Development and Validation of a Deep Learning Model for Histopathological Slide Analysis in Lung Cancer Diagnosis.

Lung cancer is the leading cause of cancer-related deaths worldwide. Two of the crucial factors contributing to these fatalities are delayed diagnosis and suboptimal prognosis. The rapid advancement of deep learning (DL) approaches provides a significant opportunity for medical imaging techniques to play a pivotal role in the early detection of lung tumors and subsequent monitoring during treatment. This study presents a DL-based model for efficient lung cancer detection using whole-slide images. Our methodology combines convolutional neural networks (CNNs) and separable CNNs with residual blocks, thereby improving classification performance. Our model improves accuracy (96% to 98%) and robustness in distinguishing between cancerous and non-cancerous lung cell images in less than 10 s. Moreover, the model's overall performance surpassed that of active pathologists, with an accuracy of 100% vs. 79%. There was a significant linear correlation between pathologists' accuracy and years of experience (r Pearson = 0.71, 95% CI 0.14 to 0.93, p = 0.022). We conclude that this model enhances the accuracy of cancer detection and can be used to train junior pathologists.

Histochemical Evidence for Reduced Immune Response in Nasal Mucosa of Patients with COVID-19.

The primary entry point of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the nasal mucosa, where viral-induced inflammation occurs. When the immune response fails against SARS-CoV-2, understanding the altered response becomes crucial. This study aimed to compare SARS-CoV-2 immunological responses in the olfactory and respiratory mucosa by focusing on epithelia and nerves. Between 2020 and 2022, we obtained post mortem tissues from the olfactory cleft from 10 patients with histologically intact olfactory epithelia (OE) who died with or from COVID-19, along with four age-matched controls. These tissues were subjected to immunohistochemical reactions using antibodies against T cell antigens CD3, CD8, CD68, and SARS spike protein for viral evidence. Deceased patients with COVID-19 exhibited peripheral lymphopenia accompanied by a local decrease in CD3+ cells in the OE. However, SARS-CoV-2 spike protein was sparsely detectable in the OE. With regard to the involvement of nerve fibers, the present analysis suggested that SARS-CoV-2 did not significantly alter the immune response in olfactory or trigeminal fibers. On the other hand, SARS spike protein was detectable in both nerves. In summary, the post mortem investigation demonstrated a decreased T cell response in patients with COVID-19 and signs of SARS-CoV-2 presence in olfactory and trigeminal fibers.

Effects of prenatal Doppler ultrasound on the retina of the chick embryo in ovo.

Currently, the effect of prenatal ultrasound on foetal development is intensively discussed and the guidelines for prenatal diagnostics have been changed. However, data supporting these concerns are scarce. Therefore, we used an established in ovo model of the chicken embryo to investigate cell proliferation and apoptosis within the retina. A total of 21 chicken eggs were fenestrated on Day 5 and allocated to either the control (n = 8) or exposition group (n = 13). The exposition group was treated with pulsed-wave Doppler ultrasound (PWD) for 10 min while controls remained without treatment. After subsequent incubation (6-48 h), the eggs were sacrificed, and chicken embryos were examined morphologically (HE-staining) and immunohistochemically. Counting of apoptotic and proliferating cells per retina was performed using antibodies specific for phospho-histone-H3 and active caspase-3 in combination with a biotin-labelled secondary antibody and peroxidase conjugated avidin-biotin complex for chromogenic detection. Due to a rather low number of specimens at each time point after ultrasound exposition, we neglected the effects of incubation time and focused on treatment effects. This approach revealed that the median number of proliferating cells is reduced after 10 min of exposure to PWD (569 vs. 766), while the number of apoptotic cells is fairly comparable between groups (5 vs. 6). Our data contribute to a better understanding of prenatal US on foetal development by suggesting that PWD could have an impact on the number of proliferating cells in the developing chicken retina and therefore justify further investigations.

Perception of Pathologists in Poland of Artificial Intelligence and Machine Learning in Medical Diagnosis-A Cross-Sectional Study.

