70-kDa Protein Research Articles

Psb35 Protein Stabilizes the CP47 Assembly Module and Associated High-Light Inducible Proteins during the Biogenesis of Photosystem II in the Cyanobacterium Synechocystis sp. PCC6803.

Photosystem II (PSII) is a large membrane protein complex performing primary charge separation in oxygenic photosynthesis. The biogenesis of PSII is a complicated process that involves a coordinated linking of assembly modules in a precise order. Each such module consists of one large chlorophyll (Chl)-binding protein, number of small membrane polypeptides, pigments and other cofactors. We isolated the CP47 antenna module from the cyanobacterium Synechocystis sp. PCC 6803 and found that it contains a 11-kDa protein encoded by the ssl2148 gene. This protein was named Psb35 and its presence in the CP47 module was confirmed by the isolation of FLAG-tagged version of Psb35. Using this pulldown assay, we showed that the Psb35 remains attached to CP47 after the integration of CP47 into PSII complexes. However, the isolated Psb35-PSIIs were enriched with auxiliary PSII assembly factors like Psb27, Psb28-1, Psb28-2 and RubA while they lacked the lumenal proteins stabilizing the PSII oxygen-evolving complex. In addition, the Psb35 co-purified with a large unique complex of CP47 and photosystem I trimer. The absence of Psb35 led to a lower accumulation and decreased stability of the CP47 antenna module and associated high-light-inducible proteins but did not change the growth rate of the cyanobacterium under the variety of light regimes. Nevertheless, in comparison with WT, the Psb35-less mutant showed an accelerated pigment bleaching during prolonged dark incubation. The results suggest an involvement of Psb35 in the life cycle of cyanobacterial Chl-binding proteins, especially CP47.

Strong interactions between Salp15 homologues from the tick I. ricinus and distinct types of the outer surface OspC protein from Borrelia

Ticks belonging to the genus Ixodes are parasites feeding on vertebrate blood and vectors for many pathogenic microbes, including Borrelia burgdorferi sensu lato spirochetes, the causative agent of Lyme borreliosis. The tick saliva contains a mixture of bioactive molecules showing a wide range of properties for efficient engorgement. One of the most extensively studied components of tick saliva is a 15-kDa salivary gland protein (Salp15) from Ixodes scapularis. This multifunctional protein suppresses the immune response of hosts through pleiotropic action on a few crucial defense pathways. Salp15 and its homologue from I. ricinus Iric1 have been also shown to bind to Borrelia burgdorferi sensu stricto outer surface protein C (OspC) permitting the spirochetes to evade antibody-mediated killing in the human host. Further studies revealed that Salp15 and Iric1 protected B. burgdorferi s. s. and B. garinii expressing OspC against the complement system. OspC is the most variable protein on the outer surface of Borrelia, which in addition to Salp15 can also bind other ligands, such as plasminogen, fibrinogen, fibronectin or complement factor 4. So far several OspC variants produced by B. burgdorferi s. l. spirochetes were shown to be capable of binding Salp15 or its homologue, but the protection against borreliacidal antibodies has only been proven in the case of B. burgdorferi s. s. The question of Salp15 contribution to Borrelia survival during the infection has been comprehensively studied during the last decades. In contrast, the organization of the OspC-Salp15 complex has been poorly explored. This report describes the binding between three Salp15 homologues from the tick Ixodes ricinus (Iric1, Iric2 and Iric3) and OspC from four B. burgdorferi sensu lato strains in terms of the binding parameters, analyzed with two independent biophysical methods – Microscale thermophoresis (MST) and Biolayer interferometry (BLI). The results of both experiments show a binding constant at the nanomolar level, which indicates very strong interactions. While the Iric1-OspC binding has been reported before, we show in this study that also Iric2 and Iric3 are capable of OspC binding with high affinity. This observation suggests that these two Salp15 homologues might be used by B. burgdorferi s. l. in a way analogous to Iric1. A comparison of the results from the two methods let us propose that N-terminal immobilization of OspC significantly increases the affinity between the two proteins. Finally, our results indicate that the Iric binding site is located in close proximity of the OspC epitopes recognized by human antibodies, which may have important biological and medical implications.

