Adeno-associated virus genome quantification with amplification-free CRISPR-Cas12a.

Efficient manufacturing of recombinant Adeno-Associated Viral (rAAV) vectors to meet rising clinical demand remains a major hurdle. One of the most significant challenges is the generation of large amounts of empty capsids without the therapeutic genome. There is no standardized analytical method to accurately quantify the viral genes, and subsequently the empty-to-full ratio, making the manufacturing challenges even more complex. We propose the use of CRISPR diagnostics (CRISPR-Dx) as a robust and rapid approach to determine AAV genome titers. We designed and developed the CRISPR-AAV Evaluation (CRAAVE) assay to maximize sensitivity, minimize time-to-result, and provide a potentially universal design for quantifying multiple transgene constructs encapsidated within different AAV serotypes. We also demonstrate an on-chip CRAAVE assay with lyophilized reagents to minimize end user assay input. The CRAAVE assay was able to detect AAV titers as low as 7e7 vg/mL with high precision (<3% error) in quantifying unknown AAV titers when compared with conventional quantitative PCR (qPCR) method. The assay only requires 30 min of assay time, shortening the analytical workflow drastically. Our results suggest CRISPR-Dx could be a promising tool for efficient rAAV genome titer quantification and has the potential to revolutionize biomanufacturing process analytical technology (PAT).

Seizures in Children with Influenza during the 2022-2023 Winter Season, a Case Series.

Influenza is a viral infection presenting with general symptoms such as fever, headache, fatigue, and involvement of airways or the gastrointestinal tract. The nervous system may be involved, but less frequently. These neurological complications remain challenging to diagnose; moreover, no guidelines for management and treatment exist. Therefore, when presenting with neurological symptoms, patients undergo invasive diagnostic procedures and empirical treatments before making the correct diagnosis. During the winter of 2022-2023, four children between nine months and nine years of age were admitted to the Lausanne University Hospital, Switzerland, complaining of influenza and neurological complications. This report presents the symptoms of neurological manifestation and the treatment management of the four patients. All the legally authorized representatives gave their written informed consent before study inclusion.

Indian Hedgehog release from TNF-activated renal epithelia drives local and remote organ fibrosis.

Progressive fibrosis is a feature of aging and chronic tissue injury in multiple organs, including the kidney and heart. Glioma-associated oncogene 1 expressing (Gli1+) cells are a major source of activated fibroblasts in multiple organs, but the links between injury, inflammation, and Gli1+ cell expansion and tissue fibrosis remain incompletely understood. We demonstrated that leukocyte-derived tumor necrosis factor (TNF) promoted Gli1+ cell proliferation and cardiorenal fibrosis through induction and release of Indian Hedgehog (IHH) from renal epithelial cells. Using single-cell-resolution transcriptomic analysis, we identified an "inflammatory" proximal tubular epithelial (iPT) population contributing to TNF- and nuclear factor κB (NF-κB)-induced IHH production in vivo. TNF-induced Ubiquitin D (Ubd) expression was observed in human proximal tubular cells in vitro and during murine and human renal disease and aging. Studies using pharmacological and conditional genetic ablation of TNF-induced IHH signaling revealed that IHH activated canonical Hedgehog signaling in Gli1+ cells, which led to their activation, proliferation, and fibrosis within the injured and aging kidney and heart. These changes were inhibited in mice by Ihh deletion in Pax8-expressing cells or by pharmacological blockade of TNF, NF-κB, or Gli1 signaling. Increased amounts of circulating IHH were associated with loss of renal function and higher rates of cardiovascular disease in patients with chronic kidney disease. Thus, IHH connects leukocyte activation to Gli1+ cell expansion and represents a potential target for therapies to inhibit inflammation-induced fibrosis.

Evaluation of novel chromatographic prototypes for supercoiled plasmid DNA polishing.