BACKGROUND: In the past vicennium, several artificial intelligence (AI) and machine learning (ML) models have been developed to assist in medical diagnosis, decision making, and design of treatment protocols. The number of active pathologists in Poland is low, prolonging tumor patients' diagnosis and treatment journey. Hence, applying AI and ML may aid in this process. Therefore, our study aims to investigate the knowledge of using AI and ML methods in the clinical field in pathologists in Poland. To our knowledge, no similar study has been conducted. METHODS: We conducted a cross-sectional study targeting pathologists in Poland from June to July 2022. The questionnaire included self-reported information on AI or ML knowledge, experience, specialization, personal thoughts, and level of agreement with different aspects of AI and ML in medical diagnosis. Data were analyzed using IBM® SPSS® Statistics v.26, PQStat Software v.1.8.2.238, and RStudio Build 351. RESULTS: Overall, 68 pathologists in Poland participated in our study. Their average age and years of experience were 38.92 ± 8.88 and 12.78 ± 9.48 years, respectively. Approximately 42% used AI or ML methods, which showed a significant difference in the knowledge gap between those who never used it (OR = 17.9, 95% CI = 3.57-89.79, p < 0.001). Additionally, users of AI had higher odds of reporting satisfaction with the speed of AI in the medical diagnosis process (OR = 4.66, 95% CI = 1.05-20.78, p = 0.043). Finally, significant differences (p = 0.003) were observed in determining the liability for legal issues used by AI and ML methods. CONCLUSION: Most pathologists in this study did not use AI or ML models, highlighting the importance of increasing awareness and educational programs regarding applying AI and ML in medical diagnosis.

Low impact of regular PCR testing on presence at work site during the COVID-19 pandemic: experiences during an open observational study in Lower Saxony 2020-21.

BACKGROUND: Since social distancing during the COVID-19-pandemic had a profound impact on professional life, this study investigated the effect of PCR testing on on-site work. METHODS: PCR screening, antibody testing, and questionnaires offered to 4,890 working adults in Lower Saxony were accompanied by data collection on demographics, family status, comorbidities, social situation, health-related behavior, and the number of work-related contacts. Relative risks (RR) with 95 % confidence intervals were estimated for the associations between regular PCR testing and other work and health-related variables, respectively, and working on-site. Analyses were stratified by the suitability of work tasks for mobile office. RESULTS: Between April 2020 and February 2021, 1,643 employees underwent PCR testing. Whether mobile working was possible strongly influenced the work behavior. Persons whose work was suitable for mobile office (mobile workers) had a lower probability of working on-site than persons whose work was not suitable for mobile office (RR = 0.09 (95 % CI: 0.07 - 0.12)). In mobile workers, regular PCR-testing was slightly associated with working on-site (RR = 1.19 (0.66; 2.14)). In those whose working place was unsuitable for mobile office, the corresponding RR was 0.94 (0.80; 1.09). Compared to persons without chronic diseases, chronically ill persons worked less often on-site if their workplace was suitable for mobile office (RR = 0.73 (0.40; 1.33)), but even more often if their workplace was not suitable for mobile office (RR = 1.17 (1.04; 1.33)). CONCLUSION: If work was suitable for mobile office, regular PCR-testing did not have a strong effect on presence at the work site. TRIAL REGISTRATION: An ethics vote of the responsible medical association (Lower Saxony, Germany) retrospectively approved the evaluation of the collected subject data in a pseudonymized form in the context of medical studies (No. Bo/30/2020; Bo/31/2020; Bo/32/2020).