Campylobacter jejuni Serine Protease HtrA Cleaves the Tight Junction Component Claudin-8.

Campylobacter jejuni express the high temperature requirement protein A (HtrA), a secreted serine protease, which is implicated in virulence properties of the pathogen. Previous studies have shown that C. jejuni HtrA can cleave the epithelial transmembrane proteins occludin and E-cadherin in the tight and adherens junctions, respectively. In the present report, we studied the interaction of HtrA with another human tight junction protein, claudin-8. Confocal immunofluorescence experiments have shown that C. jejuni infection of the intestinal polarized epithelial cells in vitro leads to a relocation of claudin-8. Wild-type C. jejuni induced the downregulation of claudin-8 signals in the tight junctions and an accumulation of claudin-8 agglomerates in the cytoplasm, which were not seen during infection with isogenic ΔhtrA knockout deletion or protease-inactive S197A point mutants. Western blotting of protein samples from infected vs. uninfected cells revealed that an 18-kDa carboxy-terminal fragment is cleaved-off from the 26-kDa full-length claudin-8 protein, but not during infection with the isogenic ΔhtrA mutant. These results were confirmed by in vitro cleavage assays using the purified recombinant C. jejuni HtrA and human claudin-8 proteins. Recombinant HtrA cleaved purified claudin-8 in vitro giving rise to the same 18-kDa sized carboxy-terminal cleavage product. Mapping studies revealed that HtrA cleavage occurs in the first extracellular loop of claudin-8. Three-dimensional modeling of the claudin-8 structure identified an exposed HtrA cleavage site between the amino acids alanine 58 and asparagine 59, which is in well agreement with the mapping studies. Taken together, HtrA operates as a secreted virulence factor targeting multiple proteins both in the tight and adherens junctions. This strategy may help the bacteria to open the cell-to-cell junctions, and to transmigrate across the intestinal epithelium by a paracellular mechanism and establish an acute infection.

Molecular Pathways of Interferon-Stimulated Gene 15: Implications in Cancer.

Human interferon-stimulated gene 15 (ISG15) is a 15-kDa ubiquitin-like protein that can be detected as either free ISG15 or covalently associated with its target proteins through a process termed ISGylation. Interestingly, extracellular free ISG15 has been proposed as a cytokinelike protein, whereas ISGylation is a posttranslational modification. ISG15 is a small protein with implications in some biological processes and pathologies that include cancer. This review highlights the findings of both free ISG15 and protein ISGylation involved in several molecular pathways, emerging as central elements in some cancer types.

Shear stress-exposed pulmonary artery endothelial cells fail to upregulate HSP70 in chronic thromboembolic pulmonary hypertension.

The pathophysiological mechanisms underlying chronic thromboembolic pulmonary hypertension (CTEPH) are still unclear. Endothelial cell (EC) remodeling is believed to contribute to this pulmonary disease triggered by thrombus and hemodynamic forces disbalance. Recently, we showed that HSP70 levels decrease by proatherogenic shear stress. Molecular chaperones play a major role in proteostasis in neurological, cancer and inflammatory/ infectious diseases. To shed light on microvascular responses in CTEPH, we characterized the expression of molecular chaperones and annexin A2, a component of the fibrinolytic system. There is no animal model that reproduces microvascular changes in CTEPH, and this fact led us to isolated endothelial cells from patients with CTEPH undergoing pulmonary endarterectomy (PEA). We exposed CTEPH-EC and control human pulmonary endothelial cells (HPAEC) to high- (15 dynes/cm2) or low- (5 dynes/cm2) shear stress. After high-magnitude shear stress HPAEC upregulated heat shock protein 70kDa (HSP70) and the HSP ER paralogs 78 and 94kDa glucose-regulated protein (GRP78 and 94), whereas CTEPH-ECs failed to exhibit this response. At static conditions, both HSP70 and HSP90 families in CTEPH-EC are decreased. Importantly, immunohistochemistry analysis showed that HSP70 expression was downregulated in vivo, and annexin A2 was upregulated. Interestingly, wound healing and angiogenesis assays revealed that HSP70 inhibition with VER-155008 further impaired CTEPH-EC migratory responses. These results implicate HSP70 as a novel master regulator of endothelial dysfunction in type 4 PH. Overall, we first show that global failure of HSP upregulation is a hallmark of CTEPH pathogenesis and propose HSP70 as a potential biomarker of this condition.

