Systemic neutrophil degranulation and emergency granulopoiesis in patients with Clostridioides difficile infection.

OBJECTIVES: Clostridioides difficile infection (CDI) is characterized by neutrophilia in blood, with a high leukocyte count accompanying severe infection. In this study, we characterized peripheral blood neutrophil activation and maturity in CDI by (i) developing a method to phenotype stored neutrophils for disease-related developmental alterations and (ii) assessing neutrophil-associated biomarkers. METHODS: We stored fixed leukocytes from blood collected within 24 h of diagnosis from a cohort of hospitalized patients with acute CDI. Additional study cohorts included recurrent CDI patients at time of and two months after FMT therapy and a control healthy cohort. We assessed levels of neutrophil surface markers CD66b, CD11b, CD16 and CD10 by flow cytometry. Plasma neutrophil elastase and lipocalin-2 were measured using ELISA, while G-CSF, GM-CSF and cytokines were measured using O-link Proteomic technology. RESULTS: CD66b+ neutrophil abundance assessed by flow cytometry correlated well with complete blood counts, establishing that neutrophils in stored blood are sufficiently well-preserved for phenotyping by flow cytometry. Neutrophil abundance was significantly increased in CDI patients compared to healthy controls. Emergency granulopoiesis in acute CDI patients was evidenced by lower neutrophil surface expression of CD10, CD11b and CD16. CD10+ staining of neutrophils started to recover within 3-7 days of CDI treatment. Neutrophil activation and degranulation were higher in acute CDI as assessed by plasma neutrophil elastase and lipocalin-2. Biomarker levels in immunocompetent subjects were associated with recurrence and fatal outcomes. CONCLUSIONS: Neutrophil activation and emergency granulopoiesis characterize the early immune response in acute CDI, with plasma degranulation biomarkers predictive of disease severity.

Novel Biomarkers, Including tcdB PCR Cycle Threshold, for Predicting Recurrent Clostridioides difficile Infection.

Traditional clinical models for predicting recurrent Clostridioides difficile infection do not perform well, likely owing to the complex host-pathogen interactions involved. Accurate risk stratification using novel biomarkers could help prevent recurrence by improving underutilization of effective therapies (i.e., fecal transplant, fidaxomicin, bezlotoxumab). We used a biorepository of 257 hospitalized patients with 24 features collected at diagnosis, including 17 plasma cytokines, total/neutralizing anti-toxin B IgG, stool toxins, and PCR cycle threshold (CT) (a proxy for stool organism burden). The best set of predictors for recurrent infection was selected by Bayesian model averaging for inclusion in a final Bayesian logistic regression model. We then used a large PCR-only data set to confirm the finding that PCR CT predicts recurrence-free survival using Cox proportional hazards regression. The top model-averaged features were (probabilities of >0.05, greatest to least): interleukin 6 (IL-6), PCR CT, endothelial growth factor, IL-8, eotaxin, IL-10, hepatocyte growth factor, and IL-4. The accuracy of the final model was 0.88. Among 1,660 cases with PCR-only data, cycle threshold was significantly associated with recurrence-free survival (hazard ratio, 0.95; P < 0.005). Certain biomarkers associated with C. difficile infection severity were especially important for predicting recurrence; PCR CT and markers of type 2 immunity (endothelial growth factor [EGF], eotaxin) emerged as positive predictors of recurrence, while type 17 immune markers (IL-6, IL-8) were negative predictors. In addition to novel serum biomarkers (particularly, IL-6, EGF, and IL-8), the readily available PCR CT may be critical to augment underperforming clinical models for C. difficile recurrence.

Protocol for Testing Yoga to Reduce Post-Lumbar Spine Surgery Pain: A Methodology Article.

