Search

The article discusses the Rare Disease Moonshot initiative, which aims to address the gaps and challenges in therapeutic development for the world's rarest conditions. It emphasizes the importance of public-private partnerships and collaboration to accelerate research and deliver safe and effective medical products. The initiative focuses on integrating regulatory science, developing sustainable infrastructures, and de-risking investments to support the development of new treatments and cures for rare and ultra-rare diseases. The authors highlight the need for multistakeholder collaborations and the dissemination of recommendations to maximize the benefits of the initiative for patients with rare diseases. [Extracted from the article]

The tropical cloud forest ecosystem in western equatorial Africa (WEA) is known to be sensitive to the presence of an extensive and persistent low-level stratiform cloud deck during the long dry season from June to September (JJAS). Here, we present a new climatology of the diurnal cycle of the low-level cloud cover from surface synoptic stations over WEA during JJAS 1971-2019. For the period JJAS 2008-19, we also utilized estimates of cloudiness from four satellite products, namely, the Satellite Application Facility on Support to Nowcasting and Very Short Range Forecasting (SAFNWC) cloud classification, the Day and Night Microphysical Schemes (DMS/NMS), and cross sections from CALIPSO and CloudSat (2B-GEOPROF-lidar). A comparison with surface stations reveals that the NMS at night together with SAFNWC at daytime yield the smallest biases. The climatological analysis reveals that low-level clouds persist during the day over the coastal plains and windward side of the low mountain ranges. Conversely, on their leeward sides, i.e., over the plateaus, a decrease of the low-level cloud frequency is observed in the afternoon, together with a change from stratocumulus to cumulus. At night, the low-level cloud deck reforms over this region with the largest cloud occurrence frequencies in the morning. Vertical profiles from 2B-GEOPROF-lidar reveal cloud tops below 3000 m even at daytime. The station data and the suitable satellite products form the basis to better understand the physical processes controlling the clouds and to evaluate cloudiness from reanalyses and models. [ABSTRACT FROM AUTHOR]

Purpose: Despite known high-risk features, accurate identification of patients at high risk of cancer recurrence in colon cancer remains a challenge. As tumour stroma plays an important role in tumour invasion and metastasis, the easy, low-cost and highly reproducible tumour-stroma ratio (TSR) could be a valuable prognostic marker, which is also believed to predict chemo resistance. Methods: Two independent series of patients with colon cancer were selected. TSR was estimated by microscopic analysis of 4 µm haematoxylin and eosin (H&E) stained tissue sections of the primary tumour and the corresponding metastatic lymph nodes. Patients were categorized as TSR-low (≤ 50%) or TSR-high (> 50%). Differences in overall survival and cancer-free survival were analysed by Kaplan–Meier curves and cox-regression analyses. Analyses were conducted for TNM-stage I–II, TNM-stage III and patients with an indication for chemotherapy separately. Results: We found that high TSR was associated with poor cancer-free survival in TNM-stage I–II colon cancer in two independent series, independent of other known high-risk features. This association was also found in TNM-stage III tumours, with an additional prognostic value of TSR in lymph node metastasis to TSR in the primary tumour alone. In addition, high TSR was found to predict chemo resistance in patients receiving adjuvant chemotherapy after surgical resection of a TNM-stage II–III colon tumour. Conclusion: In colon cancer, the TSR of both primary tumour and lymph node metastasis adds significant prognostic value to current pathologic and clinical features used for the identification of patients at high risk of cancer recurrence, and also predicts chemo resistance. [ABSTRACT FROM AUTHOR]

The use of hair as a matrix for the evaluation of chronic ethanol drinking behavior presents the advantage of a longer window of detection and higher specificity when compared to classical biochemical markers. The most recent recommendations the Society of Hair Testing (SOHT) indicate that ethyl palmitate (EtP) hair levels can be used to estimate the ethanol drinking behavior, alternatively to the combined measurement of four main fatty acid ethyl esters. In this study, solid-phase microextraction (SPME) conditions for the extraction of EtP from hair were optimized using response surface analysis, after a Box–Behnken experiment. Analyses were performed by GC-MS. The optimized HS-SPME conditions, using a PDMS-DVB (65 μm) fiber, were pre-adsorption time of 6 min, extraction time of 60 min and incubation temperature of 94°C. The linear range was 0.05 to 3 ng mg− 1 , with accuracy within 95.15–109.91%. Between-assay and within-assay precision were 8.58–12.53 and 6.12–6.82%, respectively. The extraction yield was 61.3–71.9%. The assay was applied to hair specimens obtained from 46 volunteers, all presenting EtP levels within the linear range of the assay. Using a statistically designed experiment, a sensitive SPME-GC-MS assay for the measurement of EtP in hair was developed and validated, requiring only 20 mg of hair. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Copyright of Revue de Médecine Périnatale is the property of John Libbey Eurotext Ltd. and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

