterol Hepatol 14:491–502, 2017, https://doi.org/10.1038/nrgastro.2017.75). Prebiotics are carbohydrates that may have the potential to reverse the harmful effects of gut bacterial protein fermentation. Three-stage continuous colonic model systems were inoculated with fecal samples from omnivore and vegetarian volunteers. Casein (equivalent to 105 g protein consumption per day) was used within the systems as a protein source. Two different doses of inulin-type fructans (Synergy1) were later added (equivalent to 10 g per day in vivo and 15 g per day) to assess whether this influenced protein fermentation. Bacteria were enumerated by fluorescence in situ hybridization with flow cytometry. Metabolites from bacterial fermentation (short-chain fatty acid [SCFA], ammonia, phenol, indole, and p-cresol) were monitored to further analyze proteolysis and the prebiotic effect. A significantly higher number of bifidobacteria was observed with the addition of inulin together with reduction of Desulfovibrio spp. Furthermore, metabolites from protein fermentation, such as branched-chain fatty acids (BCFA) and ammonia, were significantly lowered with Synergy1. Production of p-cresol varied among donors, as we recognized four high producing models and two low producing models. Prebiotic addition reduced its production only in vegetarian high p-cresol producers.

IMPORTANCE Dietary protein levels are generally higher in Western populations than in the world average. We challenged three-stage continuous colonic model systems containing high protein levels and confirmed the production of potentially harmful metabolites from proteolysis, especially replicates of the transverse and distal colon. Fermentations of proteins with a prebiotic supplementation resulted in a change in the human gut microbiota and inhibited the production of some proteolytic metabolites. Moreover, we observed both bacterial and metabolic differences between fecal bacteria from omnivore donors and vegetarian donors. Proteins with prebiotic supplementation showed higher Bacteroides spp. and inhibited Clostridium cluster IX in omnivore models, while in vegetarian modes, Clostridium cluster IX was higher and Bacteroides spp. lower with high protein plus prebiotic supplementation. Synergy1 addition inhibited p-cresol production in vegetarian high p-cresol-producing models while the inhibitory effect was not seen in omnivore models.

Burkholderia sp. strain SG-MS1 and Pseudomonas sp. strain SG-MS2 have previously been found to mineralize (+)-pinoresinol through a common catabolic pathway. Here, we used comparative genomics, proteomics, protein semipurification, and heterologous expression to identify a flavoprotein from the vanillyl alcohol oxidase/p-cresol methyl hydroxylase (VAO/PCMH) enzyme family in SG-MS2 that carries out the initial hydroxylation of (+)-pinoresinol at the benzylic carbon. The cognate gene is translationally coupled with a downstream cytochrome gene, and the cytochrome is required for activity. The flavoprotein has a unique combination of cofactor binding and cytochrome requirements for the VAO/PCMH family. The heterologously expressed enzyme has a K_m of 1.17 μM for (+)-pinoresinol. The enzyme is overexpressed in strain SG-MS2 upon exposure to (+)-pinoresinol, along with 45 other proteins, 22 of which were found to be encoded by genes in an approximately 35.1-kb cluster also containing the flavoprotein and cytochrome genes. Homologs of 18 of these 22 genes, plus the flavoprotein and cytochrome genes, were also found in a 38.7-kb cluster in SG-MS1. The amino acid identities of four of the other proteins within the SG-MS2 cluster suggest they catalyze conversion of hydroxylated pinoresinol to protocatechuate and 2-methoxyhydroquinone. Nine other proteins upregulated in SG-MS2 on exposure to (+)-pinoresinol appear to be homologs of proteins known to comprise the protocatechuate and 2-methoxyhydroquinone catabolic pathways, but only three of the cognate genes lie within the cluster containing the flavoprotein and cytochrome genes.