Mobile SARS­CoV­2 screening facilities for rapid deployment and university-based diagnostic laboratory.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic has created a public crisis. Many medical and public institutions and businesses went into isolation in response to the pandemic. Because SARS-CoV-2 can spread irrespective of a patient's course of disease, these institutions' continued operation or reopening based on the assessment and control of virus spread can be supported by targeted population screening. For this purpose, virus testing in the form of polymerase chain reaction (PCR) analysis and antibody detection in blood can be central. Mobile SARS-CoV-2 screening facilities with a built-in biosafety level (BSL)-2 laboratory were set up to allow the testing offer to be brought close to the subject group's workplace. University staff members, their expertise, and already available equipment were used to implement and operate the screening facilities and a certified diagnostic laboratory. This operation also included specimen collection, transport, PCR and antibody analysis, and informing subjects as well as public health departments. Screening facilities were established at different locations such as educational institutions, nursing homes, and companies providing critical supply chains for health care. Less than 4 weeks after the first imposed lockdown in Germany, a first mobile testing station was established featuring a build-in laboratory with two similar stations commencing operation until June 2020. During the 15-month project period, approximately 33,000 PCR tests and close to 7000 antibody detection tests were collected and analyzed. The presented approach describes the required procedures that enabled the screening facilities and laboratories to collect and process several hundred specimens each day under difficult conditions. This report can assist others in establishing similar setups for pandemic scenarios.

PRG3 and PRG5 C-Termini: Important Players in Early Neuronal Differentiation.

The functional importance of neuronal differentiation of the transmembrane proteins' plasticity-related genes 3 (PRG3) and 5 (PRG5) has been shown. Although their sequence is closely related, they promote different morphological changes in neurons. PRG3 was shown to promote neuritogenesis in primary neurons; PRG5 contributes to spine induction in immature neurons and the regulation of spine density and morphology in mature neurons. Both exhibit intracellularly located C-termini of less than 50 amino acids. Varying C-termini suggested that these domains shape neuronal morphology differently. We generated mutant EGFP-fusion proteins in which the C-termini were either swapped between PRG3 and PRG5, deleted, or fused to another family member, plasticity-related gene 4 (PRG4), that was recently shown to be expressed in different brain regions. We subsequently analyzed the influence of overexpression in immature neurons. Our results point to a critical role of the PRG3 and PRG5 C-termini in shaping early neuronal morphology. However, the results suggest that the C-terminus alone might not be sufficient for promoting the morphological effects induced by PRG3 and PRG5.

Differential Cellular Balance of Olfactory and Vomeronasal Epithelia in a Transgenic BACHD Rat Model of Huntington's Disease.

Background. For neurodegenerative diseases such as Huntington's disease (HD), early diagnosis is essential to treat patients and delay symptoms. Impaired olfaction, as observed as an early symptom in Parkinson´s disease, may also constitute a key symptom in HD. However, there are few reports on olfactory deficits in HD. Therefore, we aimed to investigate, in a transgenic rat model of HD: (1) whether general olfactory impairment exists and (2) whether there are disease-specific dynamics of olfactory dysfunction when the vomeronasal (VNE) and main olfactory epithelium (MOE) are compared. Methods. We used male rats of transgenic line 22 (TG22) of the bacterial artificial chromosome Huntington disease model (BACHD), aged 3 days or 6 months. Cell proliferation, apoptosis and macrophage activity were examined with immunohistochemistry in the VNE and MOE. Results. No differences were observed in cellular parameters in the VNE between the groups. However, the MOE of the 6-month-old HD animals showed a significantly increased number of mature olfactory receptor neurons. Other cellular parameters were not affected. Conclusions. The results obtained in the TG22 line suggest a relative stability in the VNE, whereas the MOE seems at least temporarily affected.

Peripheral blood derived endothelial colony forming cells as suitable cell source for pre-endothelialization of arterial vascular grafts under dynamic flow conditions.

