Search

Histologic evaluation of allograft biopsies after lung transplantation has several limitations, suggesting that molecular assessment using tissue transcriptomics could improve biopsy interpretation. This single-center, retrospective cohort study evaluated discrepancies between the histology of transbronchial biopsies (TBBs) with no rejection (NR) and T-cell mediated rejection (TCMR) by molecular diagnosis. The accuracy of diagnosis was assessed based on response to treatment. 54 TBBs from Prague Lung Transplant Program obtained between December 2015 and January 2020 were included. Patients with acute cellular rejection (ACR) grade ≥ 1 by histology received anti-rejection treatment. Response to therapy was defined as an increase in FEV1 of ≥ 10% 4 weeks post-biopsy compared to the pre-biopsy value. Among the 54 analyzed TBBs, 25 (46%) were concordant with histology, while 29 (54%) showed discrepancies. ACR grade 0 was found in 12 TBBs (22%) and grade A1 ≥ 1 in 42 TBBs (78%). Treatment response was present in 14% in the NR group and in 50% in the TCMR group (p = 0.024). Our findings suggest that low-grade acute cellular rejection is less likely to be associated with molecular TCMR, which might better identify lung transplant recipients who benefit from therapy.

The effect of soil amendment with the STOCKOSORB®500 MICRO hydrophilic polymer on the photosynthetic traits in beech seedlings (Fagus sylvatica L.) during 50 days of dehydration was investigated. Dehydration was detected through osmotic potential (Ψs) in the assimilatory organs of beech seedlings. The addition of Stockosorb positively affected the CO2 assimilation rate (A) and instantaneous water use efficiency (A/T), for severely drought-treated seedlings. In comparison with irrigated plants, the values of A of non-irrigated plants with Stockosorb substrate decreased by 50%, and in non-irrigated plants with common substrate by 88%. The fast kinetics of chlorophyll a fluorescence indicated chronic photoinhibition under drought treatment without Stockosorb, while no significant changes in maximal quantum efficiency (Fv/Fm) were recorded under drought treatment with Stockosorb. The actual quantum efficiency of PSII (ΦPSII) markedly decreased in both treatments - with and without Stockosorb, though significant differences were found only between control treatments and drought treatment without Stockosorb. Moreover, the thermal energy dissipation (NPQ) was strongly limited under severe drought stress. The capacity to down regulate PSII functionality through non-photochemical quenching was maintained under drought treatment with Stockosorb. The results indicate that an amendment with soil conditioner significantly improved the photosynthetic performance of drought-stressed beech seedlings.

Until recently, investigators had no idea how accurately cultivated microorganisms represented the overall microbial diversity. The cultivation-dependent approach is limited by the fact that the overwhelming majority of microorganisms present in soil cannot be cultivated under laboratory conditions. The development of molecular phylogenetics has recently enabled characterization of naturally occurring microbial biota without cultivation. There is a vast amount of information held within the genomes of cultivable and non-cultivable microorganisms, and new methods based on analysis of DNA allow to investigate this potential. In this work we show some aspects, advantages and disadvantages of classical and new approaches in taxonomical and functional description of bacteria present in natural microbial assemblages on the example of cultivable bacteria isolated from rhizosphere of plants, tobacco and black nightshade, planted in PCB contaminated soil. Biochemical analysis of isolates showed 8 different bacterial species. This identification was compared by discrimination using MALDI-TOF mass spectrometry and identity evaluation after sequencing of 16S rDNA. Six strains from original number of 8 were positively identified after 16S rDNA sequencing and their phylogenetic relations were compared. These analyses confirmed closed relations of all species (two of isolates exhibited the same characteristics and were discriminated as the same species Pseudomonas stutzeri) and also of Burkholderia xenovorans LB 400, a well-known PCB degrader. Nevertheless, only two isolates gave a positive reaction after amplification of the biphenyl dioxygenase gene and exhibited potential to degrade PCB. These results indicate that only a subset of the recovered molecular information, derived from active population based on molecular and functional analysis is relevant to microbial ecology.

Tobacco, Nicotiana tabacum L., var. Wisconsin 38 as the control (WSC), and four genetically modified lines of the same variety, were tested for Cd, Zn and Ni accumulation. Genetically modified lines of the same variety, bearing the transgene CUP1 (gene coding a yeast metallothionein), GUS (reporter gene for ß-glucuronidase), HisCUP (CUP combined with a polyhistidine tail), and HisGUS (reporter gene for ß-glucuronidase, combined with a polyhistidine tail) under a constitutive promoter, enabling it to follow the heavy metal tolerance and uptake changes as a function of the transgene present. Control and transgenic lines were tested for accumulation of risk elements on sand nutrient medium with the addition of cadmium, zinc and nickel. The results showed high Cd accumulation ability of HisCUP line. The Cd content in aboveground biomass was increased by 90% compared to the non-transformed control and Cd content in roots was decreased by 49%. Determination of Zn content in aboveground biomass did not confirm higher uptake by transgenic plants significant for phytoremediation. The Ni content was significantly increased in aboveground biomass of HisGUS construct. GUS construct introduced the ability to accumulate all investigated metals; the others accumulated only one in extended amount.