ISG20 and nuclear exosome promote destabilization of nascent transcripts for spliceosomal U snRNAs and U1 variants

Primary RNA transcripts are processed in a plethora of ways to become mature functional forms. In one example, human spliceosomal U snRNAs are matured at their 3'-end by an exonuclease termed TOE1. This process is important because mutations in TOE1 gene can cause a human genetic disease, pontocerebellar hypoplasia (PCH). Nevertheless, TOE1 may not be the only maturation exonuclease for U snRNAs in the cell. Here, we biochemically identify two exonucleolytic factors, Interferon-stimulated gene 20-kDa protein (ISG20) and the nuclear exosome as such candidates, using a newly developed in vitro system that recapitulates 3'-end maturation of U1 snRNA. However, extensive 3'-end sequencing of endogenous U1 snRNA of the knockdown (KD) cells revealed that these factors are not the maturation factors per se. Instead, the nascent transcripts of the spliceosomal U snRNAs as well as of unstable U1 variants were found to increase in quantity upon KD of the factors. These results indicated that ISG20 and the nuclear exosome promote the degradation of nascent spliceosomal U snRNAs and U1 variants, and therefore implied their role in the quality control of newly synthesized U snRNAs.

Characterization of Hibiscus Latent Fort Pierce Virus-Derived siRNAs in Infected Hibiscus rosa-sinensis in China.

Although limited progress have been made about pathogen system of Hibiscus rosa-sinensis and Hibiscus latent Fort Pierce virus (HLFPV), interaction between plant host and pathogen remain largely unknown, which led to deficiency of effective measures to control disease of hibiscus plants caused by HLFPV. In this study, infection of HLFPV in Hibiscus rosa-sinensis was firstly confirmed for the first time by traditional electron microscopy, modern reverse transcription polymerase chain reaction and RNA-seq methods in China (HLFPV-Ch). Sequence properties analyzing suggested that the full-length sequences (6,465 nt) of HLFPV-Ch had a high sequence identity and a similar genomic structure with other tobamoviruses. It includes a 5′-terminal untranslated region (UTR), followed by four open reading frames encoding for a 128.5-kDa replicase, a 186.5-kDa polymerase, a 31-kDa movement protein, 17.6-kDa coat protein, and the last a 3′-terminal UTR. Furthermore, HLFPV-Ch–derived virus-derived siRNAs (vsiRNAs) ant its putative target genes, reported also for the first time, were identified and characterized from disease Hibiscus rosa-sinensis through sRNA-seq and Patmatch server to investigate the interaction in this pathogen systems. HLFPV-Ch–derived vsiRNAs demonstrated several general and specific characteristics. Gene Ontology classification revealed predicted target genes by vsiRNAs are involved in abroad range of cellular component, molecular function and biological processes. Taken together, for first time, our results certified the HLFPV infection in China and provide an insight into interaction between HLFPV and Hibiscus rosa-sinensis.

Glial activation is moderated by sex in response to amyloidosis but not to tau pathology in mouse models of neurodegenerative diseases