In regenerative medicine, autologous peripheral blood derived endothelial colony forming cells (PB-derived ECFC) represent a promising source of endothelial cells (EC) for pre-endothelialization of arterial tissue engineered vascular grafts (TEVG) since they are readily attainable, can easily be isolated and possess a high proliferation potential. The aim of this study was to compare the phenotype of PB-derived ECFC with arterial and venous model cells such as human aortic endothelial cells (HAEC) and human umbilical vein endothelial cells (HUVEC) under dynamic cell culture conditions to find a suitable cell source of EC for pre-endothelialization. In this study PB-derived ECFC were cultivated over 24 h under a high pulsatile shear stress (20 dyn/cm2, 1 Hz) and subsequently analyzed. ECFC oriented and elongated in the direction of flow and expressed similar anti-thrombotic and endothelial differentiation markers compared to HAEC. There were significant differences observable in gene expression levels of CD31, CD34 and NOTCH4 between ECFC and HUVEC. These results therefore suggest an arterial phenotype for PB-derived ECFC both under static and flow conditions, and this was supported by NOTCH4 protein expression profiles. ECFC also significantly up-regulated gene expression levels of anti-thrombotic genes such as krueppel-like factor 2, endothelial nitric oxide synthase 3 and thrombomodulin under shear stress cultivation as compared to static conditions. Dynamically cultured PB-derived ECFC therefore may be a promising cell source for pre-endothelialization of arterial TEVGs.

Plasticity-Related Gene 5 Is Expressed in a Late Phase of Neurodifferentiation After Neuronal Cell-Fate Determination.

During adult neurogenesis, neuronal stem cells differentiate into mature neurons that are functionally integrated into the existing network. One hallmark during the late phase of this neurodifferentiation process is the formation of dendritic spines. These morphological specialized structures form the basis of most excitatory synapses in the brain, and are essential for neuronal communication. Additionally, dendritic spines are affected in neurological disorders, such as Alzheimer's disease or schizophrenia. However, the mechanisms underlying spinogenesis, as well as spine pathologies, are poorly understood. Plasticity-related Gene 5 (PRG5), a neuronal transmembrane protein, has previously been linked to spinogenesis in vitro. Here, we analyze endogenous expression of the PRG5 protein in different mouse brain areas, as well as on a subcellular level. We found that native PRG5 is expressed dendritically, and in high abundance in areas characterized by their regenerative capacity, such as the hippocampus and the olfactory bulb. During adult neurogenesis, PRG5 is specifically expressed in a late phase after neuronal cell-fate determination associated with dendritic spine formation. On a subcellular level, we found PRG5 not to be localized at the postsynaptic density, but at the base of the synapse. In addition, we showed that PRG5-induced formation of membrane protrusions is independent from neuronal activity, supporting a possible role in the morphology and stabilization of spines.

Olfactory Bulb D2/D3 Receptor Availability after Intrastriatal Botulinum Neurotoxin-A Injection in a Unilateral 6-OHDA Rat Model of Parkinson's Disease.

Olfactory deficits occur as early non-motor symptoms of idiopathic Parkinson's disease (PD) in humans. The first central relay of the olfactory pathway, the olfactory bulb (OB), depends, among other things, on an intact, functional crosstalk between dopaminergic interneurons and dopamine receptors (D2/D3R). In rats, hemiparkinsonism (hemi-PD) can be induced by unilateral injection of 6-hydroxydopamine (6-OHDA) into the medial forebrain bundle (MFB), disrupting dopaminergic neurons of the substantia nigra pars compacta (SNpc). In a previous study, we showed that subsequent injection of botulinum neurotoxin-A (BoNT-A) into the striatum can reverse most of the pathological motor symptoms and normalize the D2/D3R availability. To determine whether this rat model is suitable to explain olfactory deficits that occur in humans with PD, we examined the availability of D2/D3R by longitudinal [18F]fallypride-PET/CT, the density of tyrosine hydroxylase immunoreactivity in the OB, olfactory performance by an orienting odor identification test adapted for rats, and a connectome analysis. PET/CT and immunohistochemical data remained largely unchanged after 6-OHDA lesion in experimental animals, suggesting that outcomes of the 6-OHDA hemi-PD rat model do not completely explain olfactory deficits in humans. However, after subsequent ipsilateral BoNT-A injection into the striatum, a significant 8.5% increase of the D2/D3R availability in the ipsilateral OB and concomitant improvement of olfactory performance were detectable. Based on tract-tracing meta-analysis, we speculate that this may be due to indirect connections between the striatum and the OB.

