Combination of resveratrol and 5-azacytydine improves osteogenesis of metabolic syndrome mesenchymal stem cells.

Endocrine disorders have become more and more frequently diagnosed in humans and animals. In horses, equine metabolic syndrome (EMS) is characterized by insulin resistance, hyperleptinemia, hyperinsulinemia, inflammation and usually by pathological obesity. Due to an increased inflammatory response in the adipose tissue, cytophysiological properties of adipose derived stem cells (ASC) have been impaired, which strongly limits their therapeutic potential. Excessive accumulation of reactive oxygen species, mitochondria deterioration and accelerated ageing of those cells affect their multipotency and restrict the effectiveness of the differentiation process. In the present study, we have treated ASC isolated from EMS individuals with a combination of 5-azacytydine (AZA) and resveratrol (RES) in order to reverse their aged phenotype and enhance osteogenic differentiation. Using SEM and confocal microscope, cell morphology, matrix mineralization and mitochondrial dynamics were assessed. Furthermore, we investigated the expression of osteogenic-related genes with RT-PCR. We also investigated the role of autophagy during differentiation and silenced PARKIN expression with siRNA. Obtained results indicated that AZA/RES significantly enhanced early osteogenesis of ASC derived from EMS animals. Increased matrix mineralization, RUNX-2, collagen type I and osteopontin levels were noted. Furthermore, we proved that AZA/RES exerts its beneficial effects by modulating autophagy and mitochondrial dynamics through PARKIN and RUNX-2 activity.

Excessive Endoplasmic Reticulum Stress Correlates with Impaired Mitochondrial Dynamics, Mitophagy and Apoptosis, in Liver and Adipose Tissue, but Not in Muscles in EMS Horses.

Nowadays, endocrine disorders have become more frequent in both human and veterinary medicine. In horses, reduced physical activity combined with carbohydrate and sugar overload may result in the development of the so-called equine metabolic syndrome (EMS). EMS is characterized by insulin resistance, hyperinsulinemia, elevated blood triglyceride concentrations and usually obesity. Although the phenotypic features of EMS individuals are well known, the molecular mechanism underlying disease development remains elusive. Therefore, in the present study, we analyzed insulin-sensitive tissues, i.e., muscles, liver and adipose tissue in order to evaluate insulin resistance and apoptosis. Furthermore, we assessed mitochondrial dynamics and mitophagy in those tissues, because mitochondrial dysfunction is linked to the development of metabolic syndrome. We established the expression of genes related to insulin resistance, endoplasmic reticulum (ER) stress and mitochondria clearance by mitophagy using RT-PCR and Western blot. Cell ultrastructure was visualized using electron transmission microscopy. The results indicated that adipose tissue and liver of EMS horses were characterized by increased mitochondrial damage and mitophagy followed by triggering of apoptosis as mitophagy fails to restore cellular homeostasis. However, in muscles, apoptosis was reduced, suggesting the existence of a protective mechanism allowing that tissue to maintain homeostasis.

The Effect of Methyl-ß-cyclodextrin on Apoptosis, Proliferative Activity, and Oxidative Stress in Adipose-Derived Mesenchymal Stromal Cells of Horses Suffering from Metabolic Syndrome (EMS).

Methyl-ß-cyclodextrin (MßCD) is a cyclic oligosaccharide, commonly used as a pharmacological agent to deplete membrane cholesterol. In this study, we examined the effect of MßCD on adipose-derived mesenchymal stromal cells (ASCs) isolated form healthy horses (ASCCTRL) and from horses suffering from metabolic syndrome (ASCEMS). We investigated the changes in the mRNA levels of the glucose transporter 4 (GLUT4) and found that MßCD application may lead to a significant improvement in glucose transport in ASCEMS. We also showed that MßCD treatment affected GLUT4 upregulation in an insulin-independent manner via an NO-dependent signaling pathway. Furthermore, the analysis of superoxide dismutase activity (SOD) and reactive oxygen species (ROS) levels showed that MßCD treatment was associated with an increased antioxidant capacity in ASCEMS. Moreover, we indicated that methyl-ß-cyclodextrin treatment did not cause a dysfunction of the endoplasmic reticulum and lysosomes. Thereby, we propose the possibility of improving the functionality of ASCEMS by increasing their metabolic stability.