BackgroundIn vivo assessment of neuroinflammation by 18-kDa translocator protein positron-emission-tomography (TSPO-PET) ligands receives growing interest in preclinical and clinical research of neurodegenerative disorders. Higher TSPO-PET binding as a surrogate for microglial activation in females has been reported for cognitively normal humans, but such effects have not yet been evaluated in rodent models of neurodegeneration and their controls. Thus, we aimed to investigate the impact of sex on microglial activation in amyloid and tau mouse models and wild-type controls.MethodsTSPO-PET (18F-GE-180) data of C57Bl/6 (wild-type), AppNL-G-F (β-amyloid model), and P301S (tau model) mice was assessed longitudinally between 2 and 12 months of age. The AppNL-G-F group also underwent longitudinal β-amyloid-PET imaging (Aβ-PET; 18F-florbetaben). PET results were confirmed and validated by immunohistochemical investigation of microglial (Iba-1, CD68), astrocytic (GFAP), and tau (AT8) markers. Findings in cerebral cortex were compared by sex using linear mixed models for PET data and analysis of variance for immunohistochemistry.ResultsWild-type mice showed an increased TSPO-PET signal over time (female +23%, male +4%), with a significant sex × age interaction (T = − 4.171, p < 0.001). The Aβ model AppNL-G-F mice also showed a significant sex × age interaction (T = − 2.953, p = 0.0048), where cortical TSPO-PET values increased by 31% in female AppNL-G-F mice, versus only 6% in the male mice group from 2.5 to 10 months of age. Immunohistochemistry for the microglial markers Iba-1 and CD68 confirmed the TSPO-PET findings in male and female mice aged 10 months. Aβ-PET in the same AppNL-G-F mice indicated no significant sex × age interaction (T = 0.425, p = 0.673). The P301S tau model showed strong cortical increases of TSPO-PET from 2 to 8.5 months of age (female + 32%, male + 36%), without any significant sex × age interaction (T = − 0.671, p = 0.504), and no sex differences in Iba-1, CD68, or AT8 immunohistochemistry.ConclusionFemale mice indicate sex-dependent microglia activation in aging and in response to amyloidosis but not in response to tau pathology. This calls for consideration of sex difference in TSPO-PET studies of microglial activation in mouse models of neurodegeneration and by extension in human studies.

Diversity of Plant Virus Movement Proteins: What Do They Have in Common?

The modern view of the mechanism of intercellular movement of viruses is based largely on data from the study of the tobacco mosaic virus (TMV) 30-kDa movement protein (MP). The discovered properties and abilities of TMV MP, namely, (a) in vitro binding of single-stranded RNA in a non-sequence-specific manner, (b) participation in the intracellular trafficking of genomic RNA to the plasmodesmata (Pd), and (c) localization in Pd and enhancement of Pd permeability, have been used as a reference in the search and analysis of candidate proteins from other plant viruses. Nevertheless, although almost four decades have passed since the introduction of the term “movement protein” into scientific circulation, the mechanism underlying its function remains unclear. It is unclear why, despite the absence of homology, different MPs are able to functionally replace each other in trans-complementation tests. Here, we consider the complexity and contradictions of the approaches for assessment of the ability of plant viral proteins to perform their movement function. We discuss different aspects of the participation of MP and MP/vRNA complexes in intra- and intercellular transport. In addition, we summarize the essential MP properties for their functioning as “conditioners”, creating a favorable environment for viral reproduction.

Characterization of carp seminal plasma Wap65-2 and its participation in the testicular immune response and temperature acclimation

Two functionally distinct isoforms of warm-temperature acclimation related 65-kDa protein (Wap65-1 and Wap65-2) with a role in the immune response are present in fish. To our knowledge, contrary to Wap65-1, Wap65-2 has neither been isolated nor functionally characterized in carp especially in reproductive system. The aim of this study was to characterize Wap65-2 and ascertain its functions in immune response and temperature acclimation within reproductive system. Wap65-2 corresponded to one of the most abundant proteins in carp seminal plasma, with a high immunologic similarity to their counterparts in seminal plasma of other fish species and a wide tissue distribution, with predominant expression in the liver. The immunohistochemical localization of Wap65-2 to spermatogonia, Leydig cells, and the epithelium of blood vessels within the testis suggests its role in iron metabolism during spermatogenesis and maintenance of blood-testis barrier integrity. Wap65-2 secretion by the epithelial cells of the spermatic duct and its presence around spermatozoa suggests its involvement in the protection of spermatozoa against damage caused by heme released from erythrocytes following hemorrhage and inflammation. Our results revealed an isoform-specific response of Wap65 to temperature acclimation and Aeromonas salmonicida infection which alters blood-testis barrier integrity. Wap65-2 seems to be related to the immune response against bacteria, while Wap65-1 seems to be involved in temperature acclimation. This study expands the understanding of the mechanism of carp testicular immunity against bacterial challenge and temperature changes, in which Wap65-2 seems to be involved and highlights their potential usefulness as biomarkers of inflammation and temperature acclimation.