Chronic kidney disease predictors in obese adolescents.

BACKGROUND: Glomerular hyperfiltration, initiating development of obesity-related glomerulopathy, results in an enlargement of the glomeruli and unsealing of the filtration barrier. It can be followed by adaptive focal segmental glomerulosclerosis and chronic kidney disease (CKD). The aim of the study was to determine the expression pattern of lipid metabolism and selected kidney damage markers in obese adolescents and to identify potential factors which can predict CKD. METHODS: The study group consisted of 142 adolescents with a BMI z-score > 2. Sixty-two healthy and normal-weight individuals served as controls. The factors associated with the rate of glomerular filtration in obese adolescents were assessed by linear regression methods using univariate and multivariate analyses. The risk of developing CKD was estimated using the Fisher's exact test. RESULTS: The study group was divided into "elevated," "normal," and "decreased" glomerular filtration rate (GFR) patients. Increased urine galectin-3 (Gal-3) concentration was diagnosed in all patients. "Decreased GFR" subjects expressed increased urine concentration of neutrophil gelatinase-associated lipocalin (NGAL) and daily megalin excretion. Thirty-nine study participants developed CKD. Increased uric acid (UA) concentration was associated with CKD development both in "normal" and "decreased GFR" patients. Additionally, in "normal" GFR patients, increased concentrations of cholesterol (Ch), triglycerides (TG), and NGAL were associated with CKD. CONCLUSIONS: Increased serum concentrations of Ch, TG, and UA and increased urine concentration of NGAL might predict CKD development in obese adolescents with normal and decreased GFR. A higher resolution version of the Graphical abstract is available as Supplementary information.

Organ Weights in NPC1 Mutant Mice Partly Normalized by Various Pharmacological Treatment Approaches.

Niemann-Pick Type C1 (NPC1, MIM 257220) is a rare, progressive, lethal, inherited autosomal-recessive endolysosomal storage disease caused by mutations in the NPC1 leading to intracellular lipid storage. We analyzed mostly not jet known alterations of the weights of 14 different organs in the BALB/cNctr-Npc1m1N/-J Jackson Npc1 mice in female and male Npc1+/+ and Npc1-/- mice under various treatment strategies. Mice were treated with (i) no therapy, (ii) vehicle injection, (iii) a combination of miglustat, allopregnanolone, and 2-hydroxypropyl-ß-cyclodextrin (HPßCD), (iv) miglustat, and (v) HPßCD alone starting at P7 and repeated weekly throughout life. The 12 respective male and female wild-type mice groups were evaluated in parallel. In total, 351 mice (176 Npc1+/+, 175 Npc1-/-) were dissected at P65. In both sexes, the body weights of None and Sham Npc1-/- mice were lower than those of respective Npc1+/+ mice. The influence of the Npc1 mutation and/or sex on the weights of various organs, however, differed considerably. In males, Npc1+/+ and Npc1-/- mice had comparable absolute weights of lungs, spleen, and adrenal glands. In Npc1-/- mice, smaller weights of hearts, livers, kidneys, testes, vesicular, and scent glands were found. In female Npc1-/- mice, ovaries, and uteri were significantly smaller. In Npc1-/- mice, relative organ weights, i.e., normalized with body weights, were sex-specifically altered to different extents by the different therapies. The combination of miglustat, allopregnanolone, and the sterol chelator HPßCD partly normalized the weights of more organs than miglustat or HPßCD mono-therapies.

Fibroblast Growth Factor 21 as a Potential Biomarker for Improved Locomotion and Olfaction Detection Ability after Weight Reduction in Obese Mice.

