Plasmodium berghei, we show that a Plasmodium-specific Apicoplast protein plays an important role for Liver Merozoite formation (PALM). While the resulting knockout mutants develop normally for most of the life cycle, merozoite release into the blood stream and the ability to establish an infection are severely impaired. Presence of a signature blood-stage antigen, merozoite surface protein 1 and normal apicoplast morphology indicate that the inability to finalize merozoite segregation is a direct consequence of loss of PALM function. Experimental immunization of mice with as few as two doses of palm- sporozoites can elicit sterile protection up to 110 days after final immunization. Our data establish that a tailor-made arrest in the final steps of hepatic merozoite formation can induce strong protective immune responses and that malaria parasites employ a distinct apicoplast protein for efficient formation of pre-erythrocytic merozoites.Malaria parasites undergo a population expansion inside the host liver before disease onset. Developmental arrest inside host hepatocytes elicits protective immune responses. Therefore, elucidation of the molecular mechanisms leading to mature hepatic merozoites, which initiate the pathogenic blood phase, also informs anti-malaria vaccine strategies. Using targeted gene deletion in the rodent model malaria parasite  Apicomplexa. Plasmodium falciparum is a major cause of childhood mortality in sub-Saharan Africa and continues to be the most prevalent and fatal arthropod-borne infectious disease [Malaria parasites are obligate intracellular eukaryotic pathogens that belong to the phylum  disease .Plasmodium species follow a complex developmental programme of stage conversions as they progress through their life cycles  pathway lead to premature arrest, as evident from the absence of signature proteins of mature blood-stage infectious parasites (Plasmodium-specific apicoplast protein for liver merozoite formation (PALM) and its requirement for efficient transition to a blood-stage infection.Here, we demonstrate the importance of a previously uncharacterized Plasmodium genes with a specific critical function in liver merozoite formation, we systematically searched available data sets for potential liver stage-specific genes of unknown function. We focused on a small Plasmodium-specific gene (PBANKA_010110) expressed during liver-stage development . As an alternative, we generated a parasite line, using a pairment . Comparaarasites . AbsencePALM transcripts in mature blood stages  of mice remained entirely malaria-free at day 17 after infection , which localizes to the parasitophorous vacuole . PALM loss-of-function parasites retain their capacity to infect and mature inside hepatocytes. However, towards the final steps of merozoite segregation, palm- parasites display a strong impairment, resulting in a severe delay in sporozoite-induced blood-stage infections.In this study, we characterized a novel apicoplast targeted protein of the malaria parasite with an important function during liver merozoite formation; hence, the term ‘palm- parasites develop to a much later time point, as indicated by the expression of the merozoite signature protein MSP1. In this study, we were able to define many key features of this previously unrecognized protein, including the subcellular localization, the life cycle stages at which PALM is expressed, and the critical time point of PALM activity. What could be the molecular function during the process of merozoite formation?Unlike several genetically arrested parasites deficient in apicoplast-targeted proteins of the FASII pathway  demonstrated that apicoplast growth, branching and protein import of ACP are unaffected in the absence of PALM. We can, therefore, reject the hypothesis that PALM is involved in the process of apicoplast segregation itself. This is plausible, because of the observed expression of PALM in blood-stage schizonts and mosquito stage midgut oocysts and the apparent absence of PALM orthologues in other obligate intracellular parasites of the phylum Apicomplexa.Staining of developing liver-stage PALM in vivo is further supported by the differential clinical outcome of a blood stage-induced vs. a sporozoite-induced infection. We can exclude a detectable in vivo role of PALM during blood-stage replication, which in the P. berghei ANKA-C57BL/6 mouse model leads to a uniform fatal outcome.The strictly liver stage-specific role of PALM emerged in response to specific requirements for the development of malaria parasites in hepatocytes. Because pre-erythrocytic development of avian malaria species, such as Plasmodium gallinaceum, does not occur in hepatocytes but rather in cells of the mononuclear phagocyte system, it is conceivable that these parasites lack PALM. In the absence of complete genome sequences of avian Plasmodium species, cloning and sequencing of the orthologous region should reveal whether P. gallinaceum harbours a PALM orthologue or not. The sheer number of merozoites being produced during the pre-erythrocytic phase may require a yet unidentified synthetic pathway in the apicoplast that is dispensable for other life cycle stages and related apicomplexan parasites.An intriguing possibility is that palm- sporozoites and developing blood-stage parasitaemia do not develop ECM. This finding is reminiscent of the consistent findings seen in phase II clinical trials with the RTS,S vaccine . C57BL/6 mice were used for sporozoite challenges and analysis of ECM. All other parasite infections were conducted in NMRI mice, unless otherwise indicated.Mutant parasites were generated as described previously  was done overnight at 4°C. Bound antibodies were detected using donkey anti-mouse IgG Alexa Fluor 488 conjugated antibodies . Nuclei were visualized with the DNA-dye DRAQ5  and coverslips were mounted with Fluoromount-G (Southern Biotech).Midguts of PALM-mCherry-myc infected mosquitoes were dissected and liberated sporozoites were settled in RPMI medium containing 3% bovine serum albumin and fixed with 4% paraformaldehyde. Subsequently, sporozoites were permeabilized with 0.1% Triton X-100 in PBS and blocked with 3% bovine serum albumin. Incubation with mouse anti-myc antibodies  was done overnight at 4°C. Bound antibodies were detected using donkey anti-mouse IgG Alexa Fluor 546 conjugated antibodies . Coverslips were mounted with Fluoromount-G (Southern Biotech).Salivary glands of To determine sporozoite infectivity to mice, sporozoites collected from mosquito salivary glands were injected intravenously at the numbers indicated into C57BL/6 mice. Patency was determined by examination of daily Giemsa-stained thin blood smears.Plasmodium berghei mosquito stages were maintained and analysed using standard techniques . To confirm apicoplast targeting of PALM, liver-stage parasites were co-stained with rabbit anti-P. berghei ACP peptide antiserum  peptide antiserum  and a monoclonal mouse anti-P. yoelii merozoite surface protein 1 (MSP1) antibody against a 90 kDa N-terminal fragment of the protein that shares 74% identity with P. berghei MSP1 . Liver-stage parasites were visualized and quantified using monoclonal mouse anti-P. berghei heat shock protein 70 (HSP70) antibodies  treatment of sporozoite-infected hepatoma cells as described previously  seeding of 100 000 to 150 000 hepatoma cells per well in 24-well plates and inoculation with 100 000 sporozoites per well 24 h later, or by (ii) seeding of 30 000 hepatoma cells per well in eight-well chamber slides  and inoculation with 10 000 sporozoites 24 h later. Thereafter, standard procedures were used (palm- sporozoites extracted from salivary glands of infected mosquitoes. Sporozoites were injected intravenously in a volume of 100 µl. For the prime/boost protocol, animals that remained malaria-free after the first immunization were given a second dose 5–7 weeks after the first immunization. Only animals that remained blood-stage parasite-negative after the first immunization and subsequent boost were used for the challenge experiments at 4–6 weeks after the last immunization. Mice were challenged with five ANKA-GFP-infected mosquitoes, 10 000 intravenously injected ANKA-GFP sporozoites, or 10 intravenously injected ANKA-GFP blood-stage parasites. The number of ANKA-GFP salivary gland sporozoites per mosquito used for the challenge by bite ranged from 20 000 to 70 000. At least three age-matched naïve animals were included to verify infectivity of sporozoites during all challenge experiments. Parasitaemia was monitored by daily Giemsa-stained thin blood smears, starting from day 3 after immunization or ANKA-GFP challenge until at least day 17.Age-matched female C57BL/6 mice were immunized with two doses of 1000 or 10 000


HIV-1 encodes an essential regulatory protein, Tat which orchestrates viral gene expression from the HIV-1 promoter. Tat activity depends on Tat hijacking the nuclear transport system to gain access to the transcriptional machinery. The sole import factor identified for Tat to date, Importin β, directly interacts with Tat-NLS to mediate its active nuclear import. While Tat localises in the nucleoli & nucleoplasm of transformed cells, in HIV-1 infected primary T-cells, Tat accumulates at the plasma membrane, where it is secreted. The different subcellular accumulation of Tat could be due to selective enhancement of the nuclear import machinery in transformed cells or alternatively, an increase in Tat nuclear export or its specific cytosolic retention in primary T cells. Of note, Tat does not contain a classical NES and its nuclear export remains to be established. Here, we characterise the bidirectional transport receptor, Importin 13 (IPO13) as a novel transport receptor mediating the nucleo-cytoplasmic shuttling of Tat.in vitro GST pulldowns were employed using recombinant GST-YFP-Tat and corresponding mutants with 6xhis-IPO13. In vitro nuclear transport assays were performed using digitonin permeabilised HeLa cells. Tat activity was monitored using HeLa-LTR-Luc reporter cells. SiRNA knockdown of IPO13 was validated by Western blot.Co-localisation studies were performed using immunofluorescence and confocal microscopy, followed by deconvolution. For protein interaction studies, In vitro transport assays revealed IPO13 as a novel import factor for HIV-1 Tat and identified residues 49R50K51K within Tat-NLS as essential for import by IPO13. Of note, IPO13-mediated Tat nuclear import unusually resulted in Tat nucleoplasmic distribution with weak nucleolar staining, while IPO13 import of the Tat-NLS alone resulted in a typical strong nucleolar accumulation. Importantly, IPO13 could mediate the active, CRM-1 independent nuclear export of Tat, but not Tat-NLS alone, via the Nuclear Pore Complexes. Collectively, these results suggest that the Tat-NLS is sufficient for interaction with IPO13 and nuclear import, but Tat nuclear export by IPO13 requires additional domains. Interestingly, overexpression of IPO13 induced Tat redistribution to the cytoplasm in vivo. Finally, siRNA mediated knockdown of IPO13 in HeLa-LTR-Luc reporter cells inhibited Tat transactivation by 60%, compared to non-target siRNA controls, highlighting the functional significance of IPO13 for Tat activity.GST pulldowns demonstrated that Tat interacts directly with IPO13 via its NLS, as well as via additional NLS independent binding sites. We have identified IPO13 as an essential new transport factor for Tat and provided the first evidence of Tat nucleo-cytoplasmic shuttling, which could have functional implications for Tat cytoplasmic activities and its secretion by infected cells.


