Post COVID-19 Self-Care of the Therapist: A Community Project

The continuous COVID-19 pandemic has brought new expectations and higher demand in the field of mental health counseling. Many individuals began seeking therapy during this time which is a good thing however, the therapists are facing a higher risk of burnout syndrome and compassion fatigue. Therapists have not shifted their focus as much on themselves through acts of self-care during this time despite the world around them shifting. In graduate counseling programs there is discussion and perhaps a dedicated week of course material to self-care however, it is not taught the importance to implement and discover what acts of care work best for each individual. This community engagement project advocates for therapist engagement of self-care through an in-service experience with the clinical population at a therapeutic day school. Participants engaged in a discussion regarding self-care, a dance/movement therapy practice discussion and experiential and concluded with assembling their own “self-care kit”. I observed and received an overall positive response and gratitude for both reminding and providing some tools to allow this clinical team to take time to care for themselves. When the therapist takes time to do self-care, they will be more engaged and present with the individuals they help

Testing and model selection for prediction in large sets of variables

In dieser Arbeit wird die Selektion von Variablen die (in Wahrheit) einen Einfluss auf einen klinischen Endpunkt (z.B. den Ausgang einer bestimmten Therapie) haben aus einer großen Menge von Kandidatenvariablen mit Hilfe von nur kleinen Stichproben von Patienten, die auf die Therapie reagieren bzw. nicht reagieren, behandelt. Die Selektion basiert auf einer multiplen Testprozedur die die False Discovery Rate (FDR) einhält. Mit jenen, mit Hilfe der multiplen Testprozedur selektierten, Variablen soll ein prognostischer Score konstruiert werden, mit dem man den klinischen Endpunkt eines zukünftigen Patienten vorhersagen kann. Dieser lineare Score wird aufgrund der resultierenden Receiver Operating Characteristic Curve (ROC) bewertet. Die Selektionsgrenze für die FDR, welche die beste Fläche unter der ROC-Kurve (AUC) liefert ist allerdings von unbekannten Parametern wie z.B. der Effektgröße oder der Anzahl der Variablen, die tatsächlich einen Einfluss auf den klinischen Endpunkt haben stark abhängig. Um in einem spezifischen Datensatz nach der optimalen Selektionsschranke zu suchen wird die Verwendung einer Prozedur zur Kreuzvalidierung vorgeschlagen. Diese Prozedur (i) ermittelt ein adäquates Selektionskriterium für die multiple Testprozedur, (ii) berechnet einen (positiv verzerrten) Schätzer für die AUC für zukünftige Prognosen und (iii) liefert einen Schätzer für die FDR, der nahe der wahren FDR ist. Darüber hinaus geben niedrige Werte der ermittelten kreuzvalidierten AUC und große Werte der kreuzvalidierten FDR einen Hinweis darauf, dass der Einfluss der Variablen auf den klinischen Endpunkt zu gering ist und/oder dass die gegebene Stichprobengröße zu gering ist um die gegebenen Effekte zu finden.Multiple testing has been applied for selecting prognostic variables related with a clinical outcome (response to therapy) from a large number of candidates in small samples of "responding" or "non-responding" patients which are then used to estimate a score for prediction in future patients. We evaluated selection based on control of the false discovery rate (FDR) to build a linear score by considering the resulting receiver operating characteristic (ROC) for independent prediction of future patients. We simulated different scenarios with varying number of tested candidates, proportion of prognostic variables and sample sizes. Underlying effect sizes were determined such that optimal prediction, if known, would lead to a ROC-curve crossing through a benchmark point with pre-fixed values of sensitivity and specificity. We show that the "best" FDR-threshold which provides the ROC-curve with the largest area under the curve (AUC) varies largely over the different parameter constellation not known in advance. Hence, cross validation is proposed to determine the optimal selection threshold in a specific sample. This procedure (i) allows to choose an appropriate selection criterion, (ii) results in an estimate of the AUC for future prediction (though positively biased) and (iii) provides an estimate of the FDR close to the true FDR. Moreover, low estimates of the cross validated AUC and large estimates of the cross validated FDR may indicate a lack of sufficiently prognostic variables and/or too small sample sizes

