The Making of Transgenic

The complex color patterns on the wings and body of Drosophila guttifera (D. guttifera) are emerging as model systems for studying evolutionary and developmental processes. Studies regarding these processes depend on overexpression and downregulation of developmental genes, which ultimately rely upon an effective transgenic system. Methods describing transgenesis in Drosophila melanogaster (D. melanogaster) have been reported in several studies, but they cannot be applied to D. guttifera due to the low egg production rate and the delicacy of the eggs. In this protocol, we describe extensively a comprehensive method used for generating transgenic D. guttifera. Using the protocol described here, we are able to establish transgenic lines, identifiable by the expression of enhanced green fluorescent protein (EGFP) in the eye disks of D. guttifera larvae. The entire procedure, from injection to screening for transgenic larvae, can be completed in approximately 30 days and should be relatively easy to adapt to other non-model Drosophila species, for which no white-eyed mutants exist

Rna in situ hybridization for detecting gene expression patterns in the abdomens and wings of drosophila species

RNA in situ hybridization (ISH) is used to visualize spatio-temporal gene expression patterns with broad applications in biology and biomedicine. Here we provide a protocol for mRNA ISH in developing pupal wings and abdomens for model and non-model Drosophila species. We describe best practices in pupal staging, tissue preparation, probe design and synthesis, imaging of gene expression patterns, and image-editing techniques. This protocol has been successfully used to investigate the roles of genes underlying the evolution of novel color patterns in non-model Drosophila species

The modular expression patterns of three pigmentation genes prefigure unique abdominal morphologies seen among three Drosophila species

To understand how novel animal body colorations emerged, one needs to ask how the development of color patterns differs among closely related species. Here we examine three species of fruit flies – Drosophila guttifera (D. guttifera), D. palustris, and D. subpalustris – displaying a varying number of abdominal spot rows. Through in situ hybridization experiments, we examine the mRNA expression patterns for the pigmentation genes Dopa decarboxylase (Ddc), tan (t), and yellow (y) during pupal development. Our results show that Ddc, t, and y are co-expressed in modular, identical patterns, each foreshadowing the adult abdominal spots in D. guttifera, D. palustris, and D. subpalustris. We suggest that differences in the expression patterns of these three genes partially underlie the morphological diversity of the quinaria species group

physics considerations and electromagnetic field simulations up to 23.5 Tesla (1GHz)

Background Glioblastoma multiforme is the most common and most aggressive malign brain tumor. The 5-year survival rate after tumor resection and adjuvant chemoradiation is only 10 %, with almost all recurrences occurring in the initially treated site. Attempts to improve local control using a higher radiation dose were not successful so that alternative additive treatments are urgently needed. Given the strong rationale for hyperthermia as part of a multimodal treatment for patients with glioblastoma, non-invasive radio frequency (RF) hyperthermia might significantly improve treatment results. Methods A non-invasive applicator was constructed utilizing the magnetic resonance (MR) spin excitation frequency for controlled RF hyperthermia and MR imaging in an integrated system, which we refer to as thermal MR. Applicator designs at RF frequencies 300 MHz, 500 MHz and 1GHz were investigated and examined for absolute applicable thermal dose and temperature hotspot size. Electromagnetic field (EMF) and temperature simulations were performed in human voxel models. RF heating experiments were conducted at 300 MHz and 500 MHz to characterize the applicator performance and validate the simulations. Results The feasibility of thermal MR was demonstrated at 7.0 T. The temperature could be increased by ~11 °C in 3 min in the center of a head sized phantom. Modification of the RF phases allowed steering of a temperature hotspot to a deliberately selected location. RF heating was monitored using the integrated system for MR thermometry and high spatial resolution MRI. EMF and thermal simulations demonstrated that local RF hyperthermia using the integrated system is feasible to reach a maximum temperature in the center of the human brain of 46.8 °C after 3 min of RF heating while surface temperatures stayed below 41 °C. Using higher RF frequencies reduces the size of the temperature hotspot significantly. Conclusion The opportunities and capabilities of thermal magnetic resonance for RF hyperthermia interventions of intracranial lesions are intriguing. Employing such systems as an alternative additive treatment for glioblastoma multiforme might be able to improve local control by “fighting fire with fire”. Interventions are not limited to the human brain and might include temperature driven targeted drug and MR contrast agent delivery and help to understand temperature dependent bio- and physiological processes in-vivo

Preclinical Pharmacokinetic Evaluation to Facilitate Repurposing of Tyrosine Kinase Inhibitors Nilotinib and Imatinib as Antiviral Agents

