Single-leaflet tilting-disc aortic valve prostheses are known to generate eccentric transvalvular flow jets. These prostheses are routinely inserted with the major valve opening directed toward the non-coronary sinus to achieve more favorable hemodynamic performance. From the viewpoint of blood flow dynamics, the structural and functional properties of tilting-disc aortic valves resemble those of congenital bicuspid aortic valves with right- and left-coronary leaflet fusion, which have been associated with aortopathy in the ascending aorta. Here we describe the case of a patient who had undergone aortic valve replacement in 1987 with a Björk-Shiley tilting-disc valve and required reoperation for ascending aortic aneurysm 29 years later. Eccentric flow jets through the tilting-disc valve directly impinged on the posterior wall of the ascending aorta including the aortotomy suture line, possibly contributing to the development of the saccular aneurysm in the ascending aorta. Single-leaflet tilting-disc aortic valve prostheses  are known to generate eccentric transvalvular flow jets . These p2) and an elevated peak prosthetic aortic jet velocity of 300-350 cm/s had been maintained within the boundary area without worsening. Cardiac computed tomography (CT) angiography revealed no significant obstructive coronary disease, but an unexpected saccular ascending aortic aneurysm (maximum transaortic diameter: 51 mm) arising from the posterior ascending aortic wall was observed , i.e. a well-known stimulus leading to flow-induced vascular remodeling [In the present case, preoperative TEE revealed eccentric systolic flow jets directly impinging on the saccular ascending aortic aneurysm through the tilting disc valve prosthesis. Girdauskas impinged . Barker modeling . It is tet al. quantified the restricted cusp opening of BAV , and 62 ± 5° for conjoined cusps (left-coronary and right-coronary cusps)), demonstrating that the BAV’s conjoined cusp opening angle independently predicted aortic diameter and growth rate [Björk-Shiley tilting-disc heart valves, earlier-generation mechanical prostheses widely used throughout the world in the 1970–80 s, have been withdrawn from clinical use due to structural dysfunction in some models . TiltingAs for the pathogenesis of BAV-related aortopathy, there has been an ongoing nature-nurture debate between the genetic theory  and the hemodynamic theory  . If BAV-Supplementary DataClick here for additional data file.


Hepatocellular carcinoma (HCC) is the most frequent type of primary liver cancer and one of the prominent causes of cancer mortality, leading to approximately 780,000 deaths per year worldwide. Down-regulation of microRNA-125b (miR-125b) is a prognostic indicator in HCC patients. Conversely, over-expression of miR-125b in HCC cells induces cell cycle arrest, inhibits proliferation, migration and invasion. Extracellular vesicles (EVs) function as intercellular messengers transferring proteins, RNAs, DNAs, carbohydrates, and lipids. Since EVs protect their cargo from degradation, delivery of therapeutic bioactive molecules, in particular miRNAs, through EVs represents an innovative avenue for cancer therapy. In this study, we evaluated a replacement strategy for the treatment of HCC via delivery of EVs secreted from human adipose tissue-derived mesenchymal stromal/medicinal signaling cells (ASCs) genetically modified with a lentiviral vector expressing miR-125b with a specific ExoMotif sequence tag to enhance the loading into extracellular vesicles. In particular, we determined that the delivery of miR-125b-loaded EVs produced in engineered ASCs specifically reduces HCC cell proliferation in vitro modulating a series of miR-125b targets, which belong to the p53 signaling pathway. This proof-of-concept study supports the development of innovative therapeutic strategies for HCC via EV-mediated miRNA delivery. Micro RNAs (miRNAs) are a class of approximately 22 nucleotide-long non-coding RNAs predominantly involved in the regulation of target gene expression, mainly at the post transcriptional level . More thEVs are membranous vesicles originating from several cells via multivesicular bodies (exosomes), shedding from the cell membrane (microparticles), or produced by apoptotic cells (apoptotic bodies), and released in many body fluids ,6. The IHepatocellular carcinoma (HCC) is the sixth most common tumor in the world and the second highest cause of cancer mortality with increasing incidence, especially in developing nations . SurgicaMesenchymal stromal/stem cells, also referred as medicinal signaling cells (MSCs) , isolateGenetically engineering MSCs in order to overexpress miRNAs containing specific sequences  to promote target packaging into EVs , may repHuman adipose tissue-derived mesenchymal stromal cells (ASC)  were iso® Number: HB-8065), human colorectal carcinoma HCT 116 (ATCC® Number: CCL-247), human cutaneous melanoma cells MeWo (ATCC® Number: HTB-65), and human embryonic kidney cells 293T (ATCC® Number: CRL-3216) were cultured following American Type Culture Collection (ATCC) recommendations. Characteristics and culture conditions for human HuH-7  in the EV miR-125b samples by the normalized gene expression [2∧ (-Delta CT)] of the Ctrl samples.Hep G2 cells  using a two-tailed Student’s Lentiviral vectors expressing either GFP or miR-125b and GFP including a specific ExoMotif sequence tag to target the gene product into extracellular vesicles were produced. Then we transduced human HCC Hep G2 cells by the lentiviral vectors to determine the effect of ExoMotif tagged miR-125b expression. Transduction efficiency was monitored by fluorescence microscopy analysis of GFP expression a. The puMicroRNA-125b is associated with tumor suppressive signaling in several types of cancers . In part6 ASC/100 mm dishes were transduced at multiplicity of infection approximately 10 achieving transduction efficiency above 85%. Fluorescence microscopy analysis of GFP expression revealed the typical dotted pattern along the cell membrane resembling multivesicular bodies . To better investigate the potential of miR-125b-containing EVs to reduce HCC cell proliferation rate we used the IncuCyte™ live-cell imaging system, which allows for automated analysis of the kinetic of cell behavior over time. Cells were cultured and treated with 10 µg of engineered EV purified from ExoMotif-tagged miR-125b-expressing ASCs and observed for seven consecutive days. As reported in The long-term effect on Hep G2 cells of the delivery of engineered miR-125b EV purified from ASCs was also monitored by colony formation assay. Hep G2 treated with EV miR-125b presented a significant reduction in colony formation compared to control cells incubated in presence of EVs from non-engineered ASCs. Conversely, no detrimental effect on cell proliferation was observed in colon cancer cell line HCT 116 maintained in the same conditions for seven days b. These MicroRNA-125b has been proposed as a novel negative regulator of p53 . In thisIn the current manuscript, we describe the generation of a lentiviral vector for the expression of miR-125b including a specific sequence (ExoMotif) for loading into EVs, the transduction of human mesenchymal cells and the evaluation of this strategy to produce miR-125b-loaded EVs for reducing HCC cell proliferation. MicroRNA-125b is ubiquitously expressed and its role depends on cellular context. The functional importance of miR-125b dysregulation in several cancers has been extensively evaluated indicating that miR-125b may have oncogenic or tumor-suppressive functions, depending on the tumor type . This paEngineered EVs with defined miRNA content can be produced ex vivo to serve as biological therapeutic shuttles enabling targeted treatment . Two majMesenchymal stromal cells exert their regenerative action mainly by paracrine secretion of growth factors, chemokines, and cytokines and EVs; consequently, mesenchymal stromal cells are a promising source for production and engineering of EVs for therapeutic purposes. In particular, antitumor response against HCC has been observed upon administration of EVs isolated from non-genetically modified stromal cells isolated from adipose tissue ; anti-fiWe show that lentiviral-mediated miR-125b gene transfer was effective and did not induce significant detrimental effect on ASCs that produced functionally active, mature miR-125b endogenously encapsulated into EVs. The procedure has the potential for flexible large-scale, GMP-compliant production to obtain suitable amounts of purified EVs for clinical applications . InteresThe aim of our study was mainly to evaluate the feasibility of lentiviral-mediated gene transfer into ASCs to produce engineered EVs. One concern about our findings is that we relied on commercially available reagents developed to isolate EVs from ASC culture medium. Although widely accepted, this method may suffer from some limitations due to possible presence in the EV preparations of a mixed population of vesicles or contaminations of non-EV protein or protein aggregates . As a maAlbeit promising, there are several limitations to the implementation of EV-mediated miRNA replacement therapeutic approach including: The need to develop improved and standardized systems for scalable production, isolation and characterization of EVs suitable for clinical translation studies ,78; to oCollectively, our results reinforce the possibility to genetically modify mesenchymal cells in order to produce engineered EVs that can deliver their cargo for therapeutic purposes. Moreover, the results confirm the anti-proliferative role of miR-125b in HCC supporting EV-mediated miR-125b delivery as a possible therapeutic strategy.