Modeling and measuring intracellular fluxes of secreted recombinant protein in Pichia pastoris with a novel 34S labeling procedure

<p>Abstract</p> <p>Background</p> <p>The budding yeast <it>Pichia pastoris </it>is widely used for protein production. To determine the best suitable strategy for strain improvement, especially for high secretion, quantitative data of intracellular fluxes of recombinant protein are very important. Especially the balance between intracellular protein formation, degradation and secretion defines the major bottleneck of the production system. Because these parameters are different for unlimited growth (shake flask) and carbon-limited growth (bioreactor) conditions, they should be determined under "production like" conditions. Thus labeling procedures must be compatible with minimal production media and the usage of bioreactors. The inorganic and non-radioactive ³⁴S labeled sodium sulfate meets both demands.</p> <p>Results</p> <p>We used a novel labeling method with the stable sulfur isotope ³⁴S, administered as sodium sulfate, which is performed during chemostat culivations. The intra- and extracellular sulfur 32 to 34 ratios of purified recombinant protein, the antibody fragment Fab3H6, are measured by HPLC-ICP-MS. The kinetic model described here is necessary to calculate the kinetic parameters from sulfur ratios of consecutive samples as well as for sensitivity analysis. From the total amount of protein produced intracellularly (143.1 μg g^-1h^-1protein per yeast dry mass and time) about 58% are degraded within the cell, 35% are secreted to the exterior and 7% are inherited to the daughter cells.</p> <p>Conclusions</p> <p>A novel ³⁴S labeling procedure that enables <it>in vivo </it>quantification of intracellular fluxes of recombinant protein under "production like" conditions is described. Subsequent sensitivity analysis of the fluxes by using MATLAB, indicate the most promising approaches for strain improvement towards increased secretion.</p

Intravenous calcitriol normalizes insulin sensitivity in uremic patients

Intravenous calcitriol normalizes insulin sensitivity in uremic patients. Recent studies suggest that secondary hyperparathyroidism and/or vitamin D deficiency are responsible for the insulin resistance in chronic renal failure. We investigated the effect of a 12-week intravenous treatment with 1,25 dihydroxycholecalciferol on glucose metabolism in 10 hemodialysis patients compared with 10 healthy control subjects by the frequently-sampled intravenous glucose tolerance test, analyzed with the minimal model technique. Compared to control subjects, the uremic patients featured elevated levels of parathyroid hormone (432 ± 60 vs. 41 ± 4 ng/liter, P < 0.001), insulin resistance (insulin sensitivity index, SI: 4.9 ± 0.8 vs. 9.5 ± 0.9 MIN-1/(µ/m1), P < 0.002), increased posthepatic insulin delivery (6.48 ± 2.48 vs. 2.73 ± 3.14 nmol/liter in 4 hr, P < 0.001) and a reduced C-peptide fractional clearance (0.033 ± 0.004 vs. 0.085 ± 0.009 min-1, P < 0.0002). Following treatment with 1,25 dihydroxycholecalciferol, the parathyroid hormone levels decreased significantly to 237 ± 30 ng/liter (P < 0.05), the insulin sensitivity index (SI: 9.6 ± 2.2, P < 0.05) reached a value similar to that of control subjects, and posthepatic insulin delivery decreased to 4.63 ± 0.83 nmol/liter in 4 hr (P < 0.01), while all the other parameters remained unchanged. In summary, uremic patients with secondary hyperparathyroidism were found to be severely insulin resistant and hyperinsulinemic. Intravenous vitamin D treatment led to a significant reduction of parathyroid hormone levels and to a complete normalization of insulin sensitivity in the hemodialysis patients. Thus, intravenous 1,25 dihydroxycholecalciferol improves insulin resistance in uremic patients, acting per se or by reducing secondary hyperparathyroidism

Novel insights into the unfolded protein response using Pichia pastoris specific DNA microarrays

