Adventures with Particles

Despite some gender-related bumps in the road, the author had the good fortune that her career spanned the evolution of the Standard Model from its inception in the late 1960s and early 1970s to its final confirmation with the discovery of the Higgs boson in 2012. Her major contributions to these developments and other facets of her career are described.

Screening and validation of genome-edited animals

The emergence of an array of genome-editing tools in recent years has facilitated the introduction of genetic modifications directly into the embryo, increasing the ease, efficiency and catalogue of alleles accessible to researchers across a range of species. Bypassing the requirement for a selection cassette and resulting in a broad range of outcomes besides the desired allele, genome editing has altered the allele validation process both temporally and technically. Whereas traditional gene targeting relies upon selection and allows allele validation at the embryonic stem cell modification stage, screening for the presence of the intended allele now occurs in the (frequently mosaic) founder animals. Final confirmation of the edited allele can only take place at the subsequent G1 generation and the validation strategy must differentiate the desired allele from a range of unintended outcomes. Here we present some of the challenges posed by gene editing, strategies for validation and considerations for animal colony management.

Construction of PX-LmGP63 Using CRISPR-Cas9 as Primary Goal for GP63 gene Knockout in Leishmania major and Leishmanization

Background: Leishmania is an intracellular protozoan parasite that uses complex methods for destroying the innate immune response in mammalian host macrophage cells. Many factors have been identified that play a role in the severity of the parasite’s pathogenicity. One of the factors is the GP63, which is a group of metalloproteinases that disrupts the signaling mechanism of the host cell. Objectives: The aim of this study was to construct PX-LMGP63 vector through CRISPR-Cas9 for GP63 gene knockout in Leishmania major as a potential method for leishmanization. Methods: A pair of gRNAs were designed based on the mRNA sequence of the GP63. Then annealing primers were cloned into the linearized vector PX-459 and transformed into the DH5ɑ competent cells. Then, PCR assay was performed with gene-specific and vector primers to confirm the colonies. In addition, the constructed plasmid was sequenced for final confirmation. Results: The expected size band of 270 was confirmed by PCR. The plasmid sequence showed that the gRNA789 was ligated in the vector. The created structure was named PX-LMGP63 and will be transfected into the promastigote cell in the next step. Conclusions: Owing to the prevalence of cutaneous Leishman as a public health problem in most countries and the lack of an effective vaccine for leishmaniasis, the use of the CRISPR method may make it possible to achieve an effective vaccine in the future.

Current Screening Strategy Poses Risk of Spreading of Hepatitis C Virus Infection

Background: Hepatitis C virus is one of the significant causes of morbidity and mortality in the World. Surprisingly, despite national screening campaigns, new cases of HCV are still on the rise. Methods and Results: A total of 5914 healthy blood donors was included in this study after informed consent. Anti-HCV ELISA was used to check presence of antigen in participant’s plasma.using Monlisa HCV Antigen-Antibody Ultra kit. Final confirmation was done by using real time PCR considered as a gold standard. Results: 0.5% of anti-HCV ELISA negative samples showed presence of antigen in plasma, when checked through core Ag detection method. Conclusion: Our result suggested that HCV core antigen detection and/or combo testing are far safer screening methods for the detection of HCV and the use of these methods can avoid/reduce further spread of this deadly disease.

Parathyroid adenoma: a case report

<p class="abstract">The incidence of primary hyperparathyroidism in India is 2.5/1000 individuals. Primary hyperparathyroidism can be caused by a non-cancerous parathyroid adenoma, hyperplasia or rarely by parathyroid carcinoma. Most of these patients have few or no symptoms. Correct diagnosis can be reached by clinical settings, biochemical and radiological tests and final confirmation by histopathology of the specimen.</p>