Background Several tyrosine kinase inhibitors (TKIs) developed as anti-cancer drugs, also have anti-viral activity due to their ability to disrupt productive replication and dissemination in infected cells. Consequently, such drugs are attractive candidates for “repurposing” as anti-viral agents. However, clinical evaluation of therapeutics against infectious agents associated with high mortality, but low or infrequent incidence, is often unfeasible. The United States Food and Drug Administration formulated the “Animal Rule” to facilitate use of validated animal models for conducting anti-viral efficacy studies. Methods To enable such efficacy studies of two clinically approved TKIs, nilotinib, and imatinib, we first conducted comprehensive pharmacokinetic (PK) studies in relevant rodent and non-rodent animal models. PK of these agents following intravenous and oral dosing were evaluated in C57BL/6 mice, prairie dogs, guinea pigs and Cynomolgus monkeys. Plasma samples were analyzed using an LC-MS/MS method. Secondarily, we evaluated the utility of allometry-based inter-species scaling derived from previously published data to predict the PK parameters, systemic clearance (CL) and the steady state volume of distribution (Vss) of these two drugs in prairie dogs, an animal model not tested thus far. Results Marked inter-species variability in PK parameters and resulting oral bioavailability was observed. In general, elimination half-lives of these agents in mice and guinea pigs were much shorter (1–3 h) relative to those in larger species such as prairie dogs and monkeys. The longer nilotinib elimination half-life in prairie dogs (i.v., 6.5 h and oral, 7.5 h), facilitated multiple dosing PK and safety assessment. The allometry-based predicted values of the Vss and CL were within 2.0 and 2.5-fold, respectively, of the observed values. Conclusions Our results suggest that prairie dogs and monkeys may be suitable rodent and non-rodent species to perform further efficacy testing of these TKIs against orthopoxvirus infections. The use of rodent models such as C57BL/6 mice and guinea pigs for assessing pre-clinical anti-viral efficacy of these two TKIs may be limited due to short elimination and/or low oral bioavailability. Allometry-based correlations, derived from existing literature data, may provide initial estimates, which may serve as a useful guide for pre-clinical PK studies in untested animal models

Nationwide Outcome after Pancreatoduodenectomy in Patients at very High Risk (ISGPS-D) for Postoperative Pancreatic Fistula

OBJECTIVE: To assess nationwide surgical outcome after pancreatoduodenectomy (PD) in patients at very high risk for postoperative pancreatic fistula (POPF), categorized as ISGPS-D.SUMMARY BACKGROUND DATA: Morbidity and mortality after ISGPS-D PD is perceived so high that a recent randomized trial advocated prophylactic total pancreatectomy (TP) as alternative aiming to lower this risk. However, current outcomes of ISGPS-D PD remain unknown as large nationwide series are lacking.METHODS: Nationwide retrospective analysis including consecutive patients undergoing ISGPS-D PD (i.e., soft texture and pancreatic duct ≤3 mm), using the mandatory Dutch Pancreatic Cancer Audit (2014-2021). Primary outcome was in-hospital mortality and secondary outcomes included major morbidity (i.e., Clavien-Dindo grade ≥IIIa) and POPF (ISGPS grade B/C). The use of prophylactic TP to avoid POPF during the study period was assessed.RESULTS: Overall, 1402 patients were included. In-hospital mortality was 4.1% (n=57), which decreased to 3.7% (n=20/536) in the last 2 years. Major morbidity occurred in 642 patients (45.9%) and POPF in 410 (30.0%), which corresponded with failure to rescue in 8.9% (n=57/642). Patients with POPF had increased rates of major morbidity (88.0% vs. 28.3%; P&lt;0.001) and mortality (6.3% vs. 3.5%; P=0.016), compared to patients without POPF. Among 190 patients undergoing TP, prophylactic TP to prevent POPF was performed in 4 (2.1%).CONCLUSION: This nationwide series found a 4.1% in-hospital mortality after ISGPS-D PD with 45.9% major morbidity, leaving little room for improvement through prophylactic TP. Nevertheless, given the outcomes in 30% of patients who develop POPF, future randomized trials should aim to prevent and mitigate POPF in this high-risk category.</p

Nationwide Outcome after Pancreatoduodenectomy in Patients at very High Risk (ISGPS-D) for Postoperative Pancreatic Fistula