Cryptophycins: cytotoxic cyclodepsipeptides with potential for tumor targeting.

Cryptophycins are a class of 16-membered highly cytotoxic macrocyclic depsipeptides isolated from cyanobacteria. The biological activity is based on their ability to interact with tubulin. They interfere with microtubule dynamics and prevent microtubules from forming correct mitotic spindles, which causes cell-cycle arrest and apoptosis. Their strong antiproliferative activities with 100-fold to 1000-fold potency compared with those of paclitaxel and vinblastine have been observed. Cryptophycins are highly promising drug candidates, as their biological activity is not negatively affected by P-glycoprotein, a drug efflux system commonly found in multidrug-resistant cancer cell lines and solid tumors. Cryptophycin-52 had been investigated in phase II clinical trials but failed because of its high neurotoxicity. Recently, cryptophycin conjugates with peptides and antibodies have been developed for targeted delivery in tumor therapy. Copyright © 2017 European Peptide Society and John Wiley & Sons, Ltd.

The Cladophora glomerata Enriched by Biosorption Process in Cr(III) Improves Viability, and Reduces Oxidative Stress and Apoptosis in Equine Metabolic Syndrome Derived Adipose Mesenchymal Stromal Stem Cells (ASCs) and Their Extracellular Vesicles (MV's).

This study investigated in vitro effects of freshwater alga Cladophora glomerata water extract enriched during a biosorption process in Cr(III) trivalent chromium and chromium picolinate on adipose-derived mesenchymal stromal stem cells (ASCs) and extracellular microvesicles (MVs) in equine metabolic syndrome-affected horses. Chemical characterisation of natural Cladophora glomerata was performed with special emphasis on: vitamin C, vitamin E, total phenols, fatty acids, free and protein-bound amino acids as well as measured Cr in algal biomass. To examine the influence of Cladophora glomerata water extracts, in vitro viability, oxidative stress factor accumulation, apoptosis, inflammatory response, biogenesis of mitochondria, autophagy in ASCs of EMS and secretory activity manifested by MV release were investigated. For this purpose, various methods of molecular biology and microscopic observations (i.e., immunofluorescence staining, SEM, TEM, FIB observations, mRNA and microRNA expression by RT-qPCR) were applied. The extract of Cladophora glomerata enriched with Cr(III) ions reduced apoptosis and inflammation in ASCs of EMS horses through improvement of mitochondrial dynamics, decreasing of PDK4 expression and reduction of endoplastic reticulum stress. Moreover, it was found, that Cladophora glomerata and Cr(III) induce antioxidative protection coming from enhanced SOD activity Therefore, Cladophora glomerata enriched with Cr(III) ions might become an interesting future therapeutic agent in the pharmacological treatment of EMS horses.

Antioxidant and Anti-Senescence Effect of Metformin on Mouse Olfactory Ensheathing Cells (mOECs) May Be Associated with Increased Brain-Derived Neurotrophic Factor Levels-An Ex Vivo Study.

Metformin, the popular anti-diabetic drug was shown to exert multiple biological effects. The most recent metformin gained attention as an agent that mobilizes endogenous progenitor cells and enhances regenerative potential of organisms, for example by promoting neurogenesis. In the present study, we examined the role of metformin on mouse olfactory ensheathing cells (mOECs) derived from animals receiving metformin for eight weeks at a concentration equal to 2.8 mg/day. The mOECs expanded ex vivo were characterized in terms of their cellular phenotype, morphology, proliferative activity, viability and accumulation of oxidative stress factors. Moreover, we determined the mRNA and protein levels of brain-derived neurotrophic factor (BDNF), distinguishing the secretion of BDNF by mOECs in cultures and circulating serum levels of BDNF. The mOECs used in the experiment were glial fibrillary acidic protein (GFAP) and p75 neurotrophin receptor (p75NTR) positive and exhibited both astrocyte-like and non-myelin Schwann cell-like morphologies. Our results revealed that the proliferation of OECs derived from mice treated with metformin was lowered, when compared to control group. Simultaneously, we noted increased cell viability, reduced expression of markers associated with cellular senescence and a decreased amount of reactive oxygen species. We observed increased mRNA expression of BDNF and its down-stream genes. Obtained results indicate that metformin may exert antioxidant, anti-apoptotic and senolytic action on OECs expanded ex vivo.

