PFDN4 as a Prognostic Marker Was Associated with Chemotherapy Resistance through CREBP1/AURKA Pathway in Triple-Negative Breast Cancer.

Breast cancer is the most common malignancy and its incidence is increasing. It is currently mainly treated by clinical chemotherapy, but chemoresistance remains poorly understood. Prefolded proteins 4 (PFDN4) are molecular chaperone complexes that bind to newly synthesized polypeptides and allow them to fold correctly to stabilize protein formation. This study aimed to investigate the role of PFDN4 in chemotherapy resistance in breast cancer. Our study found that PFDN4 was highly expressed in breast cancer compared to normal tissues and was statistically significantly associated with stage, nodal status, subclasses (luminal, HER2 positive and triple negative), triple-negative subtype and disease-specific survival by TCGA database analysis. CRISPR knockout of PFDN4 inhibited the growth of 89% of breast cancer cell lines, and the triple-negative cell line exhibited a stronger inhibitory effect than the non-triple-negative cell line. High PFDN4 expression was associated with poor overall survival in chemotherapy and resistance to doxorubicin and paclitaxel through the CREBP1/AURKA pathway in the triple-negative MDAMB231 cell line. This study provides insightful evidence for the value of PFDN4 in poor prognosis and chemotherapy resistance in breast cancer patients.

Using Next-Generation Sequencing and Bioinformatic Methods to Predict New Genes That May Be Regulated by CD47 in Oral Squamous Cell Carcinoma.

Oral squamous cell carcinoma (OSCC) is one of the most common cancers in the world, and the incidence and death rate of OSCC in men is twice that of women. CD47 is a ubiquitous cell surface transmembrane protein, also known as integrin-related protein (IAP). Previous studies have pointed out that CD47 can inhibit the growth of OSCC, but the detailed mechanism is not clear. This study aimed to explore the effect of CD47 gene expression profiles in OSCC. The OSCC cell lines, OECM-1 and OC-2, overexpressed CD47, and the expression profiles of mRNAs were analyzed through next-generation sequencing (NGS) with a bioinformatic approach. A total of 14 differentially expressed genes (DEGs) were listed. In addition, ingenuity pathway analysis (IPA) was used to analyze the molecular function (MF), biological process (BP), and cellular component (CC) network signaling. The human protein atlas (HPA) database was used to analyze gene expression and the survivability of human cancer. The results found that HSPA5, HYOU1, and PDIA4 were involved in the IPA network and when highly expressed, mediated the survivability of cancer. In addition, HSPA5 was positively and significantly correlated with CD47 expression (p < 0.0001) and induced by CD47-overexpression in the OECM-1 and OC-2 OSCC cancer cell lines. These findings provide important insights into possible new diagnostic strategies, including unfolded protein for OSCC-targeting CD47.

Effects of Luteolin on Human Breast Cancer Using Gene Expression Array: Inferring Novel Genes.

Taraxacum officinale (dandelion) is often used in traditional Chinese medicine for the treatment of cancer; however, the downstream regulatory genes and signaling pathways mediating its effects on breast cancer remain unclear. The present study aimed to explore the effects of luteolin, the main biologically active compound of T. officinale, on gene expression profiles in MDA-MB-231 and MCF-7 breast cancer cells. The results revealed that luteolin effectively inhibited the proliferation and motility of the MDA-MB-231 and MCF-7 cells. The mRNA expression profiles were determined using gene expression array analysis and analyzed using a bioinformatics approach. A total of 41 differentially expressed genes (DEGs) were found in the luteolin-treated MDA-MB-231 and MCF-7 cells. A Gene Ontology analysis revealed that the DEGs, including AP2B1, APP, GPNMB and DLST, mainly functioned as oncogenes. The human protein atlas database also found that AP2B1, APP, GPNMB and DLST were highly expressed in breast cancer and that AP2B1 (cut-off value, 75%) was significantly associated with survival rate (p = 0.044). In addition, a Kyoto Encyclopedia of Genes and Genomes pathway analysis revealed that the DEGs were involved in T-cell leukemia virus 1 infection and differentiation. On the whole, the findings of the present study provide a scientific basis that may be used to evaluate the potential benefits of luteolin in human breast cancer. Further studies are required, however, to fully elucidate the role of the related molecular pathways.

