Comparative structural, biophysical, and receptor binding study of true type and wild type AAV2.

Adeno-associated viruses (AAV) are utilized as gene transfer vectors in the treatment of monogenic disorders. A variant, rationally engineered based on natural AAV2 isolates, designated AAV-True Type (AAV-TT), is highly neurotropic compared to wild type AAV2 in vivo, and vectors based on it, are currently being evaluated for central nervous system applications. AAV-TT differs from AAV2 by 14 amino acids, including R585S and R588T, two residues previously shown to be essential for heparan sulfate binding of AAV2. The capsid structures of AAV-TT and AAV2 visualized by cryo-electron microscopy at 3.4 and 3.0 Å resolution, respectively, highlighted structural perturbations at specific amino acid differences. Differential scanning fluorimetry (DSF) performed at different pH conditions demonstrated that the melting temperature (Tm) of AAV2 was consistently ∼5 °C lower than AAV-TT, but both showed maximal stability at pH 5.5, corresponding to the pH in the late endosome, proposed as required for VP1u externalization to facilitate endosomal escape. Reintroduction of arginines at positions 585 and 588 in AAV-TT caused a reduction in Tm, demonstrating that the lack of basic amino acids at these positions are associated with capsid stability. These results provide structural and thermal annotation of AAV2/AAV-TT residue differences, that account for divergent cell binding, transduction, antigenic reactivity, and transduction of permissive tissues between the two viruses. Specifically, these data indicate that AAV-TT may not utilize a glycan receptor mediated pathway to enter cells and may have lower antigenic properties as compared to AAV2.

Adeno-associated virus Rep proteins antagonize phosphatase PP1 to counteract KAP1 repression of the latent viral genome.

Adeno-associated virus (AAV) is a small human Dependovirus whose low immunogenicity and capacity for long-term persistence have led to its widespread use as vector for gene therapy. Despite great recent successes in AAV-based gene therapy, further improvements in vector technology may be hindered by an inadequate understanding of various aspects of basic AAV biology. AAV is unique in that its replication is largely dependent on a helper virus and cellular factors. In the absence of helper virus coinfection, wild-type AAV establishes latency through mechanisms that are not yet fully understood. Challenging the currently held model for AAV latency, we show here that the corepressor Krüppel-associated box domain-associated protein 1 (KAP1) binds the latent AAV2 genome at the rep ORF, leading to trimethylation of AAV2-associated histone 3 lysine 9 and that the inactivation of KAP1 repression is necessary for AAV2 reactivation and replication. We identify a viral mechanism for the counteraction of KAP1 in which interference with the KAP1 phosphatase protein phosphatase 1 (PP1) by the AAV2 Rep proteins mediates enhanced phosphorylation of KAP1-S824 and thus relief from KAP1 repression. Furthermore, we show that this phenomenon involves recruitment of the NIPP1 (nuclear inhibitor of PP1)-PP1α holoenzyme to KAP1 in a manner dependent upon the NIPP1 FHA domain, identifying NIPP1 as an interaction partner for KAP1 and shedding light on the mechanism through which PP1 regulates cellular KAP1 activity.

Vector-mediated l-3,4-dihydroxyphenylalanine delivery reverses motor impairments in a primate model of Parkinson's disease.