Currently, acute postoperative pain during hospitalization is primarily managed by medications, and patients must adhere to restrictive postoperative precautions for 3 months following lumbar spine surgeries. Yoga can be an alternative approach to assist in acute and subacute postoperative pain management, anxiety, and return to function. The purpose of the present work was to develop and test the feasibility and explore the effectiveness of a tailored yoga program, delivered in-person during the hospital stay and electronically after hospital discharge, as a potential new avenue for postoperative care. This pilot study will use a crossover randomized controlled design. Individuals aged between 40 and 80 years who are scheduled for lumbar laminectomy and/or fusion, and who have not practiced regular yoga within the past 6 months at the time of enrollment, will be recruited and randomized to either a tailored yoga program (intervention group) or usual care (control group) during the hospital stay (phase one). Bearing in mind postoperative precautions, all subjects will be instructed to perform a home-based tailored yoga program delivered electronically via YouTube links for 8 weeks post-hospital discharge (phase two). The primary outcome measures assessing feasibility are adherence/compliance. Secondary outcome measures include pain, anxiety, function, sleep, perceived stress, and pain-catastrophizing behavior. Length of hospital stay and pain medication use, gait distance, and overall physical activity during hospitalization will also be collected. Finally, a qualitative interview will be obtained after completion of the hospital and home-based programs. This study will determine the feasibility of a tailored yoga program for acute and subacute postoperative lumbar spine surgery pain, anxiety, and functional outcomes.

Use of Social Network Strategy Among Young Black Men Who Have Sex With Men for HIV Testing, Linkage to Care, and Reengagement in Care, Tennessee, 2013-2016.

OBJECTIVES: Tennessee was 1 of 8 states that received funding from the Care and Prevention in the United States Demonstration Project, which aimed to reduce HIV-related morbidity and mortality among racial/ethnic and sexual minority populations. The objective of this study was to describe implementation of a social network strategy (SNS) program, which leverages personal connections in social networks, to reach people with undiagnosed HIV infection for HIV testing. We targeted young black men who have sex with men (MSM) at 3 agencies in Memphis and Nashville, Tennessee, during 2013-2016. METHODS: Specialists at the 3 agencies identified MSM with and without diagnosed HIV infection (ie, recruiters) who could recruit members from their social networks for HIV testing (ie, network associates). Both recruiters and network associates received OraQuick rapid and confirmatory HIV tests. We used χ2 and Fisher exact tests to assess differences in demographic characteristics, HIV testing, and care engagement status by agency. RESULTS: Of 1752 people who were tested for HIV in the SNS program, 158 (9.0%) tested positive; of these, 80 (50.6%) were newly diagnosed with HIV. Forty-seven of the 78 (60.3%) people who were previously diagnosed with HIV were not in care in the previous 12 months; of these, 27 (57.4%) were reengaged in medical care. Of 80 people newly diagnosed with HIV, 44 (55.0%) were linked to care. CONCLUSIONS: The SNS program ascertained HIV status among a high-risk population in a heavily burdened region. Further program evaluation is needed to understand how to improve linkage to care among people with newly diagnosed HIV.

Proteomic analysis of in vivo-assembled pre-mRNA splicing complexes expands the catalog of participating factors.

Previous compositional studies of pre-mRNA processing complexes have been performed in vitro on synthetic pre-mRNAs containing a single intron. To provide a more comprehensive list of polypeptides associated with the pre-mRNA splicing apparatus, we have determined the composition of the bulk pre-mRNA processing machinery in living cells. We purified endogenous nuclear pre-mRNA processing complexes from human and chicken cells comprising the massive (>200S) supraspliceosomes (a.k.a. polyspliceosomes). As expected, RNA components include a heterogeneous mixture of pre-mRNAs and the five spliceosomal snRNAs. In addition to known pre-mRNA splicing factors, 5' end binding factors, 3' end processing factors, mRNA export factors, hnRNPs and other RNA binding proteins, the protein components identified by mass spectrometry include RNA adenosine deaminases and several novel factors. Intriguingly, our purified supraspliceosomes also contain a number of structural proteins, nucleoporins, chromatin remodeling factors and several novel proteins that were absent from splicing complexes assembled in vitro. These in vivo analyses bring the total number of factors associated with pre-mRNA to well over 300, and represent the most comprehensive analysis of the pre-mRNA processing machinery to date.

Suppression of estrogen biosynthesis by procyanidin dimers in red wine and grape seeds.