Since the world first approved gene therapeutics, nucleic acid-based therapies have gained prominence. Several strategies for DNA-based therapy have been approved, and numerous clinical trials for plasmid DNA (pDNA)-based vaccines are currently in development. Due to the rising interest in pDNA for vaccination and gene therapy, plasmid manufacturing must become more effective. One of the most critical steps is downstream processing, involving isolation and purification procedures. To comply with the regulatory guidelines, pDNA must be available as a highly purified, homogeneous preparation of supercoiled pDNA (sc pDNA). This process undertakes several challenges, primarily due to the diversity of molecules derived from the producer organism. In this study, different resins were tested for the adsorption and selective polishing of sc pDNA. To identify optimal pDNA adsorption conditions, batch and column assays were performed with different resins while promoting electrostatic and hydrophobic interactions. The effect of ionic strength, pH, and contact time were evaluated and optimized. Additionally, static and dynamic binding capacities were determined for the selected resins. Analytical chromatography and agarose gel electrophoresis were used to assess the selectivity of the most promising resins toward sc pDNA isoform. Also, genomic DNA, endotoxins, and proteins were quantified to characterize the final sc pDNA quality. At the same time, the recovery and purity yields were evaluated by quantification of sc pDNA after the purification procedure. Overall, the results of the chromatographic assays using agmatine- and arginine-based resins have shown promising potential for sc pDNA polishing. Both resins demonstrated excellent binding capacity for pDNA, with agmatine outperforming arginine-based resin in terms of capacity. However, arginine-based resin exhibited a superior pDNA recovery yield, reaching a notable 52.2% recovery compared to 10.09% from agmatine. Furthermore, both resins exhibited high relative purity levels above 90% for the sc pDNA. The comprehensive characterization of the recovered sc pDNA also revealed a significant reduction in gDNA levels, reinforcing the potential of these prototypes for obtaining high-quality and pure sc pDNA. These findings highlight the promising applications of both resins in scalable pDNA purification processes for gene therapy and biopharmaceutical applications.

The Role of Ageing and Parenchymal Senescence on Macrophage Function and Fibrosis.

In this review, we examine senescent cells and the overlap between the direct biological impact of senescence and the indirect impact senescence has via its effects on other cell types, particularly the macrophage. The canonical roles of macrophages in cell clearance and in other physiological functions are discussed with reference to their functions in diseases of the kidney and other organs. We also explore the translational potential of different approaches based around the macrophage in future interventions to target senescent cells, with the goal of preventing or reversing pathologies driven or contributed to in part by senescent cell load in vivo.

Cellular senescence inhibits renal regeneration after injury in mice, with senolytic treatment promoting repair.

The ability of the kidney to regenerate successfully after injury is lost with advancing age, chronic kidney disease, and after irradiation. The factors responsible for this reduced regenerative capacity remain incompletely understood, with increasing interest in a potential role for cellular senescence in determining outcomes after injury. Here, we demonstrated correlations between senescent cell load and functional loss in human aging and chronic kidney diseases including radiation nephropathy. We dissected the causative role of senescence in the augmented fibrosis occurring after injury in aged and irradiated murine kidneys. In vitro studies on human proximal tubular epithelial cells and in vivo mouse studies demonstrated that senescent renal epithelial cells produced multiple components of the senescence-associated secretory phenotype including transforming growth factor ß1, induced fibrosis, and inhibited tubular proliferative capacity after injury. Treatment of aged and irradiated mice with the B cell lymphoma 2/w/xL inhibitor ABT-263 reduced senescent cell numbers and restored a regenerative phenotype in the kidneys with increased tubular proliferation, improved function, and reduced fibrosis after subsequent ischemia-reperfusion injury. Senescent cells are key determinants of renal regenerative capacity in mice and represent emerging treatment targets to protect aging and vulnerable kidneys in man.

Cellular Senescence and Senotherapies in the Kidney: Current Evidence and Future Directions.

Cellular senescence refers to a cellular phenotype characterized by an altered transcriptome, pro-inflammatory secretome, and generally irreversible growth arrest. Acutely senescent cells are widely recognized as performing key physiological functions in vivo promoting normal organogenesis, successful wound repair, and cancer defense. In contrast, the accumulation of chronically senescent cells in response to aging, cell stress, genotoxic damage, and other injurious stimuli is increasingly recognized as an important contributor to organ dysfunction, tissue fibrosis, and the more generalized aging phenotype. In this review, we summarize our current knowledge of the role of senescent cells in promoting progressive fibrosis and dysfunction with a particular focus on the kidney and reference to other organ systems. Specific differences between healthy and senescent cells are reviewed along with a summary of several experimental pharmacological approaches to deplete or manipulate senescent cells to preserve organ integrity and function with aging and after injury. Finally, key questions for future research and clinical translation are discussed.

Cellular Senescence in the Kidney.