Salivary proteins related to the diversity of diets in tropical bats.

La familia Phyllostomidae presenta una gran diversidad de dietas que requieren adaptaciones fisiológicas para metabolizar los diferentes alimentos que consumen. En frugívoros de la familia Pteropodidae e insectívoros de las familias Vespertilionidae y Molossidae se han reportado proteínas salivales distintivas de cada dieta. Por ello, se planteó determinar moléculas salivales asociadas con las diferentes dietas de los filostómidos. Los organismos se encontraban en ayuno al tomar la muestra, a la cual se le adicionó un buffer inhibidor de proteasas y se almacenó a -20°C hasta su uso. Las proteínas se identificaron por medio de SDS-PAGE y se evaluó si su presencia en los individuos estaba asociada con la historia evolutiva de las especies. Además, se determinó si las proteínas encontradas estarían relacionadas con la dieta del individuo. Se capturaron 15 especies con dietas nectarívora, insectívora y frugívora. Se encontró una proteína de 60kDa en filostómidos herbívoros y una de 50kDa en vespertilionidos y filostómidos con alto consumo de insectos. Además, se registró una proteína de 30kDa en todos los filostómidos y en 2 de las 3 especies de vespertilionidos. Los análisis indicaron que la presencia de las proteínas no estaría relacionada con la cercanía filogenética y que, para las proteínas de 30 y 50kDa, tampoco sería explicada por la dieta como sí ocurre con la proteína de 60kDa. Los filostómidos habrían retenido de su dieta ancestral insectívora la proteína de 30kDa y adquirido evolutivamente la de 60kDa para procesar plantas y lograr la amplia diversificación ecológica que presentan.

Accessory Gvp Proteins Form a Complex During Gas Vesicle Formation of Haloarchaea.

Halobacterium salinarum forms gas vesicles consisting of a protein wall surrounding a gas-filled space. The hydrophobic 8-kDa protein GvpA is the major constituent of the ribbed wall, stabilized by GvpC at the exterior surface. In addition, eight accessory Gvp proteins are involved, encoded by gvpFGHIJKLM that are co-transcribed in early stages of growth. Most of these proteins are essential, but their functions are not yet clear. Here we investigate whether GvpF through GvpM interact. Pull-down experiments performed in Haloferax volcanii with the cellulose-binding-domain as tag suggested many interactions, and most of these were supported by the split-GFP analyses. The latter study indicated that GvpL attracted all other accessory Gvp, and the related GvpF bound besides GvpL also GvpG, GvpH and GvpI. A strong interaction was found between GvpH and GvpI. GvpG showed affinity to GvpF and GvpL, whereas GvpJ, GvpK and GvpM bound GvpL only. Using GvpA for similar analyses yielded GvpF as the only interaction partner. The contact site of GvpF was confined to the N-terminal half of GvpA and subsequently mapped to certain amino acids. Taken together, our results support the idea that the accessory Gvp form a complex early in gas-vesicle assembly attracting GvpA via GvpF.

Application of antigenic biomarkers for Mycobacterium tuberculosis.