Background: Plasma donor-derived cell-free DNA (dd-cfDNA) is used to screen for rejection in heart transplants. We launched the Trifecta-Heart study ( ClinicalTrials.gov No. NCT04707872), an investigator-initiated, prospective trial, to examine the correlations between genome-wide molecular changes in endomyocardial biopsies (EMBs) and plasma dd-cfDNA. The present report analyzes the correlation of plasma dd-cfDNA with gene expression in EMBs from 4 vanguard centers and compared these correlations with those in 604 kidney transplant biopsies in the Trifecta-Kidney study ( ClinicalTrials.gov No. NCT04239703)., Methods: We analyzed 137 consecutive dd-cfDNA-EMB pairs from 70 patients. Plasma %dd-cfDNA was measured by the Prospera test (Natera Inc), and gene expression in EMBs was assessed by Molecular Microscope Diagnostic System using machine-learning algorithms to interpret rejection and injury states., Results: Top transcripts correlating with dd-cfDNA were related to genes increased in rejection such as interferon gamma-inducible genes (eg, HLA-DMA ) but also with genes induced by injury and expressed in macrophages (eg, SERPINA1 and HMOX1 ). In gene enrichment analysis, the top dd-cfDNA-correlated genes reflected inflammation and rejection pathways. Dd-cfDNA correlations with rejection genes in EMB were similar to those seen in kidney transplant biopsies, with somewhat stronger correlations for TCMR genes in hearts and ABMR genes in kidneys. However, the correlations with parenchymal injury-induced genes and macrophage genes were much stronger in hearts., Conclusions: In this first analysis of Trifecta-Heart study, dd-cfDNA correlates significantly with molecular rejection but also with injury and macrophage infiltration, reflecting the proinflammatory properties of injured cardiomyocytes. The relationship supports the utility of dd-cfDNA in clinical management of heart transplant recipients., Competing Interests: The Trifecta study is an investigator-initiated study supported by a grant from Natera Inc to Transcriptome Sciences Inc/Alberta Transplant Applied Genomics Centre. P.F.H. reports having shares in Transcriptome Sciences Inc, a University of Alberta research company with an interest in molecular diagnostics and is a consultant to Natera Inc; all Natera Inc authors are employees and own equity at Natera Inc. The other authors declare no conflicts of interest., (Copyright © 2024 The Author(s). Published by Wolters Kluwer Health, Inc.)

Most kidney transplant patients who undergo biopsies are classified as having no rejection based on consensus thresholds. However, we hypothesized that because these patients have normal adaptive immune systems, T cell-mediated rejection (TCMR) and antibody-mediated rejection (ABMR) may exist as subthreshold activity in some transplants currently classified as no rejection. To examine this question, we studied genome-wide microarray results from 5086 kidney transplant biopsies (from 4170 patients). An updated molecular archetypal analysis designated 56% of biopsies as no rejection. Subthreshold molecular TCMR and/or ABMR activity molecular activity was detectable as elevated classifier scores in many biopsies classified as no rejection, with ABMR activity in many TCMR biopsies and TCMR activity in many ABMR biopsies. In biopsies classified as no rejection histologically and molecularly, molecular TCMR classifier scores correlated with increases in histologic TCMR features and molecular injury, lower estimated glomerular filtration rate, and higher risk of graft loss, and molecular ABMR activity correlated with increased glomerulitis and donor-specific antibody. No rejection biopsies with high subthreshold TCMR or ABMR activity had a higher probability of having TCMR or ABMR, respectively, diagnosed in a future biopsy. We conclude that many kidney transplant recipients have unrecognized subthreshold TCMR or ABMR activity, with significant implications for future problems., Competing Interests: Declaration of competing interest The authors of this manuscript have conflicts of interest to disclose as described by the American Journal of Transplantation. P.F. Halloran holds shares in Transcriptome Sciences Inc., a University of Alberta research company dedicated to developing molecular diagnostics, supported in part by a licensing agreement between Transcriptome Sciences Inc. and Thermo Fisher Scientific, and by a research grant from Natera, Inc. P.F. Halloran is a consultant to Natera, Inc. and Argenx BV. The other authors have declared no conflict of interest exists., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Histologic evaluation of allograft biopsies after lung transplantation has several limitations, suggesting that molecular assessment using tissue transcriptomics could improve biopsy interpretation. This single-center, retrospective cohort study evaluated discrepancies between the histology of transbronchial biopsies (TBBs) with no rejection (NR) and T-cell mediated rejection (TCMR) by molecular diagnosis. The accuracy of diagnosis was assessed based on response to treatment. 54 TBBs from Prague Lung Transplant Program obtained between December 2015 and January 2020 were included. Patients with acute cellular rejection (ACR) grade ≥ 1 by histology received anti-rejection treatment. Response to therapy was defined as an increase in FEV1 of ≥ 10% 4 weeks post-biopsy compared to the pre-biopsy value. Among the 54 analyzed TBBs, 25 (46%) were concordant with histology, while 29 (54%) showed discrepancies. ACR grade 0 was found in 12 TBBs (22%) and grade A1 ≥ 1 in 42 TBBs (78%). Treatment response was present in 14% in the NR group and in 50% in the TCMR group ( p = 0.024). Our findings suggest that low-grade acute cellular rejection is less likely to be associated with molecular TCMR, which might better identify lung transplant recipients who benefit from therapy., Competing Interests: PH holds shares in Transcriptome Sciences Inc. (TSI), a University of Alberta research company dedicated to developing molecular diagnostics, supported in part by a licensing agreement between TSI and Thermo Fisher Scientific, and by a research grant from Natera, Inc. PH is a consultant to Natera, Inc. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Zajacova, Mackova, Halloran, Gauthier, Balko, Guney, Rakita, Svorcova, Kolarik, Vachtenheim, Pozniak, Simonek, Fila, Lischke, Halloran and Havlin.)