Background DNA Microarrays are regarded as a valuable tool for basic and applied research in microbiology. However, for many industrially important microorganisms the lack of commercially available microarrays still hampers physiological research. Exemplarily, our understanding of protein folding and secretion in the yeast Pichia pastoris is presently widely dependent on conclusions drawn from analogies to Saccharomyces cerevisiae. To close this gap for a yeast species employed for its high capacity to produce heterologous proteins, we developed full genome DNA microarrays for P. pastoris and analyzed the unfolded protein response (UPR) in this yeast species, as compared to S. cerevisiae. Results By combining the partially annotated gene list of P. pastoris with de novo gene finding a list of putative open reading frames was generated for which an oligonucleotide probe set was designed using the probe design tool TherMODO (a thermodynamic model-based oligoset design optimizer). To evaluate the performance of the novel array design, microarrays carrying the oligo set were hybridized with samples from treatments with dithiothreitol (DTT) or a strain overexpressing the UPR transcription factor HAC1, both compared with a wild type strain in normal medium as untreated control. DTT treatment was compared with literature data for S. cerevisiae, and revealed similarities, but also important differences between the two yeast species. Overexpression of HAC1, the most direct control for UPR genes, resulted in significant new understanding of this important regulatory pathway in P. pastoris, and generally in yeasts. Conclusion The differences observed between P. pastoris and S. cerevisiae underline the importance of DNA microarrays for industrial production strains. P. pastoris reacts to DTT treatment mainly by the regulation of genes related to chemical stimulus, electron transport and respiration, while the overexpression of HAC1 induced many genes involved in translation, ribosome biogenesis, and organelle biosynthesis, indicating that the regulatory events triggered by DTT treatment only partially overlap with the reactions to overexpression of HAC1. The high reproducibility of the results achieved with two different oligo sets is a good indication for their robustness, and underlines the importance of less stringent selection of regulated features, in order to avoid a large number of false negative results

Buffered lidocaine 1%/epinephrine 1:100,000 with sodium bicarbonate (sodium hydrogen carbonate) in a 3:1 ratio is less painful than a 9:1 ratio: A double-blind, randomized, placebo-controlled, crossover trial

Background: Neutralizing (buffering) lidocaine 1%/epinephrine 1:100,000 solution (Lido/Epi) with sodium hydrogen carbonate (NaHCO3) (also called sodium bicarbonate) is widely used to reduce burning sensations during infiltration of Lido/Epi. Optimal mixing ratios have not been systematically investigated. Objectives: To determine whether a Lido/Epi:NaHCO3 mixing ratio of 3:1 (investigational medicinal product 1) causes less pain during infiltration than a mixing ratio of 9:1 (IMP2) or unbuffered Lido/Epi (IMP3). Methods: Double-blind, randomized, placebo-controlled, crossover trial (n = 2 × 24) with 4 investigational medicinal products (IMP1-4). Results: The 3:1 mixing ratio was significantly less painful than the 9:1 ratio (P = .044). Unbuffered Lido/Epi was more painful than the buffered Lido/Epi (P = .001 vs IMP1; P = .033 vs IMP2). IMP4 (NaCl 0.9% [placebo]) was more painful than any of the anesthetic solutions (P = .001 vs IMP1; P = .001 vs IMP2; P = .016 vs IMP3). In all cases, the anesthesia was effective for at least 3 hours. Limitations: Results of this trial cannot be generalized to other local anesthetics such as prilocaine, bupivacaine, or ropivacaine, which precipitate with NaHCO3 admixtures. Conclusions: Lido/Epi-NaHCO3 mixtures effectively reduce burning pain during infiltration. The 3:1 mixing ratio is significantly less painful than the 9:1 ratio. Reported findings are of high practical relevance, given the extensive use of local anesthesia today

Novel insights into the unfolded protein response using Pichia pastoris specific DNA microarrays