This study used CMR to evaluate patients with IHD prior to ICD implantation and correlated CMR measurements to VA inducibility and spontaneous VA events during follow-up. The results demonstrated that the gray-zone measurement using MCLE may be more sensitive in predicting appropriate ICD therapy for VA.In addition to measures of left ventricular ejection fraction (LVEF) and clinical staging of heart failure, myocardial infarct (MI) heterogeneity including MI and peri-infarct gray-zone (GZ) has the potential to predict the occurrence of inducible sustained ventricular arrhythmia (VA) and spontaneous VA events after implantation of implantable defibrillator (ICD) in patients with ischemic heart disease (IHD). Late-gadolinium (Gd)-enhancement (LGE) cardiac MR (CMR) using inversion-recovery fast-gradient-echo (IR-FGRE) is commonly used for the determination of infarct heterogeneity in these patients. Recently, a multi-contrast late-enhancement (MCLE) sequence has been developed for better infarct heterogeneity quantification. Compared to IR-FGRE, we hypothesized that MCLE may be a more sensitive method to predict the occurrence of inducible VA and severe events post-ICD implantation.This study used CMR to evaluate patients with IHD prior to ICD implantation and correlated CMR measurements to VA inducibility and spontaneous VA events during follow-up. The MRI protocol included LV functional parameter assessment using steady-state free precession (SSFP), as well as LGE-MRI using IR-FGRE and MCLE post double-dose Gd injection. LV functional parameters were measured using Q-Mass or CMR42 software. The GZ analysis in IR-FGRE used a full-width half-maximum method. For MCLE, GZ analysis used a semi-automated data clustering algorithm. An unpaired t-test with unequal variance was used for the statistical analysis of the proportion of GZ, MI core and total MI relative to LV myocardium mass.Twenty-five patients with IHD for planned ICD implantation  were enrolled. All patients completed the MRI protocol, ventricular electro-physiology study (EPS), and six months follow-up at the ICD clinic. Eight patients (32%) had inducible VA during programmed ventricular stimulation and six of these eight patients had at least one appropriate shock for VF from the ICD at follow-up. Another two patients without inducible VA had appropriate shocks from the ICD. CMR measurements of LVEF and myocardial infarct heterogeneity are shown in Tables CMR measurements of myocardial heterogeneity using MCLE and IR-FGRE are good predictors for the occurrence of inducible VA; however, the gray-zone measurement using MCLE may be more sensitive in predicting appropriate ICD therapy for VA.CIHR.


Pancreatic sections from 5- to 11-month-old littermates were examined. Histomorphometric analyses revealed significant suppression of islet mass expansion in PACAP/+:Ay/+ mice compared with Ay/+ mice at 11 months, but no significant difference between PACAP/+ and +/+ (wild-type) mice, as previously reported. The suppressed islet mass in PACAP/+:Ay/+ mice was due to a decrease in islet density but not islet size. In addition, the density of tiny islets (<0.001 mm2) and of insulin-positive clusters in ductal structures were markedly decreased in PACAP/+:Ay/+ mice compared with Ay/+ mice at 5 months of age. In contrast, PACAP overexpression caused no significant effects on the level of aldehyde-fuchsin reagent staining (a measure of β-cell granulation) or the volume and localization of glucagon-positive cells in the pancreas. These results support previously reported inhibitory effects of PACAP on pancreatic islet mass expansion, and suggest it has persistent suppressive effects on pancreatic islet density which may be related with ductal cell-associated islet neogenesis in type II diabetes.Pituitary adenylate cyclase-activating polypeptide (PACAP) is a potent insulinotropic G-protein-coupled receptor ligand, for which morphoregulative roles in pancreatic islets have recently been suggested. Here, we evaluated the effects of pancreatic overexpression of PACAP on morphometric changes of islets in a severe type II diabetes model. Following cross-breeding of obese-diabetic model KKA Pituitary adenylate cyclase-activating polypeptide (PACAP) is an extraordinarily potent insulinotropic peptide   hyperinsulinemia. We also found that PACAP overexpression retained its previously observed suppressive effects, particularly those relating to hyperinsulinemia, in HFD-fed KKAy mice , aldehyde-fuchsin (AF), and anti-insulin and anti-glucagon antibodies. Here, we used PACAP/+ mice to evaluate the direct and local action of PACAP on the islet morphology, since PACAP can exert pleiotropic actions on adipocyte and adrenal medulla in addition to islets and secondary affect islets. The results obtained show that PACAP retains its inhibitory effects on pancreatic islet mass expansion, and provide a range of evidence suggesting that pancreatic PACAP affects ductal cell-associated islet neogenesis.In the present study, we performed several morphometrical analyses of the islet phenotype of HFD-fed KKA1 mice  were obtained by mating female transgenic mice overexpressing PACAP in their pancreatic β cells . F1 males were individually housed after genotyping and weaning, and males with the Ay allele (Ay/+ and Ay/+:PACAP/+) were fed with an HFD  from 4 weeks of age, while the other males continued on a normal diet (ND) . These diets contain either 11.8% (DC-8) or 56.7% (HFD-32) of energy derived from fat.All animal care and handling procedures were approved by the Institutional Animal Care and Use Committee of Osaka University. Mice were housed in a temperature-, humidity-, and light-controlled room with a 12-h light/12-h dark cycle (lights on at 08:00 a.m.) and allowed free access to water and chow. Mating, genotyping, and feeding procedures were as previously described  or anti-glucagon  antibodies, in which signals were visualized using the diaminobenzidine method and were counterstained with cresyl violet. To investigate the architectural changes in the islets, two serial sections were prepared for immunostaining with anti-insulin and anti-glucagon antibodies, respectively.n = 4–6 for 5-month-old mice, and n = 7–9 for 11-month-old mice), total islet number, size of each islet, and total pancreatic area were counted or measured, and analyzed as previously described . Islet mass was calculated by multiplying the pancreas weight by the relative islet area per pancreas. The density per mm2 of total pancreatic area, in addition to the frequency of the six groups of optical islet size , was also determined. In AF-stained sections , blinded observers evaluated the AF reagent staining in each islet of four F1 groups. In sections stained with anti-insulin or anti-glucagon antibodies (n = 4 for each group), the positive area and the size of each insulin-positive cluster were measured using ImageJ software . The number of insulin-positive clusters, and of glucagon cell-infiltrated islets , and morphometrical parameters were examined as follows. In HE-stained sections . The statistical significance of differences was assessed using two-way ANOVA followed by the Tukey–Kramer test, χy mice fed with a ND, we previously showed that pancreatic PACAP overexpression markedly suppresses the increase of mean islet size and of islet density  islets was preferentially increased in Ay/+ mice compared with +/+ mice, and this increase was significantly inhibited in Ay/+:PACAP/+ mice. Since the density of smaller (<0.003 mm2) islets was also decreased in Ay/+:PACAP/+ mice at 5 months of age, we also compared the density of islets sized <0.001 mm2 between Ay/+ and Ay/+:PACAP/+ mice at that age , whereas they were clearly shifted right compared to +/+ mice . Collectively, these results suggest that PACAP overexpression does not affect the overall size distribution of islets in HFD-KKAy mice, but has clear inhibitory effects on islet density, particularly in the smaller islets (<0.003 mm2) at an earlier age (5 months old).We next examined which size of islets was decreased by PACAP in HFD-fed KKAe Figure C. In boty/+ and Ay/+:PACAP/+ mice, we used AF staining to examine the degranulation of β-cells, a well known phenotypic change in obese mouse islets, including those of KKAy mice  lacked AF staining, whereas those at 11 months of age (n = 3 for each genotype) showed definite AF staining. These results suggest that an unexpected recovery in β-cell degranulation could be occurring in HFD-fed KKAy mice, and that pancreatic PACAP overexpression does not affect either degranulation, or compensatory re-granulation, of β-cells in this model.To explore the possible changes of islets in Ay mice is unexpectedly enhanced at 11 months of age compared with their age-matched wild-types  was attenuated in both of Ay/+ and Ay/+:PACAP/+ compared with wild-type mice, but no significant difference was observed between two groups . These data suggest that the impaired first-phase insulin response seems to be persistently observed in both Ay/+ and Ay/+:PACAP/+ mice, and that pancreatic PACAP overexpression showed little effects on the glucose-induced insulin release at least at 11 months of age.In line with the unexpected recovery in β-cell degranulation, we previously showed that the glucose disposal in HFD-fed KKAs Figure . The resy/+ and PACAP/+:Ay/+ mice using two adjacent sections stained with anti-insulin and anti-glucagon antibodies, respectively , were often observed in these two groups. Glucagon-stained sections revealed an apparent reduction of staining in both Ay/+ and PACAP/+:Ay/+ mice compared with +/+ mice. In addition, some islets showed an altered localization of glucagon-positive cells (as indicated by arrows); namely, the cells localized not only peripherally but also in the central area of the islets. In age-matched +/+ mice, this was rarely observed and glucagon-positive cells were predominantly localized at the periphery of the islets. Based on these observations, a range of parameters were examined  in Ay/+:PACAP/+ mice compared with Ay/+ mice, whereas the total frequency of ductal clusters was not significantly different. In glucagon-stained sections, no significant difference was observed in the percent of glucagon-positive area and of glucagon cell-infiltrated islets.The above data indicate that phenotypic differences between the 5-month-old groups were more obvious and are possibly causative for the changes in 11-month-old groups. Thus, we next performed detailed analyses on the islets of 5-month-old Ay Figure B. In insy mice; Sakurai et al., y/+:PACAP/+ mice, the significant suppression of islet mass expansion at 11 but not at 5 months of age  was less than half of that observed in ND-fed KKAy mice  of islet density, but not of mean islet size, in Ae Figure , these dy/+:PACAP/+ mice fed with ND, we previously showed that the pancreatic PACAP content and the plasma insulin level are increased by 3.5- and 2.8-fold of PACAP/+ mice  is regulated via diverse processes including differentiation from ductal precursor cells, trans-differentiation from non-β-cells, fission or fusion between islets, and replication, hypertrophy, and apoptosis of the β-cells themselves (Bonner-Weir et al., In conclusion, the present study provides additional evidence for the inhibitory effects of PACAP on pancreatic β-cell mass expansion, and suggested its possible effects on ductal precursor-related islet genesis. An increased number of ductal insulin-positive cells has been reported in pancreatic biopsy samples from human type II diabetic patients, in which the increased β-cell mass was suggested to be due to increased islet neogenesis but not to islet enlargement (Butler et al., 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.