IMPORTANCE (+)-Pinoresinol is an important plant defense compound, a major food lignan for humans and some other animals, and the model compound used to study degradation of the β-β' linkages in lignin. We report a gene cluster, in one strain each of Pseudomonas and Burkholderia, that is involved in the oxidative catabolism of (+)-pinoresinol. The flavoprotein component of the α-hydroxylase which heads the pathway belongs to the 4-phenol oxidizing (4PO) subgroup of the vanillyl alcohol oxidase/p-cresol methyl hydroxylase (VAO/PCMH) enzyme family but constitutes a novel combination of cofactor and electron acceptor properties for the family. It is translationally coupled with a cytochrome gene whose product is also required for activity. The work casts new light on the biology of (+)-pinoresinol and its transformation to other bioactive molecules. Potential applications of the findings include new options for deconstructing lignin into useful chemicals and the generation of new phytoestrogenic enterolactones from lignans.

A longstanding awareness in generating resistance to common antimicrobial therapies by Gram-negative bacteria has made them a major threat to global health. The application of antimicrobial peptides as a therapeutic agent would be a great opportunity to combat bacterial diseases. Here, we introduce a new antimicrobial peptide (~8.3 kDa) from probiotic strain Lactobacillus acidophilus ATCC 4356, designated acidocin 4356 (ACD). This multifunctional peptide exerts its anti-infective ability against Pseudomonas aeruginosa through an inhibitory action on virulence factors, bacterial killing, and biofilm degradation. Reliable performance over tough physiological conditions and low hemolytic activity confirmed a new hope for the therapeutic setting. Antibacterial kinetic studies using flow cytometry technique showed that the ACD activity is related to the change in permeability of the membrane. The results obtained from molecular dynamic (MD) simulation were perfectly suited to the experimental data of ACD behavior. The structure-function relationship of this natural compound, along with the results of transmission electron microscopy analysis and MD simulation, confirmed the ability of the ACD aimed at enhancing bacterial membrane perturbation. The peptide was effective in the treatment of P. aeruginosa infection in mouse model. The results support the therapeutic potential of ACD for the treatment of Pseudomonas infections.

IMPORTANCE Multidrug-resistant bacteria are a major threat to global health, and the Pseudomonas bacterium with the ability to form biofilms is considered one of the main causative agents of nosocomial infections. Traditional antibiotics have failed because of increased resistance. Thus, finding new biocompatible antibacterial drugs is essential. Antimicrobial peptides are produced by various organisms as a natural defense mechanism against pathogens, inspiring the possible design of the next generation of antibiotics. In this study, a new antimicrobial peptide was isolated from Lactobacillus acidophilus ATCC 4356, counteracting both biofilm and planktonic cells of Pseudomonas aeruginosa. A detailed investigation was then conducted concerning the functional mechanism of this peptide by using fluorescence techniques, electron microscopy, and in silico methods. The antibacterial and antibiofilm properties of this peptide may be important in the treatment of Pseudomonas infections.

Background:

Incentive payments for chronic diseases in British Columbia were intended to support primary care physicians in providing more comprehensive care, but research shows that not all physicians bill incentives and not all eligible patients have them billed on their behalf. We investigated patient and physician characteristics associated with billing incentives for chronic diseases in BC.

At about 6300 km, Chang Jiang or Long River (长江) is the longest river in Asia and third longest in the world. It is the longest in the world to flow entirely within one country and plays a large role in the history, culture, and economy of China. For thousands of years, the river has been used for water, irrigation, sanitation, transportation, industry, boundary-marking, and war. Passing through 10 provinces, it commences its flow from the Tanggula Mountains (唐古拉山) in ethnic Tibetan Qinghai (青海) Province in northwest China and ends at the East China Sea in Shanghai (上海). Along the middle reaches of the river, between the western upstream Baidi City (白帝城) of Chongqing (重庆) Municipality and Yichang (宜昌) city of Hubei (湖北) Province downstream, the river passes through 3 adjacent gorges, known as the Three Gorges (三峡): Qutang, Wuxia, and Xiling gorge (瞿塘, 巫峡, 西陵峡) cutting through the Wu Mountains (巫山). Spanning a distance of about 120 km, these gorges are noted for their natural beauty: unusual-looking mountain peaks ranging from 800 m to 2000 m, precipitous valleys, dense forest, and spectacular landscape. Many ancient government officials, scholars, poets, and painters visited the Three Gorges and left their impressions and praises in writings. Archeological discoveries in recent years have shown for the first time that the Three Gorges area should be recognized as the birthplace of Chinese civilization.