Ever since its introduction 40 years ago l-3,4-dihydroxyphenylalanine (l-DOPA) therapy has retained its role as the leading standard medication for patients with Parkinson's disease. With time, however, the shortcomings of oral l-DOPA treatment have become apparent, particularly the motor fluctuations and troublesome dyskinetic side effects. These side effects, which are caused by the excessive swings in striatal dopamine caused by intermittent oral delivery, can be avoided by delivering l-DOPA in a more continuous manner. Local gene delivery of the l-DOPA synthesizing enzymes, tyrosine hydroxylase and guanosine-tri-phosphate-cyclohydrolase-1, offers a new approach to a more refined dopaminergic therapy where l-DOPA is delivered continuously at the site where it is needed i.e. the striatum. In this study we have explored the therapeutic efficacy of adeno-associated viral vector-mediated l-DOPA delivery to the putamen in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-treated rhesus monkeys, the standard non-human primate model of Parkinson's disease. Viral vector delivery of the two enzymes, tyrosine hydroxylase and guanosine-5'-tri-phosphate-cyclohydrolase-1, bilaterally into the dopamine-depleted putamen, induced a significant, dose-dependent improvement of motor behaviour up to a level identical to that obtained with the optimal dose of peripheral l-DOPA. Importantly, this improvement in motor function was obtained without any adverse dyskinetic effects. These results provide proof-of-principle for continuous vector-mediated l-DOPA synthesis as a novel therapeutic strategy for Parkinson's disease. The constant, local supply of l-DOPA obtained with this approach holds promise as an efficient one-time treatment that can provide long-lasting clinical improvement and at the same time prevent the appearance of motor fluctuations and dyskinetic side effects associated with standard oral dopaminergic medication.

A Comparison of Case Volume in Craniofacial Surgery by Plastic Surgery Residency Training Model.

BACKGROUND: Plastic surgeons in the United States are trained under 2 residency training models: integrated and independent. This study analyzes the variability of craniofacial surgery cases performed both between and within training models. METHODS: Case volume data from national data reports of 5 plastic surgery resident cohorts were analyzed (2011-2015). Craniofacial surgery case volumes across 4 major categories and 23 subcategories were compared between training models via t tests. Differences in intramodel variability were compared with F tests. Fold differences were calculated between mean case volumes and minimum requirements in craniofacial surgery. RESULTS: A total of 526 independent/combined (64%) and 292 integrated (36%) plastic surgery residents were included. Integrated residents reported more cases classified as congenital defect (118.8 ± 49.3 vs 110.3 ± 42.9, P = 0.013), neoplasm (202.0 ± 79.7 vs 163.2 ± 60.8, P < 0.001), and trauma (149.0 ± 61.8 vs 127.0 ± 52.0, P < 0.001), but not aesthetic (122.3 ± 68.6 vs 116.5 ± 50.5, P = 0.201). Integrated residents reported more case volume in 12 case subcategories, whereas independent/combined residents reported more cases in 3 case subcategories. Integrated residents had greater intramodel variability in 12 case subcategories, whereas independent/combined residents had greater intramodel variability in 2 case subcategories. Fold differences between mean case volumes and minimum requirements ranged from 1.8 times to 6.0 times. CONCLUSIONS: Integrated residents tended to report significantly more craniofacial surgery cases and exhibit greater intrapathway variability. More research is needed to understand the impact of disparate case volume on core competency training in craniofacial surgery during plastic surgery residency.

Single-Port Laparoscopic Treatment and Outcome of Dogs with Ovarian Remnant Syndrome: 13 Cases (2010-2018).

Ovarian remnant syndrome (ORS) is a condition resulting from incomplete removal of ovarian tissue during ovariectomy and/or ovariohysterectomy. Single-port laparoscopy (SPL) is an alternative to ventral midline laparotomy for treatment of ORS. Medical records of 13 client-owned female dogs who underwent SPL for the treatment of ORS were retrospectively reviewed to evaluate surgical technique and outcome. Dogs who had undergone a previous attempt at open ovariectomy or ovariohysterectomy were included. Major intraoperative complications did not occur and conversion to open laparotomy was not required. In 1 dog, an SPL + 1 technique was used, in which an additional port was placed cranial to the single-port device to aid in dissection and tissue manipulation. Median surgical time was 45 min (range, 30-90 min). Clinical signs related to estrus had resolved in 11 of 13 dogs with a median follow-up time of 18 mo. Two of 13 dogs were lost to follow-up at 3 mo postoperatively; however, signs of estrus had resolved at time of last follow-up. SPL treatment for ORS was feasible and successful in this cohort of dogs. Reduced surgical time was found in this study compared with previous reports investigating multiple-port laparoscopic treatment of ORS.