Copyright of Canadian Veterinary Journal / Revue Vétérinaire Canadienne is the property of Canadian Veterinary Medical Association and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

Endonucleases of the HUH family are specialized in processing single-stranded DNA in a variety of evolutionarily highly conserved biological processes related to mobile genetic elements. They share a structurally defined catalytic domain for site-specific nicking and strand-transfer reactions, which is often linked to the activities of additional functional domains, contributing to their overall versatility. To assess if these HUH domains could be interchanged, we created a chimeric protein from two distantly related HUH endonucleases, containing the N-terminal HUH domain of the bacterial conjugative relaxase TrwC and the C-terminal DNA helicase domain of the human adeno-associated virus (AAV) replicase and site-specific integrase. The purified chimeric protein retained oligomerization properties and DNA helicase activities similar to Rep68, while its DNA binding specificity and cleaving-joining activity at oriT was similar to TrwC. Interestingly, the chimeric protein could catalyse site-specific integration in bacteria with an efficiency comparable to that of TrwC, while the HUH domain of TrwC alone was unable to catalyze this reaction, implying that the Rep68 C-terminal helicase domain is complementing the TrwC HUH domain to achieve site-specific integration into TrwC targets in bacteria. Our results illustrate how HUH domains could have acquired through evolution other domains in order to attain new roles, contributing to the functional flexibility observed in this protein superfamily. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Copyright of Revue de Médecine Périnatale is the property of John Libbey Eurotext Ltd. and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

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. Declaration of interest: MYR and LLT receive salary support from MolecuLight Inc. as clinical trial managers. No other authors are associated with, receive compensation from, or own shares of the company. This study was sponsored by MolecuLight, Inc. [ABSTRACT FROM AUTHOR]

As their names imply, parvoviruses of the genus Dependovirus rely for their efficient replication on the concurrent presence of a helpervirus, such as herpesvirus, adenovirus, or papilloma virus. Adeno-associated virus 2 (AAV2) is such an example, which in turn can efficiently inhibit the replication of each helpervirus by distinct mechanisms. In a previous study we have shown that expression of the AAV2 rep gene is not compatible with efficient replication of herpes simplex virus 1 (HSV-1). In particular, the combined DNA-binding and ATPase/helicase activities of the Rep68/78 proteins have been shown to exert opposite effects on the replication of AAV2 and HSV-1. While essential for AAV2 DNA replication these protein activities account for the Rep-mediated inhibition of HSV-1 replication. Here, we describe a novel Rep mutant (Rep-D371Y), which displayed an unexpected phenotype. Rep-D371Y did not block HSV-1 replication, but still supported efficient AAV2 replication, at least when a double-stranded AAV2 genome template was used. We also found that the capacity of Rep-D371Y to induce apoptosis and a Rep-specific DNA damage response was significantly reduced compared to wild-type Rep. These findings suggest that AAV2 Rep-helicase subdomains exert diverging activities, which contribute to distinct steps of the AAV2 life cycle. More important, the novel AAV2 mutant Rep-D371Y may allow deciphering yet unsolved activities of the AAV2 Rep proteins such as DNA second-strand synthesis, genomic integration or packaging, which all involve the Rep-helicase activity. [ABSTRACT FROM AUTHOR]

Current passive optical networks (PONs) use on?off keying (OOK) modulation throughout the entire network regardless of actual path losses. Statistics of commercial gigabit passive optical network (GPON) deployments show significant path loss differences among the optical network units (ONUs). Flexible 4- and 8-level pulse amplitude modulation (4/8-PAM) allocation for the better-situated ONUs can significantly increase the PON?s aggregated data rate. By keeping to intensity modulation direct detection (IM-DD) modulation and a constant symbol rate, the introduction of adaptive, multilevel PAM does not require expensive optics investments; additionally, 8-PAM is made feasible with zero-overhead data-aided equalization. Two scenarios for distributing the extra capacity are explored: first, controlling the dynamic bandwidth allocation of the time division multiplexed PON (TDM-PON) to provide equal time-length slots to both 4/8-PAM ONUs and OOK ONUs, thereby providing maximum aggregated capacity increase; and second, setting up the dynamic bandwidth allocation to provide equal capacity slots to the various ONUs so the entire ONU population in the same PON can benefit from the extra capacity. Both scenarios result in a larger aggregated capacity of the PON and provide additional options for the network operator during the network design phase. [ABSTRACT FROM PUBLISHER]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