Recent approaches for the synthesis of modified cryptophycins.

Cryptophycins are a family of antimitotic depsipeptides with very high cytotoxicity even against multi-drug resistant (MDR) cancer cells. The first representative was isolated from cyanobacteria Nostoc sp. in 1990. Their bioactivity is based on their interaction with the protein tubulin. Cryptophycins were found to induce apoptosis due to inhibition of the microtubule dynamics. Consequently, cryptophycin analogues are considered as potential antitumour agents. Retrosynthetically, cryptophycins can be subdivided into four building blocks, namely units A-D, to be assembled in the total synthesis. Since the discovery of this compound class, numerous synthetic analogues have been designed for structure-activity relationship (SAR) studies. This review gives a critical overview on cryptophycins, while the main focus lies on the synthetic challenges of recently published cryptophycins, together with emerging concepts on cryptophycin bioconjugation and prodrug design.

The PTP1B selective inhibitor MSI-1436 mitigates Tunicamycin-induced ER stress in human hepatocarcinoma cell line through XBP1 splicing modulation.

Protein tyrosine phosphatase PTP1B is considered as a key metabolic enzyme that has been reported to be associated with insulin resistance onset, and underlying cellular metabolic malfunctions, including ER stress and mitochondrial failure. In this study, effects of selective PTP1B inhibition using MSI-1436 on cellular apoptosis, oxidative stress, mitochondrial dysfunction and ER stress have been assessed using an in vitro model of Tunicamycin induced ER stress in HepG2 cell line. Inhibition of PTP1B using MSI-1436 significantly increased cell viability and reduced the number of apoptotic cells as well as the expression of key apoptosis initiators and effectors. MSI-1436 further mitigated ER stress, by downregulating the expression of IRE1, ATF6 and PERK transcripts, all being key ER stress sensors. Interestingly, MSI-1436 inhibited the XBP1 splicing, and thus its UPR-associated transcriptional activity. PTP1B inhibition further enabled to restore proper mitochondrial biogenesis, by improving transmembrane potential, and diminishing intracellular ROS while restoring of endogenous antioxidant enzymes genes expression. PTP1B inhibition using MSI-1436 could improve cellular apoptosis and metabolic integrity through the mitigation of ER and mitochondrial stress signalling pathways, and excessive ROS accumulation. This strategy may be useful for the treatment of metabolic disorders including IR, NAFLD and diabetes.

A Missense Mutation in the Collagen Triple Helix of EDA Is Associated with X-Linked Recessive Hypohidrotic Ectodermal Dysplasia in Fleckvieh Cattle.

Mutations within the ectodysplasin A (EDA) gene have been associated with congenital hypotrichosis and anodontia (HAD/XHED) in humans, mice, dogs and cattle. We identified a three-generation family of Fleckvieh cattle with male calves exhibiting clinical and histopathological signs consistent with an X-linked recessive HAD (XHED). Whole genome and Sanger sequencing of cDNA showed a perfect association of the missense mutation g.85716041G>A (ss2019497443, rs1114816375) within the EDA gene with all three cases following an X-linked recessive inheritance, but normal EDAR and EDARADD. This mutation causes an exchange of glycine (G) with arginine (R) at amino acid position 227 (p.227G>R) in the second collagen triple helix repeat domain of EDA. The EDA variant was associated with a significant reduction and underdevelopment of hair follicles along with a reduced outgrowth of hairs, a complete loss of seromucous nasolabial and mucous tracheal and bronchial glands and a malformation of and reduction in number of teeth. Thermostability of EDA G227R was reduced, consistent with a relatively mild hair and tooth phenotype. However, incisors and canines were more severely affected in one of the calves, which correlated with the presence of a homozygous missense mutation of RNF111 (g.51306765T>G), a putative candidate gene possibly associated with tooth number in EDA-deficient Fleckvieh calves.

Total synthesis and biological evaluation of fluorinated cryptophycins.