The study and characterization of biomolecules involved in the interaction between mycobacteria and their hosts are crucial to determine their roles in the invasion process and provide basic knowledge about the biology and pathogenesis of disease. Promising new biomarkers for diagnosis and immunotherapy have emerged recently. Mycobacterium is an ancient pathogen that has developed complex strategies for its persistence in the host and environment, likely based on the complexity of the network of interactions between the molecules involved in infection. Several biomarkers have received recent attention in the process of developing rapid and reliable detection techniques for tuberculosis. Among the most widely investigated antigens are CFP-10 (10-kDa culture filtrate protein), ESAT-6 (6-kDa early secretory antigenic target), Ag85A, Ag85B, CFP-7, and PPE18. Some of these antigens have been proposed as biomarkers to assess the key elements of the response to infection of both the pathogen and host. The design of novel and accurate diagnostic methods is essential for the control of tuberculosis worldwide. Presently, the diagnostic methods are based on the identification of molecules in the humoral response in infected individuals. Therefore, these tests depend on the capacity of the host to develop an immune response, which usually is heterogeneous. In the last 20 years, special attention has been given to the design of multiantigenic diagnostic methods to improve the levels of sensitivity and specificity. In this review, we summarize the state of the art in the study and use of mycobacterium biomolecules with the potential to support novel tuberculosis control strategies.

Follicle inhibition at the primordial stage without increasing apoptosis, with a combination of everolimus, verapamil.

The aim of the present study was to test whether inhibition of ovarian primordial follicles and subsequent activation can be achieved by transient mTOR inhibition. In this preclinical investigation, forty-five female immature Wistar rats were randomized in 5 groups. The control group received subcutaneous saline injections. The other groups received Everolimus, Everolimus plus Verapamil, Everolimus plus Fisetin, and Fisetin alone. Primary and secondary outcomes were measured in the left ovary after a treatment period of 8weeks. Ten days later, animals received 35IU FSH for 4days and 35IU of hCG on the 5th day. The same parameters were examined in the right ovary. AMH, estradiol, and progesterone levels were assessed at the end of both interventions. Significantly, more primordial and less atretic follicles were observed in the Everolimus plus Verapamil group. AMH and progesterone levels were substantially lower in the Everolimus group. Interestingly, after ovarian stimulation higher levels of AMH and progesterone were observed in the Everolimus plus Verapamil group. Immunoblot analysis of ovarian extracts revealed that the administration of Everolimus led to a significant reduction in the mTORC1-mediated phosphorylation of the 70-kDa ribosomal protein S6 kinase 1. This decrease was reversed in the presence of FSH after stopping drug administration. The expression of the anti-apoptotic molecule Bcl2 as well as of LC3-II and ATG12 was increased after removal of the Everolimus plus Verapamil combination, indicating reduced apoptosis and increased autophagy, whereas the levels of the proliferation marker PCNA in the granulosa cells were elevated, consistent with initiation of follicular growth.Thus, the combination of Everolimus plus Verapamil is capable of increasing the number of competent primordial follicles while reducing atresia.

Molecular goniometers for single-particle cryo-electron microscopy of DNA-binding proteins.

Correct reconstruction of macromolecular structure by cryo-electron microscopy (cryo-EM) relies on accurate determination of the orientation of single-particle images. For small (<100 kDa) DNA-binding proteins, obtaining particle images with sufficiently asymmetric features to correctly guide alignment is challenging. We apply DNA origami to construct molecular goniometers—instruments that precisely orient objects—and use them to dock a DNA-binding protein on a double-helix stage that has user-programmable tilt and rotation angles. We construct goniometers with fourteen different stage configurations to orient and visualize the protein just above the cryo-EM grid surface. Each goniometer has a distinct barcode pattern that we use during particle classification to assign angle priors to the bound protein. We use goniometers to obtain a 6.5 Å structure of BurrH, an 82-kDa DNA-binding protein whose helical pseudosymmetry prevents accurate image orientation using traditional cryo-EM. Our approach should be adaptable to other DNA-binding proteins as well as small proteins fused to DNA-binding domains.