Multifunctional enzymes glutathione transferases (GSTs) are involved in the development of chemoresistance, thus representing a promising target for a novel approach in cancer treatment. This superfamily of polymorphic enzymes exhibits extraordinary substrate promiscuity responsible for detoxification of numerous conventional chemotherapeutics, at the same time regulating signaling pathways involved in cell proliferation and apoptosis. In addition to upregulated GST expression, different cancer cell types have a unique GST signature, enabling targeted selectivity for isoenzyme specific inhibitors and pro-drugs. As a result of extensive research, certain GST inhibitors are already tested in clinical trials. Catalytic properties of GST isoenzymes are also exploited in bio-activation of specific pro-drugs, enabling their targeted accumulation in cancer cells with upregulated expression of the appropriate GST isoenzyme. Moreover, the latest approach to increase specificity in treatment of solid tumors is development of GST pro-drugs that are derivatives of conventional anti-cancer drugs. A future perspective is based on the design of new drugs, which would selectively target GST overexpressing cancers more prone to developing chemoresistance, while decreasing side effects in off-target cells. Chemoresistance is a multifactorial phenomenon and a common problem in cancer treatment. There are several mechanisms for cancer cells to acquire resistance to anti-cancer drugs ,2. The mS-transferases or GSTs) are multifunctional enzymes involved in a number of catalytic and non-catalytic processes, still traditionally recognized as phase II cellular detoxification system enzymes. They are able to catalyze the nucleophilic addition of glutathione (GSH) to a wide variety of non-polar exogenous  and endogenous compounds, yielding more water-soluble products, hence facilitating their elimination phenyl}1-diazen-1-ium-1,2-diolate) or PABA/NO, designed to release NO more readily when catabolyzed by GSTP1-1 in comparison to other GST isoenzymes [2-diazeniumdiolates derivative named JS-K, which acts either by binding to GSTP1 with consequential release of high concentrations of NO or it binds to GST with previously bound GSH, decreasing its intracellular availability for detoxification reactions [Among the first synthesized pro-drugs is a nitric oxide (NO) pro-drug [Ooenzymes ,145. Sinoenzymes ,147. Oneeactions .A pro-drug which has already reached phase III clinical trials is a modified glutathione analogue and nitrogen mustard pro-drug, TLK286 or canfosfamide. It is bio-activated by GSTP1-1 into alkylating metabolite capable of covalently binding DNA ,150,151.A specific pro-drug has been identified even for cancer cells with upregulated GSTA1-1 expression. Namely, synthetic bombesin-sulphonamide derivatives are able to recognize bombesin receptor on cancer cell thus increasing drug uptake, which, once in the cell, undergoes GSTA1-1 catalyzed modification into GST competitive inhibitor .Surprisingly, even metformin analogues are considered as GST pro-drugs. This drug, which is originally used in diabetes mellitus treatment, also exhibits certain anti-cancer effects  and is tThe latest approach to treatment of solid tumors is development of pro-drugs that are derivatives of conventional anti-cancer drugs, such as doxorubicin (DOX). By incorporating sulfonamide moiety into existing anti-cancer drugs it becomes a pro-drug which, after being catalyzed by GSTs, releases the cytotoxic compound. In that way, a cytotoxic drug is released in high concentrations in cancer cells with upregulated GST expression, while cells with normal GST expression remain protected from afore mentioned cytotoxic effect . Among tOne of the major problems in conventional cancer therapy is the inability to selectively target cancer cells and to avoid side effects and chemoresistance to applied anti-cancer drug. Another important principle that needs to be respected is compliance with the novel approach in precision medicine, that the specific drug should be given in the specific dose to the specific patient. Glutathione transferases are responsible for both detoxification of numerous conventional chemotherapeutics, but also involved in regulation of cell proliferation and apoptosis. Due to their dual functionality and upregulated expression in various solid tumors they seem suitable for the development of novel drugs. Even more, different cancer cell types have a unique GST signature, enabling targeted selectivity for isoenzyme specific inhibitors and pro-drugs. The importance of GSTs substrate promiscuity was even contemplated based on the classical Greek aphorism “The fox knows many things, but the hedgehog knows one great thing” . Namely,


To obtain precise estimates of age, haematoma volume, secondary haematoma expansion (HE) and mortality for patients with intracerebral haemorrhage (ICH) taking oral anticoagulants [Vitamin K antagonists (VKA-ICH) or non-Vitamin K antagonist oral anticoagulants (NOAC-ICH)] and those not taking oral anticoagulants (non-OAC ICH) at ICH symptom onset.We conducted a systematic review and meta-analysis of studies comparing VKA-ICH or NOAC-ICH or both with non-OAC ICH. Primary outcomes were haematoma volume (in ml), HE, and mortality . We calculated odds ratios (ORs) using the Mantel–Haenszel random-effects method and corresponding 95% confidence intervals (95%CI) and determined the mean ICH volume difference.p < 0.0001, I2 = 92%, p < 0.001). Haematoma volume was significantly larger in VKA-ICH with a mean difference of 9.66 ml . HE occurred significantly more often in VKA-ICH . VKA-ICH was associated with significantly higher in-hospital mortality  and 3-month mortality . We did not find sufficient data for a meta-analysis comparing NOAC-ICH and non-OAC-ICH.We identified 19 studies including data from 16,546 patients with VKA-ICH and 128,561 patients with non-OAC ICH. Only 2 studies reported data on 4943 patients with NOAC-ICH. Patients with VKA-ICH were significantly older than patients with non-OAC ICH  contains supplementary material, which is available to authorized users. Intracerebral haemorrhage (ICH) is a devastating form of stroke with high mortality and morbidity . Oral anCurrent knowledge about the influence of OAC on haematoma volume, secondary haematoma expansion (HE) and mortality in ICH is mainly based on data from small single centre studies conducted in the 1990s , 5 or eaSince the introduction of NOACs, several single-center , 14 and We conducted a systematic review and meta-analysis of published studies to determine the most precise available estimates of age, haematoma volume, risk of HE and mortality for OAC-ICH (VKA-ICH or NOAC-ICH) compared to non-OAC ICH.The report was prepared with respect to the PRISMA recommendations . The anaTwo investigators (DJS and MBG) independently searched pubmed.gov/MEDLINE for relevant publications on 31 October 2018 and on 14 November 2018. We used the following search terms: [ and (ICH) AND ] including different spellings and common abbreviations .Original studies published in English,Comparing OAC-ICH (either VKA-ICH or NOAC-ICH or both) with non-OAC ICH.Recruiting patients from the same population .Reporting at least one of the following outcomes for both types of ICH (OAC-ICH and non-OAC ICH): mortality, haematoma volume or HE.We applied the following inclusion criteria:Study with less than 10 participants with OAC-ICH.Studies reporting Matched cohorts rather than consecutive patients.We applied the following exclusion criteria:ICH volume: mean ICH volume in millilitre (ml) with standard deviation (SD); if median and interquartile range were provided, we extrapolated the mean and SD using a published formula and method .Haematoma expansion (HE), usually defined as haematoma volume increase of + 6 ml or + 33% between baseline and follow-up imaging.Mortality: either in-hospital or 3-month mortality.Two author (DJS and MBG) independently performed the literature research and screened all titles and abstracts for eligibility. We read the full text of articles potentially eligible for inclusion and independently extracted data on study design, demographics and outcomes. Disagreements were resolved by collegial discussion. We included the following outcomes.VKA was defined as the use of warfarin, phenprocoumon, fluindione or acénocoumarol. NOAC was defined as the use of apixaban, dabigatran, edoxaban or rivaroxaban.Two authors (DJS and MBG) independently assessed study quality and risk of bias using the scheme suggested by the Cochrane collaboration (“Tool to assess risk of bias in cohort studies”).We used Review Manager (RevMan) Version 5.3  statistical program package. Patients with VKA-ICH and NOAC-ICH were analysed separately and compared with patients with non-OAC ICH.2 statistics and displayed the results using forest plots.We used the Mantel–Haenszel random-effects method to compare mortality and HE and calculated odds ratios (OR) and corresponding 95% confidence intervals (CI). ICH volumes as continuous variable were pooled using the difference of means comparison based on the mean ICH volume reported in each study and the corresponding SD. We reported the mean difference of ICH volume (in ml) with corresponding SD. Heterogeneity was assessed using IThe literature research identified 226 publications. After reading the titles and abstracts/full papers if applicable, we included 19 studies .All 19 studies reported data of 16,546 patients with VKA-ICH. The number of patients with VKA-ICH ranged between 21 and 208 patients in single- and multi-centre studies, with the GWTG registry reporting data of 15,036 patients with VKA-ICH. Fourteen of 19 studies recruited less than 100 patients with VKA-ICH. Patients with VKA-ICH were significantly older than patients with non-OAC ICH  and 3-month mortality  although the data showed significant heterogeneity  and more recent studies  a higher mean haematoma volume of about 10 ml; (2) nearly twice the risk of in-hospital and 3-month mortality; and (3) nearly triple the risk of HE. There are insufficient data to draw firm conclusions about NOAC-ICH compared to non-OAC ICH.This meta-analysis confirms, refines and expands current knowledge on the effect of OAC on ICH derived from prior studies. Several studies , 5, 12 dHE is also an important predictor of poor outcome and potential target for treatment , 33. In Although we found increased mortality in patients with VKA-ICH confirming prior research , 5, 8, tWhile many studies investigated differences between VKA-ICH and NOAC-ICH –17, we iOur study has several strengths: 1) combining and pooling data from multiple, independent studies with a large number of patients with VKA-ICH, we overcome limitations from prior smaller studies, refining and expanding their findings; 2) combining data from older with more recent studies allowed to highlight possible improvements in treatment of VKA-ICH underlining that further efforts to improve treatment are still important; 3) independent literature research and extraction of study data from two investigators reduced bias and increased data validity.Our study has the following limitations: (1) we used aggregate observational data which are always prone to bias; (2) we were not able to perform multivariate analysis accounting for other potential predictors of haematoma volume, HE and mortality including age (VKA-ICH patients were 5 years older), acute treatment  and other factors (e.g. comorbidities); (3) we were not able to calculate gender- or age-specific results. Furthermore, we did not have access to INR results at presentation. (4) Different methods applied to assess haematoma volumes have certainly contributed to the observed heterogeneity of results and are a source of bias. However, these methodological limitations applies to both, OAC-ICH and non-OAC-ICH. (5) Even with sophisticated measuring techniques, for large haematomas, the magnitude of haematoma volume measurement error may exceed the threshold used to dichotomize HE in the respective study . In our To summarize, this meta-analysis provided precise estimates of key prognostic variables and outcomes: we showed that VKA-ICH has larger haematoma volume, more frequent HE and higher mortality than non-VKA-ICH. Studies comparing NOAC-ICH and non-OAC ICH are scarce and current knowledge on NOAC-ICH is only available in relation to the effect of VKA-ICH. Further research on the effect of NOAC on these outcomes is needed.Supplementary file1 (DOCX 34 kb)Supplementary file2 (PNG 96 kb)Supplementary file3 (PNG 17 kb)Below is the link to the electronic supplementary material.