A novel adeno-associated virus capsid with enhanced neurotropism corrects a lysosomal transmembrane enzyme deficiency.

Recombinant adeno-associated viruses (AAVs) are popular in vivo gene transfer vehicles. However, vector doses needed to achieve therapeutic effect are high and some target tissues in the central nervous system remain difficult to transduce. Gene therapy trials using AAV for the treatment of neurological disorders have seldom led to demonstrated clinical efficacy. Important contributing factors are low transduction rates and inefficient distribution of the vector. To overcome these hurdles, a variety of capsid engineering methods have been utilized to generate capsids with improved transduction properties. Here we describe an alternative approach to capsid engineering, which draws on the natural evolution of the virus and aims to yield capsids that are better suited to infect human tissues. We generated an AAV capsid to include amino acids that are conserved among natural AAV2 isolates and tested its biodistribution properties in mice and rats. Intriguingly, this novel variant, AAV-TT, demonstrates strong neurotropism in rodents and displays significantly improved distribution throughout the central nervous system as compared to AAV2. Additionally, sub-retinal injections in mice revealed markedly enhanced transduction of photoreceptor cells when compared to AAV2. Importantly, AAV-TT exceeds the distribution abilities of benchmark neurotropic serotypes AAV9 and AAVrh10 in the central nervous system of mice, and is the only virus, when administered at low dose, that is able to correct the neurological phenotype in a mouse model of mucopolysaccharidosis IIIC, a transmembrane enzyme lysosomal storage disease, which requires delivery to every cell for biochemical correction. These data represent unprecedented correction of a lysosomal transmembrane enzyme deficiency in mice and suggest that AAV-TT-based gene therapies may be suitable for treatment of human neurological diseases such as mucopolysaccharidosis IIIC, which is characterized by global neuropathology.

InterPrEP: internet-based pre-exposure prophylaxis with generic tenofovir disoproxil fumarate/emtrictabine in London - analysis of pharmacokinetics, safety and outcomes.

OBJECTIVES: The National Health Service in England (NHS England) does not provide pre-exposure prophylaxis (PrEP) against HIV, forcing people to purchase generic versions on the internet. However, there are concerns about the authenticity of medicines purchased online. We established an innovative service offering plasma tenofovir (TFV) and emcitrabine (FTC) therapeutic drug monitoring for people buying generic PrEP online, to ensure that drug concentrations in vivo were consistent with those of propriety brands and previously published data. METHODS: TFV/FTC concentrations were measured by ultra-performance liquid chromatography ultraviolet detection. Evaluation of renal function and testing for HIV, hepatitis B virus (HBV) and hepatitis C virus (HCV) were also carried out, at baseline and every 3-6 months, with risk reduction advice. RESULTS: A total of 293 individuals presented having purchased PrEP on the internet: 85% were white, 84% were taking daily PrEP, and 16% were event-driven. Most were on generic TFV disoproxil fumarate (TDF)/FTC from Cipla Ltd. Median (range) TFV and FTC plasma concentrations were 104 (21-597) ng/mL and 140 (17-1876) ng/mL, respectively. All concentrations were above our established plasma TFV and FTC targets, based on previously published data. Renal function was normal in all evaluable individuals and no new cases of HIV, HBV or HCV infection were seen. CONCLUSIONS: In a population at high risk of HIV acquisition, who cannot yet access PrEP on the NHS, concentrations of TFV and FTC in generic formulations purchased over the internet were similar to (or slightly higher than) those measured in phase I studies with the original formulation from Gilead (Truvada™), which has demonstrated high levels of protection against HIV infection in previous PrEP clinical trials.

Massive uterine lipoleiomyoma and leiomyoma in a miniature poodle bitch.