Background DNA Microarrays are regarded as a valuable tool for basic and applied research in microbiology. However, for many industrially important microorganisms the lack of commercially available microarrays still hampers physiological research. Exemplarily, our understanding of protein folding and secretion in the yeast Pichia pastoris is presently widely dependent on conclusions drawn from analogies to Saccharomyces cerevisiae. To close this gap for a yeast species employed for its high capacity to produce heterologous proteins, we developed full genome DNA microarrays for P. pastoris and analyzed the unfolded protein response (UPR) in this yeast species, as compared to S. cerevisiae. Results By combining the partially annotated gene list of P. pastoris with de novo gene finding a list of putative open reading frames was generated for which an oligonucleotide probe set was designed using the probe design tool TherMODO (a thermodynamic model-based oligoset design optimizer). To evaluate the performance of the novel array design, microarrays carrying the oligo set were hybridized with samples from treatments with dithiothreitol (DTT) or a strain overexpressing the UPR transcription factor HAC1, both compared with a wild type strain in normal medium as untreated control. DTT treatment was compared with literature data for S. cerevisiae, and revealed similarities, but also important differences between the two yeast species. Overexpression of HAC1, the most direct control for UPR genes, resulted in significant new understanding of this important regulatory pathway in P. pastoris, and generally in yeasts. Conclusion The differences observed between P. pastoris and S. cerevisiae underline the importance of DNA microarrays for industrial production strains. P. pastoris reacts to DTT treatment mainly by the regulation of genes related to chemical stimulus, electron transport and respiration, while the overexpression of HAC1 induced many genes involved in translation, ribosome biogenesis, and organelle biosynthesis, indicating that the regulatory events triggered by DTT treatment only partially overlap with the reactions to overexpression of HAC1. The high reproducibility of the results achieved with two different oligo sets is a good indication for their robustness, and underlines the importance of less stringent selection of regulated features, in order to avoid a large number of false negative results

The response to unfolded protein is involved in osmotolerance of Pichia pastoris

Background The effect of osmolarity on cellular physiology has been subject of investigation in many different species. High osmolarity is of importance for biotechnological production processes, where high cell densities and product titers are aspired. Several studies indicated that increased osmolarity of the growth medium can have a beneficial effect on recombinant protein production in different host organisms. Thus, the effect of osmolarity on the cellular physiology of Pichia pastoris, a prominent host for recombinant protein production, was studied in carbon limited chemostat cultures at different osmolarities. Transcriptome and proteome analyses were applied to assess differences upon growth at different osmolarities in both, a wild type strain and an antibody fragment expressing strain. While our main intention was to analyze the effect of different osmolarities on P. pastoris in general, this was complemented by studying it in context with recombinant protein production. Results In contrast to the model yeast Saccharomyces cerevisiae, the main osmolyte in P. pastoris was arabitol rather than glycerol, demonstrating differences in osmotic stress response as well as energy metabolism. 2D Fluorescence Difference Gel electrophoresis and microarray analysis were applied and demonstrated that processes such as protein folding, ribosome biogenesis and cell wall organization were affected by increased osmolarity. These data indicated that upon increased osmolarity less adaptations on both the transcript and protein level occurred in a P. pastoris strain, secreting the Fab fragment, compared with the wild type strain. No transcriptional activation of the high osmolarity glycerol (HOG) pathway was observed at steady state conditions. Furthermore, no change of the specific productivity of recombinant Fab was observed at increased osmolarity. Conclusion These data point out that the physiological response to increased osmolarity is different to S. cerevisiae. Increased osmolarity resulted in an unfolded protein response (UPR) like response in P. pastoris and lead to pre-conditioning of the recombinant Fab producing strain of P. pastoris to growth at high osmolarity. The current data demonstrate a strong similarity of environmental stress response mechanisms and recombinant protein related stresses. Therefore, these results might be used in future strain and bioprocess engineering of this biotechnologically relevant yeast

Open access to sequence: Browsing the Pichia pastoris genome

The first genome sequences of the important yeast protein production host Pichia pastoris have been released into the public domain this spring. In order to provide the scientific community easy and versatile access to the sequence, two web-sites have been installed as a resource for genomic sequence, gene and protein information for P. pastoris: A GBrowse based genome browser was set up at and a genome portal with gene annotation and browsing functionality at . Both websites are offering information on gene annotation and function, regulation and structure