Novel approaches such as fractal dimension analysis on conventional MRI (cMRI) and advanced MR techniques such as diffusion tensor imaging (DTI) reveal significant differences between ALS-CST+ and the aforementioned ALS subtypes. Our unbiased neuroimaging studies demonstrate that the ALS-CST+ group, which can be initially identified by T2-, PD-, and FLAIR-weighted cMRI, is distinctive and distinguishable from other ALS subtypes with possible differences in disease pathogenesis.Amyotrophic lateral sclerosis (ALS) is an incurable and progressively fatal neurodegenerative disease that manifests with distinct clinical phenotypes, which are seen in neuroimaging, and clinical studies. T2- and proton density (PD)-weighted magnetic resonance imaging (MRI) displays hyperintense signal along the corticospinal tract (CST) in some ALS patients with upper motor neuron (UMN)-predominant signs. These patients tend to be younger and have significantly faster disease progression. We hypothesize that such ALS patients with CST hyperintensity (ALS-CST+) comprise a clinical subtype distinct from other ALS subtypes, namely patients with UMN-predominant ALS  Etiology and site of origin of amyotrophic lateral sclerosis (ALS) within the central nervous system (CNS) are unknown . ALS diaIn previous studies, we have evaluated brain MRI changes in patients with ALS based on their clinical phenotype and extent of UMN or cognitive impairment, including in those with UMN-predominant ALS, classic ALS [expressing relatively equal amounts of UMN and lower motor neuron (LMN) dysfunction], or ALS with frontotemporal dementia (ALS-FTD). Although an ALS diagnosis relies on the clinical presence of both UMN and LMN signs, a proportion of patients with ALS present with evidence of only UMN abnormalities and develop LMN signs later. A hyperintense signal is visible along both corticospinal tracts (CST’s) on conventional T2-, proton density (PD)-, and FLAIR- weighted MRI in some patients with predominant or exclusive UMN signs , while oUnlike LMN abnormalities, which can be identified by routinely used electromyography (EMG), even if such signs are subclinical, objectively identifying UMN abnormalities  is more in vivo between such ALS patient groups and possibly to gain insights into disease pathogenesis and progression.The focus of this review is to summarize our previously published findings of how non-biased conventional MRI sequences acquired at 1.5T have identified differences between ALS patients with specific clinical phenotypes. Our goal was to demonstrate the utility of widely accessible routine clinical MRI in revealing unique macropathologic differences with CST hyperintensity (ALS-CST+), (2) UMN-predominant ALS patients without CST hyperintensity (ALS-CST-), (3) patients with classic ALS (ALS-Cl), and (4) ALS patients with frontotemporal dementia (ALS-FTD), and (5) neurological controls. UMN-predominant ALS patients were defined as those with LMN signs that were either absent, or if present, were restricted to only one neuraxial level  at time of MRI. UMN-predominant patients with CST hyperintensity were those in whom hyperintense signal was observed along the CST bilaterally in T2-, FLAIR-, and especially PD-weighted images. Patients with ALS-FTD displayed cognitive or behavioral impairment during clinical evaluation, as assessed by EP Pioro, including disturbances of language, executive function and impulse control. Such patients underwent bedside evaluation, including MoCA testing, extensive formal neuropsychometric testing by an experienced neuropsychologist, and usually both.Groups of individuals evaluated by conventional clinical T2/PD/FLAIR-weighted MRI included: (1) UMN-predominant ALS patients not have CST hyperintensity. Since diffusion tensor imaging (DTI) could provide more insight with its diverse metrics (which are based on diffusion of water molecules), we studied the DTI metrics: fractional anisotropy (FA), axial diffusivity (AD), radial diffusivity (RD), and mean diffusivity (MD) along the CST in ALS patients of ALS-CST+ and ALS-CST- groups, compared to neurologic controls  at the SubPMC level, which is between the PMC and CSoLV levels in several patients of both ALS-CST+ and ALS-CST- groups, as shown from a representative patient in P = 0.049). Further, the frequency of virtual CST truncation was significantly (P = 0.018) higher in all ALS patients (both ALS-CST+ and ALS-CST- groups combined) than in the control group. To determine if this truncation was specific to descending motor fibers, we identified virtual non-motor fiber tracts connecting the primary sensory cortex (PSC) and subcortical white matter. Because most of these sensory fibers are afferents to the PSC, they should generally be unaffected by corticomotoneuron degeneration. In fact, truncation of such virtual non-motor (sensory) tracts occurred in only one subject from each of the ALS patient groups: 1 of 21 (4.7%) in ALS-CST+, and 1 of 24 (4.1%) in ALS-CST- groups. Our DTT findings of subcortical truncation of essentially only virtual motor (and not sensory) fibers, as shown from a representative patient in The above studies demonstrated distinct pathological changes in regions of interest (ROIs) along the CST in the ALS-CST+ group compared to other ALS groups and neurologic controls. However, the ROI approach is limited because of operator bias where voxels are placed, and evaluation of the CST only where voxels are placed, rather than along its entire length. In order to more accurately and objectively identify areas along the tract’s entire length, we used diffusion tensor tractography (DTT) to reconstruct a “virtual” CST .DTT idenImportantly, the truncation of motor fibers more frequently in one ALS phenotype than another, no truncation in neurologic controls, and differential involvement of motor but not sensory fibers all suggest that virtual CST truncation is and disease- and fiber-type specific. Therefore, these results further support unique pathologies along the CST in these two UMN-predominant ALS patient groups.P < 0.001), as previously reported (P = 0.001), indicating a significantly faster decline of motor function in the former group of patients. Of note, duration of disease in the ALS-CST- group averaged 3 years prior to MRI , reflecting their slow progression, and was over 48 months in a one-third of them. This suggests that some of these slowest progressing patients without CST hyperintensity may have, in fact, represented a group with primary lateral sclerosis (PLS)  compared to ALS-CST- group  .in vivo by certain MRI techniques  is a non-integer number that characterizes the morphometric variability of a complex and irregular shape. FD analysis can quantitatively measure the internal shape complexity of brain WM from MRI by characterizing multifractal behavior of different textures instead of using only pixel intensity values . Higher P = 0.001) and general structure (P = 0.02) were significantly higher in ALS-CST+ patients compared to ALS-FTD patients, as shown in P = 0.10) and ALS-Cl (P = 0.10) groups. However, neurologic controls and ALS patients revealed no significant differences in FD values. These results indicate that shape complexity in the ALS-CST+ patient group was significantly greater than in the ALS-FTD group, and trended higher than in the other two patient subtypes. Although the significance of this higher FD in ALS-CST+ patients is unclear, it is likely related to differences in integrity of axons, myelin, and other changes within the neuropil, including inflammatory processes.We used FD analysis to evaluate WM structural degeneration in each of the four ALS patient groups: ALS-CST+, ALS-CST-, ALS-Cl, and ALS-FTD. In this study , we estiP < 0.05) (p < 0.001) and ALS-FTD groups (p < 0.05), indicating earlier neurologic evaluation after symptom onset. Also, disease progression rate was significantly faster in ALS-CST+ patients than in ALS-CST-, ALS-Cl, and ALS-FTD groups, as had been observed in a preliminary study of another group of ALS patients (Patients in the ALS-CST+ group were younger when compared to those in the ALS-CST- group ( < 0.05) . In contpatients . The revpatients , was sigWe hypothesize that ALS-CST+ patients comprise a distinct phenotype from ALS-CST-, ALS-Cl, and ALS-FTD with unique micropathology of the CST and potentially important differences in ALS pathogenesis. Prescreening ALS patients for the presence of CST hyperintensity may be useful when enrolling or stratifying into clinical trials.The aforementioned brain MRI studies uniformly revealed objective differences in patients with the various ALS subtypes: UMN-predominant ALS with CST hyperintensity (ALS-CST+), UMN-predominant ALS without CST hyperintensity (ALS-CST-), classic ALS (ALS-Cl), and ALS with FTD (ALS-FTD). Specifically, patients in the ALS-CST+ group show distinctive and distinguishable changes from the others, including patients in the ALS-CST- group, which appear phenotypically similar, at least in relation to extent of UMN dysfunction. Coupled with the patients’ distinct clinical characteristics, these neuroimaging abnormalities strongly suggest that CST hyperintensity, as revealed by conventional MRI (cMRI) T2/PD, and FLAIR sequences used during routine clinical evaluation, is not artefactual or non-specific but identifies a unique ALS patient group. VR and EPP conceptualized the idea for the manuscript. VR wrote the manuscript. EPP made substantive revisions to the manuscript.The 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.