Cryptophycins are cytotoxic natural products that exhibit considerable activities even against multi-drug-resistant tumor cell lines. As fluorinated pharmaceuticals have become more and more important during the past decades, fluorine-functionalized cryptophycins were synthesized and evaluated in cell-based cytotoxicity assays. The unit A trifluoromethyl-modified cryptophycin proved to be highly active against KB-3-1 cells and exhibited an IC(50) value in the low picomolar range. However, the replacement of the 3-chloro-4-methoxyphenyl-substituent in unit B by a pentafluorophenyl moiety resulted in a significant loss of activity.

Strategies for Isolating and Propagating Circulating Tumor Cells in Men with Metastatic Prostate Cancer.

Selecting a well-suited method for isolating/characterizing circulating tumor cells (CTCs) is challenging. Evaluating sensitive and specific markers for prostate cancer (PCa)-specific CTC identification and analysis is crucial. We used the CellCollector EpCAM-functionalized system (CC-EpCAM) and evaluated and developed a PCa-functionalized version (CC-PCa); we then compared CTC isolation techniques that exploit the physical and biological properties of CTCs. We established two cohorts of metastatic PCa patients (mPCa; 15 in cohort 1 and 10 in cohort 2). CTC cultivation experiments were conducted with two capturing methods (Ficoll and ScreenCell). The most sensitive detection rates and highest CTC counts were reached with the CC-PCa and ScreenCell system. Patients with ≥5 CTCs isolated with CC-EpCAM had an overall survival (OS) of 0.93 years, and patients with ≥5 CTCs isolated with CC-PCa had an OS of 1.5 years in cohort 1. Nevertheless, we observed the highest sensitivity and specificity for 24-month survival by the Ficoll with CD45 depletion and ScreenCell system with May-Grunwald Giemsa (MGG) staining. The EpCAM molecule is an essential factor related to OS for CTC isolation based on biological properties in mPCa patients. The best-suited CTC capture system is not limited to one characteristic of cells but adapted to downstream analysis.

Copper impurity of iron raw material contributes to improved cell culture performance.

Cell culture medium (CCM) formulations are chemically defined to reduce lot-to-lot variability and complexity of the medium while still providing all essential nutrients supporting cell growth and productivity of various cell lines. However, raw material impurities may still introduce variations and inconsistencies to final CCM formulations. In one of our previous studies (Weiss et al. Biotechnol Prog. 2021;37(4):e3148), we have demonstrated the impact of iron raw material impurity on Chinese hamster ovary (CHO) cell performance and critical quality attributes (CQAs) of recombinant proteins within the Cellvento® 4CHO CCM platform by identifying manganese impurity as the main root cause for improved cell performance and altered glycosylation profiles. This study sought to investigate the impact of iron raw material impurities within another medium platform, namely EX-CELL® Advanced CHO Fed-Batch-Medium. As opposed to previously published results, in this platform, copper instead of manganese impurity present within the used ferric ammonium citrate (FAC) iron source was responsible for an improved cell performance of a CHOZN® cell line and a slight difference in CQAs of the produced recombinant protein. The use of tightly controlled raw material specifications or the use of low impurity iron sources is therefore crucial to minimize the impact of impurities on cell performance in any CCM platform and thereby guarantee consistent and reproducible cell culture processes.

Synthesis and application of a chiral diborate.

A chiral diborate with different counterions is reported whose structure was unambiguously confirmed by X-ray analysis. This chiral dianion was used in the resolution of trans-1,2-diamines and also as a chiral shift reagent for NMR analysis of triphenylphosphonium salts.

Impact of iron raw materials and their impurities on CHO metabolism and recombinant protein product quality.

Cell culture medium (CCM) composition affects cell growth and critical quality attributes (CQAs) of monoclonal antibodies (mAbs) and recombinant proteins. One essential compound needed within the medium is iron because of its central role in many cellular processes. However, iron is also participating in Fenton chemistry leading to the formation of reactive oxygen species (ROS) causing cellular damage. Therefore, this study sought to investigate the impact of iron in CCM on Chinese hamster ovary (CHO) cell line performance, and CQAs of different recombinant proteins. Addition of either ferric ammonium citrate (FAC) or ferric citrate (FC) into CCM revealed major differences within cell line performance and glycosylation pattern, whereby ammonium was not involved in the observed differences. Inductively coupled plasma mass spectrometry (ICP-MS) analysis identified varying levels of impurities present within these iron sources, and manganese impurity rather than iron was proven to be the root cause for increased cell growth, titer, and prolonged viability, as well as altered glycosylation levels. Contrary effects on cell performance and protein glycosylation were observed for manganese and iron. The use of low impurity iron raw material is therefore crucial to control the effect of iron and manganese independently and to support and guarantee consistent and reproducible cell culture processes.