Anomalous origin of the left coronary artery from the pulmonary artery (ALCAPA) is a rare coronary artery anomaly.Purpose: The aim of this study is to show the role of cardiovascular magnetic resonance (CMR) in assessing these patients.6 patients with repaired ALCAPA  underwent CMR to assess clinical suspicion of myocardial ischemia - short axis and long axis cine images (assess ventricular function), late-gadolinium enhancement , adenosine stress perfusion scan (detection of reversible ischaemia) and 3D whole-heart imaging .The LV function was preserved in all patients (mean LVEF= 62.7% ± 4.23%). The LV volumes were within the normal ranges LV  In one patient, hypokinesia of the anterior segments was visualized. 5 of the 6 patients showed sub-endocardial late gadolinium enhancement involving the antero-lateral wall and the anterior papillary muscle. 4 of the 6 patients presented areas of inducible ischemia, reversible at rest. In 3 of the 6 patients the proximal course of the left coronary artery wasn't clearly visualized. In these patients, proximal the LCA obstruction was confirmed during invasive coronary angiography.CMR is a good, non-invasive, radiation-free investigation in the post-surgical evaluation of ALCAPA. We show that antero-lateral sub-endocardial myocardial fibrosis is a characteristic finding. Furthermore, stress adensoine CMR perfusion, can identify reversible ischaemia in this group. Such imaging may be a promising tool in the future clinical decision -making.


The Middle East Respiratory Syndrome Coronavirus (MERS-CoV) causes symptoms similar to Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV), yet involving an additional component of acute renal failure (ARF) according to several published case reports. Impairment of the kidney is not typically seen in Coronavirus infections. The role of kidney infection in MERS is not understood.In-situ histological staining of the respective cellular receptors for MERS- and SARS-Coronavirus showed highly similar staining patterns with a focus of a receptor-specific signal in kidney epithelial cells. Comparative infection experiments with SARS- and MERS-CoV in primary human kidney cells versus primary human bronchial epithelial cells showed cytopathogenic infection only in kidney cells, and only if infected with MERS-CoV. Kidney epithelial cells produced almost 1000-fold more infectious MERS-CoV progeny than bronchial epithelial cells, while only a small difference was seen between cell types when infected with SARS-CoV.A systematic review of communicated and peer-reviewed case reports revealed differences in descriptions of kidney involvement in MERS versus SARS patients. In particular, ARF in MERS patients occurred considerably earlier after a median time to onset of 11 days  as opposed to 20 days for SARS, according to the literature. Epidemiological studies should analyze kidney impairment and its characteristics in MERS-CoV. Virus replication in the kidney with potential shedding in urine might constitute a way of transmission, and could explain untraceable transmission chains leading to new cases. Individual patients might benefit from early induction of renoprotective treatment. Due to its distribution in several countries of the Arabian Peninsula there is a risk of global spread through travel and pilgrimage, posing a potential threat to global public health.Coronaviruses (CoVs) cause human disease with symptoms ranging from mild respiratory symptoms to severe pneumonia . In SeptThe clinical picture of MERS-CoV infection is characterized by acute atypical pneumonia and respiratory failure, resembling symptoms caused by SARS-CoV -5. Acutehttp://www.promedmail.org) using the search term “coronavirus” on items published between 15th September 2012 and 16th September 2013. The broad search term was chosen due to inconsistencies of nomenclature before the consensus name MERS-CoV was announced [It has been shown recently that MERS-CoV enters target cells not via the SARS-CoV receptor Angiotensin-converting-enzyme-2 (ACE-2), but via binding to Dipeptidyl-peptidase 4 (DPP-4) ,10. BothIn total, the search revealed 655 publications, including 508 articles in PubMed, and 147 reports in ProMed  and polyclonal goat-anti-human DPP-4 immunoglobulin (R&D systems) showed that both receptors are abundantly expressed in the healthy human kidney, and both receptors are predominantly found in the epithelial layer of the renal ducts . As the bronchoalveolar epithelium of the lung constitutes the primary target compartment for both viruses, infection of human primary bronchial epithelial cells  was studied in parallel. As summarized in Figure 5/mL and inoculated with either MERS-CoV strain EMC/2012, or SARS-CoV strain Frankfurt-1 at multiplicities of infection (MOI) of 0.5 for 1 hour. Cells were washed twice and supernatants were harvested immediately after washing, as well as 20 and 40 hours later. Cells were checked regularly for appearance of cytopathic effects (CPE) by light microscopy. Replication of both viruses was quantified by titration of supernatants in duplicate plaque assays on Vero cells as described previously, as well as by real-time RT-PCR [To test whether kidney epithelial cells can actually be infected e RT-PCR ,16.Pronounced CPE consisting of visible and strong cell lysis occurred only in HREpC, and only upon infection with MERS-CoV Figure C. Neithein-vitro infection experiments suggest differences in kidney cell involvement might exist between SARS- and MERS-CoV infection. In a retrospective analysis of 536 SARS cases, only in 36 (6.7%) patients acute renal impairment was diagnosed. ARF occurred rather late in the course of disease after a median of 20 days from the appearance of first symptoms [Our results from symptoms . In our symptoms -20. Onlypost mortem kidney tissue of infected patients [In-vitro studies of SARS-CoV with immortalized human proximal tubular epithelial cells showed replication without cell impairment as seen in our study, while no infection of podocyte cell lines and only low-level replication in glomerular mesangial cells (MC) was seen, providing further evidence against specific involvement of the renal tract in SARS-CoV infection [in-vivo. Nevertheless, our results have some congruence with clinical observations in a well-investigated MERS case [While no autopsy data have become available for patients infected with MERS-CoV, post-mortem studies have been done systematically in SARS victims. Histopathological findings revealed mainly acute tubular necrosis without evidence of glomerular pathology, which is considered to be a consequence of systemic inflammatory response in the context of multi-organ failure, rather than a specific effect of viral infection of the kidney . SARS-Copatients . In-vitrnfection . In contnfection . The abunfection . The cleERS case . Virus iERS case . The autERS case ,6,7. CliThe authors declare that they have no competing interests.IE, MAM designed the experiments, IE, MAM, SK performed experiments and analysis, IE, CD wrote the manuscript, IE, DNG, CD contributed to the final version of the manuscript. All authors read and approved the final manuscript.