Background: We explored the changes in gene expression correlating with dysfunction and graft failure in endomyocardial biopsies., Methods: Genome-wide microarrays (19,462 genes) were used to define mRNA changes correlating with dysfunction (left ventricular ejection fraction [LVEF] ≤ 55) and risk of graft loss within 3 years postbiopsy. LVEF data was available for 1,013 biopsies and survival data for 779 patients (74 losses). Molecular classifiers were built for predicting dysfunction (LVEF ≤ 55) and postbiopsy 3-year survival., Results: Dysfunction is correlated with dedifferentiation-decreased expression of normal heart transcripts, for example, solute carriers, along with increased expression of inflammation genes. Many genes with reduced expression in dysfunction were matrix genes such as fibulin 1 and decorin. Gene ontology (GO) categories suggested matrix remodeling and inflammation, not rejection. Genes associated with the risk of failure postbiopsy overlapped dysfunction genes but also included genes affecting microcirculation, for example, arginase 2, which reduces NO production, and endothelin 1. GO terms also reflected increased glycolysis and response to hypoxia, but decreased VEGF and angiogenesis pathways. T cell-mediated rejection was associated with reduced survival and antibody-mediated rejection with relatively good survival, but the main determinants of survival were features of parenchymal injury. Both dysfunction and graft loss were correlated with increased biopsy expression of BNP (gene NPPB). Survival probability classifiers divided hearts into risk quintiles, with actuarial 3-year postbiopsy survival >95% for the highest versus 50% for the lowest., Conclusions: Dysfunction in transplanted hearts reflects dedifferentiation, decreased matrix genes, injury, and inflammation. The risk of short-term loss includes these changes but is also associated with microcirculation abnormalities, glycolysis, and response to hypoxia., (Copyright © 2024 International Society for the Heart and Lung Transplantation. Published by Elsevier Inc. All rights reserved.)

In lung transplantation, antibody-mediated rejection (AMR) diagnosed using the International Society for Heart and Lung Transplantation criteria is uncommon compared with other organs, and previous studies failed to find molecular AMR (ABMR) in lung biopsies. However, understanding of ABMR has changed with the recognition that ABMR in kidney transplants is often donor-specific antibody (DSA)-negative and associated with natural killer (NK) cell transcripts. We therefore searched for a similar molecular ABMR-like state in transbronchial biopsies using gene expression microarray results from the INTERLUNG study (#NCT02812290). After optimizing rejection-selective transcript sets in a training set (N = 488), the resulting algorithms separated an NK cell-enriched molecular rejection-like state (NKRL) from T cell-mediated rejection (TCMR)/Mixed in a test set (N = 488). Applying this approach to all 896 transbronchial biopsies distinguished 3 groups: no rejection, TCMR/Mixed, and NKRL. Like TCMR/Mixed, NKRL had increased expression of all-rejection transcripts, but NKRL had increased expression of NK cell transcripts, whereas TCMR/Mixed had increased effector T cell and activated macrophage transcripts. NKRL was usually DSA-negative and not recognized as AMR clinically. TCMR/Mixed was associated with chronic lung allograft dysfunction, reduced one-second forced expiratory volume at the time of biopsy, and short-term graft failure, but NKRL was not. Thus, some lung transplants manifest a molecular state similar to DSA-negative ABMR in kidney and heart transplants, but its clinical significance must be established., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: P F Halloran reports financial support was provided by Thermo Fisher Scientific. P F Halloran reports financial support was provided by Natera Inc. P F Halloran reports a relationship with Natera, Inc. that includes: consulting or advisory. Disclosure P F Halloran holds shares in Transcriptome Sciences Inc (TSI), a University of Alberta research company dedicated to developing molecular diagnostics, supported in part by a licensing agreement between TSI and Thermo Fisher Scientific, and by a research grant from Natera Inc. P.F. Halloran is a consultant to Natera Inc. The other authors have nothing to disclose., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Background: The SARS-Cov-2 (COVID-19) pandemic affected the delivery of health and social care services globally. However, little is known about how palliative care social work services were impacted., Aim: The aim of this study was to capture and analyse data from palliative care social workers who provided professional support in a range of settings across 21 countries during the COVID-19 pandemic., Design: A cross-sectional survey-based design was used for this empirical study and this paper primarily focuses on the quantitative responses., Setting/participants: Participants, palliative care social workers, were drawn internationally via members of the EAPC Social Work Task Force and the World Hospice Palliative Care Social Work network., Results: We received 362 survey responses from 21 countries. Most (79%) respondents worked with adults in in-patient units or hospitals. The number of referrals during COVID-19 increased more in non-European countries, compared to European countries. The full range of social work services could no longer be delivered, existing services changed and 65.3% of participants reported higher levels of pressure during the pandemic, which was linked to higher levels of staff absence and additional duties. For many respondents (40.8%), this included facilitating online communication between patients and their families., Conclusions: Our findings indicate that restrictions to limit the spread of COVID-19 resulted in adaptations to service delivery, increased pressure on staff and moral distress, like other health and social care professions. All members of the palliative team need support and supervision to ensure effective interdisciplinary working and team cohesion.