OBJECTIVE: To assess nationwide surgical outcome after pancreatoduodenectomy (PD) in patients at very high risk for postoperative pancreatic fistula (POPF), categorized as ISGPS-D.SUMMARY BACKGROUND DATA: Morbidity and mortality after ISGPS-D PD is perceived so high that a recent randomized trial advocated prophylactic total pancreatectomy (TP) as alternative aiming to lower this risk. However, current outcomes of ISGPS-D PD remain unknown as large nationwide series are lacking.METHODS: Nationwide retrospective analysis including consecutive patients undergoing ISGPS-D PD (i.e., soft texture and pancreatic duct ≤3 mm), using the mandatory Dutch Pancreatic Cancer Audit (2014-2021). Primary outcome was in-hospital mortality and secondary outcomes included major morbidity (i.e., Clavien-Dindo grade ≥IIIa) and POPF (ISGPS grade B/C). The use of prophylactic TP to avoid POPF during the study period was assessed.RESULTS: Overall, 1402 patients were included. In-hospital mortality was 4.1% (n=57), which decreased to 3.7% (n=20/536) in the last 2 years. Major morbidity occurred in 642 patients (45.9%) and POPF in 410 (30.0%), which corresponded with failure to rescue in 8.9% (n=57/642). Patients with POPF had increased rates of major morbidity (88.0% vs. 28.3%; P&lt;0.001) and mortality (6.3% vs. 3.5%; P=0.016), compared to patients without POPF. Among 190 patients undergoing TP, prophylactic TP to prevent POPF was performed in 4 (2.1%).CONCLUSION: This nationwide series found a 4.1% in-hospital mortality after ISGPS-D PD with 45.9% major morbidity, leaving little room for improvement through prophylactic TP. Nevertheless, given the outcomes in 30% of patients who develop POPF, future randomized trials should aim to prevent and mitigate POPF in this high-risk category.</p

How has the OSD affected our state hospitals?

The long-awaited occupation-specific dispensation (OSD) process for state-employed doctors has now been concluded. The final offer, signed and accepted in the bargaining chamber despite being rejected by 92% of doctors in a SAMA survey, has not received much attention or fanfare. At the conclusion of this process, which has been drawn out over several years, many points have emerged that are extremely worrying for the future of health care in this country

EMU Detection of a Large and Low Surface Brightness Galactic SNR G288.8-6.3

We present the serendipitous detection of a new Galactic Supernova Remnant (SNR), G288.8-6.3 using data from the Australian Square Kilometre Array Pathfinder (ASKAP)-Evolutionary Map of the Universe (EMU) survey. Using multi-frequency analysis, we confirm this object as an evolved Galactic SNR at high Galactic latitude with low radio surface brightness and typical SNR spectral index of $\alpha = -0.41\pm0.12$. To determine the magnetic field strength in SNR G288.8-6.3, we present the first derivation of the equipartition formulae for SNRs with spectral indices $\alpha>-0.5$. The angular size is 1.\!^\circ 8\times 1.\!^\circ 6 (107.\!^\prime 6 \times 98.\!^\prime 4) and we estimate that its intrinsic size is $\sim40$pc which implies a distance of $\sim1.3$kpc and a position of $\sim140$pc above the Galactic plane. This is one of the largest angular size and closest Galactic SNRs. Given its low radio surface brightness, we suggest that it is about 13000 years old.Comment: Accepted for publication in The Astrophysical Journa

Long-Term Outcome of Immediate Versus Postponed" Intervention in Patients With Infected Necrotizing Pancreatitis" (POINTER)" Multicenter Randomized Trial

Objective: To compare the long-term outcomes of immediate drainage versus the postponed-drainage approach in patients with infected necrotizing pancreatitis. Background: In the randomized POINTER trial, patients assigned to the postponed-drainage approach using antibiotic treatment required fewer interventions, as compared with immediate drainage, and over a third were treated without any intervention. Methods: Clinical data of those patients alive after the initial 6-month follow-up were re-evaluated. The primary outcome was a composite of death and major complications. Results: Out of 104 patients, 88 were re-evaluated with a median followup of 51 months. After the initial 6-month follow-up, the primary outcome occurred in 7 of 47 patients (15%) in the immediate-drainage group and 7 of 41 patients (17%) in the postponed-drainage group (RR 0.87, 95% CI 0.33-2.28; P=0.78). Additional drainage procedures were performed in 7 patients (15%) versus 3 patients (7%) (RR 2.03; 95% CI 0.56-7.37; P=0.34). The median number of additional interventions was 0 (IQR 0-0) in both groups (P=0.028). In the total follow-up, the median number of interventions was higher in the immediate-drainage group than in the postponed-drainage group (4 vs. 1, P=0.001). Eventually, 14 of 15 patients (93%) in the postponed-drainage group who were successfully treated in the initial 6-month follow-up with antibiotics and without any intervention remained without intervention. At the end of follow-up, pancreatic function and quality of life were similar. Conclusions: Also, during long-term follow-up, a postponed-drainage approach using antibiotics in patients with infected necrotizing pancreatitis results in fewer interventions as compared with immediate drainage and should therefore be the preferred approach.</p