The Impact of Pharmacist-Managed Service on Warfarin Therapy in Patients after Mechanical Valve Replacement.

Objective: To evaluate the impact of pharmacist interventions on international normalized ratio (INR) control during the warfarin initiation phase after mechanical valve replacement. Methods: This was a retrospective cohort study conducted in a cardiovascular surgery ward in a tertiary hospital from August 1, 2015, to July 31, 2019. Patients aged ≥20 years who were admitted for mechanical valve replacement were enrolled in this study and further classified into conventional and pharmacist-managed warfarin therapy (PMWT) groups. All participants were prospectively followed up until the first outpatient appointment after valve replacement. The effectiveness outcomes were time in therapeutic range (TTR), time to therapeutic INR, number of patients with therapeutic INR at discharge and at first outpatient appointment, and length of hospital stay. The safety outcome was the number of patients with any supratherapeutic INR during the hospital stay. Multivariate logistic regression analyses were also used to determine the predictors of a therapeutic INR at discharge or with any supratherapeutic INR during admission. Results: A total of 39 and 33 patients were enrolled in the conventional and PMWT groups, respectively. At discharge, 18 patients (46.2%) in the conventional group and 24 patients (72.7%) in the PMWT group had achieved the therapeutic INR (P=0.023). Compared to the conventional group, fewer patients in the PMWT group had supratherapeutic INR during hospital stay (35.9% vs. 9.0%, P=0.008). No significant differences were found in TTR, time to therapeutic INR, number of patients with therapeutic INR at return appointment, and length of stay between the study groups. In the multivariate regression analyses, PMWT predicted achieving therapeutic INR at discharge (odds ratio (OR) and 95% confidence interval (CI), 3.14 [1.08-9.14]) and was inversely associated with supratherapeutic INRs during admission (OR = 0.21 [0.05-0.82]). Conclusions: Among patients admitted for mechanical valve replacement, the implementation of PMWT was associated with optimal therapeutic INR at discharge and no supratherapeutic INR during admission. Therefore, pharmacist participation is essential for improving the quality of warfarin therapy.

A Novel Mucosal Adjuvant System for Immunization against Avian Coronavirus Causing Infectious Bronchitis.

Infectious bronchitis (IB) caused by infectious bronchitis virus (IBV) is currently a major threat to chicken health, with multiple outbreaks being reported in the United States over the past decade. Modified live virus (MLV) vaccines used in the field can persist and provide the genetic material needed for recombination and emergence of novel IBV serotypes. Inactivated and subunit vaccines overcome some of the limitations of MLV with no risk of virulence reversion and emergence of new virulent serotypes. However, these vaccines are weakly immunogenic and poorly protective. There is an urgent need to develop more effective vaccines that can elicit a robust, long-lasting immune response. In this study, we evaluate a novel adjuvant system developed from Quil-A and chitosan (QAC) for the intranasal delivery of nucleic acid immunogens to improve protective efficacy. The QAC adjuvant system forms nanocarriers (<100 nm) that efficiently encapsulate nucleic acid cargo, exhibit sustained release of payload, and can stably transfect cells. Encapsulation of plasmid DNA vaccine expressing IBV nucleocapsid (N) protein by the QAC adjuvant system (pQAC-N) enhanced immunogenicity, as evidenced by robust induction of adaptive humoral and cellular immune responses postvaccination and postchallenge. Birds immunized with pQAC-N showed reduced clinical severity and viral shedding postchallenge on par with protection observed with current commercial vaccines without the associated safety concerns. Presented results indicate that the QAC adjuvant system can offer a safer alternative to the use of live vaccines against avian and other emerging coronaviruses.IMPORTANCE According to 2017 U.S. agriculture statistics, the combined value of production and sales from broilers, eggs, turkeys, and chicks was $42.8 billion. Of this number, broiler sales comprised 67% of the industry value, with the production of >50 billion pounds of chicken meat. The economic success of the poultry industry in the United States hinges on the extensive use of vaccines to control infectious bronchitis virus (IBV) and other poultry pathogens. The majority of vaccines currently licensed for poultry health include both modified live vaccine and inactivated pathogens. Despite their proven efficacy, modified live vaccine constructs take time to produce and could revert to virulence, which limits their safety. The significance of our research stems from the development of a safer and potent alternative mucosal vaccine to replace live vaccines against IBV and other emerging coronaviruses.