A 15-year-old, intact, female miniature poodle was presented for further evaluation of a large abdominal mass. Computed tomography was conducted to determine the origin of the mass and 2 large uterine masses were discovered. Ovariohysterectomy was performed and histopathological evaluation revealed a massive uterine lipoleiomyoma (27 × 17 × 15 cm), the largest recorded in the veterinary literature, and a smaller leiomyoma (7 × 5 × 4 cm).

Lipoléiomyome utérin massif et léiomyome chez une chienne Caniche miniature. Une chienne Caniche miniature intacte âgée de 15 ans a été présentée pour une évaluation approfondie d'une grosse masse abdominale. Une analyse par tomodensitométrie a été réalisée afin de déterminer l'origine de la masse et deux grandes masses utérines ont été découvertes. L'ovariohystérectomie a été réalisée et l'évaluation histopathologique a révélé un lipoléimomyome utérin massif (27 × 17 × 15 cm), le plus gros jamais consigné dans la littérature vétérinaire et un plus petit léiomyome (7 × 5 × 4 cm).(Traduit par Isabelle Vallières).

Identification of a Functionally Relevant Adeno-Associated Virus Rep68 Oligomeric Interface.

UNLABELLED: The life cycle of the human parvovirus adeno-associated virus (AAV) is orchestrated by four Rep proteins. The large Rep proteins, Rep78 and Rep68, are remarkably multifunctional and display a range of biochemical activities, including DNA binding, nicking, and unwinding. Functionally, Rep78 and Rep68 are involved in transcriptional regulation, DNA replication, and genomic integration. Structurally, the Rep proteins share an AAA(+) domain characteristic of superfamily 3 helicases, with the large Rep proteins additionally containing an N-terminal origin-binding domain (OBD) that specifically binds and nicks DNA. The combination of these domains, coupled with dynamic oligomerization properties, is the basis for the remarkable multifunctionality displayed by Rep68 and Rep78 during the AAV life cycle. In this report, we describe an oligomeric interface formed by Rep68 and demonstrate how disruption of this interface has drastic effects on both the oligomerization and functionality of the Rep proteins. Our results support a role for the four-helix bundle in the helicase domain of Rep68 as a bona fide oligomerization domain (OD). We have identified key residues in the OD that are critical for the stabilization of the Rep68-Rep68 interface; mutation of these key residues disrupts the enzymatic activities of Rep68, including DNA binding and nicking, and compromises viral DNA replication and transcriptional regulation of the viral promoters. Taken together, our data contribute to our understanding of the dynamic and substrate-responsive Rep78/68 oligomerization that is instrumental in the regulation of the DNA transitions that take place during the AAV life cycle. IMPORTANCE: The limited genome size of small viruses has driven the evolution of highly multifunctional proteins that integrate different domains and enzymatic activities within a single polypeptide. The Rep68 protein from adeno-associated virus (AAV) combines a DNA binding and endonuclease domain with a helicase-ATPase domain, which together support DNA replication, transcriptional regulation, and site-specific integration. The coordination of the enzymatic activities of Rep68 remains poorly understood; however, Rep68 oligomerization and Rep68-DNA interactions have been suggested to play a crucial role. We investigated the determinants of Rep68 oligomerization and identified a hydrophobic interface necessary for Rep68 activity during the AAV life cycle. Our results provide new insights into the molecular mechanisms underlying the regulation of the versatile Rep proteins. Efficient production of AAV-based gene therapy vectors requires optimal Rep expression levels, and studies such as the one presented here could contribute to further optimization of AAV production schemes.

Point-of-care fluorescence imaging predicts the presence of pathogenic bacteria in wounds: a clinical study.

