Development and application of novel cloning strategies for analysis of genes controlling embryo development.

Initially, we aimed to identify novel genes regulating vertebrate neurogenesis and somitogenesis by screening cDNAs derived from gastrulation/neurulation stage zebrafish embryos for clones revealing corresponding genes with expression patterns suggestive of roles in these processes. The lack of suitable cDNA libraries prompted us to devise a simplified method for producing randomly-primed, directionally cloned cDNA libraries from small amounts of embryonic tissue. To achieve this, several techniques were combined, including cDNA synthesis on a solid carrier, random priming of 1st cDNA strand synthesis, non-specific priming of 2nd cDNA strand synthesis and amplification of initially small amounts of cDNAs by suppression-PCR. A pilot-scale in situ screen using a cDNA library produced by the above method identified a gene, spadetail, that is expressed in presomitic mesoderm and in unidentified, apparently irregularly distributed cells of the spinal cord. spt functions in mesodermal development, yet its role in neural tissue remains unknown. Analysis of the spadetail-expressing neural cells' gene co-expression profile and dorsoventral location implied that they are Dorsal Longitudinal Ascending interneurons. Quantitative analysis of these cells' rostrocaudal distribution showed that there is a tendency to higher cell numbers in rostral spinal segments. The observation that spadetail-expressing neurons are frequently juxtaposed to somitic cells expressing spadetail at low levels suggests that the distribution of spadetail-expressing neurons may be 'inefficiently' patterned by spadetail-expressing somitic cells or that the expression of spadetail in both tissues is induced by a common positional cue. The strategy for non-specific was then extended to develop a simple technique for cloning unknown DNA sequences flanking known DNA. An initial nonspecific PCR amplification was performed with a single primer that binds specifically within known sequence and non-specifically in the unknown DNA region. In a second reaction, the sequences of interest were amplified from the primary reaction mixture (that also contains undesired sequences) with nested PCR using a primer that had been extended further downstream from the primer used in the initial PCR. This enabled isolation of a 0.5 kb region of amphioxus Notch cDNA, that, in turn, contributed to the subsequent analysis of the evolution of vertebrate Notch genes.Thesis (Ph.D.) -- University of Adelaide, School of Molecular and Biomedical Science, 200

The identity and distribution of neural cells expressing the mesodermal determinant spadetail

BACKGROUND: The spadetail (spt) gene of zebrafish is expressed in presomitic mesoderm and in neural cells previously suggested to be Rohon-Beard neurons. The mechanism(s) generating the apparently irregular rostrocaudal distribution of spt-expressing cells in the developing CNS is unknown. RESULTS: spt-expressing neural cells co-express huC, a marker of neurons. These cells also co-express the genes islet-1, -2 and -3 but not valentino. The islet-1 gene expression, irregular distribution and dorsolateral position of spt-expressing cells in the developing CNS are characteristic of dorsal longitudinal ascending (DoLA) interneurons. Shortly after their birth, these neurons extend processes rostrally into which spt mRNA is transported. At 24 hours post fertilisation(hpf), spt-expressing neurons occur most frequently at rostral levels caudal of the 5(th)-formed somite pair. There is no apparent bias in the number of spt-expressing cells on the left or right sides of embryos. Extended staining for spt-transcription reveals expression in the dorsocaudal cells of somites at the same dorsoventral level as the spt-expressing neurons. There is frequent juxtaposition of spt-expression in newly formed somites and in neurons. This suggests that both types of spt-expressing cell respond to a common positional cue or that neurons expressing spt are patterned irregularly by flanking somitic mesoderm. CONCLUSIONS: spt-expressing cells in the developing CNS appear to be DoLA interneurons. The irregular distribution of these cells along the rostrocaudal axis of the spinal cord may be due to "inefficient" patterning of neural spt expression by a signal(s) from flanking, regularly distributed somites also expressing spt