BRAF protein immunoprecipitation, elution, and digestion from cell extract using a microfluidic mixer for mutant BRAF protein quantification by mass spectrometry.

This study utilized a microfluidic mixer for the sample pretreatment of cell extracts for target protein quantification by mass spectrometers, including protein immunoprecipitation and protein enzymatic digestion. The time of sample pretreatment was reduced and thus the throughput of quantitative mutant proteins was increased by using the proposed method. Whole cell lysates of the cancer cell line HT-29 with gene mutations were used as the sample. The target protein BRAF was immunoprecipitated using magnetic beads in a pneumatic micromixer. Purified protein was then eluted and digested by trypsin in another two micromixers to yield peptide fragments in the solution. Using stable isotope-labeled standard as the internal control, wild-type and mutant BRAF proteins were quantified using mass spectrometry, which could be used for cancer screening. Compared with conventional methods in which protein immunoprecipitation lasts overnight, the micromixer procedure takes only 1 h, likely improving the throughput of mutant BRAF protein quantification by mass spectrometry. Graphical abstract Three micromixers were used to reduce the sample pretreatment time of cell extracts for target protein quantification by mass spectrometers, including protein immunoprecipitation, protein elution, and protein enzymatic digestion.

Circulating Interleukin-6 is Associated with Prognosis and Genetic Polymorphisms of MIR608 in Patients with Esophageal Squamous Cell Carcinoma.

BACKGROUND: No effective targeted therapy exists for esophageal squamous cell carcinoma (ESCC), the major cell type of esophageal cancer. The pleiotropic cytokine interleukin (IL)-6 is associated with adverse prognosis of some cancers, and the open reading frame of IL-6 contains an miR-608 microRNA-targeted site. We investigated the correlation of circulating IL-6 levels with prognosis and with the mir608:rs4919510 genetic polymorphism in ESCC. METHODS: A total of 213 patients with primary ESCC were enrolled. Plasma IL-6 levels of ESCC patients were analyzed by enzyme-linked immunosorbent assay (ELISA). The patients' genotypes of mir608:rs4919510 were analyzed using the MassARRAY system, and functional assays were performed by transient overexpression in cells. The cytotoxicity of IL-6 signaling blockers in ESCC cells was analyzed by MTT assay. RESULTS: We found that plasma IL-6 levels significantly correlated with overall survival (p = 0.019), disease recurrence (p = 0.003), and postoperative complications (p =0.002). Patients with the GG genotype of mir608:rs4919510 had a 4.56-fold increased risk of high expression of IL-6 compared with patients with the CC genotype (odds ratio 4.56, 95% confidence interval 1.87-11.09; p =0.001). Transient overexpression of the miR-608 C (miR-608_C) and G variants (miR-608_G) in cancer cells revealed that the miR-608_G variant was less efficient in regulating the expression of IL-6 compared with miR-608_C. Finally, the IL-6 signaling blocker ruxolitinib exhibited effective cytotoxicity in ESCC cells. CONCLUSIONS: The results of this study provide a novel direction for a biomarker-based targeted therapy for ESCC.

Effect of Extracorporeal Membrane Oxygenation on the New Vancomycin Dosing Regimen in Critically Ill Patients Receiving Continuous Venovenous Hemofiltration.

BACKGROUND: The optimal dosing regimen of vancomycin for critically ill patients receiving continuous venovenous hemofiltration (CVVH) remains controversial, not to mention those with concurrent use of extracorporeal membrane oxygenation (ECMO). We aimed to determine if a new dosing regimen can achieve the target vancomycin trough concentration (Ctrough) of 10-20 mcg/mL in patients receiving CVVH with or without ECMO. METHODS: We conducted a retrospective study by enrolling patients who received vancomycin while undergoing CVVH. The vancomycin dosing regimen was 15-20 mg/kg as the loading dose and 7.5 mg/kg every 12 hours as the maintenance doses. Serum concentration was determined after at least 4 doses of vancomycin were given. RESULTS: A total of 38 patients were enrolled, of which 21 were also on ECMO. The ultrafiltration rate of CVVH was 30.6 ± 5.5 mL·kg·h with the Ctrough of 14.7 ± 3.5 mcg/mL. Ctrough was within the target range in 82% of patients. All CVVH-only patients achieved the target concentration, whereas only 76.2% of those with concurrent ECMO did (P = 0.031). CONCLUSIONS: All patients receiving CVVH achieved the target Ctrough with this new dosing regimen, but those with concurrent ECMO did not. Ctrough must be more closely monitored in patients using ECMO simultaneously.