[Investigations on suckling piglets castrated under automated isoflurane anesthesia]. / Untersuchungen zur automatisierten Isoflurannarkose bei der Saugferkelkastration.

OBJECTIVE: Investigation of defensive reactions, piglet losses, post-bleeding and recovery time of suckling piglets castrated under automated isoflurane anesthesia as well as measurements of isoflurane concentrations in ambient air and estimation of the cleaning and disinfection success of anesthesia masks. MATERIAL AND METHODS: A total of 955 suckling piglets (age: 4.0 ± 1.2 days; weight: 2.0 ± 0.5 kg) were castrated under automated isoflurane anesthesia (PorcAnest 3000®) after the administration of a non-steroidal anti-inflammatory drug. Reactions during the procedure were assessed before castration using the interdigital claw reflex and during castration by defensive movements and vocal response. The piglets' recovery time was evaluated in the stable corridor (pass 1) and the farrowing pen (passes 4-5). In 73 animals, the spermatic cord was cut with a scalpel and the post-operative bleeding visually evaluated. In addition, exposure measurements of isoflurane were performed and anesthetic masks were examined for total bacterial contamination and indicator bacteria before and after the anesthetic procedure and following their disinfection. RESULTS: Following insufflation of isoflurane for 90 seconds 94.3 % of the piglets and after prior testing of the interdigital claw reflex and possible extension of the anesthetic supply 95.3 % of the piglets showed no or minimal defensive movements during castration. An anesthetic incident occurred in 0.9 % of the piglets, but no animal died. The recovery time lasted 7.3 ± 4.7 minutes (pass 1) and 6.2 ± 3.3 minutes (passes 4-5). Cutting the spermatic cord with a scalpel led to a higher post-bleeding score (p < 0.001) compared to use of the emasculator. Isoflurane concentrations in ambient air ranged between 4.5 und 28.1 mg/m3. Masks disinfection reduced the total germ count by 99.8 %. Contaminations with Escherichia coli and MRSA were no longer detectable in 4 of 6 cases after disinfection. CONCLUSION: Isoflurane anesthesia led in over 94 % of the piglets to no or minimal defensive reactions during castration. Anesthetic incidents occurred rarely and no piglet losses were recorded. Therefore, automated isoflurane anesthesia is associated with a low risk for suckling piglets. Measurements of isoflurane concentrations on persons involved were below the internationally lowest limit value. Disinfection of the anesthesia masks may prevent germ transmission between animal groups via this potential vector.

Approaches for the synthesis of functionalized cryptophycins.

The first syntheses of bioactive cryptophycins functionalized at unit D were accomplished in a one-pot Staudinger reduction/cyclization step. An azido precursor for the lower part of the backbone was introduced to minimize protective group chemistry and enable a very convenient synthesis of cryptophycin-52 and unit D cryptophycin analogues containing an ester or a free carboxylic acid for bioconjugations. Both new cryptophycin derivatives show high biological activity in cytotoxicity assays.

Influences of Immunocastration on Endocrine Parameters, Growth Performance and Carcass Quality, as Well as on Boar Taint and Penile Injuries.