Freezing-thawing induces deprotamination, cryocapacitation-associated changes; DNA fragmentation; and reduced progesterone sensitivity in buck spermatozoa

In the present study, there was evaluation of cryocapacitation-associated changes, apoptotic-like changes, deprotamination, total antioxidant capacity (TAC), and in vitro sperm functional attributes in Barbari bucks after freezing-thawing. The correlation between deprotamination and sperm functional characteristics was established. Using immunoblotting procedures, there was detection of the presence of a single 28-kDa protein band corresponding to protamine-1. The localization in the head region of the spermatozoa was further validated by an immunofluorescence test. Capacitated (B-) and acrosome-reacted (AR-) pattern spermatozoa, spermatozoa with the externalization of phosphatidylserine and a relatively lesser mitochondrial transmembrane potential, and deprotamination and DNA fragmentation was greater (P < 0.05) after freezing-thawing and indicated there were cryocapacitation- and apoptotic-like changes, respectively. Furthermore, the detection of phosphorylation of tyrosine-containing proteins with use of immunoblotting and immunofluorescence procedures confirmed there were cryocapacitation-like changes in the buck spermatozoa after freezing-thawing. Total antioxidant capacity (TAC), in vitro thermal resistance response, Vanguard distance, progesterone sensitivity, and in vitro capacitation response were less (P < 0.05) in the spermatozoa after freezing-thawing compared with spermatozoa after initial dilution and equilibration. Deprotamination (chromomycin A3-positive cells, CMA3+) and DNA fragmentation (TUNEL+ve) were positively correlated with B- and AR-pattern spermatozoa, while other values for other variables were negatively correlated. In conclusion, the results of this study indicated there was protamine-1 in buck spermatozoa and after freezing-thawing there was a loss of protamine-1 combined with cryocapacitation-associated changes and apoptotic-like changes in buck spermatozoa. Spermatozoa deprotamination might be attributed to increased DNA fragmentation, resulting in compromised fertilizing capacity of buck spermatozoa.

During bacteremia, Pseudomonas aeruginosa PAO1 adapts by altering the expression of numerous virulence genes including those involved in quorum sensing.

Pseudomonas aeruginosa is a Gram-negative opportunistic pathogen that produces numerous virulence factors and causes serious infections in trauma patients and patients with severe burns. We previously showed that the growth of P. aeruginosa in blood from severely burned or trauma patients altered the expression of numerous genes. However, the specific influence of whole blood from healthy volunteers on P. aeruginosa gene expression is not known. Transcriptome analysis of P. aeruginosa grown for 4 h in blood from healthy volunteers compared to that when grown in laboratory medium revealed that the expression of 1085 genes was significantly altered. Quorum sensing (QS), QS-related, and pyochelin synthesis genes were downregulated, while genes of the type III secretion system and those for pyoverdine synthesis were upregulated. The observed effect on the QS and QS-related genes was shown to reside within serum fraction: growth of PAO1 in the presence of 10% human serum from healthy volunteers significantly reduced the expression of QS and QS-regulated genes at 2 and 4 h of growth but significantly enhanced their expression at 8 h. Additionally, the production of QS-regulated virulence factors, including LasA and pyocyanin, was also influenced by the presence of human serum. Serum fractionation experiments revealed that part of the observed effect resides within the serum fraction containing <10-kDa proteins. Growth in serum reduced the production of many PAO1 outer membrane proteins but enhanced the production of others including OprF, a protein previously shown to play a role in the regulation of QS gene expression. These results suggest that factor(s) within human serum: 1) impact P. aeruginosa pathogenesis by influencing the expression of different genes; 2) differentially regulate the expression of QS and QS-related genes in a growth phase- or time-dependent mechanism; and 3) manipulate the production of P. aeruginosa outer membrane proteins.

Thioredoxin-albumin fusion protein prevents urban aerosol-induced lung injury via suppressing oxidative stress-related neutrophil extracellular trap formation.