LacI Transcriptional Regulatory Networks in Clostridium thermocellum DSM1313.

Organisms regulate gene expression in response to the environment to coordinate metabolic reactions. Clostridium thermocellum expresses enzymes for both lignocellulose solubilization and its fermentation to produce ethanol. One LacI regulator termed GlyR3 in C. thermocellum ATCC 27405 was previously identified as a repressor of neighboring genes with repression relieved by laminaribiose (a ß-1,3 disaccharide). To better understand the three C. thermocellum LacI regulons, deletion mutants were constructed using the genetically tractable DSM1313 strain. DSM1313 lacI genes Clo1313_2023, Clo1313_0089, and Clo1313_0396 encode homologs of GlyR1, GlyR2, and GlyR3 from strain ATCC 27405, respectively. Growth on cellobiose or pretreated switchgrass was unaffected by any of the gene deletions under controlled-pH fermentations. Global gene expression patterns from time course analyses identified glycoside hydrolase genes encoding hemicellulases, including cellulosomal enzymes, that were highly upregulated (5- to 100-fold) in the absence of each LacI regulator, suggesting that these were repressed under wild-type conditions and that relatively few genes were controlled by each regulator under the conditions tested. Clo1313_2022, encoding lichenase enzyme LicB, was derepressed in a ΔglyR1 strain. Higher expression of Clo1313_1398, which encodes the Man5A mannanase, was observed in a ΔglyR2 strain, and α-mannobiose was identified as a probable inducer for GlyR2-regulated genes. For the ΔglyR3 strain, upregulation of the two genes adjacent to glyR3 in the celC-glyR3-licA operon was consistent with earlier studies. Electrophoretic mobility shift assays have confirmed LacI transcription factor binding to specific regions of gene promoters.IMPORTANCE Understanding C. thermocellum gene regulation is of importance for improved fundamental knowledge of this industrially relevant bacterium. Most LacI transcription factors regulate local genomic regions; however, a small number of those genes encode global regulatory proteins with extensive regulons. This study indicates that there are small specific C. thermocellum LacI regulons. The identification of LacI repressor activity for hemicellulase gene expression is a key result of this work and will add to the small body of existing literature on the area of gene regulation in C. thermocellum.

Clopidogrel-Associated Neutropenia: Case Report and Review of the Literature.

Clopidogrel is a second generation of thienopyridine, which has antiplatelet effect by inhibiting P2Y12 receptor. Hematologic adverse effect is very uncommon during clopidogrel use, but some cases of clopidogrel-associated neutropenia were reported in the past decade. Until now, there was no summary data to delineate the clinical course and safe alternatives of this event. We report 2 cases of clopidogrel-associated neutropenia and review other 10 case reports from 2000 to 2014. The median onset of neutropenia was 22 days, and the recovery time was 4 days after receiving granulocyte-colony-stimulating factor. Bone marrow studies in 6 cases all showed hypocellular or toxic damage. Six cases used cilostazol, prasugrel, or ticagrelor as safe alternatives. Closely monitoring blood cell counts is highly suggested in the first month after using clopidogrel. Newer P2Y12 inhibitors, especially ticagrelor, could be effective and safe alternatives if patients had a history of clopidogrel-associated neutropenia.

Uracil DNA glycosylase BKRF3 contributes to Epstein-Barr virus DNA replication through physical interactions with proteins in viral DNA replication complex.