Maintaining proper adhesion between neighboring cells depends on the ability of cells to mechanically respond to tension at cell-cell junctions through the actin cytoskeleton. Thus, identifying the molecules involved in responding to cell tension would provide insight into the maintenance, regulation, and breakdown of cell-cell junctions during various biological processes. Vinculin, an actin-binding protein that associates with the cadherin complex, is recruited to cell-cell contacts under increased tension in a myosin II-dependent manner. However, the precise role of vinculin at force-bearing cell-cell junctions and how myosin II activity alters the recruitment of vinculin at quiescent cell-cell contacts have not been demonstrated.We generated vinculin knockdown cells using shRNA specific to vinculin and MDCK epithelial cells. These vinculin-deficient MDCK cells form smaller cell clusters in a suspension than wild-type cells. In wound healing assays, GFP-vinculin accumulated at cell-cell junctions along the wound edge while vinculin-deficient cells displayed a slower wound closure rate compared to vinculin-expressing cells. In the presence of blebbistatin (myosin II inhibitor), vinculin localization at quiescent cell-cell contacts was unaffected while in the presence of jasplakinolide (F-actin stabilizer), vinculin recruitment increased in mature MDCK cell monolayers.These results demonstrate that vinculin plays an active role at adherens junctions under increased tension at cell-cell contacts where vinculin recruitment occurs in a myosin II activity-dependent manner, whereas vinculin recruitment to the quiescent cell-cell junctions depends on F-actin stabilization. Cells experience force and, therefore, need to mechanically respond to stabilize cell junctions with both neighboring cells and the underlying extracellular matrix. Cadherins are the adhesion proteins composing the adherens junctions at cell-cell contacts while cadherin-associated proteins and the actin cytoskeleton provide stability and structural support between neighboring cells. The E-cadherin complex was identified as a mechanosensor at cell-cell contacts where applying force on the extracellular domain of E-cadherin resulted in vinculin-dependent cell stiffening . VinculiVinculin was originally identified as a protein associated at the ends of actin fibers terminating at the plasma membrane . Along wThe association of vinculin with integrins at focal adhesions has been well examined, where vinculin binds to talin and paxillin, two integrin-binding proteins -9. At foIn biochemical assays with purified proteins, a direct interaction occurs between the vinculin head domain and the cadherin-associated protein α-catenin -25, withMDCK cells are the prototypical polarized epithelial cell model, yet the interaction of vinculin with the E-cadherin complex in MDCK cells is different from other cell lines. This has been attributed to low tension in MDCK cell monolayers under normal conditions possibly due to the unique cadherin distribution of MDCK cell-cell contacts , thus reThe aim of this study was to investigate the role of vinculin at force-bearing adherens junctions and its recruitment to sites of cell-cell contact. In this report, using vinculin knockdown and GFP-vinculin expressing cells in wound-healing assays, we demonstrate that vinculin is required for proper wound closure and vinculin-deficiency results in a reduced rate of wound closure. Additionally, we show that recruitment of vinculin to cell-cell contacts is increased with F-actin stabilization.We generated stable cell lines of vinculin knockdown cells using three shRNA target sequences for canine vinculin. For analysis of vinculin in cell-cell adhesion formation, two vinculin knockdown MDCK cell lines were chosen as these subclones represented the intermediate  and least  total vinculin level compared to wild-type Figure  and 1B. To further analyze the relative distribution of vinculin, we immuno-labeled vinculin in wild-type and vinculin knockdown cells. The immuno-labeled vinculin localized to the cytoplasm and focal adhesions in wild-type cells, but significantly less in knockdown cells Figure  and 1D. Since the residual vinculin in knockdown cells accumulated at cell-cell contacts, albeit much less than wild-type cells, we tested whether the residual vinculin is sufficient to support cell-cell contact formations using the hanging drop adhesion assay. The wild-type and knockdown cells were suspended in a media to allow cell clustering and cell cluster sizes were analyzed over time. The vinculin knockdown cells displayed small cell clusters Figure , suggestDespite the reduced vinculin level at cell-cell contacts, vinculin knockdown cells form a cell monolayer without any phenotypic defects Figure . Since vIn the wound-healing assay, vinculin accumulated at the wound edge in both wild-type and knockdown cells. However, vinculin intensity in the knockdown cells was reduced compared to the wild-type cells Figure . MDCK ceVinculin localizes along the length of cell-cell contacts in mature MDCK cell monolayers ,25 Figu. Since bCalyculin-A is a serine/threonine phosphatase inhibitor that inhibits Myosin Light Chain (MLC) phosphatase. The inhibition of MLC phosphatase is thought to induce myosin II activity by minimizing the de-phosphorylation of MLC, thus myosin II remains in a phosphorylated and active state. In the presence of calyculin-A, numerous actin bundles formed at the basal surface Figure , a sign One factor that may be involved in recruiting vinculin to cell-cell contacts is the amount or dynamic state of F-actin, a binding partner to which vinculin contains F-actin binding domains ,6. To deTo test whether stabilizing F-actin affects vinculin recruitment to cell-cell contacts under tension, jasplakinolide treatment was included in the MDCK wound-healing assay. In the presence of jasplakinolide, vinculin accumulated at cell-cell contacts along the wound edge similarly to control Figure . AlthougOn the other hand, vinculin accumulation was either reduced or absent along the wound edge in blebbistatin-treated cells Figure , likely Our data suggests the existence of two vinculin pools at the sites of cell-cell adhesion: myosin II dependent and independent pools. In MDCK cells, the myosin II dependent pool is revealed only when cell-cell contacts are under high tension  supplemented with 10% fetal bovine serum, penicillin, streptomycin, and kanamycin. MDCK cells stably expressing GFP-vinculin was previously described . PrimaryCanine vinculin-specific oligonucleotide sequences  were inserted into the pSUPER.gfp/neo expression vector (Oligoengine), and transfected into MDCK cells using Lipofectamine 2000 (Invitrogen). Stable vinculin knockdown cells were subcloned and screened for low vinculin levels using Western blot . The total number of subclones was 25 (shRNA1), 19 (shRNA2), and 16 (shRNA3) clones. Stable cell lines were maintained with 100 μg/ml G418 treatment.Cells were imaged in a 37°C temperature-controlled chamber using a Zeiss AxioObserver equipped with a Yokogawa spinning disk confocal system, 40× and 63× objectives, 488 and 561 nm solid-state lasers, and a Cool SNAP HQ camera. The microscope system was controlled by Slidebook software (Intelligent Imaging Innovations).A photo-ablation system (Intelligent Imaging Innovation) was used to ablate a single cell within a cell monolayer. The subsequent wound area was imaged every minute for 1 hr and the area of the wound opening was quantified using ImageJ. All images were acquired and generated using the same exposure, laser power, and gain. Wound size measurements within the first 10 min following cell ablation were used to analyze the initial velocity of wound closure.5 cells/ml medium. 25 μl of cell suspension was seeded onto glass-bottom dishes, inverted upside-down, and incubated at 37°C. Cell suspensions were then triturated through a pipette tip 30 times and the cluster sizes were quantified using ImageJ.Cells were suspended at a density of 2.5 × 10MDCK cells were seeded onto collagen-coated coverslips. Cells were fixed in 3% para-formaldehyde containing 0.3% TritonX-100 for 10 min and blocked with 1% BSA containing 0.3% TritonX-100. Following primary antibody incubation, immuno-labeled cells were detected with AlexaFluor 488-conjugated secondary antibodies (Molecular Probes). Cells were counter-stained with AlexaFluor 568-phalloidin (Molecular Probes) to detect F-actin. Fluorescence intensities were quantified using ImageJ. All images were acquired and generated using the same exposure and laser power. Fluorescence intensities were measured at cell-cell junctions by obtaining selection areas specific to F-actin staining at cell-cell contacts. The same selection areas obtained with F-actin images were overlaid onto corresponding vinculin images to obtain vinculin intensities along the cell-cell contacts  and vinculin knockdown (KD) cells using vinculin (vin) and tubulin (tub) antibodies. Three independent shRNA sequences were used (see Methods for detail). Figure S2. Vinculin recruitment to focal adhesions occurs with F-actin stabilization in MDCK cells. Confluent MDCK cell monolayers on collagen-coated coverslips were incubated with media containing 200 nM jasplakinolide (Jas) or media alone (Control), fixed, and immune-stained for vinculin and F-actin  at focal adhesions. The same exposure and laser power were used to acquire and generate the images. Scale bar 10 μm. Figure S3. Fluorescence intensity analysis of vinculin and actin at cell-cell contacts. Selection areas for fluorescence intensity for analysis were generated using F-actin  staining at cell-cell contacts. Selection areas were then overlaid onto vinculin images to measure vinculin fluorescence intensities at cell-cell contacts.Click here for file


Calcitonin gene-related peptide (CGRP) is a potent vasodilator and sensory neuropeptide implicated in the pathophysiology of migraine headache. CGRP receptor (CGRP-R) antagonists, including telcagepant, have shown clinical efficacy in treating migraine. CGRP-Rs are expressed in the CNS, particularly in the brainstem and cerebellum as well as in the periphery on vascular smooth muscle cells. To investigate whether central CGRP-Rs were likely to be involved in the anti-migraine effects of CGRP-R antagonists we examined central CGRP-R occupancy (CGRP RO) at an efficacious dose of telcagepant in healthy volunteers and in migraineurs during ictal and interictal periods using the novel PET tracer [11C]MK-4232. CGRP RO was evaluated in healthy subjects (n=3) at the lowest clinically efficacious dose  of telcagepant ~2h after dosing, coinciding with the time point of efficacy evaluation in clinical migraine studies. PET imaging showed only low CGRP RO (4% - 10%) which is within test-retest variability, suggesting that central activity is not a pre-requisite for anti-migraine efficacy of CGRP-R antagonists. A supratherapeutic telcagepant dose  produced only moderate  CGRP RO at ~Tmax (3h after administration). Subsequently, the possibility that brain penetration of telcagepant, a P-gp brain efflux pump substrate, may be increased in migraine patients or during migraine by blood brain barrier opening, was investigated by PET studies in the ictal and interictal periods in migraineurs (n=3) ~2 h after telcagepant dosing . Comparison of [11C]MK-4232 PET study results from ictal and interictal periods in migraineurs to healthy volunteers, suggests similar low RO and no significant differences between states. In conclusion, PET studies with the CGRP-R PET tracer [11C]MK-4232 after therapeutic doses of telcagepant in healthy volunteers and migraineurs suggest that central antagonism of CGRP-R is not necessary for therapeutic efficacy in migraine pain relief and that migraine pain is therefore at least in part peripheral in origin.


