Testing anabolic activity, potency and mechanisms of action of the phyto-derived beta 2 agonist higenamine.

Higenamine (Hige), a plant derived alkaloid is classified as ß2 agonist by the World Anti-Doping Agency (WADA). However, pharmacologic mechanisms of its performance-enhancing activity have not been investigated so far. Therefore, we investigate the anabolic activity and associated molecular mechanisms of Hige in C2C12 myotubes. In differentiated C2C12 cells dose-dependent effects of Hige on myotube size were analyzed. The mRNA expression of genes involved in hypertrophy was measured. For mechanistic studies, ß2-adrenoceptor (ADRB2), androgen receptor (AR), and estrogen receptor (ER) inhibitors and dexamethasone (Dexa) were co-incubated and myotube diameter was evaluated. The interaction of Hige with the AR and ER was investigated. Hige treatment significantly increased myotube diameters and stimulated the mRNA expression of hypertrophy-involved genes. In contrast to the ADRB2 inhibitor (ICI 118551), the ER inhibitor ZK 191703, the AR inhibitor Flutamide (Flu), and treatment with Dexa were able to antagonize the Hige-induced increase of myotube diameter. Hige has antagonistic activity in the AR and ER yeast transactivation assay. Our results demonstrate that Hige induces anabolic effects in C2C12 cells but not via the ADRB2. There are indications for a cross talk between Hige and the AR and ER. Future studies are necessary to investigate the involved molecular mechanisms.

Combinatory in vitro effects of the ß2-agonists salbutamol and formoterol in skeletal muscle cells.

ß2-agonists are used for the treatment of bronchoconstriction, but also abused in doping. Beside an ergogenic activity ß2-agonists may have also anabolic activity. Therefore, we investigated the anabolic activity and associated molecular mechanisms of Salbutamol (SAL) and Formoterol (FOR) alone, as well as in combination in C2C12 myotubes. In differentiated C2C12 cells, dose-dependent effects of SAL and FOR (alone/in combination) on myotube diameter, myosin heavy chain (MHC) protein expression and the mRNA expression of genes involved in hypertrophy were analyzed. ß2-adrenoceptor 2 (ADRB2), androgen receptor (AR) and estrogen receptor (ER) inhibitors, as well as dexamethasone (Dexa) were co-incubated with the ß2-agonists and myotube diameter was determined. SAL and FOR treatment significantly induced hypertrophy and increased MHC expression and the mRNA expression of Igf1, mTOR, PIk3r1 and AMpKa2. In contrast to an ER inhibitor, the ADRB2 and AR inhibitors, as well as Dexa antagonized FOR and SAL induced hypertrophy. Combined treatment with SAL and FOR resulted in significant additive effects on myotube diameter and MHC expression. Future clinical studies are needed to prove this effect in humans and to evaluate this finding with respect to antidoping regulations.

Resuscitation highlights in 2021.

BACKGROUND: This review is the latest in a series of regular annual reviews undertaken by the editors and aims to highlight some of the key papers published in Resuscitation during 2021. METHODS: Hand-searching by the editors of all papers published in Resuscitation during 2021. Papers were selected based on then general interest and novelty and were categorised into themes. RESULTS: 98 papers were selected for brief mention. CONCLUSIONS: Resuscitation science continues to evolve and incorporates all links in the chain of survival.

Resuscitation highlights in 2020.

BACKGROUND: This review is the latest in a series of regular annual reviews undertaken by the editors and aims to highlight some of the key papers published in Resuscitation during 2020. The number of papers submitted to the Journal in 2020 increased by 25% on the previous year.MethodsHand-searching by the editors of all papers published in Resuscitation during 2020. Papers were selected based on then general interest and novelty and were categorised into general themes.ResultsA total of 103 papers were selected for brief mention in this review.ConclusionsResuscitation science continues to evolve rapidly and incorporate all links in the chain of survival.

Airway management during in-hospital cardiac arrest: An international, multicentre, retrospective, observational cohort study.