UNLABELLED: Epstein-Barr virus (EBV) BKRF3 shares sequence homology with members of the uracil-N-glycosylase (UNG) protein family and has DNA glycosylase activity. Here, we explored how BKRF3 participates in the DNA replication complex and contributes to viral DNA replication. Exogenously expressed Flag-BKRF3 was distributed mostly in the cytoplasm, whereas BKRF3 was translocated into the nucleus and colocalized with the EBV DNA polymerase BALF5 in the replication compartment during EBV lytic replication. The expression level of BKRF3 increased gradually during viral replication, coupled with a decrease of cellular UNG2, suggesting BKRF3 enzyme activity compensates for UNG2 and ensures the fidelity of viral DNA replication. In immunoprecipitation-Western blotting, BKRF3 was coimmuno-precipitated with BALF5, the polymerase processivity factor BMRF1, and the immediate-early transactivator Rta. Coexpression of BMRF1 appeared to facilitate the nuclear targeting of BKRF3 in immunofluorescence staining. Residues 164 to 255 of BKRF3 were required for interaction with Rta and BALF5, whereas residues 81 to 166 of BKRF3 were critical for BMRF1 interaction in glutathione S-transferase (GST) pulldown experiments. Viral DNA replication was defective in cells harboring BKRF3 knockout EBV bacmids. In complementation assays, the catalytic mutant BKRF3(Q90L,D91N) restored viral DNA replication, whereas the leucine loop mutant BKRF3(H213L) only partially rescued viral DNA replication, coupled with a reduced ability to interact with the viral DNA polymerase and Rta. Our data suggest that BKRF3 plays a critical role in viral DNA synthesis predominantly through its interactions with viral proteins in the DNA replication compartment, while its enzymatic activity may be supplementary for uracil DNA glycosylase (UDG) function during virus replication. IMPORTANCE: Catalytic activities of both cellular UDG UNG2 and viral UDGs contribute to herpesviral DNA replication. To ensure that the enzyme activity executes at the right time and the right place in DNA replication forks, complex formation with other components in the DNA replication machinery provides an important regulation for UDG function. In this study, we provide the mechanism for EBV UDG BKRF3 nuclear targeting and the interacting domains of BKRF3 with viral DNA replication proteins. Through knockout and complementation approaches, we further demonstrate that in addition to UDG activity, the interaction of BKRF3 with viral proteins in the replication compartment is crucial for efficient viral DNA replication.

Recombinant lipoprotein-based vaccine candidates against C. difficile infections.

BACKGROUND: Opportunistically nosocomial infections in hospitalized patients are often related to Clostridium difficile infections (CDI) due to disruption of the intestinal micro-flora by antibiotic therapies during hospitalization. Clostridial exotoxins A and B (TcdA and TcdB) specifically bind to unknown glycoprotein(s) in the host intestine, disrupt the intestinal barrier leading to acute inflammation and diarrhea. The C-terminal receptor binding domain of TcdA (A-rRBD) has been shown to elicit antibody responses that neutralize TcdA toxicity in Vero cell cytotoxicity assays, but not effectively protect hamsters against a lethal dose challenge of C. difficile spores. To develop an effective recombinant subunit vaccine against CDI, A-rRBD was lipidated (rlipoA-RBD) as a rational design to contain an intrinsic adjuvant, a toll-like receptor 2 agonist and expressed in Escherichia coli. RESULTS: The purified rlipoA-RBD was characterized immunologically and found to have the following properties: (a) mice, hamsters and rabbits vaccinated with 3 µg of rlipoA-RBD produced strong antibody responses that neutralized TcdA toxicity in Vero cell cytotoxicity assays; furthermore, the neutralization titer was comparable to those obtained from antisera immunized either with 10 µg of TcdA toxoid or 30 µg of A-rRBD; (b) rlipoA-RBD elicited immune responses and protected mice from TcdA challenge, but offered insignificant protection (10 to 20 %) against C. difficile spores challenge in hamster models; (c) only rlipoA-RBD formulated with B-rRBD consistently confers protection (90 to 100 %) in the hamster challenge model; and (d) rlipoA-RBD was found to be 10-fold more potent than A-rRBD as an adjuvant to enhancing immune responses against a poor antigen such as ovalbumin. CONCLUSION: These results indicate that rlipoA-RBD formulated with B-rRBD could be an excellent vaccine candidate for preclinical studies and future clinical trials.

Germinal center kinase-like kinase overexpression in T cells as a novel biomarker in rheumatoid arthritis.