Mass spectrometry imaging (MSI) is an enabling technology for label-free drug disposition studies at high spatial resolution in life science- and pharmaceutical research. We present the first extensive clinical matrix-assisted laser desorption/ionization (MALDI) quantitative mass spectrometry imaging (qMSI) study of drug uptake and distribution in clinical specimen, analyzing 56 specimens of tumor and corresponding non-tumor tissues from 27 imatinib-treated patients with the biopsy-proven rare disease gastrointestinal stromal tumors (GIST). For validation, we compared MALDI-TOF-qMSI with conventional UPLC-ESI-QTOF-MS-based quantification from tissue extracts and with ultra-high resolution MALDI-FTICR-qMSI. We introduced a novel generalized nonlinear calibration model of drug quantities based on computational evaluation of drug-containing areas that enabled better data fitting and assessment of the inherent method nonlinearities. Imatinib tissue spatial maps revealed striking inefficiency in drug penetration into GIST liver metastases even though the corresponding healthy liver tissues in the vicinity showed abundant imatinib levels beyond the limit of quantification (LOQ), thus providing evidence for secondary drug resistance independent of mutation status. Taken together, these findings underscore the important application of MALDI-qMSI in studying the spatial distribution of molecularly targeted therapeutics in oncology, namely to serve as orthogonal post-surgical approach to evaluate the contribution of anticancer drug disposition to resistance against treatment. In the latter case, MALDI-MSI has found its way into pharmaceutical research and development, where disposition of drugs and their carriers can be effectively monitored alongside their pharmacodynamics and toxic effects8.Matrix-assisted laser desorption/ionization (MALDI) mass spectrometry imaging (MSI) has emerged as a powerful label-free technology for studying spatial distributions of analytes in tissues. Owing to its high chemical specificity and ability to track hundreds of analytes simultaneously, MALDI-MSI has gained traction in various areas of biomolecular research such as quantitative profiling of metabolites, lipids, peptides, proteins and drugs5. In contrast, MALDI-MSI provides spatial information, and much effort has recently been devoted to establish quantitative MSI (qMSI) techniques12. Linear calibration based on tissue mimetic models or compound dilution series spotted onto tissue, as well as signal normalization against stable isotope-labeled internal standards (IS)11 and the calculation of tissue extinction coefficients (TEC)10 were introduced to compensate for the inherently high technical variability of MALDI-MSI12. Furthermore, advanced rational testing criteria for evaluating linearity of response, variability, reproducibility and limits of detection of qMSI have been suggested13. Despite these technical advances, MALDI-qMSI has not yet been widely adapted to clinical pharmacology.Traditionally, quantitative assessment of drug distribution in patient tissues employs bioanalytical techniques such as UPLC-ESI-MS, which assume homogenous distribution of analytes and require tissue homogenization resulting in a complete loss of spatial context14. However, tumor heterogeneity and the poorly understood spatial organization of the tumor microenvironment present major challenges for drug uptake and, hence, effective cancer treatment16. This challenge has prompted qMSI studies of drug disposition and of pharmacological/toxic effects in tumor tissues and their surroundings in mice18. Most mouse studies and pioneering qualitative MSI studies of the tyrosine kinase inhibitor (TKI) erlotinib in patient tissue report high degrees of variability and intratumor heterogeneity as well as highly heterogeneous drug distribution21. However, clinical qMSI proof-of-concept studies of drug distribution are still lacking.In clinical oncology, the ability to monitor drug penetration into tumors constitutes a key medical need22. Imatinib is the standard first line treatment for GIST, effectively stopping autophosphorylation and tumor proliferation particularly in exon 11 mutated GIST25. However, tumors are prone to imatinib resistance, which is mainly attributed to secondary somatic mutations in the receptor tyrosine kinases KIT (CD117 or stem cell factor receptor), the gene product of the protooncogene c-kit, or the platelet-derived growth factor receptor alpha (PDGFRA)27. In this study, we introduce generalized nonlinear regression as a superior calibration method based on imatinib-containing pixels, report MALDI-qMSI of three full technical replicates, and compare these results [fast time of flight MALDI-TOF-qMSI and ultra-high-resolution Fourier-Transform Ion Cyclotron Resonance (FTICR-qMSI)] with conventional UPLC-ESI-QTOF-MS quantification. Taken together, our data suggests that MALDI-qMSI compares well with UPLC-ESI-QTOF-MS quantification, and that spatially resolved MS has utility in clinical pharmacology. We can demonstrate that independent of mutation status of the tumor, imatinib failed to penetrate or to be retained in tumor tissue in all GIST liver metastasis cases tested.Here, we present the first extensive clinical MALDI-qMSI drug disposition study of a TKI, imatinib, in 56 resection specimens of tumor and surrounding non-tumor tissues from 27 patients with the biopsy-proven rare disease gastrointestinal stromal tumor (GIST). GISTs can occur throughout the gastrointestinal tract but also as a metastatic tumor elsewhere in the abdominal cavity, as well as in liver, but rarely in lungs and distant peripheral sites. GISTs being a rare disease have been estimated to occur with a frequency of 14–20 per million28, thus enabling investigation of the drug’s distribution in resection specimens. Due to the limited quantities of tissue available for these samples and since all measurements were conducted in triplicates, some samples were used up completely before reaching the next round of measurements such that only samples from 18 and 5 patients were available for UPLC-ESI-QTOF-MS and MALDI-FTICR-qMSI measurements, respectively. All slides for MALDI-qMSI featured an imatinib dilution series spotted onto porcine liver tissue and a spray-coating of deuterated imatinib-D8 for lock-mass calibration and normalization, respectively  of the therapeutic TKI, imatinib, into GIST tissue. We systematically examined GIST and corresponding non-tumor samples from 27 patients who received the TKI imatinib as first line treatment but presented with refractory disease. Some patients had developed metastatic lesions, mostly in the liver  for all three full replicates using gold-standard UPLC-ESI-QTOF-MS and MALDI-TOF-qMSI Fig. . Also foMost tissue sections with imatinib levels above LOQ for both MS methods corresponded to normal liver tissue. In these cases, imatinib quantified by MALDI-TOF-qMSI displayed acceptable correlation with results from UPLC-ESI-QTOF-MS quantification. However, MALDI-TOF-qMSI tended to underestimate imatinib. Nevertheless, 78% of all samples, in which imatinib could be quantified, were inside a window of a 2-fold difference  in tumor tissue sections and their corresponding non-tumor  control tissues by MALDI-TOF-qMSI revealed that despite continuous administration of the prescribed dosage of imatinib until surgery (see Supplementary Table m/z 480.25), a known metabolite of imatinib31, was detected and visualized  within surrounding normal liver tissue. Sample A appears to be a notable exception, as comparable amounts of imatinib were present in both normal and tumor tissue , a proven MALDI-MSI marker of vasculature , based on a small cohort of patient-derived tissues, are in line with results from several MALDI-qMSI mouse studies on various chemotherapeutic drug agents in different cell line-based and patient-derived xenograft models39. It should be noted that while the presence of sufficient quantities of a drug in the target tissue is invariably a prerequisite for its efficacy, MSI in clinical pharmacology may not be limited to drug distribution studies. In the future, it may be even more intriguing to use MSI for spatial assessment of drug-response markers. For instance, in a preclinical rodent model of gastric cancer we have demonstrated earlier that polyacetylated histone H4 can be imaged by MS as a pharmacodynamic marker of action of the histone deacetylase inhibitor panobinostat40.The observed discrepancy between imatinib amounts in normal hepatic versus metastatic tissue is striking . FurtheIn summary, while providing additional spatial information on imatinib distribution, MALDI-qMSI based on generalized nonlinear calibration and focused computational analysis of drug-bearing pixels provided quantitative measures of imatinib that deviated from reference analysis by UPLC-ESI-QTOF-MS by less than two-fold in 78% of cases (87% of cases by FTICR) and provided a generalized method for modeling analytes dilution series as well as assessing the degree of the apparent nonlinearity of the system. While further refinement of the method and testing in larger patient cohorts will be crucial, our data provides a proof-of-concept for clinical utility of MALDI-qMSI. Furthermore, spatial mapping of imatinib