P1557EFFECTIVENESS OF INTERVENTION FOR SAFETY CONFIRMATION OF HEMODIALYSIS PREPARATION PROCESS

Background and Aims It has been determined that more than half of the medical errors related to haemodialysis treatment are due to the preparation process at the initial stage of treatment, and that the errors are mainly caused by insufficient confirmation behaviour of healthcare professionals. Pointing confirmation and double check have been proven effective as safety treatments, but no specific strategy has been established to verify these insufficient confirmation behaviours. This study clarified the actual behaviour of healthcare professionals engaged in haemodialysis during the preparation process at the initial stage of treatment and defined the confirmation behaviour necessary to start safe haemodialysis based on the results. Moreover, the aim was to assess the effectiveness of an intervention program for enhancing adherence to confirmation practice. Method A first study was conducted to investigate the actual state of behaviour, and a second study to verify the effectiveness of an intervention program. In the first study, we monitored and analysed the actual behaviours of 23 clinical engineering technicians and 14 registered nurses at a dialysis centre during the preparation process (218 steps) at the initial stage of treatment, and calculated their adherence rates. In the second study, the final confirmation process at the initial stage of treatment was determined (13 steps), and an intervention program aimed at adherence was devised. To verify its effectiveness, 18 clinical engineering technicians and 8 registered nurses were randomly divided into an experimental group (E) and a control group (C). The intervention program for Group E comprised baseline without intervention, intervention 1 (verbal explanation of confirmatory behaviour), intervention 2 (adding graphic feedback), intervention 3 (adding video feedback), and follow-up without intervention. That of Group C comprised only baseline and intervention 1. The adherence rate to the final confirmation process in both groups was calculated. Results In the first study, the adherence rate to the preparation process at the initial stage of treatment was 67.2%, and the adherence rate in the confirmation process immediately after the start of dialysis was 21.0%, which was remarkably low. In the second study, the adherence rate to the final confirmation process in Group E was 17.1% at baseline, 45.5% after intervention 1, 65.5% after intervention 2, 90.4% after intervention 3, and 93.9% at follow-up. In contrast, the adherence rate in Group C was 8.6% at baseline and 50.4% after intervention 1. Conclusion The adherence rate to the preparation process at the initial stage of treatment was less than 70%. Furthermore, the intervention program using graphic feedback and video feedback increased the adherence rate to the final confirmation process. Intervention programs aimed at the behaviour modification of healthcare professionals involved in haemodialysis have increased the adherence rate to confirmation actions in the final confirmation step of haemodialysis, and it has become clear that verbal teaching and visualized feedback are effective.

Reviewing the identity of the Maltese Polypodium (Polypodiaceae) – new evidence from morphology and flow cytometry

The first record of Polypodium from Gozo (Maltese Islands) was described as a new endemic taxon, Polypodium vulgare subsp. melitense, based on its unique set of morphological characters. It was treated as a novelty and designated as a subspecies of P. vulgare mainly due to the lack of paraphyses, the presence of 10–16 annular cells, and a mean spore length of 64 μm. The fern was reassessed by us employing a more rigid morphological analysis and the application of flow cytometry. The absence of paraphyses was confirmed, but the number of annular cells (5–11) and the spore length (70–79 μm) differed from the previous study. These and other morphological traits, the phenology (leaf-shedding in spring), the calcareous growth habitat, and the southern distribution implied that the fern is P. cambricum. Final confirmation was obtained from flow cytometry; the genome size of 17 pg corresponds perfectly with the range obtained for other accessions of this diploid species. The Polypodium reported from Malta does not merit taxonomic distinction despite being a rare form of P. cambricum, which lacks paraphyses.

Diagnostic yield of commercial immunodots to diagnose paraneoplastic neurologic syndromes

ObjectiveTo investigate the diagnostic yield of commercial immunodots to detect onconeural antibodies associated with paraneoplastic neurologic syndromes (PNSs), we analyzed the proportion of confirmed positive results using alternative techniques.MethodsSera (n = 5,300) of patients with suspected PNS were tested by PNS+2 blot (Ravo Diagnostika; January 2016–May 2017) or EUROLINE PNS 12 Ag (Euroimmun; July 2017–November 2018). Positive samples were further explored by in-house indirect immunofluorescence and a third in-house technique (Western blot or cell-based assay) using recombinant protein. Those found negative by these 2 techniques were considered as nonconfirmed. We analyzed the relationship between band intensity and final confirmation. Clinical data were collected for all confirmed results and nonconfirmed EUROLINE immunodots.ResultsPNS+2 blot was positive in 128/1,658 (7.7%) sera and confirmed in 47/128 (36.7%). EUROLINE was positive in 186/3,626 (5.1%) and confirmed in 56/186 (30.1%). Confirmation was highly variable among the antibodies tested, from 7.2% (PNS+2 blot) and 5.8% (EUROLINE) for anti-Yo to 88.2% (PNS+2 blot) and 65.0% (EUROLINE) for anti-Hu. None of the 27 weak positive sera by EUROLINE was confirmed. Band intensity in confirmed cases was variable among the antibodies from strong positive for all anti-Yo (n = 3) and anti-Hu (n = 11) to positive (n = 19) or strong positive (n = 9) for anti-SOX1. Among patients with a nonconfirmed EUROLINE result and available clinical information, all had an alternative diagnosis, and only 6.7% had cancer.ConclusionsImmunodots may be useful for PNS screening, but a threshold should be established for each antibody, and clinical information and confirmation by other techniques are essential.Classification of evidenceThe study provides Class IV evidence that immunodot assays for onconeural antibodies accurately identify patients with paraneoplastic neurologic syndromes.

Clopidogrel: An Original Drug or a Generic?

Objective: To analyse studies assessing the therapeutic equivalence of the original Clopidogrel and its generics. Key Points. Most studies assessing the therapeutic equivalence (impact on the platelets aggregation and the risk of myocardial infarction) of the original Clopidogrel and its generics demonstrated their interchangeability. Still, most of the results were generated in small observational studies, therefore they are not reliable enough. Conclusion. For the final confirmation of the therapeutic equivalence of the Clopidogrel and its generics, larger studies are required. Keywords: Clopidogrel, original drug, generic, therapeutic equivalence.