Obesity is one of the most challenging diseases of the 21st century and is accompanied by behavioural disorders. Exercise, dietary adjustments, or time-restricted feeding are the only successful long-term treatments to date. Fibroblast growth factor 21 (FGF21) plays a key role in dietary regulation, but FGF21 resistance is prevalent in obesity. The aim of this study was to investigate in obese mice whether weight reduction leads to improved behaviour and whether these behavioural changes are associated with decreased plasma FGF21 levels. After establishing a model for diet-induced obesity, mice were subjected to three different interventions for weight reduction, namely dietary change, treadmill exercise, or time-restricted feeding. In this study, we demonstrated that only the combination of dietary change and treadmill exercise affected all parameters leading to a reduction in weight, fat, and FGF21, as well as less anxious behaviour, higher overall activity, and improved olfactory detection abilities. To investigate the interrelationship between FGF21 and behavioural parameters, feature selection algorithms were applied designating FGF21 and body weight as one of five highly weighted features. In conclusion, we concluded from the complementary methods that FGF21 can be considered as a potential biomarker for improved behaviour in obese mice after weight reduction.

Antidepressant-Like Properties of Intrastriatal Botulinum Neurotoxin-A Injection in a Unilateral 6-OHDA Rat Model of Parkinson's Disease.

Parkinson's patients often suffer from depression and anxiety, for which there are no optimal treatments. Hemiparkinsonian (hemi-PD) rats were used to test whether intrastriatal Botulinum neurotoxin-A (BoNT-A) application could also have antidepressant-like properties in addition to the known improvement of motor performance. To quantify depression- and anxiety-like behavior, the forced swim test, tail suspension test, open field test, and elevated plus maze test were applied to hemi-PD rats injected with BoNT-A or vehicle. Furthermore, we correlated the results in the forced swim test, open field test, and elevated plus maze test with the rotational behavior induced by apomorphine and amphetamine. Hemi-PD rats did not show significant anxiety-like behavior as compared with Sham 6-OHDA- + Sham BoNT-A-injected as well as with non-injected rats. However, hemi-PD rats demonstrated increased depression-like behaviors compared with Sham- or non-injected rats; this was seen by increased struggling frequency and increased immobility frequency. Hemi-PD rats intrastriatally injected with BoNT-A exhibited reduced depression-like behavior compared with the respective vehicle-receiving hemi-PD animals. The significant effects of intrastriatally applied BoNT-A seen in the forced swim test are reminiscent of those found after various antidepressant drug therapies. Our data correspond with the efficacy of BoNT-A treatment of glabellar frown lines in treating patients with major depression and suggest that also intrastriatal injected BoNT-A may have some antidepressant-like effect on hemi-PD.

Clinical applicability of optogenetic gene regulation.

The field of optogenetics is rapidly growing in relevance and number of developed tools. Among other things, the optogenetic repertoire includes light-responsive ion channels and methods for gene regulation. This review will be confined to the optogenetic control of gene expression in mammalian cells as suitable models for clinical applications. Here optogenetic gene regulation might offer an excellent method for spatially and timely regulated gene and protein expression in cell therapeutic approaches. Well-known systems for gene regulation, such as the LOV-, CRY2/CIB-, PhyB/PIF-systems, as well as other, in mammalian cells not yet fully established systems, will be described. Advantages and disadvantages with regard to clinical applications are outlined in detail. Among the many unanswered questions concerning the application of optogenetics, we discuss items such as the use of exogenous chromophores and their effects on the biology of the cells and methods for a gentle, but effective gene transfection method for optogenetic tools for in vivo applications.

Gender-Specific Effects of Two Treatment Strategies in a Mouse Model of Niemann-Pick Disease Type C1.