OBJECTIVE: Germinal center kinase-like kinase (GLK; also called MAPKKKK-3) activates protein kinase Cθ (PKCθ) during T cell activation and controls autoimmunity in lupus patients. Intracellular kinases are involved in the pathogenesis of rheumatoid arthritis (RA). We undertook this study to determine the role of GLK in RA. METHODS: The severity of collagen-induced arthritis (CIA) was studied in GLK-deficient mice. Expression levels of GLK from RA patients were determined by Western blotting, flow cytometry, real-time polymerase chain reaction, and immunohistochemical staining. Localization of GLK in T cells was identified by confocal microscopy. RA disease activity was assessed using the Disease Activity Score in 28 joints. RESULTS: GLK-deficient mice displayed impaired CIA development and decreased inflammatory cytokine levels. Local T cell infiltration and collagen restimulation responses were impaired by GLK deficiency. RA patients showed significantly higher GLK protein and messenger RNA levels in peripheral blood T cells than did healthy controls. GLK-overexpressing T cells in synovial fluid and synovial tissue samples from RA patients were increased compared with those from osteoarthritis patients. Confocal microscopy and flow cytometry showed that GLK colocalized and coexisted with phosphorylated PKCθ in T cells from RA patients. Frequencies of GLK-expressing T cells were significantly correlated with RA disease activity. CONCLUSION: GLK overexpression in T cells contributes to the pathogenesis of RA, indicating that GLK is a novel biomarker for autoimmune disease severity and a potential therapeutic target for RA.

Vascular Endothelial Growth Factor Inhibitors and the Risk of Aortic Aneurysm and Aortic Dissection.

Importance: Vascular endothelial growth factor pathway inhibitors (VPIs) pose a concern for aortic aneurysm (AA) and aortic dissection (AD), signaling potential vascular disease development. Objective: To investigate VPI-associated AA and AD. Design, Setting, and Participants: This case-control study with a nested design used full population data from a national claims database in Taiwan between 2011 and 2019. Eligible participants were aged 20 years or older with kidney, hepatic, gastrointestinal, or pancreatic cancer diagnosed between January 1, 2012, and December 31, 2019. The first cancer diagnosis date was defined as the cohort entry date. Cases were patients who received a diagnosis of AA or AD in hospitalizations or emergency visits between the cohort entry date and December 31, 2019. Controls were matched by ratio (up to 1:5) based on age, sex, cancer type, cohort entry date, and the index date (ie, the first AA or AD event date). Data analysis was performed between January 2022 and December 2023. Exposures: Use of the oral VPIs sorafenib, sunitinib, and pazopanib between cohort entry date and index date. Main Outcomes and Measures: In the primary analysis, AA and AD were evaluated compositely, while in the secondary analyses, they were evaluated separately. Adjusted odds ratios (aORs) were calculated using conditional logistic regression to assess the association with VPI use (sorafenib, sunitinib, and pazopanib) considering various VPI exposure windows and cumulative use. Results: A total of 1461 cases were included (mean [SD] age, 73.0 [12.3] years; 1118 male patients [76.5%]), matched to 7198 controls. AA or AD risk increased with a VPI exposure of 100 days or less before the index date (aOR, 2.10; 95% CI, 1.40-3.15), mainly from VPI-associated AD (aOR, 3.09; 95% CI, 1.77-5.39). Longer VPI duration (68 days or more: aOR, 2.64; 95% CI, 1.66-4.19) and higher cumulative dose (61 or more defined daily doses: aOR, 2.65; 95% CI, 1.66-4.23) increased the risk. Conclusions and Relevance: The use of the 3 study VPIs (sorafenib, sunitinib, and pazopanib) was associated with an increased risk of AA and AD in patients with cancer, essentially all of the risk from VPI-associated AD. Future studies are needed to determine the risk factors of VPI-associated AA and AD, as well as to establish a class effect.

Key role for the alternative sigma factor, SigH, in the intracellular life of Mycobacterium avium subsp. paratuberculosis during macrophage stress.