Background: The Molecular Microscope Diagnostic System (MMDx) may overcome histology shortcomings. Previous studies have simply examined discrepant findings but have not attempted to determine clinical endpoints. To measure performance, clinical outcomes are strongly required., Methods: This single-center cohort study described discrepancies between MMDx and histology from 51 kidney transplant recipients (KTRs) and analyzed 72 indication biopsies, including 21 follow-up biopsies. Clinical performance was assessed by a combined endpoint of graft failure, rejection on follow-up biopsy, de novo donor-specific antibody, and improvement of kidney allograft function upon antirejection treatment., Results: MMDx agreed in 33 (65%) and differed in 18 (35%) of 51 KTRs. Most discrepancies occurred in biopsies called no rejection by MMDx and rejection by histology (15/24, 63%). In contrast, in biopsies called rejection by MMDx, 3 were classified as no rejection by histology (3/27, 11%). Discrepant findings between MMDx and histology occurred following delayed graft function and MMDx from biopsies with a low percentage of cortex. Among 15 biopsies classified as no rejection by MMDx but rejection by histology, the clinical course suggested no rejection in 9 cases. Six KTRs reached the endpoint, showing predominant t ≥ 2 lesions., Conclusions: The most often occurring discrepancy is rejection by histology but no rejection by MMDx. As more KTRs do not meet the combined endpoint for rejection, MMDx might be clinically useful in these discrepant cases. Although strong histological findings have priority in indicating the treatment, clinical implementation of MMDx could strengthen treatment strategies., Competing Interests: P.F.H. has shares in Transcriptome Sciences Inc (TSI), a University of Alberta research company with an interest in molecular diagnostics. The other authors declare no conflicts of interest., (Copyright © 2022 The Author(s). Published by Wolters Kluwer Health, Inc.)

Background: Many lung transplants fail due to chronic lung allograft dysfunction (CLAD). We recently showed that transbronchial biopsies (TBBs) from CLAD patients manifest severe parenchymal injury and dedifferentiation, distinct from time-dependent changes. The present study explored time-selective and CLAD-selective transcripts in mucosal biopsies from the third bronchial bifurcation (3BMBs), compared to those in TBBs., Methods: We used genome-wide microarray measurements in 324 3BMBs to identify CLAD-selective changes as well as time-dependent changes and develop a CLAD classifier. CLAD-selective transcripts were identified with linear models for microarray data (limma) and were used to build an ensemble of 12 classifiers to predict CLAD. Hazard models and random forests were then used to predict the risk of graft loss using the CLAD classifier, transcript sets associated with rejection, injury, and time., Results: T cell-mediated rejection and donor-specific antibody were increased in CLAD 3BMBs but most had no rejection. Like TBBs, 3BMBs showed a time-dependent increase in transcripts expressed in inflammatory cells that was not associated with CLAD or survival. Also like TBBs, the CLAD-selective transcripts in 3BMBs reflected severe parenchymal injury and dedifferentiation, not inflammation or rejection. While 3BMBs and TBBs did not overlap in their top 20 CLAD-selective transcripts, many CLAD-selective transcripts were significantly increased in both for example LOXL1, an enzyme controlling matrix remodeling. In Cox models for one-year survival, the 3BMB CLAD-selective transcripts and CLAD classifier predicted graft loss and correlated with CLAD stage. Many 3BMB CLAD-selective transcripts were also increased by injury in kidney transplants and correlated with decreased kidney survival, including LOXL1., Conclusions: Mucosal and transbronchial biopsies from CLAD patients reveal a diffuse molecular injury and dedifferentiation state that impacts prognosis and correlates with the physiologic disturbances. CLAD state in lung transplants shares features with failing kidney transplants, indicating elements shared by the injury responses of distressed organs., (Copyright © 2022 International Society for Heart and Lung Transplantation. Published by Elsevier Inc. All rights reserved.)