In this work we describe a technique developed to improve medium-term prediction methods of monthly smoothed sunspot numbers. Each month, the predictions are updated using the last available observations (see the monthly output in real time at ). The improvement of the predictions is provided by applying an adaptive Kalman filter to the medium-term predictions obtained by any other method, using the six-monthly mean values of sunspot numbers covering the six months between the last available value of the 13-month running mean (the starting point for the predictions) and the 'current time' ( i.e. now). Our technique provides an effective estimate of the sunspot index at the current time. This estimate becomes the new starting point for the updated prediction that is shifted six months ahead in comparison with the last available 13-month running mean, and it provides an increase of prediction accuracy. Our technique has been tested on three medium-term prediction methods that are currently in real-time operation: The McNish-Lincoln method (NGDC), the standard method (SIDC), and the combined method (SIDC). With our technique, the prediction accuracy for the McNish-Lincoln method is increased by 17 - 30%, for the standard method by 5 - 21% and for the combined method by 6 - 57%. [ABSTRACT FROM AUTHOR]

Background: Tick-borne encephalitis (TBE) is caused by a RNA-virus and is in about 50% of cases characterized by a biphasic clinical course in adults. Different clinical syndromes have been described, including meningitis, meningoencephalitis, meningoencephalomyelitis and meningoencephaloradiculomyelitis. The latter seems to be the most disabling and severe form of TBE virus infection. Methods: Here we report five cases with meningoencephaloradiculomyelitis. Only in three patients a tick prick was remembered. Results: Only two patients could be weaned successfully from assisted ventilation; only one patient was able to return to self-dependent life without nursing support. The youngest patient in this case series showed the most favourable outcome. Conclusions: Polyradiculopathy and/or myelopathy as verified by electrophysiological examination within 4 weeks from symptom onset were indicative of a more severe disease course and a greater likelihood of moderate to serious sequelae even after long-term rehabilitation. Older age at symptom onset seems to be associated with a less favourable outcome. Because of frequent long-term hospitalization with immobilization and invasive ventilation, secondary complications, such as ventilation associated pulmonary infections and decubiti, must be avoided. [ABSTRACT FROM AUTHOR]

Recent developments in space instrumentation for solar observations and increased telemetry have necessitated the creation of advanced pattern recognition tools for different classes of solar events. The Extreme Ultraviolet Imaging Telescope (EIT) onboard the SOHO spacecraft has uncovered a new class of eruptive events on the solar disk, which are often identified as signatures of the initiation of coronal mass ejections (CMEs). The development of an automatic detection tool of these signatures is an important task. The Novel EIT Wave Machine Observing (NEMO) code () is an operational tool that automatically detects EUV waves using a sequence of EUV images. NEMO applies techniques based on the general statistical properties of the underlying physical mechanisms of eruptive events. Originally, the technique was applied to images taken with the EIT telescope. In this work, the most recent updates of the NEMO code are presented. These updates include calculations of the area of the dimming region, a novel clustering technique for the extraction of dimming regions, and new criteria to identify eruptive dimmings based on their complex characteristics. The efficiency of NEMO has been significantly increased and now permits the extraction of dimming regions observed near the solar limb and also the detection of small-scale events. Furthermore, the catalogs of solar eruptive events based on the updated NEMO may include a larger number of physical parameters associated with the dimming regions. [ABSTRACT FROM AUTHOR]

It has been proposed that the million-degree temperature of the corona is due to the combined effect of barely detectable energy releases, called nanoflares, that occur throughout the solar atmosphere. Unfortunately, the nanoflare density and brightness implied by this hypothesis means that conclusive verification is beyond present observational abilities. Nevertheless, we investigate the plausibility of the nanoflare hypothesis by constructing a magnetohydrodynamic (MHD) model that can derive the energy of a nanoflare from the nature of an ideal kink instability. The set of energy-releasing instabilities is captured by an instability threshold for linear kink modes. Each point on the threshold is associated with a unique energy release; thus we can predict a distribution of nanoflare energies. When the linear instability threshold is crossed, the instability enters a nonlinear phase as it is driven by current sheet reconnection. As the ensuing flare erupts and declines, the field transitions to a lower energy state, which is modelled by relaxation theory; i.e., helicity is conserved and the ratio of current to field becomes invariant within the loop. We apply the model so that all the loops within an ensemble achieve instability followed by energy-releasing relaxation. The result is a nanoflare energy distribution. Furthermore, we produce different distributions by varying the loop aspect ratio, the nature of the path to instability taken by each loop and also the level of radial expansion that may accompany loop relaxation. The heating rate obtained is just sufficient for coronal heating. In addition, we also show that kink instability cannot be associated with a critical magnetic twist value for every point along the instability threshold. [ABSTRACT FROM AUTHOR]