Mycobacterium avium subsp. paratuberculosis causes Johne's disease, an enteric infection in cattle and other ruminants, greatly afflicting the dairy industry worldwide. Once inside the cell, M. avium subsp. paratuberculosis is known to survive harsh microenvironments, especially those inside activated macrophages. To improve our understanding of M. avium subsp. paratuberculosis pathogenesis, we examined phagosome maturation associated with transcriptional responses of M. avium subsp. paratuberculosis during macrophage infection. Monitoring cellular markers, only live M. avium subsp. paratuberculosis bacilli were able to prevent phagosome maturation and reduce its acidification. On the transcriptional level, over 300 M. avium subsp. paratuberculosis genes were significantly and differentially regulated in both naive and IFN-γ-activated macrophages. These genes include the sigma factor H (sigH) that was shown to be important for M. avium subsp. paratuberculosis survival inside gamma interferon (IFN-γ)-activated bovine macrophages. Interestingly, an sigH-knockout mutant showed increased sensitivity to a sustained level of thiol-specific oxidative stress. Large-scale RNA sequence analysis revealed that a large number of genes belong to the sigH regulon, especially following diamide stress. Genes involved in oxidative stress and virulence were among the induced genes in the sigH regulon with a putative consensus sequence for SigH binding that was recognized in a subset of these genes (n = 30), suggesting direct regulation by SigH. Finally, mice infections showed a significant attenuation of the ΔsigH mutant compared to its parental strain, suggesting a role for sigH in M. avium subsp. paratuberculosis virulence. Such analysis could identify potential targets for further testing as vaccine candidates against Johne's disease.

The Immunogenicity and Safety of Mycobacterium tuberculosis-mosR-Based Double Deletion Strain in Mice.

Mycobacterium tuberculosis (M. tuberculosis) remains a significant global health threat, accounting for ~1.7 million deaths annually. The efficacy of the current vaccine, M. bovis BCG, ranges from 0 to 80% in children and does not prevent adulthood tuberculosis. We explored the immune profile and safety of a live-attenuated M. tuberculosis construct with double deletions of the mosR and echA7 genes, where previously, single mutations were protective against an M. tuberculosis aerosol challenge. Over 32 weeks post-vaccination (WPV), immunized mice with M. tuberculosisΔmosRΔechA7 (double mutant) were sacrificed to evaluate the vaccine persistence, histopathology, and immune responses. Interestingly, despite similar tissue colonization between the vaccine double mutant and wild-type M. tuberculosis, the vaccine construct showed a greater reaction to the ESAT-6, TB.10, and Ag85B antigens with peptide stimulation. Additionally, there was a greater number of antigen-specific CD4 T cells in the vaccine group, accompanied by significant polyfunctional T-cell responses not observed in the other groups. Histologically, mild but widely distributed inflammatory responses were recorded in the livers and lungs of the immunized animals at early timepoints, which turned into organized inflammatory foci via 32WPV, a pathology not observed in BCG-immunized mice. A lower double-mutant dose resulted in significantly less tissue colonization and less tissue inflammation. Overall, the double-mutant vaccine elicited robust immune responses dominated by antigen-specific CD4 T cells, but also triggered tissue damage and vaccine persistence. The findings highlight key features associated with the immunogenicity and safety of the examined vaccine construct that can benefit the future evaluation of other live vaccines.

A DNA Prime and MVA Boost Strategy Provides a Robust Immunity against Infectious Bronchitis Virus in Chickens.

Infectious bronchitis (IB) is an acute respiratory disease of chickens caused by the avian coronavirus Infectious Bronchitis Virus (IBV). Modified Live Virus (MLV) vaccines used commercially can revert to virulence in the field, recombine with circulating serotypes, and cause tissue damage in vaccinated birds. Previously, we showed that a mucosal adjuvant system, QuilA-loaded Chitosan (QAC) nanoparticles encapsulating plasmid vaccine encoding for IBV nucleocapsid (N), is protective against IBV. Herein, we report a heterologous vaccination strategy against IBV, where QAC-encapsulated plasmid immunization is followed by Modified Vaccinia Ankara (MVA) immunization, both expressing the same IBV-N antigen. This strategy led to the initiation of robust T-cell responses. Birds immunized with the heterologous vaccine strategy had reduced clinical severity and >two-fold reduction in viral burden in lachrymal fluid and tracheal swabs post-challenge compared to priming and boosting with the MVA-vectored vaccine alone. The outcomes of this study indicate that the heterologous vaccine platform is more immunogenic and protective than a homologous MVA prime/boost vaccination strategy.

Vorinostat decrease M2 macrophage polarization through ARID1A6488delG/HDAC6/IL-10 signaling pathway in endometriosis-associated ovarian carcinoma.