distribution within GIST patient tissues revealed striking inefficiency in its penetration into liver metastases irrespective of their mutation status. Our study therefore strongly suggests that also mechanisms other than driver mutations in receptor tyrosine kinases such as alterations in drug uptake due to decreased intra-tumor vasculature may play an important role in imatinib resistance in GIST. This finding underlines the important application of MALDI-qMSI, a methodology that can probably be translated to clinical drugs other than the proof-of-concept case imatinib, for studying the spatial distribution of molecularly targeted therapeutics in oncology. More specifically, MALDI-qMSI can enter clinical application as an orthogonal post-surgical approach to evaluate disposition of administered drugs and their metabolites in resected specimens to cast light into possible treatment resistance as a function of anticancer drug uptake, metabolism and/or retention.Human GIST and corresponding non-tumor control tissues  had been surgically removed from 27 patients  in triplicate  were deposited on the slides using a SunCollect sprayer : 2.5 pmol/µL solution, flow rate 15 µL/min, spray head distance 45 mm and speed 300 mm/min. Slides were stored in a desiccator at room temperature for one hour to allow drying of the IS. For positive-ion mode measurements, five layers  of 2,5-Dihydroxybenzoic acid (DHB) matrix  were subsequently sprayed onto the tissue at 300 mm/min as described previouslym/z 150–3000; R: 260,000 at m/z 300) was recorded in positive-ion mode on a solariX 7T XR FTICR mass spectrometer . The raster width was 200 μm for calibration curves for quantification and 50 μm for all human samples. The instrument was calibrated externally using quadratic mass calibration with peptide calibration standard II including dasatinib (m/z 488.21). For imatinib calibration curves, equal square-sized measurement areas containing identical pixel numbers were defined over each calibration spot by an in-house MATLAB tool.MALDI-TOF-qMSI data acquisition was performed on an ultrafleXtreme MS using flexControl 3.4 . High-resolving-power MSI  with a binning rate of 120,000. The imzML datasets were subsequently imported into R 3.3.1 43 using MALDIquantForeign and processed using MALDIquant packages44. Mass spectra were normalized to the maximum peak intensity over the mass range m/z 502.32 ± 100 ppm (MALDI-TOF-qMSI) and ±10 ppm (MALDI-FTICR-qMSI) of the sprayed IS imatinib-D8. Neither baseline correction nor further pre-processing of spectra were performed. All subsequent analysis and visualization (mainly with ggplot2 and fmsb packages) were performed using R.A total of 48 MALDI-TOF- and three MALDI-FTICR- qMSI-datasets were acquired and exported into imzML format10. Nonlinear regression was performed by fitting a power function as a calibration curve in the form of y = axb + c model . For MALDI-qMSI of imatinib in tissue, the mean intensity of signal-bearing pixels, i.e. imatinib signal with signal to noise ratio (S/N) ≥ 3, of each tissue section was plugged into the fitted nonlinear regression model. The limits of detection (LOD) and quantification (LOQ) in this study were computed as mean of all individually fitted calibration curves  within each tissue section to its mean intensity in the IS and matrix control area. RSE was used for quality of fit comparison, as R2 is not defined for nonlinear fits45.Linear regression analysis for calibration of the spotted imatinib dilution series was performed as described inSupporting Information


These drug-resistant microbes (or superbugs) are non-responsive to most of the commonly used antibiotics leaving us with few treatment options and escalating mortality-rates and treatment costs. The problem is further aggravated by the drying-pipeline of new and potent antibiotics effective particularly against the drug-resistant strains. Multidrug efflux pumps (EPs) are established as principal determinants of AMR, extruding multiple antibiotics out of the cell, mostly in non-specific manner and have therefore emerged as potent drug-targets for combating AMR. Plants being the reservoir of bioactive compounds can serve as a source of potent EP inhibitors (EPIs). The phyto-therapeutics with noteworthy drug-resistance-reversal or re-sensitizing activities may prove significant for reviving the otherwise fading antibiotics arsenal and making this combination-therapy effective. Contemporary attempts to potentiate the antibiotics with plant extracts and pure phytomolecules have gained momentum though with relatively less success against Gram-negative bacteria. Plant-based EPIs hold promise as potent drug-leads to combat the EPI-mediated AMR. This review presents an account of major bacterial multidrug EPs, their roles in imparting AMR, effective strategies for inhibiting drug EPs with phytomolecules, and current account of research on developing novel and potent plant-based EPIs for reversing their AMR characteristics. Recent developments including emergence of  By definition, MDR stands for acquisition of non-susceptibility to at least one agent in three or more antimicrobial classes, while XDR shows the non-susceptibility to at least one agent in all, except two or fewer antimicrobial classes, while PDR implies non-susceptibility to all antimicrobial agents from all available classes  or ineffectiveness of commonly used drugs/antibiotics against specific bacteria has emerged as one of the most threatening human health concerns and a major challenge for global drug discovery programs. AMR  has been reported at three increasing levels, Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species) pathogens have emerged with high degree of AMR and are major cause of life-threatening nosocomial infections  and vancomycin resistant S. aureus (VRSA), coagulase negative Staphylococci members including S. epidermidis and S. haemolyticus, Streptococcus pneumonia, E. faecalis, E. faecium, and Clostridium difficile  proposing the important physiological roles the EPs play during cell development, stress adaptations and bacterial pathogenesis  in E. coli, facilitating the biofilm formation , chlorpromazine and PAβN were reported to prevent biofilm formation in E. coli  multidrug transporters and secondary transporters using proton motive force (PMF) as an energy source : class K tetracyclin transporter from S. aureus], and TetA(B) is one of the most extensively studied members of the family  of test antibiotics via inhibiting the ethidium bromide (EtBr) pump , E. coli (ATCC 700928) and P. mirabilis (HI4320) confirmed via monitoring the EtBr efflux  to test its EP inhibitory potentials, and strong activities (higher than the standard reserpine) were exhibited by lysergol and its derivative, 17-O-3″,4″,5″-trimethoxybenzoyllysergol , two promising semi-synthetic, esterified derivatives of ursolic acid, 3-O-acetyl-urs-12-en-28-isopropyl ester and 3-O-acetyl-urs-12-en-28-n-butyl ester and the parent compound exhibited better EP inhibitory potencies than the standard reserpine  averting them from external impacts , which may help in understanding the structural templates and actual binding events achieved via these transporters. Molecular dynamic simulation (MDS) studies of VMAT2 multidrug transporter (MFS family) revealed the presence of two domains of six trans-membrane helices . The compound successfully blocked the disruption of Na+-coordination along with an equilibrium state bias toward occlude state of NorM transporter, ultimately blocking the extrusion of antimicrobial drugs via inhibiting the NorM transporter in drug-resistant N. gonorrhea.Recently, Kesherwani et al.  used higE. coli. Authors used in silico approaches and molecule docking approaches. The docking analysis of quercetin with EP-protein showed the importance of residues for function and stability, and notably quercetin showed best interactions as compared to the compounds like verapamil, reserpine, chlorpromazine, and carbonyl cyanide m-chloro phenylhydrazone. MDS confirmed the stability of quercetin-Mmr complex, which insights the potential of quercetin as a non-antibiotics adjuvant for treatment of bacterial infection via reducing the drug efflux from bacteria.Similarly, Suriyanarayanan and Sarojini  analyzedin silico high-throughput virtual screening. Molecular docking revealed these two compounds morelloflavone and pregnan-20-one derivative as inhibitors of MexAB-OprM EPs of P. aeruginosa and supportive in vitro assays confirmed their synergism with ciprofloxacin  with Arg141 residue and piperine provided stable protein-ligand interaction. The findings confirmed the role of piperine in augmenting rifampicin sensitivity in M. tuberculosis -Rh2 and Gln51/Asn340/Ser226 residues at active binding site in the central cavity of protein was attributed for the inhibition of NorA pump, thus promoting accumulation of ciprofloxacin inside the bacterial cell  pair. More comprehensive and deeper investigations are therefore needed that involve the exploring the high-throughput screening assisted by VK conceived the idea. VS, TK, RB, RS, and VK wrote the manuscript. All the authors made substantial contribution to the work and approved it for publication.The 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.