Various physicochemical properties of mPEG-PLGA-PLL NPs, including morphology, size, surface charge, siRNA encapsulation efficiency, and in vitro release profile of siRNA from NPs, were characterized by scanning electron microscope, particle size and zeta potential analyzer, and high performance liquid chromatography. The levels of siRNA uptake and targeted gene inhibition were detected in human lung cancer SPC-A1-GFP cells stably expressing green fluorescent protein. Examination of the cultured SPC-A1-GFP cells with fluorescent microscope and flow cytometry showed NPs loading Cy3-labeled siRNA had much higher intracellular siRNA delivery efficiencies than siRNA alone and Lipofectamine-siRNA complexes. The gene silencing efficiency of mPEG-PLGA-PLL NPs was higher than that of commercially available transfecting agent Lipofectamine while showing no cytotoxicity. Thus, the current study demonstrates that biodegradable NPs of mPEG-PLGA-PLL triblock copolymers can be potentially applied as novel non-viral vectors for improving siRNA delivery and gene silencing.Degradation of mRNA by RNA interference is one of the most powerful and specific mechanisms for gene silencing. However, insufficient cellular uptake and poor stability have limited its usefulness. Here, we report efficient delivery of siRNA via the use of biodegradable nanoparticles (NPs) made from monomethoxypoly(ethylene glycol)-poly(lactic- Then, the MTT containing medium was aspirated, and the formazan crystals formed by the living cells were dissolved in 150 μL DMSO. The absorbance was measured spectrophotometrically in an ELISA plate reader at 570 nm. Untreated cells were taken as control with 100% viability and cells without the addition of MTT were used as blank to calibrate the spectrophotometer to zero absorbance. The relative cell viability (%) compared to control cells was calculated by [abs]sample/[abs]control × 100.The cell viability in the different treatment groups and control group was evaluated by MTT method. SPC-A1-GFP cells were seeded onto 96-well plates at a density of 1 × 10P < 0.05. Statistical analyses were performed with a software package .Data were expressed as the means and standard deviation (mean ± SD). Independent samples t-test was used to determine the significance of the difference between two groups. Analysis of variance (ANOVA) was used to test for significance by multiple comparisons. Differences were considered significant at In conclusion, development of safe and efficient non-viral vectors has received tremendous attention in the hope of finding a substitute for viral vectors. The novel biodegradable mPEG-PLGA-PLL NPs could successfully transfer siRNA into lung cancer cells, and produced higher gene inhibition efficiency than commercially available transfecting agent Lipofectamine, while showing no cytotoxicity. The results of this study suggest that biodegradable NPs of mPEG-PLGA-PLL triblock copolymers can be potentially applied as novel non-viral vectors for improving siRNA delivery and gene silencing. Our future study will focus on the surface-modification of mPEG-PLGA-PLL triblock copolymers with various kinds of ligands to achieve active targeting to tissues and cells, thereby building the “perfect vector” for systemic gene therapy against cancer.


Although the mechanisms by which the HTLV-I Tax oncoprotein activates the two NF-κB signaling pathways have been well studied, there is still no convincing evidence demonstrating a functional role for NF-κB in the Tax-mediated tumorigenesis or HTLV-I-mediated adult T-cell leukemia/lymphoma (ATL). Moreover, how Tax is negatively regulated by cellular factors has not yet been explored. Using various transgenic mice and cells deficient in different key IKK/NF-κB signaling components, we have defined the differential but cooperative roles of the canonical and noncanonical NF-κB pathways in Tax-mediated tumorigenesis. One function of non-canonical NF-κB activation is to repress expression of the wwox tumor suppressor gene, which in turn facilitates Tax-induced canonical NF-κB activation. Mechanistic studies indicate that WWOX blocks Tax-induced IKKalpha recruitment and subsequent RelA phosphorylation. Furthermore, we have identified PDLIM2, an essential terminator of canonical NF-κB activation, as a negative regulator of Tax. PDLIM2 directly binds to and shuttles Tax from its activation sites to the nuclear matrix for ubiquitination-mediated degradation, thereby suppressing the tumorigenicities of HTLV-I- or Tax-transduced cells. Interestingly, PDLIM2 expression is epigenetically downregulated in HTLV-I-transformed T cells and primary ATL cells. These studies suggest that the counterbalance between PDLIM2 and HTLV-I may determine the ATL leukemogenesis and provide the first line of genetic evidence demonstrating the functional significance of NF-κB in Tax-mediated tumorigenesis. These studies also define, for the first time, a delicate cooperation of the two pro-oncogenic NF-κB pathways in tumorigenesis and a functional role of the WWOX tumor suppressor in NF-κB regulation and viral tumorigenesis.


Increased cancer risk is associated with select dietary factors. Dietary lifestyles can alter systemic acid-base balance over time. Acidogenic diets, which are typically high in animal protein and salt and low in fruits and vegetables, can lead to a sub-clinical or low-grade state of metabolic acidosis. The relationship between diet and cancer risk prompts questions about the role of acidosis in the initiation and progression of cancer. Cancer is triggered by genetic and epigenetic perturbations in the normal cell, but it has become clear that microenvironmental and systemic factors exert modifying effects on cancer cell development. While there are no studies showing a direct link between diet-induced acidosis and cancer, acid-base disequilibrium has been shown to modulate molecular activity including adrenal glucocorticoid, insulin growth factor (IGF-1), and adipocyte cytokine signaling, dysregulated cellular metabolism, and osteoclast activation, which may serve as intermediary or downstream effectors of carcinogenesis or tumor promotion. In short, diet-induced acidosis may influence molecular activities at the cellular level that promote carcinogenesis or tumor progression. This review defines the relationship between dietary lifestyle and acid-base balance and discusses the potential consequences of diet-induced acidosis and cancer occurrence or progression. Dieta+ and lactic acid production [Acidity is a well known factor associated with cancer. Lower pH levels in the extracellular space promote the invasive and metastatic potential of cancer cells -14. Extroduction . This phoduction -18. The oduction . Cationioduction ,18. Otheoduction ,20. Convoduction ,22.+ surplus and chronically lower levels of serum bicarbonate if compensatory processes become less efficient and are unresolved by dietary adjustments. Potential long-term effects of acidogenic diets are further compounded by the reduction of renal function typically from ageing [Acidogenic dietary intake such as high protein consumption can have an immediate effect on increasing net acid production while low protein lacto-vegetarian consumption can result in significantly reduced net acid excretion ,24. Shorm ageing ,25-28.+ perturbations, typically resulting in blood pH below 7.35 [+ accumulation from chronic diet-induced acidosis can be stored at the cellular level if it does not play a role in lowering blood pH or is compensated by competent renal or respiratory function. Studies of the impact of clinical metabolic acidosis on biological systems may still be informative towards understanding the effects of diet-induced acidosis because they examine how acid-base disequilibrium causes physiological stress and influences molecular pathways active in disease processes [Blood pH from prolonged or chronic acidogenic diets is reported to be near the lower physiological range (7.36-7.38) rather than the higher end (7.42-7.44). Specifically, persistent acidogenic diets have the potential to cause small decreases in blood pH and plasma bicarbonate, but not beyond the normal physiological range. This condition is described as ‘diet-induced’, ‘low-grade’, or ‘chronic metabolic acidosis’ -30 or solow 7.35 . The patlow 7.35 , while trocesses .It is generally understood that the cancer condition evolves from genetic and epigenetic changes in the normal cell. Both microenvironmental and systemic factors exert selective pressures that aid in the initiation or aggravation of tumors. Acid-base disequilibrium is considered a type of systemic stress. With the understanding that long-term acidogenic diets potentially exert chronic physiological stress, the question proposed here is: Can diet-induced acidosis increase cancer risk or promote existing tumors?3- levels are low the kidneys upregulate glutaminase activity and trigger cortisol production [Acid-base balance in the body influences adrenal hormone production of cortisol. When bicarbonate [HCOoduction -37. Studoduction . In healoduction ,40.3- levels increased while serum pH remained stable [The converse to these findings is shown in a study designed to neutralize the acidogenic effect of the ‘Western’ diet, characterized by a high consumption of meat, salt, sugar and fat, and proportionately lower intake of fruit, vegetables, and whole grains. The relationship to cortisol levels and acid-base status were examined in six healthy men and three women measuring serum and urine cortisol concentrations along with cortisol metabolite levels (tetrahydrocortisone and tetrahydrocortisol) in the urine of individuals with sodium and potassium chloride replaced with equimolar amounts of sodium and potassium bicarbonate in an otherwise similar diet under “metabolic ward conditions”. Within 24 hours, urinary and plasma cortisol and corresponding metabolites were significantly lower, signaling lower cortisol production and activity. Urinary pH and serum [NaHCOd stable .Not all studies report a positive correlation between high protein, potentially acidogenic diets, and cortisol levels. These studies did not assess acid-base balance in their experimental populations so it is difficult to confirm if these studies are directly comparable to findings linking acidogenic dietary intake and increased cortisol production. It is likely that factors such as gender and body mass index are relevant inconsistencies between various reports -44.Many of these studies suggest there may be a role for diet-induced acidosis in modulating systemic cortisol levels, and that neutralization of acid loading through alkalinization may reduce cortisol levels. Moreover, most of the studies evaluating the role of acidogenic intake on cortisol demonstrate that the interventions had an acute and dose-dependent effect on cortisol levels, suggesting a direct or closely linked dynamic between acid-base status and system cortisol levels. Finally, the studies show that diet-induced acidosis is mild and subsequent induction of cortisol activity, although higher in serum concentration, is sub-clinical and within the normal serum range . If therThere is no clear mechanism linking cortisol bioactivity directly with carcinogenesis, but studies have reported that cortisol signaling may exert biological influence on existing tumors. Androgen-independent prostate tumors expressing high levels of androgen receptor can be stimulated by cortisol and its metabolite cortisone, resulting in growth promotion and proliferation. The mechanism