Endometriosis is a common disease in women and may be one of the factors that induces malignant epithelial ovarian tumors. Previous studies suggested that endometriosis is related to ARID1A mutation mediating the expression of HDAC6, but the detailed pathogenic mechanism is still unclear. First, we collected endometriosis-associated ovarian carcinoma (EAOC) clinical samples and examined the expression of HDAC6. Our results found that the high HDAC6 expression group was positively correlated with EAOC histology (P = 0.015), stage (P < 0.000), and tumor size (P < 0.000) and inversely correlated with survival (P < 0.000). We also found that ARID1A6488delG/HDAC6 induced M2 polarization of macrophages through IL-10. In addition, the HDAC inhibitor (HDACi) vorinostat inhibited cell growth and blocked the effect of HDAC6. Tomographic microscopy was used to monitor the live cell morphology of these treated cells, and we found that vorinostat treatment resulted in substantial cell apoptosis by 3 h 42 min. Next, we established a transgenic mouse model of EAOC and found that vorinostat significantly reduced the size of ovarian tumors by inhibiting M2 macrophage polarization in mice. Together, these data demonstrate that the signaling pathway of E4F1/ARID1A6488delG/HDAC6/GATA3 mediates macrophage polarization and provides a novel immune cell-associated therapeutic strategy targeting IL-10 in EAOC.

Cost-Benefit Analysis of Involving Pharmacist for Medication Therapy Management in a Heart Transplant Clinic.

BACKGROUND: Drug-related problems (DRPs) are common in recipients of solid organ transplants. Pharmacist-led medication therapy management (MTM) has cost benefits in kidney and liver transplants; however, whether MTM is also beneficial in heart transplants remains unclear. This study explored the cost benefits of involving pharmacists in the heart transplant clinic. METHODS: This retrospective study evaluated DRPs for 1 year after implementation of pharmacist-led MTM in a heart transplant clinic. The DRPs were compared between patients receiving transplantation for <1 and >1 year. The risk matrix method was used to assess each DRP in terms of the estimated probability and severity of consequent adverse drug events (ADEs). For cost analysis, both estimated cost savings and avoidance were calculated. RESULTS: During the 1-year MTM, 372 DRPs were identified by the pharmacist, among which 169 (45%) and 203 (55%) were from patients at <1-year and ≥1-year post-transplant periods, respectively. The 2 post-transplant periods (<1 year and ≥1 year) exhibited significant differences in the distribution of the dosage or frequency problems (30% vs 18%, P = .005) and the suggestion of more appropriate medication (4% vs 10%, P = .024). In all, 92 (29%) DRPs had an ADE probability of >10%; and 63 (17%) DRPs were estimated to cause ADEs with moderate severity or higher. The estimated cost savings and cost avoidance were US $4902 and US $4519, which equaled a cost-benefit ratio of 2.39. CONCLUSION: Integration of pharmacists into heart transplant clinics could help address DRPs and may have cost benefits.

Genome sequencing of ovine isolates of Mycobacterium avium subspecies paratuberculosis offers insights into host association.

BACKGROUND: The genome of Mycobacterium avium subspecies paratuberculosis (MAP) is remarkably homogeneous among the genomes of bovine, human and wildlife isolates. However, previous work in our laboratories with the bovine K-10 strain has revealed substantial differences compared to sheep isolates. To systematically characterize all genomic differences that may be associated with the specific hosts, we sequenced the genomes of three U.S. sheep isolates and also obtained an optical map. RESULTS: Our analysis of one of the isolates, MAP S397, revealed a genome 4.8 Mb in size with 4,700 open reading frames (ORFs). Comparative analysis of the MAP S397 isolate showed it acquired approximately 10 large sequence regions that are shared with the human M. avium subsp. hominissuis strain 104 and lost 2 large regions that are present in the bovine strain. In addition, optical mapping defined the presence of 7 large inversions between the bovine and ovine genomes (~ 2.36 Mb). Whole-genome sequencing of 2 additional sheep strains of MAP (JTC1074 and JTC7565) further confirmed genomic homogeneity of the sheep isolates despite the presence of polymorphisms on the nucleotide level. CONCLUSIONS: Comparative sequence analysis employed here provided a better understanding of the host association, evolution of members of the M. avium complex and could help in deciphering the phenotypic differences observed among sheep and cattle strains of MAP. A similar approach based on whole-genome sequencing combined with optical mapping could be employed to examine closely related pathogens. We propose an evolutionary scenario for M. avium complex strains based on these genome sequences.