Senescent cells have undergone permanent growth arrest, adopt an altered secretory phenotype, and accumulate in the kidney and other organs with ageing and injury. Senescence has diverse physiologic roles and experimental studies support its importance in nephrogenesis, successful tissue repair, and in opposing malignant transformation. However, recent murine studies have shown that depletion of chronically senescent cells extends healthy lifespan and delays age-associated disease-implicating senescence and the senescence-associated secretory phenotype as drivers of organ dysfunction. Great interest is therefore focused on the manipulation of senescence as a novel therapeutic target in kidney disease. In this review, we examine current knowledge and areas of ongoing uncertainty regarding senescence in the human kidney and experimental models. We summarize evidence supporting the role of senescence in normal kidney development and homeostasis but also senescence-induced maladaptive repair, renal fibrosis, and transplant failure. Recent studies using senescent cell manipulation and depletion as novel therapies to treat renal disease are discussed, and we explore unanswered questions for future research.

Do evidence summaries increase health policy-makers' use of evidence from systematic reviews? A systematic review.

This review summarizes the evidence from six randomized controlled trials that judged the effectiveness of systematic review summaries on policymakers' decision making, or the most effective ways to present evidence summaries to increase policymakers' use of the evidence. This review included six randomized controlled studies. A randomized controlled study is one in which the participants are divided randomly (by chance) into separate groups to compare different treatments or other interventions. This method of dividing people into groups means that the groups will be similar and that the effects of the treatments they receive will be compared more fairly. At the time the study is done, it is not known which treatment is the better one. The researchers who did these studies invited people from Europe, North America, South America, Africa, and Asia to take part in them. Two studies looked at "policy briefs," one study looked at an "evidence summary," two looked at a "summary of findings table," and one compared a "summary of findings table" to an evidence summary. None of these studies looked at how policymakers directly used evidence from systematic reviews in their decision making, but two studies found that there was little to no difference in how they used the summaries. The studies relied on reports from decision makers. These studies included questions such as, "Is this summary easy to understand?" Some of the studies looked at users' knowledge, understanding, beliefs, or how credible (trustworthy) they believed the summaries to be. There was little to no difference in the studies that looked at these outcomes. Study participants rated the graded entry format higher for usability than the full systematic review. The graded entry format allows the reader to select how much information they want to read. The study participants felt that all evidence summary formats were easier to understand than full systematic reviews. Plain language summary: Policy briefs make systematic reviews easier to understand but little evidence of impact on use of study findings: It is likely that evidence summaries are easier to understand than complete systematic reviews. Whether these summaries increase the use of evidence from systematic reviews in policymaking is not clear.What is this review about?: Systematic reviews are long and technical documents that may be hard for policymakers to use when making decisions. Evidence summaries are short documents that describe research findings in systematic reviews. These summaries may simplify the use of systematic reviews.Other names for evidence reviews are policy briefs, evidence briefs, summaries of findings, or plain language summaries. The goal of this review was to learn whether evidence summaries help policymakers use evidence from systematic reviews. This review also aimed to identify the best ways to present the evidence summary to increase the use of evidence.What are the main findings of this review?: This review included six randomized controlled studies. A randomized controlled study is one in which the participants are divided randomly (by chance) into separate groups to compare different treatments or other interventions. This method of dividing people into groups means that the groups will be similar and that the effects of the treatments they receive will be compared more fairly. At the time the study is done, it is not known which treatment is the better one.The researchers who did these studies invited people from Europe, North America, South America, Africa, and Asia to take part in them. Two studies looked at "policy briefs," one study looked at an "evidence summary," two looked at a "summary of findings table," and one compared a "summary of findings table" to an evidence summary.None of these studies looked at how policymakers directly used evidence from systematic reviews in their decision making, but two studies found that there was little to no difference in how they used the summaries. The studies relied on reports from decision makers. These studies included questions such as, "Is this summary easy to understand?"Some of the studies looked at users' knowledge, understanding, beliefs, or how credible (trustworthy) they believed the summaries to be. There was little to no difference in the studies that looked at these outcomes. Study participants rated the graded entry format higher for usability than the full systematic review. The graded entry format allows the reader to select how much information they want to read.. The study participants felt that all evidence summary formats were easier to understand than full systematic reviews.What do the findings of this review mean?: Our review suggests that evidence summaries help policymakers to better understand the findings presented in systematic reviews. In short, evidence summaries should be developed to make it easier for policymakers to understand the evidence presented in systematic reviews. However, right now there is very little evidence on the best way to present systematic review evidence to policymakers.How up to date is this review?: The authors of this review searched for studies through June 2016. Executive summary/Abstract: Background: Systematic reviews are important for decision makers. They offer many potential benefits but are often written in technical language, are too long, and do not contain contextual details which makes them hard to use for decision-making. Strategies to promote the use of evidence to decision makers are required, and evidence summaries have been suggested as a facilitator. Evidence summaries include policy briefs, briefing papers, briefing notes, evidence briefs, abstracts, summary of findings tables, and plain language summaries. There are many organizations developing and disseminating systematic review evidence summaries for different populations or subsets of decision makers. However, evidence on the usefulness and effectiveness of systematic review summaries is lacking. We present an overview of the available evidence on systematic review evidence summaries.Objectives: This systematic review aimed to 1) assess the effectiveness of evidence summaries on policy-makers' use of the evidence and 2) identify the most effective summary components for increasing policy-makers' use of the evidence.Search methods: We searched several online databases (Medline, EMBASE, CINAHL, Cochrane Central Register of Controlled Trials, Global Health Library, Popline, Africa-wide, Public Affairs Information Services, Worldwide Political Science Abstracts, Web of Science, and DfiD), websites of research groups and organizations which produce evidence summaries, and reference lists of included summaries and related systematic reviews. These databases were searched in March-April, 2016.Selection criteria: Eligible studies included randomised controlled trials (RCTs), non-randomised controlled trials (NRCTs), controlled before-after (CBA) studies, and interrupted time series (ITS) studies. We included studies of policymakers at all levels as well as health system managers. We included studies examining any type of "evidence summary", "policy brief", or other product derived from systematic reviews that presented evidence in a summarized form. These interventions could be compared to active comparators (e.g. other summary formats) or no intervention.The primary outcomes were: 1) use of systematic review summaries decision-making (e.g. self-reported use of the evidence in policy-making, decision-making) and 2) policymaker understanding, knowledge, and/or beliefs (e.g. changes in knowledge scores about the topic included in the summary). We also assessed perceived relevance, credibility, usefulness, understandability, and desirability (e.g. format) of the summaries.Results: Our database search combined with our grey literature search yielded 10,113 references after removal of duplicates. From these, 54 were reviewed in full text and we included 6 studies (reported in 7 papers, 1661 participants) as well as protocols from 2 ongoing studies. Two studies assessed the use of evidence summaries in decision-making and found little to no difference in effect. There was also little to no difference in effect for knowledge, understanding or beliefs (4 studies) and perceived usefulness or usability (3 studies). Summary of Findings tables and graded entry summaries were perceived as slightly easier to understand compared to complete systematic reviews. Two studies assessed formatting changes and found that for Summary of Findings tables, certain elements, such as reporting study event rates and absolute differences were preferred as well as avoiding the use of footnotes. No studies assessed adverse effects. The risks of bias in these studies were mainly assessed as unclear or low however, two studies were assessed as high risk of bias for incomplete outcome data due to very high rates of attrition.Authors' conclusions: Evidence summaries may be easier to understand than complete systematic reviews. However, their ability to increase the use of systematic review evidence in policymaking is unclear.