for this interaction is made possible due to a mutation in the androgen receptor that favors ‘promiscuous’ binding of additional signaling molecules like glucocorticoids . In someCortisol activates the tryptophan metabolism pathway which is carried out by rate-limiting enzymes of tryptophan catabolism, 2,3-dioxygenase (TDO) and indoleamine 2,3-dioxygenase (IDO). Cortisol directly stimulates TDO activation and may augment IDO activity indirectly through inflammatory cytokine signaling such as interferon gamma ,50. ExceThe product of TDO and IDO activity, kynurenine, has several roles in promoting tumorigenesis. Kynurenine inhibits the activation of effector T-cells when tryptophan levels are low. Incapacitating effector T-cell function is suggested as an important component in increasing vulnerability to tumor development -53. TrypUpregulated cortisol bioactivity driven by diet-induced acidosis may be a factor in metabolic syndrome by promoting insulin resistance. Chronic hyperglucocorticoidism upregulates visceral obesity while reducing insulin sensitivity mainly in visceral adipocytes which appear to be more responsive to cortisol than subcutaneous adipocytes due to higher expression levels of glucocorticoid receptors ,59. ViscGlucocorticoid signaling promotes insulin resistance through other signaling pathways as well. Insulin stimulated glucose transporter-4 (GLUT-4) translocation to the cell surface of adipose tissue is suppressed by glucocorticoids. Cortisol directly inhibits insulin secretion from pancreatic beta cells. Finally, cortisol can reduce insulin mediated vasodilation of endothelial cells, and suppresses peripheral insulin driven glucose uptake -70.Acidosis associated insulin resistance through cortisol activity may result in compensatory pancreatic insulin secretion and higher levels of circulating insulin in the serum, a condition known as hyperinsulinemia. Epidemiology studies have shown a positive correlation between circulating insulin levels and increased risk and pathogenesis of colorectal and pancreatic cancers -76, cancInsulin is a pleiotropic hormone with both mitogenic and metabolic properties. It binds with the highest affinity to its own receptor and with lower affinity to the insulin growth factor-1 (IGF-1) receptor. The insulin receptor exists in two isoforms, IR-A and IR-B. IR-A is expressed at lower levels than IR-B, but has greater mitogenic activity when stimulated by insulin. Additionally, both insulin receptor isoforms can form heterodimeric complexes with the IGF-1 receptor. The IR-A/IGF hybrid receptor is expressed in all human tissues and binds to insulin with high affinity . Activat4Cl) causes a significant reduction in serum IGF-1 levels [Studies examining the relationship between diet-induced acidosis and insulin growth factor (IGF-1) levels have varied outcomes. Acute induction of systemic acidosis appears to reduce serum IGF-1 levels. Short-term (5-7 days) induction of metabolic acidosis in healthy male subjects using ammonium chloride . The study also reported that even while protein consumption increased IGF-1 serum levels, there was an age dependent decline in IGF-1 levels overall [High protein consumption over long-term periods (months to years), which promotes greater net acid production and subsequent latent or low-grade metabolic acidosis, appears to have the opposite outcome from short term studies on IGF-1 levels. Studies conducted for 12 weeks or longer revealed a strong correlation between increased dietary protein and higher serum IGF-1 levels, suggesting at least long-term dietary habits, not short-term perturbations, significantly impact IGF-1 serum concentrations -99. Anot overall . The fin overall ,89.IGF-1 binding to the insulin receptor has been shown to inhibit apoptosis and increase target cell proliferation, thus linking its signaling activity to the risk of different forms of cancer -103. SevLeptin is an adipocyte derived hormone cytokine that plays a role in regulating body weight and energy balance in the hypothalamus . Metabolin vitro, and animal investigations in that it reported the protein-rich diet was associated with significantly lower levels of serum leptin than in individuals on the vegetarian diet, independent of age [th quintile of the study population [Studies comparing serum leptin between healthy individuals consuming acidogenic type diets and those consuming more alkaline types of diets present mixed and variable findings. A study in men and women compared serum leptin levels between a group of 279 people consuming a diet rich in fish and a group of 329 people consuming a strictly vegetarian diet. Fish consumption is a high net acid producing diet . Both grt of age . Howeverpulation . Other spulation ,118. ThePhysiological acidosis may indirectly influence leptin activity through cortisol signaling in obesity which is a condition predicted to be associated with dysregulated acid-base balance . As discin vitro studies have demonstrated that leptin is associated with breast cancer, prostate cancer, gynecological cancers, gastrointestinal cancers, and leukemia [Elevated plasma leptin levels in obesity may contribute to cancer incidence . Leptin leukemia -133. Lepleukemia -138. Lepleukemia ,140. Lepleukemia ,142. Othleukemia -145, apoleukemia , and celleukemia .4Cl), resulting in reduced serum bicarbonate and subsequent reduction in adiponectin mRNA and serum protein adiponectin. This was further confirmed in cultured adipocytes where acidosis inhibited gene transcription of adiponectin, suggesting a pH sensing mechanism at the cellular level may influence the regulation of adiponectin production [Acid-base balance may play a role in modulating serum levels of adipokine hormone adiponectin. Adiponectin regulates multiple metabolic processes and is expressed exclusively in mature adipocytes and circulates in the plasma . Numerouoduction .Low serum adiponectin levels are considered to be permissive for development of cancer ,150. RedIn addition to its interference with proliferative signaling, adiponectin mediates its regulatory effects through two receptors, AdipoR1 and AdipoR2 . Signali+ accumulates persistently in the mitochondrial matrix without contributing to ATP production. This dynamic is theorized to inhibit mitochondrial energy production (MEP) through inhibition of the TCA cycle. MEP inhibition results in the diversion of electrons away from completion of the electron transport chain and toward the reduction of oxygen (O2) into reactive oxygen species (ROS) such as free radical oxygen species or peroxides [A very recent discussion about the role of diet-induced acidosis and pathophysiology introduces the hypothesis that persistent acidogenic or ‘Western’ diets lead to latent or low-grade metabolic acidosis, subsequent acid-base balance disequilibrium, and production of lactic acid at the cellular level. These events appear to be critical upstream precursors to a host of ill-conditions, diseases, and ageing. The premise further explains that increased [Heroxides ,157. As The oxidative stress generated by ROS has multiple effects causing damage to cellular and organelle membranes, sulphydryl groups in proteins, and cross-linking or fragmenting ribonucleoproteins and DNA. DNA mutagenesis through persistent oxidative stress is generally accepted as a major mechanism behind carcinogenesis and cancer progression . Oxidati3- concentrations may only partially account for neutralization of acidity, and may be supplemented further by alkaline stores from the soft tissue and bone [In vitro findings demonstrating the mechanisms of excess [H+ on bone tissue is the most reliable evidence currently driving the concept of compensatory buffering through acidosis-induced bone resorption. In cultured osteoclasts, lower pH conditions induce the breaking up of mineralized bone tissue matrix [3- deficiency may be sufficient to acidify media and promote net [H+ influx into bone [2+ efflux from bone [Although not fully understood, the long-term effect of diet-induced acidosis is considered to have an impact on bone osteoclasts . Serum [and bone . Osteocland bone ,170. In e matrix -175. Bicnto bone , and approm bone .Stimulation of osteoclastic resorption by diet-induced acidosis is mediated through receptor activator of NFкB ligand (RANKL) signaling . RANKL s2+ stores in osteoclasts increase intracellular localization of nuclear transcription factor NFATc1 through calcineurin signaling. The ovarian cancer G-protein coupled proton-sensing receptor (OGR1), which is induced during osteoclast differentiation, is thought of as the primary mediator between acidosis and NFATc1 activation. Calcineurin signaling is not required however, to maintain NFATc1 activation under extracellular acidic conditions and NFATc1 activity is reversed by extracellular alkaline conditions, suggesting that acidosis directly prevents NFATc1 inactivation by kinases [One of the strongest RANK-stimulated transcription factors in osteoclasts is the nuclear factor of activated T-cells (NFATc1) protein ,186. Onc kinases . NFATc1  kinases  and has  kinases -193. Alt+ produced from protein metabolism [This work examines the potential for cancer risk or tumor promoting consequences of diet-induced acidosis. Although protein is a major factor involved in promoting endogenous acid production, it should be made clear that attenuation of protein consumption is not a recommended dietary strategy for attaining improved acid-base balance. There is scientific evidence supporting the concept that appropriate alkali supplementation in the form of fruits and vegetables serves aptly to neutralize excess [Htabolism ,194. Thetabolism  and canctabolism .In conclusion, there are numerous systemic pathways affected by diet-induced acidosis that may be cancer promoting, but a causal role is poorly defined. Moreover, the contribution of diet-induced acidosis in driving carcinogenesis would be difficult to measure especially since the effects appear to accumulate for a long period of time. Nonetheless, exploring the role of dietary induced acidosis involvement in molecular pathways that promote carcinogenesis will raise new questions and foster ideas to improve our understanding on the role of acid-base balance in human disease.IGF-1: Insulin growth factor; GLUT: Glucose transporter; IDO: Indoleamine 2,3-dioxygenase; TDO: 2,3-dioxygenase; APC: Antigen presenting cell; AHR: Aryl hydrocarbon receptor; ROS: Reactive oxygen species; CKD: Chronic kidney disease; BMI: Body mass index; PDGF-BB: Platelet derived growth factor-BB; OGR1: Ovarian cancer G-protein coupled proton-sensing receptor; NFATc1: Nuclear factor of activated T-cells; AMP-k: Adenosine monophosphate-activated protein kinase; PPARa: Peroxisome proliferator-activated receptor alpha; MEP: Mitochondrial energy production; MAPK: Mitogen-activated protein kinase; 11ßHSD2: 11ß-hydroxysteroid dehydrogenase 2; HB EGF: Heparin-binding epidermal growth factor-like growth factor; RANKL: Receptor activator of NFкB ligand; OGR1: Ovarian cancer G-protein coupled proton-sensing receptor; NFATc1: Nuclear factor of activated T-cells; AMP-k: Adenosine monophosphate-activated protein kinase; PPARa: Peroxisome proliferator-activated receptor alpha; MEP: Mitochondrial energy production; MAPK: Mitogen-activated protein kinase; 11ßHSD2: 11ß-hydroxysteroid dehydrogenase 2; AICR: American Institute for Cancer Research.The author declares that he has no competing interests.IR conceived and drafted the manuscript in its entirety and approves the final manuscript.