Metabolic reprogramming for adaptation to the local environment has been recognized as a hallmark of cancer. Although alterations in fatty acid (FA) metabolism in cancer cells have received less attention compared to other metabolic alterations such as glucose or glutamine metabolism, recent studies have uncovered the importance of lipid metabolic reprogramming in carcinogenesis. Obesity and nonalcoholic steatohepatitis (NASH) are well-known risk factors of hepatocellular carcinoma (HCC), and individuals with these conditions exhibit an increased intake of dietary FAs accompanied by enhanced lipolysis of visceral adipose tissue due to insulin resistance, resulting in enormous exogenous FA supplies to hepatocytes via the portal vein and lymph vessels. This “lipid-rich condition” is highly characteristic of obesity- and NASH-driven HCC. Although the way in which HCC cells adapt to such a condition and exploit it to aid their progression is not understood, we recently obtained new insights into this mechanism through lipid metabolic reprogramming. In addition, accumulating evidence supports the importance of lipid metabolic reprogramming in various situations of hepatocarcinogenesis. Thus, in this review, we discuss the latest findings regarding the role of FA metabolism pathways in hepatocarcinogenesis, focusing on obesity- and NASH-driven lipid metabolic reprogramming. Hepatocellular carcinoma (HCC) is the second most frequent cause of cancer-related death worldwide . Recent Cellular adaptations caused by metabolic reprogramming can be clonally selected during tumorigenesis . MetabolObese persons exhibit an increased intake of dietary FAs accompanied by enhanced lipolysis of visceral adipose tissue due to insulin resistance, resulting in enormous exogenous FA supplies to hepatocytes via the portal vein and lymph vessels. This “lipid-rich condition” is highly characteristic of obesity-driven liver cancer. Although the way in which HCC cells adapt to this condition and exploit it to aid their progression is not understood, we recently provided new insights into this debate through lipid metabolic reprogramming . In addiFAs are taken up from an extracellular source through membrane-associated transport proteins, including fatty acid transport protein-1 (FATP1), fatty acid translocase (FAT/CD36), and liver fatty acid binding protein (L-FABP), or are produced through de novo FA synthesis. The de novo FA synthesis pathway converts citrate to acetyl-coenzyme A (CoA), malonyl-CoA and, eventually, bioactive FAs through multiple enzymatic reactions catalyzed by ATP citrate lyase (ACLY), acetyl-CoA carboxylase (ACC) and fatty acid synthase (FASN) . FAs proWhereas most normal cells prefer exogenous FA sources, cancer cells often exhibit a shift toward de novo FA synthesis. Furthermore, cancer cells sometimes use an enhanced FAO pathway, even in cells exhibiting high lipogenic activity . TherefoA recent study analyzing the global gene expression profile of HCC revealed that genes involved in FA biosynthesis are universally upregulated in HCC compared to noncancerous liver tissues . In thisACC converts acetyl-CoA to malonyl-CoA as the first rate-limiting step in de novo lipogenesis. ACC has two isoforms, ACC1 and ACC2. ACC1 catalyzes the formation of malonyl-CoA and maintains the regulation of FA synthesis, whereas ACC2 is localized in the mitochondria and mainly regulates FAO by malonyl-CoA-mediated inhibition of CPT1. Their enzymatic activities can be inhibited by AMP-activated protein kinase (AMPK)-mediated phosphorylation. Recently, ACC has attracted attention as a therapeutic target for NASH, and an ACC inhibitor that inhibits both ACC1 and ACC2, GS-0976, was reported to reduce hepatic steatosis and fibrosis markers in patients with NASH in a phase 2 clinical trial . In regaFASN, the most extensively investigated lipogenic enzyme in cancer, is responsible for the synthesis of palmitate (C16:0) from acetyl-CoA and malonyl-CoA in the presence of NADPH . FASN isSCD catalyzes the insertion of a cis double bond at the delta-9 position in saturated FAs and generates MUFAs, mainly palmitoleate (C16:1) and oleate (C18:1) from palmitate (C16:0) and stearate (C18:0), respectively. Whereas four SCD isoforms (SCD1–SCD4) have been identified in mice, only two isoforms (SCD1 and SCD5) have been identified in humans, with human SCD1 being co-orthologous to the four murine isoforms. Budhu et al. performed comprehensive gene expression and metabolic profiling of HCC and reported that the lipogenic network involving SCD signaling was significantly associated with HCC progression and patient outcome . They shThe transcription factor SREBP-1 plays a central role in cellular FA metabolism and controls the expression of various lipogenic enzymes, including ACC, FASN, and SCD. SREBP-1 is located at the ER membrane as an inactive precursor and is transported to the Golgi apparatus by the escort protein SREBP cleavage-activating protein (SCAP) in response to insulin and sterol depletion, and is then proteolytically cleaved in the Golgi to generate an active form that activates the transcription of target genes in the nucleus . SREBP-1Several chemical inhibitors targeting FA biosynthesis pathways discussed in this section are currently in preclinical and clinical trials for cancer treatment. We summarized these inhibitors in A lipid-rich condition, especially a non-esterified FAs-rich condition, is a characteristic environment for obesity- and NASH-driven HCC , but howMUP-uPA mice [PIK3CA transgenic mice [We first carried out liquid chromatography–mass spectrometry (LC–MS)-based untargeted metabolomic profiling using paired nontumor and HCC tissues obtained from DEN-injected 8-month-old mice maintained on a normal diet or a high-fat diet (HFD), and found extensive accumulation of long-chain acylcarnitine species in HFD-fed HCC tissues (HFD-HCC). Among the acylcarnitine metabolism-related genes, increased expression of CPT1A (a hepatic isoform of CPT1) and ACSL4 (a member of the ACS family), and decreased expression of CACT and CPT2, were detected in HFD-HCC, indicating that despite the enhanced conversion of FAs to acylcarnitine, the reconversion of acylcarnitine to acyl-CoA was suppressed, which could account for the marked accumulation of acylcarnitine species A. A simiuPA mice  and PIK3nic mice . In partnic mice , suggestWe further analyzed the significance of CPT2 downregulation in obesity-mediated hepatocarcinogenesis. HCC cells in which CPT2 was knocked down acquired resistance to saturated FA-induced lipotoxicity by inhibiting excessive FAO and subsequent Src-mediated c-jun NH2-terminal kinase (JNK) activation, which plays a key role in lipotoxic cell death ,64,65,66MUP-uPA mice [In our previous study, we also showed that the downregulation of CPT2 in obesity- and NASH-driven HCC was, at least in part, attributed to decreased peroxisome proliferator-activated receptor alpha (PPARα). A more recent study by Senni et al. uncovered a pivotal role for β-catenin in determining which energy source to use (glycolysis or FAO) for tumor growth by regulating the expression of PPARα . EnhanceuPA mice ,72. TherHypoxia arises in HCCs through the accelerated growth rates of cancer cells in the absence of an efficient blood supply , and recHuang et al. reported the critical role of hypoxia inducible factor-1α (HIF-1α)-mediated suppression of FAO in the growth of HCC under hypoxic conditions . HIF-1α Bjornson et al. proposed another pathway to lipid metabolic reprogramming in HCC under hypoxic conditions . They reOn the other hand, Iwamoto et al. recently reported that HCC cells rather utilize FAO for their survival under antiangiogenic drug-induced hypoxic conditions . They shTaken together, the role of FAO in the growth and survival of HCC may be context-dependent, and more extensive investigations are needed to understand the effect of cancer metabolic reprogramming on FAO.Recent studies have suggested the existence of a subpopulation of cells within the tumor, so-called cancer stem cells (CSCs), which are typically slow-cycling, but under certain conditions capable of proliferating to self-renew and provide tumor cells. CSCs are generally resistant to chemotherapy and responsible for rapid recurrence owing to their high tumor-forming potential. In recent years, metabolic features including lipid metabolism of CSCs of various tumor types have been identified ,78,79,80The expression of FASN and de novo lipogenesis are increased in glioma CSCs compared to non-CSCs . FASN inRecent next-generation sequencing technology has enabled comprehensive mutational and transcriptome profiling of HCC ,84. Howe