OBJECTIVE: Bacteria in chronic wounds are invisible to the naked eye and can lead to delayed wound healing. Point-of-care bacterial fluorescence imaging illuminates a wound with 405nm light, triggering bacteria to produce red fluorescence and enabling real-time bacterial localisation. Prospective, single-blind clinical trials (clinicaltrials.gov #NCT02682069, #NCT03091361) were conducted to determine the positive predictive value (PPV) of this red fluorescence for detecting bacteria in chronic wounds. METHOD: Lower limb chronic wounds were imaged for bacterial fluorescence using the MolecuLight i:X imaging device. Regions positive for red fluorescence were discretely sampled using either biopsy or curettage to correlate red fluorescence signals to bacterial presence and analysed via gold standard quantitative polymerase chain reaction (qPCR) or via semi-quantitative culture analysis respectively. RESULTS: A total of 60 lower limb chronic wounds were imaged. Quantitative PCR analysis of wound tissue biopsies obtained from regions of red fluorescence yielded a PPV of 100%. Total bacterial load in these areas was ≥104 CFU/g. Semi-quantitative culture analysis of curettage scrapings from regions of red fluorescence yielded a PPV of 100%, with predominately moderate or heavy bacterial growth. There were nine distinct bacterial species detected, all common pathogens in chronic wounds. Staphylococcus aureus was the most prevalent species. CONCLUSION: Bacterial fluorescence image-guided curettage or biopsy sampling positively predicts bacterial presence in wounds at potentially harmful levels, entirely eliminating the risk of false negative sampling. Fluorescence imaging of wounds offers clinicians real-time information on a wound's bacterial burden, insight which can influence treatment decisions at the point-of care.

Presence of a trs-Like Motif Promotes Rep-Mediated Wild-Type Adeno-Associated Virus Type 2 Integration.

High-throughput integration site (IS) analysis of wild-type adeno-associated virus type 2 (wtAAV2) in human dermal fibroblasts (HDFs) and HeLa cells revealed that juxtaposition of a Rep binding site (RBS) and terminal resolution site (trs)-like motif leads to a 4-fold-increased probability of wtAAV integration. Electrophoretic mobility shift assays (EMSAs) confirmed binding of Rep to off-target RBSs. For the first time, we show Rep protein off-target nicking activity, highlighting the importance of the nicking substrate for Rep-mediated integration.

Expression of CD105 on expanded mesenchymal stem cells does not predict their chondrogenic potential.

OBJECTIVE: Total bone marrow-derived mesenchymal stem cell (BMSC) populations differ in their potential to undergo chondrogenesis, with individual BMSCs differing in their chondrogenic capacity. The aim of this study was to explore the use of CD105 as a marker to isolate a chondrogenic subpopulation of BMSCs from the total, heterogeneous population. DESIGN: BMSCs were isolated from patients undergoing total hip replacement and following expansion (Passage 1-Passage 5), CD105 expression was investigated by FACS analysis. FACS was also used to sort BMSCs based on the presence of CD105 (CD105(+)/CD105(-)) or their amount of CD105 expression (CD105(Bright)/CD105(Dim)). After 3 or 5 weeks of differentiation, chondrogenic potential was determined by thionine staining for glycosaminoglycan (GAG) content and by detection of collagen type II using immunohistochemistry. RESULTS: Expanded total BMSC populations were composed almost exclusively of CD105(+) cells, the percentage of which did not correlate to subsequent chondrogenic potential; chondrogenic potential was observed to diminish with culture although CD105 expression remained stable. Similarly, differences in chondrogenic potential were observed between donors despite similar levels of CD105(+) BMSCs. Comparison of CD105(Bright) and CD105(Dim) BMSCs did not reveal a subpopulation with superior chondrogenic potential. CONCLUSIONS: Chondrogenic potential of BMSCs is often linked to CD105 expression. This study demonstrates that CD105 expression on culture expanded BMSC populations does not associate with a chondroprogenitor phenotype and CD105 should not be pursued as a marker to obtain a chondroprogenitor population from BMSCs.

Structural Studies of AAV2 Rep68 Reveal a Partially Structured Linker and Compact Domain Conformation.