Cardiovascular (CV) disease remains an important cause of morbidity and mortality for patients with chronic kidney disease (CKD). Although clustering of traditional risk factors with CKD is well recognized, kidney-specific mechanisms are believed to drive the disproportionate burden of CV disease. One perturbation that is frequently observed at high rates in patients with CKD is vascular calcification, which may be a central mediator for an array of CV sequelae. This review summarizes the pathophysiological bases of intimal and medial vascular calcification in CKD, current strategies for diagnosis and management, and posits vascular calcification as a risk marker and therapeutic target.

This work established a new murine venous thromboembolism (VTE) model. This model has multiple novel features representing clinical VTE that include the following: 1) deep venous thrombosis (DVT) was formed and extended in the long axis of femoral/saphenous vein; 2) thrombus was formed in a venous valve pocket; 3) deligation of suture-induced spontaneous pulmonary emboli of fibrin-rich DVT; and 4) cardiac motion-free femoral/saphenous vein allowed high-resolution intravital microscopic imaging of fibrin-rich DVT. This new model requires only commercially available epifluorescence microscopy. Therefore, this model has significant potential for better understanding of VTE pathophysiology.

Combined pre-and post-capillary hypertension (CpcPH) is a relatively common complication of heart failure (HF) associated with a poor prognosis. Currently, there is no specific therapy approved for this entity. Recently, treatment with beta-3 adrenergic receptor (β3AR) agonists was able to improve pulmonary hemodynamics and right ventricular (RV) performance in a translational, large animal model of chronic PH. The authors present the design of a phase II randomized clinical trial that tests the benefits of mirabegron (a clinically available β3AR agonist) in patients with CpcPH due to HF. The effect of β3AR treatment will be evaluated on pulmonary hemodynamics, as well as clinical, biochemical, and advanced cardiac imaging parameters. (Beta3 Agonist Treatment in Chronic Pulmonary Hypertension Secondary to Heart Failure [SPHERE-HF]; NCT02775539)

In 2016, the proportion of Neisseria gonorrhoeae isolates with reduced susceptibility to azithromycin rose to 3.6%. A phylogenetic analysis of 334 N. gonorrhoeae isolates collected in 2016 revealed a single, geographically diverse lineage of isolates with MICs of 2 to 16 μg/ml that carried a mosaic-like mtr locus, whereas the majority of isolates with MICs of ≥16 μg/ml appeared sporadically and carried 23S rRNA mutations. Continued molecular surveillance of N. gonorrhoeae isolates will identify new resistance mechanisms.