Pneumocystis jirovecii pneumonia (PcP) prophylaxis in kidney transplant recipients (KTR). Post-transplant management balances preventing PcP with managing TMP-SMX-related adverse effects. TMP-SMX dose reduction addresses adverse effects but its implications to incident PcP are unclear.Trimethoprim-sulfamethoxazole (TMP-SMX) is the drug of choice for anti-We performed a retrospective review of all patients transplanted between 2011 and 2015 prescribed daily single strength TMP-SMX for twelve months post-transplantation as PcP prophylaxis. Actual TMP-SMX dose and duration, adverse effects, number of dose reductions and reasons, and PcP events were captured. Multivariate logistic regression analyses for risk factors associated with dose reduction were performed.p < 0.01) while acute rejection risked leukopenia .Of 438 KTR, 233 (53%) maintained daily TMP-SMX and 205 (47%) sustained ≥1 dose reduction, with the point prevalence of a reduced dose regimen being between 18 and 25%. Median duration for daily TMP-SMX was 8.45/12 months, contributing 4137 patient-months daily TMP-SMX and 1110 patient-months with a reduced dose. PcP did not occur in any patients. There were 84 documented dose reductions for hyperkalemia and 102 for leukopenia, with 12 and 7 patients requiring TMP-SMX cessation. In multivariate analysis, a living donor transplant protected against hyperkalemia (Odds Ratio 0.46, 95% CI 0.26–0.83, TMP-SMX dose reduction is frequent in the first post-transplant year but PcP does not occur. To limit the need for TMP-SMX dose reduction due to adverse effects, a clinical trial comparing daily to thrice weekly single strength TMP-SMX in de-novo KTR is justified. Pneumocystis jirovecii (P. jirovecii) is an important pneumonia-causing opportunistic fungus in kidney transplant recipients (KTR), causing disease  through reactivation, as well as direct or indirect person-to-person transmission [smission . PcP asssmission , 3. The smission . Howeversmission . Post-trAll other PcP prophylactic agents are considered second choice to TMP-SMX because of their relatively unfavorable breadth of coverage, tolerability, cost, and efficacy . Even wiThere are no prospective randomized controlled trials (RCT) related to optimizing TMP-SMX use in KTR. As one step toward designing a clinical trial to determine the ideal TMP-SMX dose and duration for PcP prophylaxis, we retrospectively examined the efficacy and safety of daily single strength TMP-SMX prescribed as PcP prophylaxis for twelve months post-kidney transplantation. Our hypothesis was that thrice weekly single strength TMP-SMX is at least as effective as daily TMP-SMX for PcP prophylaxis in KTR.St. Michael’s Hospital (SMH) in Toronto, Canada is a university-affiliated tertiary care medical-surgical centre that currently performs 120–150 adult single-organ kidney transplant procedures annually and provides long-term follow-up to approximately 1700 KTR. We performed a retrospective review of the electronic records of all patients transplanted at SMH between January 1, 2011 and June 30, 2015, who were prescribed daily single strength TMP-SMX during their initial hospital admission with the intent to continue TMP-SMX for twelve months post-transplantation as PcP prophylaxis. If TMP-SMX dose reduction is deemed necessary, the frequency of TMP-SMX administration is typically reduced to thrice weekly, and then stopped entirely if further reduction is deemed necessary. Since 2009 all KTR receive two induction doses of perioperative basiliximab, once-daily tacrolimus (Advagraf®), mycophenolate mofetil or mycophenolic acid, and prednisone as part of their initial immunosuppressive medication regimen. Anti-thymocyte globulin is used sparingly, even in recipients deemed to be at high immunological risk. Post-transplant laboratory monitoring in the first post-transplant year is performed twice weekly for the first three months, weekly for the next three months, and then once every two weeks to the end of 9 months and monthly thereafter.Patients were excluded from this analysis if they received a multi-organ transplant, were transplanted at another centre, or were initially prescribed a different strength of TMP-SMX or another drug for PcP prophylaxis. Patient demographics as well as the occurrence of post-transplant events such as delayed graft function , acute rejection (based on Banff criteria), cytomegalovirus IgG antibodies and new-onset diabetes, were collected by chart review. The dose and duration of TMP-SMX prophylaxis, other medications prescribed during the first year, adverse effects attributed to TMP-SMX as documented by the clinician, number of dose reductions of TMP-SMX and the corresponding reasons including adverse effects attributed to TMP-SMX, and PcP events were captured from the electronic medical record. All data were verified by at least two investigators.The primary outcome was the incidence of PcP per 100 patient-months during periods of daily TMP-SMX or reduced-dose TMP-SMX. Secondary outcomes included adverse effects attributable to TMP-SMX that necessitated dose reduction including hyperkalemia, defined as serum potassium ≥5.5 mmol/L; total leukopenia, defined as total WBC ≤ 3000/cu mm; absolute neutropenia, defined as neutrophil count ≤500/cu mm; thrombocytopenia, defined as platelet count ≤100,000/cu mm; anemia, defined as hemoglobin ≤100 g/L. and hepatitis, defined as alkaline phosphatase ≥125 IU/mL, alanine aminotransferase ≥45 IU/mL, and/or aspartate aminotransferase ≥40 IU/mL. The number and timing of dose reductions or alterations in TMP-SMX over the first year post-transplant were recorded. Since the dose per se of TMP-SMX was always single strength, dose reduction was equivalent functionally to a reduction in dose frequency. All patterns of dose reduction  were combined into a single outcome variable.p-value< 0.05 was considered significant for all comparisons. SAS version 9.2  was the statistical software used for all analyses. The study was approved by the Research Ethics Board at St. Michael’s Hospital, Toronto, protocol number REB 16–210.Comparisons were made by chi-square or Fisher exact testing for categorical variables and by paired or unpaired student t-test or analysis of variance for continuous variables as appropriate, followed by multivariate logistic regression analysis. Since patients may have been switched back-and-forth between the full-dose and reduced-dose regimes during the first year post-transplant, patients contributed follow-up time to both groups. A p = 0.009) and 6 months  but this difference had resolved at 12 months . There were no cases of PcP in either group and there were no patient hospitalizations or deaths directly attributed to TMP-SMX-related adverse effects. There were also no documented cases of infection with Nocardia, Listeria, or Toxoplasma in either group.There were 438 KTR who met entry criteria for the study, after the exclusion of 33 patients receiving either dapsone or atovaquone at any time in the first 12 months post-transplantation. Of 438 KTR, 233 (53%) maintained a full dose of TMP-SMX for 12 months and 205 (47%) sustained at least one dose reduction of TMP-SMX over the 12-month follow-up period ; all other conventional risk factors for hyperkalemia were insignificant. However, there was a strong interaction between hyperkalemia and leukopenia. Patients with leukopenia were more likely to develop hyperkalemia as well .There were 84 documented dose reductions for hyperkalemia, corresponding to an overall risk for dose reduction of 0.39/100 patient-months. Risk for dose reduction was highest in the first 3 months post-transplantation at 0.54/100 patient-months. Twelve patients required a further TMP-SMX dose reduction due to persistent hyperkalemia. A living donor transplant was protective against hyperkalemia in multivariate analysis . Modifying age (e.g. to greater than 50 years) did not affect the multivariate result analysis.There were 102 documented dose reductions for leukopenia, corresponding to an overall risk for dose reduction of 2.32/100 patient-months. The risk for dose reduction was again highest in the first 3 months, at 4.05/100 patient-months. Seven patients required a further TMP-SMX dose reduction due to persistent leukopenia. Only acute rejection was independently associated with leukopenia (Odds Ratio 3.31, 95% CI 1.39–7.90, Other reasons for TMP-SMX dose reduction included thrombocytopenia and/or anemia in 5 patients and hepatitis in 7 patients. These smaller numbers of these events were not analyzed further. There were no documented cases of TMP-SMX dose reduction or discontinuation due to skin rash or allergic interstitial nephritis.In this retrospective single-centre cohort analysis of contemporary KTR, both the daily and reduced-frequency administration of single strength TMP-SMX effectively prevented PcP in the first post-transplant year. However, a protocol of initiating daily single strength TMP-SMX associates with significant adverse effects. Almost one-half of KTR required a TMP-SMX dose reduction, mostly due to hyperkalemia and leukopenia. The only discernible risk factor for hyperkalemia was donor source while that for leukopenia was acute rejection; those at risk for one were more likely to be at risk for the other. Such KTR were at additional risk for requiring further dose reductions, but fortunately there was no hospitalization or mortality directly attributable to TMP-SMX. Nonetheless, the efforts required to monitor for TMP-SMX-related adverse effects to prevent such serious outcomes mandates efforts toward evaluating TMP-SMX prophylactic regimens in KTR with less dose exposure than once daily single strength TMP-SMX, while still effectively preventing PcP.Pneumocystis infection was first described in the lungs of rats in 1909 and in humans in 1942 . The risTMP causes hyperkalemia by blocking apical membrane sodium channels in