Adeno-associated virus (AAV) nonstructural proteins Rep78 and Rep68 carry out all DNA transactions that regulate the AAV life cycle. They share two multifunctional domains: an N-terminal origin binding/nicking domain (OBD) from the HUH superfamily and a SF3 helicase domain. A short linker of ∼20 amino acids that is critical for oligomerization and function connects the two domains. Although X-ray structures of the AAV5 OBD and AAV2 helicase domains have been determined, information about the full-length protein and linker conformation is not known. This article presents the solution structure of AAV2 Rep68 using small-angle X-ray scattering (SAXS). We first determined the X-ray structures of the minimal AAV2 Rep68 OBD and of the OBD with the linker region. These X-ray structures reveal novel features that include a long C-terminal α-helix that protrudes from the core of the protein at a 45° angle and a partially structured linker. SAXS studies corroborate that the linker is not extended, and we show that a proline residue in the linker is critical for Rep68 oligomerization and function. SAXS-based rigid-body modeling of Rep68 confirms these observations, showing a compact arrangement of the two domains in which they acquire a conformation that positions key residues in all domains on one face of the protein, poised to interact with DNA.

Adeno-associated virus Rep represses the human integration site promoter by two pathways that are similar to those required for the regulation of the viral p5 promoter.

UNLABELLED: Adeno-associated virus serotype 2 (AAV2) can efficiently replicate in cells that have been infected with helper viruses, such as adenovirus or herpesvirus. However, in the absence of helper virus infection, AAV2 establishes latency by integrating its genome site specifically into PPP1R12C, a gene located on chromosome 19. This integration target site falls into one of the most gene-dense regions of the human genome, thus inviting the question as to whether the virus has evolved mechanisms to control this complex transcriptional environment in order to facilitate integration, maintain an apparently innocuous latency, and/or establish conditions that are conducive to the rescue of the integrated viral genome. The viral replication (Rep) proteins control and direct every known aspect of the viral life cycle and have been shown to tightly control all AAV2 promoters. In addition, a number of heterologous promoters are repressed by the AAV2 Rep proteins. Here, we demonstrate that Rep proteins efficiently repress expression from the target site PPP1R12C promoter. We find evidence that this repression employs mechanisms similar to those described for Rep-mediated AAV2 p5 promoter regulation. Furthermore, we show that the repression of the cellular target site promoter is based on two distinct mechanisms, one relying on the presence of a functional Rep binding motif within the 5' untranslated region (UTR) of PPP1R12C, whereas the second pathway requires only an intact nucleoside triphosphate (NTP) binding site within the Rep proteins, indicating the possible reliance of this pathway on interactions of the Rep proteins with cellular proteins that mediate or regulate cellular transcription. IMPORTANCE: The observation that repression of transcription from the adeno-associated virus serotype 2 (AAV2) p5 and integration target site promoters is mediated by shared mechanisms highlights the possible coevolution of virus and host and could lead to the identification of host factors that the virus exploits to navigate its life cycle.

Oligomeric properties of adeno-associated virus Rep68 reflect its multifunctionality.

The adeno-associated virus (AAV) encodes four regulatory proteins called Rep. The large AAV Rep proteins Rep68 and Rep78 are essential factors required in almost every step of the viral life cycle. Structurally, they share two domains: a modified version of the AAA(+) domain that characterizes the SF3 family of helicases and an N-terminal domain that binds DNA specifically. The combination of these two domains imparts extraordinary multifunctionality to work as initiators of DNA replication and regulators of transcription, in addition to their essential role during site-specific integration. Although most members of the SF3 family form hexameric rings in vitro, the oligomeric nature of Rep68 is unclear due to its propensity to aggregate in solution. We report here a comprehensive study to determine the oligomeric character of Rep68 using a combination of methods that includes sedimentation velocity ultracentrifugation, electron microscopy, and hydrodynamic modeling. We have determined that residue Cys151 induces Rep68 to aggregate in vitro. We show that Rep68 displays a concentration-dependent dynamic oligomeric behavior characterized by the presence of two populations: one with monomers and dimers in slow equilibrium and a second one consisting of a mixture of multiple-ring structures of seven and eight members. The presence of either ATP or ADP induces formation of larger complexes formed by the stacking of multiple rings. Taken together, our results support the idea of a Rep68 molecule that exhibits the flexible oligomeric behavior needed to perform the wide range of functions occurring during the AAV life cycle.