Neurotroofsed tegurid

Neurotroofsed tegurid on sekreteeritavad valgulised kasvutegurid, mis osalevad väga mitmetes närvisüsteemi arengu ja talitluse jaoks olulistes protsessides, sh neuronite arvu, närvijätkete väljakasvu ning harunemise, neuronitevaheliste sünaptiliste ühenduste tekke ja plastilisuse ning kognitiivsete protsesside regulatsioonis. Kahe peamise rühmana eristatakse närvide kasvuteguri (NGF, BDNF, NT-3, NT-4) ja gliia päritolu neurotroofsete tegurite perekonda (GDNF, neurturiin, artemiin, persefiin). Tulenevalt neurotroofsete tegurite võimest hoida elus spetsiifilisi neuronipopulatsioone ja mõjutada närvijätkete kasvu uuritakse intensiivselt nende valkude või nende toimet mimikeerivate madalmolekulaarsete ühendite kliinilisi kasutus -võimalusi närvisüsteemi haiguste, k.a neurodegeneratiivsete haiguste, meeleolu- ning ärevushäirete, neuropaatiate ja seljaaju traumade ravis. Eesti Arst 2007; 86(9):614-62

Effect of angiotensin-converting enzyme inhibitor and angiotensin receptor blocker initiation on organ support-free days in patients hospitalized with COVID-19

IMPORTANCE Overactivation of the renin-angiotensin system (RAS) may contribute to poor clinical outcomes in patients with COVID-19. Objective To determine whether angiotensin-converting enzyme (ACE) inhibitor or angiotensin receptor blocker (ARB) initiation improves outcomes in patients hospitalized for COVID-19. DESIGN, SETTING, AND PARTICIPANTS In an ongoing, adaptive platform randomized clinical trial, 721 critically ill and 58 non–critically ill hospitalized adults were randomized to receive an RAS inhibitor or control between March 16, 2021, and February 25, 2022, at 69 sites in 7 countries (final follow-up on June 1, 2022). INTERVENTIONS Patients were randomized to receive open-label initiation of an ACE inhibitor (n = 257), ARB (n = 248), ARB in combination with DMX-200 (a chemokine receptor-2 inhibitor; n = 10), or no RAS inhibitor (control; n = 264) for up to 10 days. MAIN OUTCOMES AND MEASURES The primary outcome was organ support–free days, a composite of hospital survival and days alive without cardiovascular or respiratory organ support through 21 days. The primary analysis was a bayesian cumulative logistic model. Odds ratios (ORs) greater than 1 represent improved outcomes. RESULTS On February 25, 2022, enrollment was discontinued due to safety concerns. Among 679 critically ill patients with available primary outcome data, the median age was 56 years and 239 participants (35.2%) were women. Median (IQR) organ support–free days among critically ill patients was 10 (–1 to 16) in the ACE inhibitor group (n = 231), 8 (–1 to 17) in the ARB group (n = 217), and 12 (0 to 17) in the control group (n = 231) (median adjusted odds ratios of 0.77 [95% bayesian credible interval, 0.58-1.06] for improvement for ACE inhibitor and 0.76 [95% credible interval, 0.56-1.05] for ARB compared with control). The posterior probabilities that ACE inhibitors and ARBs worsened organ support–free days compared with control were 94.9% and 95.4%, respectively. Hospital survival occurred in 166 of 231 critically ill participants (71.9%) in the ACE inhibitor group, 152 of 217 (70.0%) in the ARB group, and 182 of 231 (78.8%) in the control group (posterior probabilities that ACE inhibitor and ARB worsened hospital survival compared with control were 95.3% and 98.1%, respectively). CONCLUSIONS AND RELEVANCE In this trial, among critically ill adults with COVID-19, initiation of an ACE inhibitor or ARB did not improve, and likely worsened, clinical outcomes. TRIAL REGISTRATION ClinicalTrials.gov Identifier: NCT0273570

Prevalence, associated factors and outcomes of pressure injuries in adult intensive care unit patients: the DecubICUs study