the mammalian distal nephron to reduce transepithelial voltage and potassium secretion . AlthougTMP-SMX also associates with leukopenia that can be prolonged . LeukopeDue to the retrospective nature of the study and the fact that all patients were initially started on daily TMP-SMX, however, we cannot exclude the possibility that the beneficial effects of preventing PcP by daily oral TMP-SMX administration persisted for a length of time after conversion to a reduced frequency regime. This study provides justification for proceeding with a randomized trial in de-novo KTR to evaluate daily versus reduced frequency oral single-strength TMP-SMX to prevent PcP in the first year after kidney transplantation, after consideration of immunological risk and the consequent need for enhanced immunosuppression, as well as the possibility of a hybrid strategy requiring different dosing regimens in the early and late part of the first post-transplant year. The present study is also limited by the lack of information about drug non-adherence related to TMP-SMX, or other possible benefits of TMP-SMX prophylaxis including the occurrence of bacterial urinary tract infections. Pneumocystis colonization can also be a challenging problem in transplant recipients despite the use of TMP-SMX prophylaxis, but we were unable to ascertain potentially valuable information concerning asymptomatic infection in this population. Based on the current study, however, reduced TMP-SMX dosing may be efficient enough to prevent PcP after solid organ transplantation. There were also very few patients whose TMP-SMX dose was reduced to less than thrice weekly to allow for an efficacy comparison to a twice weekly or full dose TMP-SMX regimen.Dose reductions of TMP-SMX require a considerable amount of staff time, leading to increased overall costs for transplant programs, and the adverse effects that necessitated dose reduction in the first place will entail potentially increased patient morbidity and mortality unless they are promptly addressed. Starting with a reduced dose of TMP-SMX, such as single-strength TMP-SMX thrice weekly, carries advantages to both clinicians and patients provided PcP can be prevented at the same time. The transplant community has established with reasonable certainty that daily double-strength TMP-SMX is not necessary; what still requires further investigation is whether even daily single-strength TMP-SMX is needed. If thrice weekly single-strength TMP-SMX for a year is proven sufficient for preventing PcP, then extending such therapy beyond the first year post-transplant, when patients are seen less frequently, can be tested as a strategy to minimize the occurrence of late-onset PcP as well.In conclusion, TMP-SMX dose reduction is frequent in the first post-transplant year, but PcP does not occur after dose reduction at least during the rest of the first post-transplant year. To confirm that we can limit the need for such frequent TMP-SMX dose reduction resulting from adverse effects, a clinical trial in KTR comparing daily single strength to thrice weekly single strength TMP-SMX in de-novo KTR is justified.


Begomovirus, family Geminiviridae) and causes severe damage to tomatoes throughout tropical and sub-tropical regions of the world. Among others, the Tomato yellow leaf curl Sardinia virus (TYLCSV) species causes damage in the Mediterranean Basin since early outbreaks occurred. Nevertheless, scarce information is available about the diversity of TYLCSV. Here, we study this aspect based on the sequence information accessible in databases. Isolates of two taxonomically differentiated TYLCSV strains can be found in natural epidemics. Their evolution is mostly associated with mutation combined with selection and random genetic drift and also with inter-species recombination which is frequent in begomoviruses. Moreover, a novel putative inter-strain recombinant is reported. Although no significantly new biological behaviour was observed for this latter recombinant, its occurrence supports that as shown for other related begomoviruses, recombination continues to play a central role in the evolution of TYLCD-associated viruses and the dynamism of their populations. The confrontation of resistant tomatoes with isolates of different TYLCD-associated viruses including the novel recombinant demonstrates the existence of a variable virus x plant genotype interaction. This has already been observed for other TYLCD-associated viruses and is a challenge for the control of their impact on tomato production.The tomato leaf curl disease (TYLCD) is associated with infections of several species of begomoviruses (genus  Begomovirus (family Geminiviridae) are economically important phytopathogens that are emerging in warm regions causing severe losses in economically important crops worldwide  to TYLCD-associated viruses based on genes Ty-1 and Ty-3 . Also, the common bean cv. Donna  and a wild Solanum nigrum (from IHSM-La Mayora germplasm collection) were used in this study.The tomato cultivars, tomato breeding lines and tomato relatives used in this study were tomato cv. Moneymaker used as susceptible control, the quasi-isogenic tomato lines homozygous ty-1S  and isolates of the type   and Mild (Mld)  strains of TYLCV ,50. AddiBamHI restriction endonuclease and tandem repeat fragments were purified from agarose gel  and ligated into the BamHI cloning site of pCAMBIA 0380 vector . A virus-specific clone (pMur-TY2-Tom-11) containing an insert of about 5.6 kb (a putative dimeric 2-mer clone with a tandem repeat of two full-length copies) was selected to derive the full-length sequence of the cloned isolate named [Spain-Murcia-TY2-Tomato-2011] (acronym ES-Mur-TY2-Tom-11). A primer-walking strategy was employed to obtain the full-length viral genomic sequence (GenBank Accession No. KC953604). An infectious clone of the isolate [ES-Mur-TY2-Tom-11] was obtained by transferring the pCAMBIA clone pMur-TY2-Tom2011 into the Agrobacterium tumefaciens strain LBA4404.The total DNA extracted from young leaf tissues of the sample ES-Mur-TY2-Tom-11 using a modified CTAB method  was usedA. tumefaciens-mediated inoculation (agroinoculation), liquid cultures of A. tumefaciens were pelleted and resuspended in an agroinoculation buffer  to a final OD of 1.0 at 600 nm before stem puncture inoculation  was obtained.A dimeric infectious clone of isolate [ES-Mur-TY2-Tom-11] was obtained by transferring the clone pMur-TY2-Tom2011 into rformed)  and deteS. nigrum plants was studied because differences have been reported in these host plants for TYLCD-associated viruses and derived recombinants  reported here when compared with an isolate of TYLCSV previously reported in SpainTherefore, in conclusion, the results obtained here suggested that no significant differences were observed in the behaviour of resistant tomato cultivars/lines and Geminiviridae Study Group of the ICTV [https://talk.ictvonline.org/ictv-reports/), the existence of different strains in the TYLCSV species was proposed in previous reports [As for TYLCV and other begomoviruses ,72,73, pthe ICTV  or ICTV  reports . Here, t reports  indicate reports . Thus, w reports ,69. This reports ,44. Also reports .Ty-1 resistance [It is interesting to note that we have shown here that in addition to point mutation probably combined with selection and genetic drift, the evolution of the TYLCSV species seems to be driven by genetic exchanges achieved through inter-species recombination. Thus, isolates of strain Sar/Group 1 were shown to derive from a genetic exchange involving the IR and the 5′-end of the Rep gene, a region prone to recombination events in begomoviruses , for whisistance ,40. Simisistance  have alssistance .We have characterised here a TYLCSV isolate collected from Spanish epidemics for which a mosaic composition of the genome putatively driven by a TYLCSV inter-strain recombination is suggested. The phylogenetic relationship incongruences detected depending on the genomic region compared supported a modular evolution , with twTy-3 and Ty-1 of the same resistance locus to control TYLCD-associated viruses as already suggested [Ty-1-based resistances that might suggest additional allele differences. Nevertheless, as isogenic lines were not available, background effect differences cannot be discarded as resistance phenotype might depend on the genetic background [Ty-1/Ty-3 line TY-Sk13 respect to the Ty-1 heterozygous line TY-Sk1 might suggest negative interaction of Ty-1 and Ty-3 genes which might merit further study. But, again, the effect of background differences cannot be discarded.Recombination has been reported that can have relevant phenotypic consequences, such as increased virulence or changes in host range ,82. No suggested ,87. Moreckground . The lowWe conclude, therefore, that genetically complex relationships are observed in the TYLCSV population in which recombination events are driving evolution. Novel TYLCSV recombinants can appear such as the one characterised here, with new biological properties of unknown phytopathological consequences that can complicate TYLCSV control. All this reinforces the importance of monitoring virus populations present in the TYLCD epidemics to detect relevant alterations that could challenge control strategies.