The interdomain linker of AAV-2 Rep68 is an integral part of its oligomerization domain: role of a conserved SF3 helicase residue in oligomerization.

The four Rep proteins of adeno-associated virus (AAV) orchestrate all aspects of its viral life cycle, including transcription regulation, DNA replication, virus assembly, and site-specific integration of the viral genome into the human chromosome 19. All Rep proteins share a central SF3 superfamily helicase domain. In other SF3 members this domain is sufficient to induce oligomerization. However, the helicase domain in AAV Rep proteins (i.e. Rep40/Rep52) as shown by its monomeric characteristic, is not able to mediate stable oligomerization. This observation led us to hypothesize the existence of an as yet undefined structural determinant that regulates Rep oligomerization. In this document, we described a detailed structural comparison between the helicase domains of AAV-2 Rep proteins and those of the other SF3 members. This analysis shows a major structural difference residing in the small oligomerization sub-domain (OD) of Rep helicase domain. In addition, secondary structure prediction of the linker connecting the helicase domain to the origin-binding domain (OBD) indicates the potential to form α-helices. We demonstrate that mutant Rep40 constructs containing different lengths of the linker are able to form dimers, and in the presence of ATP/ADP, larger oligomers. We further identified an aromatic linker residue (Y224) that is critical for oligomerization, establishing it as a conserved signature motif in SF3 helicases. Mutation of this residue critically affects oligomerization as well as completely abolishes the ability to produce infectious virus. Taken together, our data support a model where the linker residues preceding the helicase domain fold into an α-helix that becomes an integral part of the helicase domain and is critical for the oligomerization and function of Rep68/78 proteins through cooperative interaction with the OBD and helicase domains.

The diagnostic value of one-step nucleic acid amplification (OSNA) for sentinel lymph nodes in colon cancer patients.

BACKGROUND: Lymph node status in colon cancer is critical for prognosis estimation and treatment allocation. The purpose of this study was to compare the performance of one-step nucleic acid amplification (OSNA) through detection of cytokeratin 19 mRNA levels with routine pathological examination (RP) and multilevel fine pathological examination (FP) in sentinel lymph nodes (SLN), detected using the ex vivo SLN mapping (SLNM) procedure, in presurgically defined nonmetastatic colon cancer patients. METHODS: In this prospective study, 325 SLNs of 128 patients from the Jeroen Bosch Hospital in 's-Hertogenbosch and the Leiden University Medical Center were investigated by RP (H&E), FP (H&E and Keratin Pan immunohistochemical staining), and OSNA. The SLNs were harvested by the SLNM procedure, using Patent blue or Indocyanine green. SLNs were divided and separate parts were used for RP, FP, and the OSNA assay. RESULTS: The diagnostic value of OSNA was 82.1 and 100 % for both FP and combined method (OSNA and FP) compared with RP. An upstaging rate of 20.2 % was obtained with the use of OSNA only and 36.4 % with the use of FP only. An upstaging rate of 46.5 % was obtained by combining the two methods together. CONCLUSIONS: OSNA and FP appeared to be promising tools for the detection of lymph node micro- and macrometastases in SLNs after SLNM. The performances of OSNA and FP in this study were superior to RP. Because OSNA allows analysis of the whole lymph node, sampling bias can be avoided. OSNA therefore may improve tumor staging.

Human cardiovascular progenitor cells develop from a KDR+ embryonic-stem-cell-derived population.