Development of rational therapeutic treatments of Alzheimer disease (AD) requires the elucidation of the etiopathogenic mechanisms of neurofibrillary degeneration and β-amyloidosis, the two hallmarks of this disease. Here we show, employing an adeno-associated virus serotype 1 (AAVl)-induced expression of the C-terminal fragment (I2CTF of Due to image rights restrictions, multiple line equation(s) cannot be graphically displayed, also called SET, in rat brain, decrease in protein phosphatase 2A (PP2A) activity, abnormal hyperphosphorylation of tau, and neurodegeneration; littermates treated identically but with vector only, i.e., AAV1-enhanced green fluorescent protein (GFP), served as a control. Furthermore, there was an increase in the level of activated glycogen synthase kinase-3β and enhanced expression of intraneuronal Aβ in AAV1-I2CTF animals. Morris water maze behavioral test revealed that infection with AAV1-I2CTF induced spatial reference memory and memory consolidation deficits and a decrease in the brain level of pSerl33-CREB. These findings suggest a novel etiopathogenic mechanism of AD, which is initiated by the cleavage of Due to image rights restrictions, multiple line equation(s) cannot be graphically displayed, producing I2CTF, describe a novel disease-relevant nontransgenic animal model of AD. [ABSTRACT FROM AUTHOR]

Viral vectors derived from adeno-associated viruses (AAVs) are widely used for gene transfer both in vitro and in vivo. The increasing use of AAV as a gene transfer vector, as well as recently shown immunological complications in clinical trials, highlight the necessity to define the specific activity of vector preparations beyond current standards. In this report, we determined the infectious, physical and genome-containing particle titers of several wild-type AAV type 2 (wtAAV2) and recombinant AAV type 2 (rAAV2) preparations that were produced and purified by standard methods. We found that the infectivity of wtAAV2 approaches a physical-to-infectious particle ratio of one. This near-perfect physical-to-infectious particle ratio defines a ‘ceiling’ for the theoretically achievable quality of recombinant AAV vectors. In comparison, for rAAV2, only approximately 50 out of 100 viral particles contained a genome and, more strikingly, only approximately 1 of the 100 viral particles was infectious. Our findings suggest that current strategies for rAAV vector design, production and/or purification should be amenable to improvements. Ultimately, this could result in the generation of near-perfect vector particles, a prospect with significant implications for gene therapy. [ABSTRACT FROM AUTHOR]

Rep68 is a multifunctional protein of the adeno-associated virus (AAV), a parvovirus that is mostly known for its promise as a gene therapy vector. In addition to its role as initiator in viral DNA replication, Rep68 is essential for site-specific integration of the AAV genome into human chromosome 19. Rep68 is a member of the superfamily 3 (SF3) helicases, along with the well-studied initiator proteins simian virus 40 large T antigen (SV40-LTag) and bovine papillomavirus (BPV) E1. Structurally, SF3 helicases share two domains, a DNA origin interaction domain (OID) and an AAA+ motor domain. The AAA+ motor domain is also a structural feature of cellular initiators and it functions as a platform for initiator oligomerization. Here, we studied Rep68 oligomerization in vitro in the presence of different DNA substrates using a variety of biophysical techniques and cryo-EM. We found that a dsDNA region of the AAV origin promotes the formation of a complex containing five Rep68 subunits. Interestingly, non-specific ssDNA promotes the formation of a double-ring Rep68, a known structure formed by the LTag and E1 initiator proteins. The Rep68 ring symmetry is 8-fold, thus differing from the hexameric rings formed by the other SF3 helicases. However, similiar to LTag and E1, Rep68 rings are oriented head-to-head, suggesting that DNA unwinding by the complex proceeds bidirectionally. This novel Rep68 quaternary structure requires both the DNA binding and AAA+ domains, indicating cooperativity between these regions during oligomerization in vitro. Our study clearly demonstrates that Rep68 can oligomerize through two distinct oligomerization pathways, which depend on both the DNA structure and cooperativity of Rep68 domains. These findings provide insight into the dynamics and oligomeric adaptability of Rep68 and serve as a step towards understanding the role of this multifunctional protein during AAV DNA replication and site-specific integration. [ABSTRACT FROM AUTHOR]

Copyright of Revue de Médecine Périnatale is the property of John Libbey Eurotext Ltd. and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)