Lung transplantion (LTx) recipients have low long-term survival and a high incidence of bronchiolitis obliterans syndrome (BOS), an inflammation of the small airways in chronic rejection of a lung allograft. There is great clinical need for a minimally invasive biomarker of BOS. Here, 644 different proteins were analyzed to detect biomarkers that distinguish BOS grade 0 from grades 1-3. The plasma of 46 double lung transplant patients was analyzed for proteins using a high-component, multiplex immunoassay that enables analysis of protein biomarkers. Proximity Extension Assay (PEA) consists of antibody probe pairs which bind to targets. The resulting polymerase chain reaction (PCR) reporter sequence can be quantified by real-time PCR. Samples were collected at baseline and 1-year post transplantation. Enzyme-linked immunosorbent assay (ELISA) was used to validate the findings of the PEA analysis across both time points and microarray datasets from other lung transplantation centers demonstrated the same findings. Significant decreases in the plasma protein levels of CRH, FERC2, IL-20RA, TNFB, and IGSF3 and an increase in MMP-9 and CTSL1 were seen in patients who developed BOS compared to those who did not. In this study, CRH is presented as a novel potential biomarker in the progression of disease because of its decreased levels in patients across all BOS grades. Additionally, biomarkers involving the remodeling of the extracellular matrix (ECM), such as MMP-9 and CTSL1, were increased in BOS patients., (© 2022. The Author(s).)

The tubulitis with/without interstitial inflammation not meeting criteria for T-cell-mediated rejection (minimal allograft injury) is the most frequent histological findings in early transplant biopsies. The course of transcriptional changes in sequential kidney graft biopsies has not been studied yet. Molecular phenotypes were analyzed using the Molecular Microscope ® Diagnostic System (MMDx) in 46 indication biopsies (median 13 postoperative days) diagnosed as minimal allograft injury and in corresponding follow-up biopsies at 3 months. All 46 patients with minimal injury in early biopsy received steroid pulses. MMDx interpreted indication biopsies as no-rejection in 34/46 (74%), T-cell-mediated rejection (TCMR) in 4/46 (9%), antibody-mediated rejection in 6/46 (13%), and mixed rejection in 2/46 (4%) cases. Follow-up biopsies were interpreted by MMDx in 37/46 (80%) cases as no-rejection, in 4/46 (9%) as TCMR, and in 5/46 (11%) as mixed rejection. Follow-up biopsies showed a decrease in MMDx-assessed acute kidney injury (P = 0.001) and an increase of atrophy-fibrosis (P = 0.002). The most significant predictor of MMDx rejection scores in follow-up biopsies was the tubulitis classifier score in initial biopsies (AUC = 0.84, P = 0.002), confirmed in multivariate binary regression (OR = 16, P = 0.016). Molecular tubulitis score at initial biopsy has the potential to discriminate patients at risk for molecular rejection score at follow-up biopsy., (© 2021 Steunstichting ESOT. Published by John Wiley & Sons Ltd.)

The Banff 2019 kidney allograft pathology update excluded isolated tubulitis without interstitial inflammation (ISO-T) from the category of borderline (suspicious) for acute T cell-mediated rejection due to its proposed benign clinical outcome. In this study, we explored the molecular assessment of ISO-T. ISO-T or interstitial inflammation with tubulitis (I + T) was diagnosed in indication biopsies within the first 14 postoperative days. The molecular phenotype of ISO-T was compared to I + T either by using RNA sequencing (n = 16) or by Molecular Microscope Diagnostic System (MMDx, n = 51). RNA sequencing showed lower expression of genes related to interferon-y (p = 1.5 *10 -16 ), cytokine signaling (p = 2.1 *10 -20 ) and inflammatory response (p = 1.0*10 -13 ) in the ISO-T group than in I + T group. Transcripts with increased expression in the I + T group overlapped significantly with previously described pathogenesis-based transcript sets associated with cytotoxic and effector T cell transcripts, and with T cell-mediated rejection (TCMR). MMDx classified 25/32 (78%) ISO-T biopsies and 12/19 (63%) I + T biopsies as no-rejection. ISO-T had significantly lower MMDx scores for interstitial inflammation (p = 0.014), tubulitis (p = 0.035) and TCMR (p = 0.016) compared to I + T. Fewer molecular signals of inflammation in isolated tubulitis suggest that this is also a benign phenotype on a molecular level.

We previously reported a system for assessing rejection in kidney transplant biopsies using microarray-based gene expression data, the Molecular Microscope ® Diagnostic System (MMDx). The present study was designed to optimize the accuracy and stability of MMDx diagnoses by replacing single machine learning classifiers with ensembles of diverse classifier methods. We also examined the use of automated report sign-outs and the agreement between multiple human interpreters of the molecular results. Ensembles generated diagnoses that were both more accurate than the best individual classifiers, and nearly as stable as the best, consistent with expectations from the machine learning literature. Human experts had ≈93% agreement (balanced accuracy) signing out the reports, and random forest-based automated sign-outs showed similar levels of agreement with the human experts (92% and 94% for predicting the expert MMDx sign-outs for T cell-mediated (TCMR) and antibody-mediated rejection (ABMR), respectively). In most cases disagreements, whether between experts or between experts and automated sign-outs, were in biopsies near diagnostic thresholds. Considerable disagreement with histology persisted. The balanced accuracies of MMDx sign-outs for histology diagnoses of TCMR and ABMR were 73% and 78%, respectively. Disagreement with histology is largely due to the known noise in histology assessments (ClinicalTrials.gov NCT01299168)., (© 2019 The American Society of Transplantation and the American Society of Transplant Surgeons.)