Infections with nontuberculous mycobacteria (NTM) have a poor prognosis in patients with underlying respiratory diseases. Clofazimine (CFZ) showed both experimental and clinical promising results against clinically relevant NTM. However, there are no data on CFZ in combination with the current recommended treatment; therefore, we aimed to study its in vivo activity in an aerosol mouse model of Mycobacterium avium. In an aerosol infection BALB/c mouse model using M. avium strain Chester, we treated 58 mice with four combinations of rifampin (RIF) at 10 mg/kg, CFZ at 25 mg/kg, and clarithromycin (CLR) and ethambutol (EMB) at 100 mg/kg. Treatment efficacy was assessed on the basis of lung CFU counts after 2 (M2) and 4 (M4) months of treatment. At M2, CLR-RIF-EMB was slightly but significantly more efficient than CFZ-RIF-EMB (3.02 ± 0.12 versus 3.55 ± 0.28, respectively, P < 0.01), whereas CLR-CFZ-EMB and CLR-CFZ-RIF-EMB dramatically decreased lung CFU counts by 4.32 and 4.47 log₁₀, respectively, compared to untreated group. At M4, CLR-RIF-EMB was significantly more efficient than CFZ-RIF-EMB (2 ± 0.53 versus 2.66 ± 0.22, respectively, P = 0.01). The addition of CLZ to CLR dramatically decreased the lung CFU count, with CFU counts 5.41 and 5.79 log₁₀ lower in the CLR-CFZ-EMB and CLR-CFZ-RIF-EMB groups, respectively, than in the untreated group. The addition of CFZ to CLR seems to improve the efficacy of CLR as early as M2 and was confirmed at M4. CFZ, in addition to RIF and EMB, on the other hand, is less effective than CLR-RIF-EMB. These results need to be confirmed by similar studies along with CFZ potential for shortening treatment.

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.

Chronic obstructive pulmonary disease (COPD) is an inflammatory lung condition, causing progressive decline in lung function leading to premature death. Acute exacerbations in COPD patients are predominantly associated with respiratory viruses. Ribavirin is a generic broad-spectrum antiviral agent that could be used for treatment of viral respiratory infections in COPD. Using the Particle Replication In Nonwetting Templates (PRINT) technology, which produces dry-powder particles of uniform shape and size, two new inhaled formulations of ribavirin (ribavirin-PRINT-CFI and ribavirin-PRINT-IP) were developed for efficient delivery to the lung and to minimize bystander exposure. Ribavirin-PRINT-CFI was well tolerated in healthy participants after single dosing and ribavirin-PRINT-IP was well tolerated in healthy and COPD participants after single and repeat dosing. Ribavirin-PRINT-CFI was replaced with ribavirin-PRINT-IP since the latter formulation was found to have improved physicochemical properties and it had a higher ratio of active drug to excipient per unit dose. Ribavirin concentrations were measured in lung epithelial lining fluid in both healthy and COPD participants and achieved target concentrations. Both formulations were rapidly absorbed with approximately dose proportional pharmacokinetics in plasma. Exposure to bystanders was negligible based on both the plasma and airborne ribavirin concentrations with the ribavirin-PRINT-IP formulation. Thus, ribavirin-PRINT-IP allowed for an efficient and convenient delivery of ribavirin to the lungs while minimizing systemic exposure. Further clinical investigations would be required to demonstrate ribavirin-PRINT-IP antiviral characteristics and impact on COPD viral-induced exacerbations. (The clinical trials discussed in this study have been registered at ClinicalTrials.gov under identifiers NCT03243760 and NCT03235726.)

The new complexes Zn(ITZ)₂Cl₂ (1) and Zn(ITZ)₂(OH)₂ (2) were synthetized by a reaction of itraconazole with their respective zinc salts under reflux. These Zn-ITZ complexes were characterized by elemental analyses, molar conductivity, mass spectrometry, ¹H and ¹³C{¹H} nuclear magnetic resonance, and UV-vis and infrared spectroscopies. The antiparasitic and antifungal activity of Zn-ITZ complexes was evaluated against three protozoans of medical importance, namely, Leishmania amazonensis, Trypanosoma cruzi, and Toxoplasma gondii, and two fungi, namely, Sporothrix brasiliensis and Sporothrix schenckii. The Zn-ITZ complexes exhibited a broad spectrum of action, with antiparasitic and antifungal activity in low concentrations. The strategy of combining zinc with ITZ was efficient to enhance ITZ activity since Zn-ITZ-complexes were more active than the azole alone. This study opens perspectives for future applications of these Zn-ITZ complexes in the treatment of parasitic diseases and sporotrichosis.

This protocol describes a simple single-step column purification (SSCP) of rAAV2 by gravity flow based on its affinity to heparin, without ultracentrifugation.