Adipose stem cells (ASCs) secrete many trophic factors that can stimulate tissue repair, including angiogenic factors, but little is known about how ASCs and their secreted factors influence cartilage regeneration. Therefore, the aim of this study was to determine the effects ASC-secreted factors have in repairing chondral defects.ASCs isolated from male Sprague Dawley rats were cultured in monolayer or alginate microbeads supplemented with growth (GM) or chondrogenic medium (CM). Subsequent co-culture, conditioned media, and in vivo cartilage defect studies were performed.col2, comp, and runx2. Chondrocytes treated for 12 or 24 hours with conditioned medium from GM-cultured ASCs had reduced sox9, acan, and col2 mRNAs; reduced proliferation and proteoglycan synthesis; and increased apoptosis. ASC-conditioned medium also increased endothelial cell tube lengthening whereas conditioned medium from CM-cultured ASCs had no effect. Treating ASCs with CM reduced or abolished these deleterious effects while adding a neutralizing antibody for VEGF-A eliminated ASC-conditioned medium induced chondrocyte apoptosis and restored proteoglycan synthesis. FGF-2 also mitigated the deleterious effects VEGF-A had on chondrocyte apoptosis and phenotype. When GM-grown ASC pellets were implanted in 1 mm non-critical hyaline cartilage defects in vivo, cartilage regeneration was inhibited as evaluated by radiographic and equilibrium partitioning of an ionic contrast agent via microCT imaging. Histology revealed that defects with GM-cultured ASCs had no tissue ingrowth from the edges of the defect whereas empty defects and defects with CM-grown ASCs had similar amounts of neocartilage formation.ASC monolayers and microbeads cultured in CM had decreased FGF-2 gene expression and VEGF-A secretion compared to ASCs cultured in GM. Chondrocytes co-cultured with GM-cultured ASCs for 7 days had decreased mRNAs for ASCs must be treated to reduce the secretion of VEGF-A and other factors that inhibit cartilage regeneration, which can significantly influence how ASCs are used for repairing hyaline cartilage. Adult stem cells, such as adipose stem cells (ASCs) are an emerging clinical option for treating tissue damage and diseases because of their accessibility and ability to differentiate into multiple cell lineages ,2. More Hyaline cartilage is the most prevalent cartilaginous tissue found in the body, with adult stem cell therapies used to regenerate hyaline cartilage in the knee and trachea of humans ,5 and toAlthough the secretory profile of ASCs for cartilage regeneration has been investigated , an in-dFor each experiment, ASCs were isolated from inguinal fat pads of six 125-g male Sprague Dawley rats  under a protocol approved by the Institutional Animal Care and Use Committee of the Georgia Institute of Technology as described in detail previously . ASCs fr6 cells/mL using a Nisco Encapsulator VAR V1 LIN-0043  as previously described -thymidine incorporation as described previously [3H]-thymidine was added to a final concentration of 0.25 μCi/mL. Radioactivity in trichloroacetic acid-insoluble cell precipitates was measured by liquid scintillation spectroscopy.DNA synthesis was assayed by measuring [eviously . Forty%-35S]-sulfate incorporation as previously described [35S]-sulfate was added to a final concentration of 18 μCi/mL for the final 4 hours of culture. Only [35S]-sulfate incorporation in the monolayer was measured since less than 15% of total radiolabeled proteoglycan production is secreted into the medium [35S]-sulfate incorporation was normalized to protein content measured with a Pierce Macro BCA protein kit .Proteoglycan synthesis was assayed by measuring [escribed . The [35e medium . The [35Alkaline phosphatase-specific activity in chondrocyte lysates was measured as a function of release of p-nitrophenol from p-nitrophenylphosphate as previously described  and norm3H]-thymidine for 4 hours prior to treatment with ASC-conditioned medium. Chondrocytes were lysed and centrifuged at 13,000 g for 15 minutes to separate intact DNA from fragmented DNA as previously described [3H]-thymidine was determined in each fraction to establish the total amount of fragmented DNA.To assess the effects of ASC-secreted factors on DNA fragmentation, confluent chondrocytes were pulsed with -sulfate incorporation, caspase-3 activity, and incorporation of [3H]-thymidine were measured as described above. DMEM + 10% FBS and conditioned medium from ASC monolayers treated with CM served as controls.To determine the effect exogenous VEGF-A and FGF-2 have on chondrocytes, 1 ng/mL and 20 ng/mL of recombinant human VEGF-Aem cells , and bec6 ASCs in size [6 ASCs/pellet and implanted into the defect. Empty defects and autografts (excised cartilage is re-implanted) served as controls. To maintain groups in the defect and to serve as an adhesion barrier, SepraFilm®  was then placed on the dorsal and ventral sides of the defect. Xiphoids were excised 35 days post-surgery and examined as described below.To assess if ASCs would inhibit cartilage regeneration, non-critically sized chondral defects were made in the xiphoids of 125-g male Sprague-Dawley rats as previously described [Radiographic imaging  was performed in the coronal plane at a voltage of 22 mV and exposure time of 16 s to visualize soft tissue penetration as previously described . Four obEquilibrium partitioning of an ionic contrast agent via micro-computed tomography  was used to visualize the distribution of proteoglycans within the xiphoid defects as previously described ,26. XiphAfter scanning, samples were washed in PBS for four hours, fixed in 10% phosphate-buffered formalin for 48 hours, and embedded in paraffin. Serial sections, 7-μm thick, were stained with H&E to highlight cells and extracellular matrix on microscope images .in vitro experiments included six independent cultures per treatment group to ensure sufficient power to detect statistically significant differences. All in vitro experiments were conducted multiple times to validate the observations, but only data from a single representative experiment are shown and are expressed as means ± standard error (SE). A power analysis determined that seven samples per group were needed for the in vivo study based on results from a previous study [P-value was less than 0.05.All us study . Statistfgf2 and vegfa compared to chondrocytes while CM reduced both vegfa and fgf2 to levels similar to that of chondrocytes .ASC-conditioned medium with a goat-anti-rat IgG and FGF-2 neutralizing antibody decreased [% Figure , and inc% Figure  comparedm Figure . As prevScoring of radiographic images showed that all seven defects with autografts had partial or full healing and three of seven empty defects had partial or full healing; however, no more than two of seven defects with ASC pellets had partial healin, whereas four of seven defects with pellets of ASCs treated with CM had partial or full healing Figure . Scores Proteoglycan was visible in EPIC-μCT images of empty defects and defects with pellets of CM-treated ASCs but absent from defects with pellets of GM-cultured ASCs Figure . EPIC-μCIn histological sections of defects with autografts, early-stage cartilage and infiltrating cells were starting to integrate the graft with the surrounding xiphoid Figure . Similarin vivo. Despite these deleterious effects, treating ASCs with chondrogenic medium can reduce the expression and production of VEGF-A, eliminate the damaging effects ASC-secreted factors have on chondrocytes, and prevent the adverse effects ASCs have on regenerating cartilage in vivo.This study is the first to definitively show that ASCs secrete angiogenic factors that are detrimental for chondrocytes and can prevent cartilage regeneration. Specifically, ASCs secrete a relatively large amount of VEGF-A, which can inhibit chondrocyte phenotype and lead to chondrocyte apoptosis. Additionally, ASCs secrete factors that negatively influence chondrocyte proliferation. Finally, ASCs prevent cartilage regeneration in a non-critical, hyaline cartilage defect In addition to chondrogenic medium, microencapsulation had a significant effect on VEGF-A and FGF-2 secretions. Specifically, microencapsulation had the opposite effect of chondrogenic medium and increased the secretion of these angiogenic factors. Hypoxia due to the high cell density within the microbead may have increased VEGF-A and FGF-2 secretions. Hypoxic conditions can increase VEGF-A and FGF-2 secretion from ASCs  since thPrevious studies have extensively studied the secretory profiles of bone marrow-derived mesenchymal stem cells (MSCs) and ASCs, showing that they not only secrete several factors that can facilitate cartilage regeneration ,31, but Although VEGF-A alone did not prevent chondrocyte proliferation, it mitigated the ability of FGF-2 to stimulate chondrocyte proliferation at a high concentration. Additionally, FGF-2 