In kidney transplant biopsies, inflammation in areas of atrophy-fibrosis (i-IFTA) is associated with increased risk of failure, presumably because inflammation is evoked by recent parenchymal injury from rejection or other insults, but some cases also have rejection. The present study explored the frequency of rejection in i-IFTA, by using histology Banff 2015 and a microarray-based molecular diagnostic system (MMDx). In unselected indication biopsies (108 i-IFTA, 73 uninflamed IFTA [i0-IFTA], and 53 no IFTA), i-IFTA biopsies occurred later, showed more scarring, and had more antibody-mediated rejection (ABMR) based on histology (28%) and MMDx (45%). T cell-mediated rejection (TCMR) was infrequent in i-IFTA based on histology (8%) and MMDx (16%). Twelve i-IFTA biopsies (11%) had molecular TCMR not diagnosed by histology, although 6 were called borderline and almost all had histologic TCMR lesions. The prominent feature of i-IFTA biopsies was molecular injury (eg, acute kidney injury [AKI] transcripts). In multivariate analysis of biopsies >1 year posttransplant, the strongest associations with graft loss were AKI transcripts and histologic atrophy-scarring; i-IFTA was not significant when molecular AKI was included. We conclude that i-IFTA in indication biopsies reflects recent/ongoing parenchymal injury, often with concomitant ABMR but few with TCMR. Thus, the application of Banff i-IFTA in the population of late biopsies needs to be reconsidered., (© 2018 The American Society of Transplantation and the American Society of Transplant Surgeons.)

Cytomegalovirus (CMV) infects 40⁻70% of women, but infection has been reported in >95% of breast cancer patients. We investigated the consequences of these observations by infecting mice with mCMV or a negative control medium for 4 days, 11 days or 10 weeks to establish active, intermediate or latent infections, respectively. Syngeneic 4T1 or E0771 breast cancer cells were then injected into a mammary fat pad of BALB/c or C57BL/6 mice, respectively. Infection did not affect tumor growth in these conditions, but latently infected BALB/c mice developed more lung metastases. The latent mCMV infection of MMTV-PyVT mice, which develop spontaneous breast tumors, also did not affect the number or sizes of breast tumors. However, there were more tumors that were multilobed with greater blood content, which had enhanced vasculature and decreased collagen content. Most significantly, mCMV infection also increased the number and size of lung metastases, which showed a higher cell proliferation. Viral DNA was detected in breast tumors and lung nodules although viral mRNA was not. These novel results have important clinical implications since an increased metastasis is prognostic of decreased survival. This work provides evidence that treating or preventing HCMV infections may increase the life expectancy of breast cancer patients by decreasing metastasis.

The placenta is the physiological bridge between mother and fetus and has life-sustaining functions during pregnancy, including metabolic regulation, fetal protection and hormone secretion. Nucleobindin-2 (NUCB2) is a calcium- and DNA-binding protein and precursor of nesfatin-1, a signalling peptide with multiple functions, including regulation of energy homeostasis and glucose transport. These are also key functions of the placenta, yet NUCB2/nesfatin-1 expression has never been comprehensively studied in this organ. In the present study, mouse placental samples from Embryonic Day (E) 7.5 to E17.5 and human chorionic villi from the first and second trimester, as well as term pregnancy, were analysed for NUCB2/nesfatin-1 expression by immunohistochemistry with an antiserum that recognised both NUCB2 and nesfatin-1. From E7.5 to E9.5, NUCB2/nesfatin-1 was expressed in the ectoplacental cone, then parietal trophoblast giant cells and early spongiotrophoblast. At E10.5-12.5, NUCB2/nesfatin-1 expression became detectable in the developing labyrinth. From E12.5 and onwards, NUCB2/nesfatin-1 was expressed in the glycogen trophoblast cells, as well as highly expressed in syncytiotrophoblast, sinusoidal trophoblast giant cells and fetal capillary endothelial cells of the labyrinth. In all trimesters of human pregnancy, NUCB2/nesfatin-1 was highly expressed in syncytiotrophoblast. In addition, there was a significant increase in NUCB2 expression in human primary trophoblast cells induced to syncytialise. Thus, the haemochorial mammalian placenta is a novel source of NUCB2/nesfatin-1 and likely a site of its action, with potential roles in glucose homeostasis and/or nutrient sensing.