In a mouse model of Niemann-Pick disease type C1 (NPC1), a combination therapy (COMBI) of miglustat (MIGLU), the neurosteroid allopregnanolone (ALLO) and the cyclic oligosaccharide 2-hydroxypropyl-ß-cyclodextrin (HPßCD) has previously resulted in, among other things, significantly improved motor function. The present study was designed to compare the therapeutic effects of the COMBI therapy with that of MIGLU or HPßCD alone on body and brain weight and the behavior of NPC1-/- mice in a larger cohort, with special reference to gender differences. A total of 117 NPC1-/- and 123 NPC1+/+ mice underwent either COMBI, MIGLU only, HPßCD only, or vehicle treatment (Sham), or received no treatment at all (None). In male and female NPC1-/- mice, all treatments led to decreased loss of body weight and, partly, brain weight. Concerning motor coordination, as revealed by the accelerod test, male NPC1-/- mice benefited from COMBI treatment, whereas female mice benefited from COMBI, MIGLU, and HPßCD treatment. As seen in the open field test, the reduced locomotor activity of male and female NPC1-/- mice was not significantly ameliorated in either treatment group. Our results suggest that in NPC1-/- mice, each drug treatment scheme had a beneficial effect on at least some of the parameters evaluated compared with Sham-treated mice. Only in COMBI-treated male and female NPC+/+ mice were drug effects seen in reduced body and brain weights. Upon COMBI treatment, the increased dosage of drugs necessary for anesthesia in Sham-treated male and female NPC1-/- mice was almost completely reduced only in the female groups.

Monokaryotic Pleurotus sapidus Strains with Intraspecific Variability of an Alkene Cleaving DyP-Type Peroxidase Activity as a Result of Gene Mutation and Differential Gene Expression.

The basidiomycete Pleurotus sapidus produced a dye-decolorizing peroxidase (PsaPOX) with alkene cleavage activity, implying potential as a biocatalyst for the fragrance and flavor industry. To increase the activity, a daughter-generation of 101 basidiospore-derived monokaryons (MK) was used. After a pre-selection according to the growth rate, the activity analysis revealed a stable intraspecific variability of the strains regarding peroxidase and alkene cleavage activity of PsaPOX. Ten monokaryons reached activities up to 2.6-fold higher than the dikaryon, with MK16 showing the highest activity. Analysis of the PsaPOX gene identified three different enzyme variants. These were co-responsible for the observed differences in activities between strains as verified by heterologous expression in Komagataella phaffii. The mutation S371H in enzyme variant PsaPOX_high caused an activity increase alongside a higher protein stability, while the eleven mutations in variant PsaPOX_low resulted in an activity decrease, which was partially based on a shift of the pH optimum from 3.5 to 3.0. Transcriptional analysis revealed the increased expression of PsaPOX in MK16 as reason for the higher PsaPOX activity in comparison to other strains producing the same PsaPOX variant. Thus, different expression profiles, as well as enzyme variants, were identified as crucial factors for the intraspecific variability of the PsaPOX activity in the monokaryons.

A pre-conditioning protocol of peripheral blood derived endothelial colony forming cells for endothelialization of tissue engineered constructs.

In regenerative medicine, autologous endothelial colony forming cells (ECFCs) bear the greatest potential to be used for surface endothelialization of tissue engineered constructs, as they are easily attainable and possess a high proliferation rate. The aim of this study was to develop a standardized pre-conditioning protocol under dynamic conditions simulating the physiology of human circulation to improve the formation of a flow resistant monolayer of ECFCs and to enhance the antithrombogenicity of the endothelial cells. The main focus of the study was to consequently compare the cellular behavior under a steady laminar flow against a pulsatile flow. Mononuclear cells were isolated out of peripheral blood (PB) buffy coats and plated on uncoated tissue culture flasks in anticipation of guidelines for Advanced Therapy Medicinal Products. ECFCs were identified by typical surface markers such as CD31, CD146 and VE-Cadherin. To explore the effects of dynamic cultivation, ECFCs and human umbilical vein endothelial cells were comparatively cultured under either laminar or pulsatile (1 Hz) flow conditions with different grades of shear stress (5 dyn/cm2versus 20 dyn/cm2). High shear stress of 20 dyn/cm2 led to a significant upregulation of the antithrombotic gene marker thrombomodulin in both cell types, but only ECFCs orientated and elongated significantly after shear stress application forming a confluent endothelial cell layer. The work therefore documents a suitable protocol to pre-condition PB-derived ECFCs for sustainable endothelialization of blood contacting surfaces and provides essential knowledge for future cultivations in bioreactor systems.