Funder: European Society of Intensive Care Medicine; doi: http://dx.doi.org/10.13039/501100013347Funder: Flemish Society for Critical Care NursesAbstract: Purpose: Intensive care unit (ICU) patients are particularly susceptible to developing pressure injuries. Epidemiologic data is however unavailable. We aimed to provide an international picture of the extent of pressure injuries and factors associated with ICU-acquired pressure injuries in adult ICU patients. Methods: International 1-day point-prevalence study; follow-up for outcome assessment until hospital discharge (maximum 12 weeks). Factors associated with ICU-acquired pressure injury and hospital mortality were assessed by generalised linear mixed-effects regression analysis. Results: Data from 13,254 patients in 1117 ICUs (90 countries) revealed 6747 pressure injuries; 3997 (59.2%) were ICU-acquired. Overall prevalence was 26.6% (95% confidence interval [CI] 25.9–27.3). ICU-acquired prevalence was 16.2% (95% CI 15.6–16.8). Sacrum (37%) and heels (19.5%) were most affected. Factors independently associated with ICU-acquired pressure injuries were older age, male sex, being underweight, emergency surgery, higher Simplified Acute Physiology Score II, Braden score 3 days, comorbidities (chronic obstructive pulmonary disease, immunodeficiency), organ support (renal replacement, mechanical ventilation on ICU admission), and being in a low or lower-middle income-economy. Gradually increasing associations with mortality were identified for increasing severity of pressure injury: stage I (odds ratio [OR] 1.5; 95% CI 1.2–1.8), stage II (OR 1.6; 95% CI 1.4–1.9), and stage III or worse (OR 2.8; 95% CI 2.3–3.3). Conclusion: Pressure injuries are common in adult ICU patients. ICU-acquired pressure injuries are associated with mainly intrinsic factors and mortality. Optimal care standards, increased awareness, appropriate resource allocation, and further research into optimal prevention are pivotal to tackle this important patient safety threat

Correction to: Prevalence, associated factors and outcomes of pressure injuries in adult intensive care unit patients: the DecubICUs study

The original version of this article unfortunately contained a mistake

Prevalence, associated factors and outcomes of pressure injuries in adult intensive care unit patients: the DecubICUs study

Funder: European Society of Intensive Care Medicine; doi: http://dx.doi.org/10.13039/501100013347Funder: Flemish Society for Critical Care NursesAbstract: Purpose: Intensive care unit (ICU) patients are particularly susceptible to developing pressure injuries. Epidemiologic data is however unavailable. We aimed to provide an international picture of the extent of pressure injuries and factors associated with ICU-acquired pressure injuries in adult ICU patients. Methods: International 1-day point-prevalence study; follow-up for outcome assessment until hospital discharge (maximum 12 weeks). Factors associated with ICU-acquired pressure injury and hospital mortality were assessed by generalised linear mixed-effects regression analysis. Results: Data from 13,254 patients in 1117 ICUs (90 countries) revealed 6747 pressure injuries; 3997 (59.2%) were ICU-acquired. Overall prevalence was 26.6% (95% confidence interval [CI] 25.9–27.3). ICU-acquired prevalence was 16.2% (95% CI 15.6–16.8). Sacrum (37%) and heels (19.5%) were most affected. Factors independently associated with ICU-acquired pressure injuries were older age, male sex, being underweight, emergency surgery, higher Simplified Acute Physiology Score II, Braden score 3 days, comorbidities (chronic obstructive pulmonary disease, immunodeficiency), organ support (renal replacement, mechanical ventilation on ICU admission), and being in a low or lower-middle income-economy. Gradually increasing associations with mortality were identified for increasing severity of pressure injury: stage I (odds ratio [OR] 1.5; 95% CI 1.2–1.8), stage II (OR 1.6; 95% CI 1.4–1.9), and stage III or worse (OR 2.8; 95% CI 2.3–3.3). Conclusion: Pressure injuries are common in adult ICU patients. ICU-acquired pressure injuries are associated with mainly intrinsic factors and mortality. Optimal care standards, increased awareness, appropriate resource allocation, and further research into optimal prevention are pivotal to tackle this important patient safety threat

iPSC-derived mesenchymal stromal cells are less supportive than primary MSCs for co-culture of hematopoietic progenitor cells

Abstract In vitro culture of hematopoietic stem and progenitor cells (HPCs) is supported by a suitable cellular microenvironment, such as mesenchymal stromal cells (MSCs)—but MSCs are heterogeneous and poorly defined. In this study, we analyzed whether MSCs derived from induced pluripotent stem cells (iPS-MSCs) provide a suitable cellular feeder layer too. iPS-MSCs clearly supported proliferation of HPCs, maintenance of a primitive immunophenotype (CD34+, CD133+, CD38-), and colony-forming unit (CFU) potential of CD34+ HPCs. However, particularly long-term culture-initiating cell (LTC-IC) frequency was lower with iPS-MSCs as compared to primary MSCs. Relevant genes for cell-cell interaction were overall expressed at similar level in MSCs and iPS-MSCs, whereas VCAM1 was less expressed in the latter. In conclusion, our iPS-MSCs support in vitro culture of HPCs; however, under the current differentiation and culture conditions, they are less suitable than primary MSCs from bone marrow