Elderly people often suffer adverse health because of inflammation associated with poor metabolism and cardiovascular dysfunction, but these conditions present differently in men and women. We performed experiments in aged male and female mice to understand this sexual dimorphism. We focused on sphingosine 1-phosphate (S1P) signaling, which has both protective and detrimental effects on vascular and metabolic function. We examined vascular function of mesenteric (resistance) arteries from aged male and female wild-type (WT) mice compared to littermate S1P receptor 3 (S1PR3) knockouts (KO). We also measured plasma glucose, insulin, triglycerides, adiponectin, corticosterone and inflammatory cytokines. The novel results of this study are: 1) methacholine-induced vasodilation relied completely on S1PR3 in both sexes, but was dependent on nitric oxide synthase (NOS) only in arteries from aged female mice; 2) S1P-induced vasoconstriction depended solely on S1PR3 in arteries from males, but only partly in females; 3) vasoconstriction to a thromboxane mimetic was decreased by endogenous NOS activity only in arteries from females, regardless of genotype; 4) myogenic responses were lower in arteries from aged WT males compared to females and responses in arteries from KO females were lower than WT females, while the opposite was true of arteries from male mice; 5) aged male mice showed higher fasting glucose and triglycerides with lower plasma adiponectin compared to females and 6) lack of signaling through S1PR3 in females was associated with decreased plasma adiponectin and increased inflammatory mediators. This study showed that there is considerable sexual dimorphism in the vascular and metabolic responses of aged mice and that reduced signaling through S1PR3 could be one mechanism to explain these effects. These results also emphasize that different treatments for mitigating the deleterious effects on vascular health in aged males versus females should be considered., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Background: Papillary thyroid carcinoma (PTC) is the most common malignant thyroid tumour. A common mutation of papillary thyroid carcinoma (PTC) is the somatic mutation of the BRAF (V600E) gene., Aim: The aim was to 1) determine the association of lymph node metastases of PTC with the BRAF gene mutation of primary tumour; 2) evaluate association of the BRAF mutation in the -primary tumour with clinicopathological para-meters; 3) examine the extent of genetic heterogeneity by monitoring the BRAF mutation in multicentric tumours., Subjects and Methods: Retrospective analysis of the BRAF (V600E) mutation in PTC and PTC neck lymph node metastases in 156 patients operated from 2003 to 2012 in Prague and Zlín, the Czech Republic, using a qPCR assay. The results were correlated with clinicopathological factors., Results: DNA was successfully extracted from 137 samples. The BRAF (V600E) mutation was detected in 78 cases (56.9%). The patients with BRAF p.Val600Glu mutation of primary tumour had only non-significantly higher risk of cervical lymph node metastases [OR=2.39 (95%) CI 1.00-5.75, p=0.052]. In the classic papillary variant, the BRAF (V600E) mutation was found significantly more often than in other PTC subtypes (p=0.022). We did not confirm any significant association between the BRAF (V600E) mutation and other clinicopathological findings., Conclusion: Except for the higher prevalence in papillary variant of PTC, BRAF p.Val600Glu mutation was not associated with other prognostic clinicopathological factors of PTC. BRAF mutation cannot be regarded as a reliable marker of node metastases in patients with PTC., (© J. A. Barth Verlag in Georg Thieme Verlag KG Stuttgart · New York.)

In hemolymph of insect species, compounds with remarkable properties for pharmaceutical industry are present. At the first line, there were found compounds of low molecular mass, less than 1 kDa. One of such compounds, β-alanyl-tyrosine (252 Da), was isolated from larval hemolymph of some species of holometabolous insects (e.g. Neobellieria bullata). Its paralytic activity and antimicrobial properties were described until now. In this study, we present the effect of elongation of β-alanyl-tyrosine by repeating of this motive on the biological and physical properties of prepared analogues. For assessment of antimicrobial properties of these new compounds strains of Gram-positive, Gram-negative bacteria and fungi were used, we also followed the haemolytic activity and toxic effect on human cell culture HepG2. On the base of ECD spectroscopy measurement, subsequent molecular modelling and known secondary structure of original β-alanyl-tyrosine dipeptide, the secondary structures of repeating sequences of β-AY were specified. The repeating structures of β-alanyl-tyrosine show increase in antimicrobial activity; for Escherichia coli, Staphylococcus aureus and Pseudomonas aeruginosa, minimal inhibitory concentration was decreased from 30 to 15 mM for 2xβ-AY, 0.4 mM for 4xβ-AY and 0.25 mM for 6xβ-AY., (© 2013 John Wiley & Sons A/S.)

Genetically modified plants can serve as an efficient tool for remediation of diverse dangerous pollutants of the environment such as pesticides, heavy metals, explosives and persistent organic compounds. Transgenic lines of Nicotiana tabacum containing bacterial bphC gene from the degradation pathway of polychlorinated biphenyls (PCBs) were tested. The product of the bphC gene - enzyme 2,3-dihydroxybiphenyl-1,2-dioxygenase is responsible for cleaving of the biphenyl ring. The presence of bphC gene in transgenic plants was detected on DNA, RNA and protein level. The expression of the bphC/His gene was verified afterpurification of the enzyme from plants by affinity chromatography followed by a Western blot and immunochemical assay. The enzyme activity of isolated protein was detected. Efficient transformation of 2,3-DHB by transgenic plants was achieved and the lines also exhibited high production of biomass. The transgenic plants were more tolerant to the commercial PCBs mixture Delor 103 than non-transgenic tobacco. And finally, the higher decrease of total PCB content and especially congener 28 in real contaminated soil from a dumpsite was determined after cultivation of transgenic plant in comparison with nontransgenic tobacco. The substrate specificity of transgenic plants was the same as substrate specificity of BphC enzyme.