The functional heart is comprised of distinct mesoderm-derived lineages including cardiomyocytes, endothelial cells and vascular smooth muscle cells. Studies in the mouse embryo and the mouse embryonic stem cell differentiation model have provided evidence indicating that these three lineages develop from a common Flk-1(+) (kinase insert domain protein receptor, also known as Kdr) cardiovascular progenitor that represents one of the earliest stages in mesoderm specification to the cardiovascular lineages. To determine whether a comparable progenitor is present during human cardiogenesis, we analysed the development of the cardiovascular lineages in human embryonic stem cell differentiation cultures. Here we show that after induction with combinations of activin A, bone morphogenetic protein 4 (BMP4), basic fibroblast growth factor (bFGF, also known as FGF2), vascular endothelial growth factor (VEGF, also known as VEGFA) and dickkopf homolog 1 (DKK1) in serum-free media, human embryonic-stem-cell-derived embryoid bodies generate a KDR(low)/C-KIT(CD117)(neg) population that displays cardiac, endothelial and vascular smooth muscle potential in vitro and, after transplantation, in vivo. When plated in monolayer cultures, these KDR(low)/C-KIT(neg) cells differentiate to generate populations consisting of greater than 50% contracting cardiomyocytes. Populations derived from the KDR(low)/C-KIT(neg) fraction give rise to colonies that contain all three lineages when plated in methylcellulose cultures. Results from limiting dilution studies and cell-mixing experiments support the interpretation that these colonies are clones, indicating that they develop from a cardiovascular colony-forming cell. Together, these findings identify a human cardiovascular progenitor that defines one of the earliest stages of human cardiac development.

Novel Blood-Based Biomarkers and Disease Modifying Therapies for Alzheimer's Disease. Are We Ready for the New Era?

Recent positive trials for novel disease modifying therapies of anti-amyloid monoclonal antibodies represent a paradigm shift in the prevention and management of Alzheimer's disease, a relentlessly progressive and debilitating disease of old age. The reported efficacy of these new agents when given early in the disease trajectory is dependent on an early and accurate disease diagnosis, which is currently based on cerebrospinal fluid tests or/and neuro-imaging studies such as positron emission tomography. These confirmatory tests provide in vivo evidence of the pathological signature of Alzheimer's disease, of increased cerebral amyloid and tau burden and neurodegeneration. The emergence of blood-based biomarkers represents another breakthrough, offering a less invasive and scalable diagnostic tool that could be applied in both primary and specialist care settings, potentially revolutionizing Alzheimer's disease clinical pathways. However, healthcare systems face challenges in the adoption of these new technologies and therapies due to diagnostic and treatment capacity constraints, as well as financial and infrastructure requirements.

Evaluation of oxidative stress markers in ethanol users.

Ethanol is a central nervous system depressant that is widely consumed worldwide. When consumed chronically, it may have several consequences to the organism, such as oxidative stress. Ethanol metabolism increases the production of oxidant molecules and its consumption may cause changes in enzymatic and non-enzymatic systems that maintain cellular homeostasis. The activity of endogenous enzymes and lipid peroxidation are altered in alcohol consumers. Therefore, this study aimed to evaluate oxidative stress parameters in ethanol users compared to a control group. For that, the activity of the enzymes superoxide dismutase, catalase, and glutathione peroxidase, the ferric reducing/antioxidant power (FRAP), and malondialdehyde were evaluated. The influence of the amount of ethanol consumed on the analyzed parameters was also verified. The group of alcohol users consisted of 52 volunteers, 85% male and 15% female, with a mean age of 41±13 years. The control group consisted of 50 non-drinkers, 40% male and 60% female, with a mean age of 50±10 years. There was a significant difference in superoxide dismutase (P<0.001) and malondialdehyde (P=0.007) measurements between groups, as both parameters were increased in the group of ethanol users. Because of the higher amount of ethanol consumed, there was an increase of the catalase activity parameters and gradual reduction of FRAP. Thus, the ethanol-consuming participants were most likely under oxidative stress.