Castration of male pigs without anesthesia is a significant welfare issue. Improvac®, a GnRH vaccine induces an endogenous immune response leading to a decrease in testicular steroids. Consequences of different vaccination schemes on testicular function and carcass quality were evaluated in immunocastrated boars (IC), surgical castrates (SC), and entire males (EM). Therefore, 128 male piglets were assigned to five treatment-groups and a long term follow-up group. IC groups received two vaccinations (V1, V2) with Improvac® at 8 and 12, 12 and 16, or 12 and 18 weeks. Testosterone-concentrations decreased significantly two weeks after V2 in feces and dropped in serum from V2 to slaughter (S) except IC-8/12 without differing significantly. GnRH-binding results indicated the highest values for IC-12/18 animals. While IC-12/16 and IC-12/18 animals showed boar taint compounds below the threshold levels, two IC-8/12 animals had concentrations above the threshold level. Feed-efficiency was higher in EM than in SC with IC in between. In IC compared to EM, a decreasing amount of polyunsaturated-fatty-acids was obvious and GnRH-vaccination reduced penile injuries. The examined vaccination protocols reduce penile injuries, improve feed efficiency and carcass quality, and reliably prevents boar taint, if manufacturer's recommendations concerning vaccination schedules are applied. Therefore immunocastration offers a reliable, animal friendly alternative to surgical castration.

Local Anesthesia in Piglets Undergoing Castration-A Comparative Study to Investigate the Analgesic Effects of Four Local Anesthetics Based on Defensive Behavior and Side Effects.

The aim of the present study was to investigate the effect of four local anesthetics on pain relief during surgical castration under standardized conditions in conscious piglets. Therefore, 71 male piglets (three to seven days) were distributed into control groups (handling, castration without anesthesia or analgesia) and local anesthetic trial groups (procaine, lidocaine, bupivacaine, mepivacaine). Then, 20 min prior to castration, animals of the treatment groups, except piglets in the handling group, received an injection of a local anesthetic or sodium chloride of 0.5 mL intratesticularly and 0.5 mL subscrotally. During injection and castration, defensive behavior was evaluated. Locomotor activity, as well as postoperative bleeding, wound healing and average daily weight gain were assessed to detect side effects. The injection caused increased defensive movements, significantly in the bupivacaine group. Lidocaine and mepivacaine significantly reduced defensive movements during castration, and procaine and bupivacaine only during severing of the spermatic cord. Impairments of locomotor activity were found in piglets injected with lidocaine, bupivacaine or sodium chloride. Considering healing, bleeding and weight gain, no negative impacts were observed. In conclusion, lidocaine and mepivacaine were able to achieve significant pain relief during the castration procedure, whereas procaine and bupivacaine only during the severing of the spermatic cord. Moreover, the injection of bupivacaine seemed to be painful itself.

Local anesthesia in piglets undergoing castration-A comparative study to investigate the analgesic effects of four local anesthetics on the basis of acute physiological responses and limb movements.

Surgical castration of male piglets without analgesia is a painful procedure. This prospective, randomized and double-blinded study aimed to evaluate the analgesic effects of four different local anesthetics for piglet castration during the first week of life. In total, 54 piglets aged 3 to 7 days were distributed into 6 treatment groups: handling (H); castration without pain relief (sodium chloride, NaCl); and castration with a local anesthetic: 4% procaine (P), 2% lidocaine (L), 0.5% bupivacaine (B) or 20 mg/ml mepivacaine (M). By excluding stress and fear as disruptive factors via a minimum anesthesia model, all piglets received individual minimum alveolar concentration (MAC) isoflurane anesthesia. Twenty minutes before castration, all treatment groups except group H received one injection per testis. Then, 0.5 ml of a local anesthetic or NaCl was injected intratesticularly (i.t.), and 0.5 ml was administered subscrotally. Acute physiological responses to noxious stimuli at injection and castration were evaluated by measuring blood pressure (BP), heart rate (HR), cortisol, epinephrine, norepinephrine and chromogranin A (CgA); limb movements were quantified. The results confirm that castration without analgesia is highly painful. Surgical castration without pain relief revealed significant changes in mean arterial blood pressure (MAP) and HR. Local anesthetic administration significantly reduced changes in BP and HR associated with castration. Piglets receiving a preoperative local anesthetic exhibited the fewest limb movements during castration, while the NaCl group exhibited the most. Injection itself was not associated with significant changes in MAP or HR. However, many piglets exhibited limb movements during injection, indicating that the injection itself causes nociceptive pain. No significant differences were found between groups regarding parameters of plasma cortisol, catecholamines and CgA. In conclusion, all four local anesthetics administered are highly effective at reducing signs of nociception during castration under light isoflurane anesthesia. However, injection of a local anesthetic seems to be painful.