Do evidence summaries increase health policy-makers use of evidence from systematic reviews?

Systematic reviews are important for decision makers. They offer many potential benefits but are often written in technical language, are too long, and do not contain contextual details which makes them hard to use for decision-making. Strategies to promote the use of evidence to decision makers are required, and evidence summaries have been suggested as a facilitator. Evidence summaries include policy briefs, briefing papers, briefing notes, evidence briefs, abstracts, summary of findings tables, and plain language summaries. There are many organizations developing and disseminating systematic review evidence summaries for different populations or subsets of decision makers. However, evidence on the usefulness and effectiveness of systematic review summaries is lacking. We present an overview of the available evidence on systematic review evidence summaries.

Constipation complication: lung injury following inadvertent intravenous injection of liquid paraffin.

Liquid paraffin is a highly refined petroleum derivative commonly used medicinally as an oral laxative in Lesotho. We present the case of a 22-year-old Basotho woman admitted under the care of gynaecology in a rural hospital in Lesotho. She was inadvertently administered 10 mL of intravenous liquid paraffin. There were no immediate complications. After 48 h, the patient became unwell with frank haemoptysis and features of systemic inflammation. A chest X-ray demonstrated new bilateral pulmonary infiltrates. She made a full clinical and radiological recovery with a 5-day course of high-dose oral prednisolone and broad-spectrum antibiotics. She was discharged home in a stable condition.