restored proteoglycan production in chondrocytes treated with VEGF-A, and high concentrations of FGF-2 eliminated the apoptotic effect VEGF-A had on chondrocytes. While both FGF-2 and VEGF-A are important facilitators for angiogenesis ,39 and tRUNX2 or increased alkaline phosphatase activity, and cartilage defects with ASC pellets did not have signs of vascularization, hypertrophic differentiation, or bone formation, in vitro conditioned media studies longer than 24 hours or in vivo studies longer than 35 days may have revealed these effects. Additionally, ASCs may secrete trophic factors that prevent hypertrophic differentiation.VEGF enhances catabolic pathways in chondrocytes, and VEGF signaling has been associated with osteophyte formation and progression of osteoarthritis in articular cartilage -46. MoreSOX9 and aggrecan, which are earlier stage markers of chondrogenesis, whereas 7 days of ASC co-culture exerted the greatest effects on mRNA levels corresponding to RUNX2, a later stage chondrogenic marker [In addition to affecting hypertrophic differentiation, the length of exposure to ASC-secreted factors and the time points measured appear to be important variables in regulating chondrocyte phenotype. Twelve hour exposure to ASC-conditioned medium appeared to have the most significant effects on mRNA levels corresponding to c marker . Becausec marker ,19,21. Aet al. showed that co-culturing chondrocytes with MSCs within the same gel reduced hypertrophy and increased compressive moduli, but it appeared that these improvements were independent of long-range MSC-paracrine signaling [et al. suggested that co-culturing ASCs and chondrocytes in the same matrix moderately increased SOX9, aggrecan, and cartilage oligomeric protein mRNA expression when normalized to the initial percentage of chondrocytes, but the findings provided little insight into the role of ASC-secreted factors [in vitro. This absence of improvement over baseline may be due to the short 5-day chondrogenic medium-treatment time as several studies have shown optimal ASC and stem cell chondrogenesis to require at least 2 to 6 weeks of chondrogenic treatment [Several studies have investigated the effects chondrocyte and stem cell co-cultures and chondrocyte-secreted factors have on stem cell differentiation ,52,53, bignaling . Additio factors . Althougreatment ,57. Howein vivo after 35 days. In vitro, ASC monolayers or microbeads cultured in growth medium secreted a large amount of VEGF-A, which caused chondrocyte apoptosis and reduced proteoglycan synthesis after 24 hours of conditioned media treatments. Additionally, these ASCs secreted factors that reduced chondrogenic gene expression and proliferation after 12 and 24 hours of conditioned media treatments respectively. Treating ASCs with chondrogenic medium for 5 days reduced the secretion of VEGF-A, significantly reduced the deleterious effects ASC-secreted factors have on chondrocytes, and eliminated the inhibitory effect ASCs have on cartilage regeneration. Blocking VEGF-A in ASC-conditioned medium eliminated the deleterious effects ASC-secreted factors had on chondrocyte phenotype and apoptosis after 24 hours. Adding a high concentration of FGF-2 eliminated the apoptotic effect VEGF-A had on chondrocytes, and adding both low and high concentrations of FGF-2 eliminated the detrimental effects VEGF-A had on chondrocyte phenotype after 24 hours. These results have significant implications on how ASCs and possibly other stem cell therapies are used for repairing cartilage. Specifically, these therapies must be pre-treated or modified to reduce the inhibitory effects of VEGF-A and other secreted factors on cartilage regeneration.This study showed that ASCs can inhibit cartilage regeneration within a focal hyaline cartilage defect ACAN: aggrecan; ANOVA: analysis of variance; ASCs: adipose stem cells; BMP-6: bone morphogenetic protein-6; cDNA: complementary deoxyribonucleic acid; CM: chondrogenic medium; COL2: type-II collagen; COMP: cartilage oligomeric matrix protein; ELISA: enzyme-linked immunosorbent assay; EPIC-μCT: equilibrium partitioning of an ionic contrast agent via micro-computed tomography; FBS: fetal bovine serum; FGF-2: fibroblast growth factor-2; GM: growth medium; IL: interleukin; MSCs: mesenchymal stem cells; PBS: phosphate-buffered saline; RPS18: ribosomal protein S18; RUNX2: runt-related transcription factor 2; SE: standard error; SOX9: sex determining region Y-box containing gene 9; TGF-β1: transforming growth factor-beta 1; TNF: tumor necrosis factor; VEGF-A: vascular endothelial growth factor-A.CSD Lee, BD Boyan, and Z Schwartz are co-inventors of the cell culture and microbead technologies described in this research, which is licensed to SpherIngenics, Inc. BD Boyan and Z Schwartz are co-founders and own stock in SpherIngenics, Inc. OA Burnsed, V Raghuram, and J Kalisvaart have no competing interests.All authors have read and approved the manuscript for publication. Authors have contributed to the conception and design , collection and assembly of data , data analysis and interpretation , writing , editing , financial and administrative support (BDB), and final approval  associated with this manuscript.


Hypertension puts continuous strain on arteries, resulting in arterial wall alterations such as vessel wall thickening and vessel wall stiffening. Due to the availability of suitable acoustic windows, vessel wall thickness (VWT) is studied traditionally by ultrasound from intima-media thickness in the carotid arteries. Arterial vessel wall stiffness can be expressed by Pulse Wave Velocity (PWV), the propagation speed of the systolic wave front through the aorta. For studying direct associations between PWV and VWT, it is desired to sample VWT in the aorta.To evaluate associations between aortic and carotid wall thickness and aortic PWV and also with age in subjects with and without hypertension using a comprehensive MRI-approach.Fifteen patients  with hypertension defined as systolic blood pressure>140mmHg and/or diastolic blood pressure>90mmHg before antihypertensive medication was instituted, were included. Fifteen age- and gender-matched healthy volunteers without history of cardiovascular disease were included for comparison.2 in-plane resolution, spectral-selective fat suppression and navigator respiratory-compensation (for aorta). Ten slices (aorta) and eight slices (common carotid artery) were analyzed using in-house developed software VesselMass. Luminal and outer wall boundaries were manually segmented. Mean VWT was computed and indexed for Body Surface Area (BSA):PWV-assessment was performed on 1.5T MRI-scanner  according to transit-time method by applying velocity-encoded MRI with high temporal resolution at the aortic arch. At the same day, aortic and carotid vessel wall imaging was performed on 3T MRI-scanner  using three-dimensional (for aorta) and multi-two-dimensional (for carotid artery) dual-inversion black-blood gradient-echo sequences with 0.5×0.5mm∑ [ / total number of slices]/BSA.Hypertensive patients presented with higher PWV and aortic and carotid VWT than healthy volunteers Table . AssociaMRI presents associations between aortic and carotid VWT and aortic PWV, and all parameters are increased in patients with hypertension versus age- and gender-matched healthy volunteers.


MicroRNAs (miRNAs) negatively regulate gene expression and its deregulation is involved in cancer progression. Our aim was to verify which miRNAs may play a role in breast cancer progression, especially metastasis.vs. non metastatic tumors) by hierarchical clustering of breast cancer expression signatures. We performed a second analysis using the ROC curve method that allowed us to identify miRNAs with greater predictive potential of malignancy. Furthermore, by Venn diagram analysis, we were able to identify 8 miRNAs that were differentially expressed in all metastatic tumors of different clinical staging, compared to the primary non-metastatic tumors. SPSS 19 software was used to generate a Kaplan-Meier curve (p<0.05) to estimate the disease-specific survival (considering the specific event such as death by cancer), based on miRNA expression patterns.MicroRNA expression profiles were generated by microarray analysis (Agilent) of 70 samples from primary breast tumors with different clinical staging. Data were analyzed using bioinformatics software , which allowed sample discrimination  for breast cancer, being hsa-let7a, hsa-let7b and hsa-miR-21 under-expressed in the metastatic vs non-metastatic tumors, and four new miRNAs candidates for markers of metastasis , the first two were over-expressed while the latest ones were under-expressed in metastatic vs non-metastatic tumors.We identified two groups of miRNAs whose expression level were associated with worse prognosis. These findings are important for better understanding of the role of miRNAs in breast cancer progression.FAPESP.