In breast cancer, in situ estrogen production has been demonstrated to play a major role in promoting tumor growth. Aromatase is the enzyme responsible for the conversion of androgen substrates into estrogens. This enzyme is highly expressed in breast cancer tissue compared with normal breast tissue. A wine extract fraction was recently isolated from red wine that exhibited a potent inhibitory action on aromatase activity. Using UV absorbance analysis, high-performance liquid chromatography profiling, accurate mass-mass spectrometry, and nanospray tandem mass spectrometry, most of the compounds in our red wine fraction were identified as procyanidin B dimers that were shown to be aromatase inhibitors. These chemicals have been found in high levels in grape seeds. Inhibition kinetic analysis on the most potent procyanidin B dimer has revealed that it competes with the binding of the androgen substrate with a K(i) value of 6 micro M. Because mutations at Asp-309, Ser-378, and His-480 of aromatase significantly affected the binding of the procyanidin B dimer, these active site residues are thought to be important residues that interact with this phytochemical. The in vivo efficacy of procyanidin B dimers was evaluated in an aromatase-transfected MCF-7 breast cancer xenograft model. The procyanidin B dimers were able to reduce androgen-dependent tumor growth, indicating that these chemicals suppress in situ estrogen formation. These in vitro and in vivo studies demonstrated that procyanidin B dimers in red wine and grape seeds could be used as chemopreventive agents against breast cancer by suppressing in situ estrogen biosynthesis.

Gingipain RgpB is excreted as a proenzyme in the vimA-defective mutant Porphyromonas gingivalis FLL92.

We have previously shown that the unique vimA (virulence-modulating) gene could modulate proteolytic activity in Porphyromomas gingivalis. Although a reduction in cysteine protease activity was observed in the vimA-defective mutant, P. gingivalis FLL92, compared to that of the wild-type strain, no changes were seen in the expression of the gingipain genes. This result might suggest posttranscriptional regulation of protease expression. To determine whether there was a defect in the translation, transport, or maturation of the gingipains, P. gingivalis FLL92 was further characterized. In contrast to the wild-type strain, a 90% reduction was seen in both Rgp and Kgp protease activities in strain FLL92 during the exponential growth phase. These activities, however, increased to approximately 60% of that of the wild-type strain during stationary phase. Throughout all the growth phases, Rgp and Kgp activities were mostly soluble, in contrast to those of the wild-type strain. Western blot analyses identified unique Rgp- and Kgp-immunoreactive bands in extracellular protein fractions from FLL92 grown to late exponential phase. Also, the RgpB proenzyme was identified in this fraction by mass spectrometry. In addition, in vitro protease activity could be induced by a urea denaturation-renaturation cycle in this fraction. These results indicate that protease activity in P. gingivalis may be growth phase regulated, possibly by multiple mechanisms. Furthermore, the gingipain RgpB is excreted in an inactive form in the vimA mutant. In addition, these results provide the first evidence of posttranslational regulation of protease activity in P. gingivalis and may suggest an important role for the vimA gene in protease activation in this organism.

A multiprotein nuclear complex connects Fanconi anemia and Bloom syndrome.

Bloom syndrome (BS) is a genetic disorder associated with dwarfism, immunodeficiency, reduced fertility, and an elevated risk of cancer. To investigate the mechanism of this disease, we isolated from human HeLa extracts three complexes containing the helicase defective in BS, BLM. Interestingly, one of the complexes, termed BRAFT, also contains five of the Fanconi anemia (FA) complementation group proteins (FA proteins). FA resembles BS in genomic instability and cancer predisposition, but most of its gene products have no known biochemical activity, and the molecular pathogenesis of the disease is poorly understood. BRAFT displays a DNA-unwinding activity, which requires the presence of BLM because complexes isolated from BLM-deficient cells lack such an activity. The complex also contains topoisomerase IIIalpha and replication protein A, proteins that are known to interact with BLM and could facilitate unwinding of DNA. We show that BLM complexes isolated from an FA cell line have a lower molecular mass. Our study provides the first biochemical characterization of a multiprotein FA complex and suggests a connection between the BLM and FA pathways of genomic maintenance. The findings that FA proteins are part of a DNA-unwinding complex imply that FA proteins may participate in DNA repair.

Qscore: an algorithm for evaluating SEQUEST database search results.

A scoring procedure is described for measuring the quality of the results for protein identifications obtained from spectral matching of MS/MS data using the Sequest database search program. The scoring system is essentially probabilistic and operates by estimating the probability that a protein identification has come about by chance. The probability is based on the number of identified peptides from the protein, the total number of identified peptides, and the fraction of distinct tryptic peptides from the database that are present in the identified protein. The score is not strictly a probability, as it also incorporates information about the quality of the individual peptide matches. The result of using Qscore on a large test set of data was similar to that achieved using approaches that validate individual spectral matches, with only a narrow overlap in scores between identified proteins and false positive matches. In direct comparison with a published method of evaluating Sequest results, Qscore was able to identify an equivalent number of proteins without any identifiable false positive assignments. Qscore greatly reduces the number of Sequest protein identifications that have to be validated manually.