Bacteria and bacterial communities in sites contaminated with polychlorinated biphenyls have been extensively studied in the past decades. However, there are still major gaps in the knowledge of environmental processes, especially in the behavior of previously described bacteria in vitro, their real degradation abilities and the enzymes that are involved in the degradation processes. In this work we analyzed actively degrading bacterial populations by stable isotope probing with (13)C biphenyl and (13)C-4-chlorobiphenyl as labeled substrates in the environment of sediment contaminated with polychlorinated biphenyls. We performed analysis of populations which degrade biphenyl and 4-chlorobiphenyl at concentrations similar to those of the original site. Several bacterial genera were revealed to actively participate in biphenyl and 4-chlorobiphenyl removal, some of which had not previously been described to take part in this process. We also found there are few differences in the communities metabolizing biphenyl and 4-chlorobiphenyl. Analysis of the genes responsible for substrate removal proved most of the genes to be closely related to Pseudomonas pseudoalcaligenes KF707 genes giving bacteria the ability of transforming di-para-chlorinated biphenyls., (Crown Copyright © 2013. Published by Elsevier Ltd. All rights reserved.)

The aim of the study was to investigate how selected natural compounds (naringin, caffeic acid, and limonene) induce shifts in both bacterial community structure and degradative activity in long-term polychlorinated biphenyl (PCB)-contaminated soil and how these changes correlate with changes in chlorobiphenyl degradation capacity. In order to address this issue, we have integrated analytical methods of determining PCB degradation with pyrosequencing of 16S rRNA gene tag-encoded amplicons and DNA-stable isotope probing (SIP). Our model system was set in laboratory microcosms with PCB-contaminated soil, which was enriched for 8 weeks with the suspensions of flavonoid naringin, terpene limonene, and phenolic caffeic acid. Our results show that application of selected plant secondary metabolites resulted in bacterial community structure far different from the control one (no natural compound amendment). The community in soil treated with caffeic acid is almost solely represented by Proteobacteria, Acidobacteria, and Verrucomicrobia (together over 99 %). Treatment with naringin resulted in an enrichment of Firmicutes to the exclusion of Acidobacteria and Verrucomicrobia. SIP was applied in order to identify populations actively participating in 4-chlorobiphenyl catabolism. We observed that naringin and limonene in soil foster mainly populations of Hydrogenophaga spp., caffeic acid Burkholderia spp. and Pseudoxanthomonas spp. None of these populations were detected among 4-chlorobiphenyl utilizers in non-amended soil. Similarly, the degradation of individual PCB congeners was influenced by the addition of different plant compounds. Residual content of PCBs was lowest after treating the soil with naringin. Addition of caffeic acid resulted in comparable decrease of total PCBs with non-amended soil; however, higher substituted congeners were more degraded after caffeic acid treatment compared to all other treatments. Finally, it appears that plant secondary metabolites have a strong effect on the bacterial community structure, activity, and associated degradative ability.

Microbial biodegradation and biotransformation reactions are essential to most bioremediation processes, yet the specific organisms, genes, and mechanisms involved are often not well understood. Stable isotope probing (SIP) enables researchers to directly link microbial metabolic capability to phylogenetic and metagenomic information within a community context by tracking isotopically labeled substances into phylogenetically and functionally informative biomarkers. SIP is thus applicable as a tool for the identification of active members of the microbial community and associated genes integral to the community functional potential, such as biodegradative processes. The rapid evolution of SIP over the last decade and integration with metagenomics provide researchers with a much deeper insight into potential biodegradative genes, processes, and applications, thereby enabling an improved mechanistic understanding that can facilitate advances in the field of bioremediation., (Copyright © 2012 Elsevier Inc. All rights reserved.)

During the second half of the last century a large amount of substances toxic for higher organisms was released to the environment. Physicochemical methods of pollutant removal are difficult and prohibitively expensive. Using biological systems such as microorganisms, plants, or consortia microorganisms-plants is easier, cheaper, and more environmentally friendly. The aim of this study was to isolate, characterize and identify microorganisms from contaminated soil and to find out the effect of plants on microbial diversity in the environment. Microorganisms were isolated by two approaches with the aim to find all cultivable species and those able to utilise biphenyl as a sole source of carbon and energy. The first approach was direct extraction and the second was isolation of bacteria after enrichment cultivation with biphenyl. Isolates were biochemically characterized by NEFERMtest 24 and then the composition of ribosomal proteins in bacterial cells was determined by MALDI-TOF mass spectrometry. Ribosomal proteins can be used as phylogenetic markers and thus MALDI-TOF MS can be exploited also for taxonomic identification because the constitution of ribosomal proteins in bacterial cells is specific for each bacterial species. Identification of microorganisms using this method is performed with the help of database Bruker Daltonics MALDI BioTyper. Isolated bacteria were analyzed from the point of the bphA gene presence. Bacteria with detected bphA gene were then taxonomically identified by 16S rRNA sequence. The ability of two different plant species, tobacco (Nicotiana tabacum) and nightshade (Solanum nigrum), to accumulate PCBs was studied as well. It was determined that various plant species differ in the PCBs accumulation from the contaminated soil. Also the content of PCBs in various plant tissues was compared. PCBs were detected in roots and aboveground biomass including leaves and berries., (Copyright © 2012. Published by Elsevier B.V.)