The number of deaths from air pollution worldwide is estimated at 8.8 million per year, more than the number of deaths from smoking. Air pollutants, such as PM2.5, are known to induce respiratory and cardiovascular diseases by inducing oxidative stress. Thioredoxin (Trx) is a 12-kDa endogenous protein that exerts antioxidant activity by promoting dithiol disulfide exchange reactions. We previously synthesized human serum albumin-fused thioredoxin (HSA-Trx), which has a longer half-life in plasma compared with Trx, and demonstrated its efficacy against various diseases including respiratory diseases. Here, we examined the effect of HSA-Trx on urban aerosol-induced lung injury in mice. Urban aerosols induced lung injury and inflammatory responses in ICR mice, but intravenous administration of HSA-Trx markedly inhibited these responses. We next analyzed reactive oxygen species (ROS) production in murine lungs using an in vivo imaging system. The results show that intratracheal administration of urban aerosols induced ROS production that was inhibited by intravenously administered HSA-Trx. Finally, we found that HSA-Trx inhibited the urban aerosol-induced increase in levels of neutrophilic extracellular trap (NET) indicators (i.e., double-stranded DNA, citrullinated histone H3, and neutrophil elastase) in bronchoalveolar lavage fluid (BALF). Together, these findings suggest that HSA-Trx prevents urban aerosol-induced acute lung injury by suppressing ROS production and neutrophilic inflammation. Thus, HSA-Trx may be a potential candidate drug for preventing the onset or exacerbation of lung injury caused by air pollutants.

Brain TSPO-PET predicts later disease progression independent of relapses in multiple sclerosis.

Overactivation of microglia is associated with most neurodegenerative diseases. In this study we examined whether PET-measurable innate immune cell activation predicts multiple sclerosis disease progression. Activation of microglia/macrophages was measured using the 18-kDa translocator protein (TSPO)-binding radioligand 11C-PK11195 and PET imaging in 69 patients with multiple sclerosis and 18 age- and sex-matched healthy controls. Radioligand binding was evaluated as the distribution volume ratio from dynamic PET images. Conventional MRI and disability measurements using the Expanded Disability Status Scale were performed for patients at baseline and 4.1 ± 1.9 (mean ± standard deviation) years later. Fifty-one (74%) of the patients were free of relapses during the follow-up period. Patients had increased activation of innate immune cells in the normal-appearing white matter and in the thalamus compared to the healthy control group (P = 0.033 and P = 0.003, respectively, Wilcoxon). Forward-type stepwise logistic regression was used to assess the best variables predicting disease progression. Baseline innate immune cell activation in the normal-appearing white matter was a significant predictor of later progression when the entire multiple sclerosis cohort was assessed [odds ratio (OR) = 4.26; P = 0.048]. In the patient subgroup free of relapses there was an association between macrophage/microglia activation in the perilesional normal-appearing white matter and disease progression (OR = 4.57; P = 0.013). None of the conventional MRI parameters measured at baseline associated with later progression. Our results strongly suggest that innate immune cell activation contributes to the diffuse neural damage leading to multiple sclerosis disease progression independent of relapses.

Association of Leptin with Total and Free Testosterone: Results from the National Health and Nutrition Examination Surveys.

Introduction:Obese men can have testosterone deficiency (TD) but the etiology is uncertain. Leptin is a 16-kDa protein produced primarily by adipose tissue and, therefore, is positively associated with the amount of body fat and can affect testosterone (T) production. We hypothesized that increased leptin can be independently associated with low T.Materials and Methods:We performed a cross-sectional analysis of men from National Health and Nutrition Examination III database to evaluate the association of leptin with serum T and calculated free testosterone (cFT). Linear regression was performed with leptin, age, waist circumference, hypertension, and diabetes as independent variables predicting cFT/T. Multiple linear regression was used to determine predictors for cFT and T using variables previously significant in the univariate analysis.Results:A total of 1193 men were analyzed. As expected, older and obese men were associated with having lower T. Interestingly, increasing leptin levels were an independent predictor of decreasing T and cFT on multivariable analysis. Increasing 1ng/mL in leptin resulted in a decrease of 5.13 and 0.11 ng/dL of T and cFT, respectively (p < 0.05). Also, every additional year of life led to a T and cFT reduction of 2.87 and 0.13 ng/dL, respectively, and increasing 1 cm in waist circumference corresponded to decrease of 4ng/dL in T (p < 0.05).Conclusions:We concluded that increasing leptin, age, and waist circumference were associated with decreasing of T and cFT. Elevated leptin levels could be one of the potential etiologies of TD.