AIM: To determine the type of airway devices used during in-hospital cardiac arrest (IHCA) resuscitation attempts. METHODS: International multicentre retrospective observational study of in-patients aged over 18 years who received chest compressions for cardiac arrest from April 2016 to September 2018. Patients were identified from resuscitation registries and rapid response system databases. Data were collected through review of resuscitation records and hospital notes. Airway devices used during cardiac arrest were recorded as basic (adjuncts or bag-mask), or advanced, including supraglottic airway devices, tracheal tubes or tracheostomies. Descriptive statistics and multivariable regression modelling were used for data analysis. RESULTS: The final analysis included 598 patients. No airway management occurred in 36 (6%), basic airway device use occurred at any time in 562 (94%), basic airway device use without an advanced airway device in 182 (30%), tracheal intubation in 301 (50%), supraglottic airway in 102 (17%), and tracheostomy in 1 (0.2%). There was significant variation in airway device use between centres. The intubation rate ranged between 21% and 90% while supraglottic airway use varied between 1% and 45%. The choice of tracheal intubation vs. supraglottic airway as the second advanced airway device was not associated with immediate survival from the resuscitation attempt (odds ratio 0.81; 95% confidence interval 0.35-1.8). CONCLUSION: There is wide variation in airway device use during resuscitation after IHCA. Only half of patients are intubated before return of spontaneous circulation and many are managed without an advanced airway. Further investigation is needed to determine optimal airway device management strategies during resuscitation following IHCA.

Metabolism of formestane in humans: Identification of urinary biomarkers for antidoping analysis.

Formestane (4-hydroxyandrost-4-ene-3,17-dione, 4OH-AED) is an aromatase inhibitor prohibited in sports. In recent years, it has been demonstrated that it can also originate endogenously by the hydroxylation in C4 position of androstenedione. Thus, the use of isotope ratio mass spectrometry (IRMS) is mandatory according to the World Antidoping Agency (WADA) to discriminate endogenous from synthetic origin. In a previous work and after oral administrations of formestane (4OH-AED), the ratio between the main formestane metabolite (4α-hydroxyepiandrosterone; 4OH-EA) and formestane parent compound could help to identify the endogenous origin, avoiding unnecessary and costly IRMS confirmations. In the present work, we investigated whether the same criteria could also be applied after transdermal applications. Six volunteers were transdermally treated once with formestane. Urine samples were collected for 120 h postadministration and analyzed by gas chromatography coupled to mass spectrometry (GC-MS and GC-MS/MS). Formestane and its major metabolites were monitored. The kinetic profile of formestane and its main metabolites was found different between oral and transdermal application. A shift on the excretion of the metabolites compared to formestane itself that can be observed after the oral administration, is absent after the transdermal one. This makes that a simple criteria cannot be applied to differentiate the endogenous from the synthetic origin based on metabolic ratios. The ratio between 4-hydroxyepiandrosterone and 4-hydroxyandrosterone (4OH-A) can be used to differentiate the route of administration. Ratios higher than one (4OH-EA/4OH-A > 1) are diagnostic of an oral administration. This allows to correctly interpret the 4OH-EA/4OH-AED ratio as proposed in our previous investigation. The results of this work demonstrate that the use of appropriate biomarkers (metabolic ratios) helps to reach correct conclusions without using complex and costly instrumentation approaches.

Effect of dietary n-3 polyunsaturated fatty acid supplementation and post-insemination plane of nutrition on systemic concentrations of metabolic analytes, progesterone, hepatic gene expression and embryo development and survival in beef heifers.

Nutrition, and particularly dietary energy intake, plays a fundamental role in reproductive function in cattle. There is some evidence that supplemental omega-3 dietary polyunsaturated fatty acids (n-3 PUFA) can exert positive effects on fertility. The objectives of this study were to evaluate the effect of dietary n-3 PUFA supplementation, post-insemination energy plane of nutrition and their interaction on embryo survival in cattle. Crossbred beef heifers (n = 185) were individually offered barley straw ad libitum and 6 kg DM of concentrate supplemented with either a rumen-protected source of saturated fatty acid (palmitic; control, CON) or a partially rumen-protected n-3 PUFA-enriched supplement (n-3 PUFA). Estrous was synchronised using two injections of PG administered at 11-d intervals and following artificial insemination (AI = Day 0) 179 heifers exhibiting oestrus were inseminated and assigned to one of two dietary treatments: (i) remain on their pre-insemination high dietary plane of nutrition (High) or (ii) restricted to 0.6 × estimated maintenance energy requirements (Low) in a 2 × 2 factorial design. The heifers were then maintained on their assigned diets until slaughter and embryo recovery on Day 16 (n = 92) or pregnancy diagnosis by ultrasound scanning at Day 30 post-AI (n = 87). Plasma concentrations of fatty acids, metabolites, insulin, progesterone (P4) and insulin-like growth factor 1 (IGF-1) were measured at appropriate intervals. Hepatic expression of mRNA for aldo-keto reductase (AKR1C), cytochrome P450 2C (CYP 2C) and cytochrome P450 3A (CYP 3A) was examined. The n-3 PUFA supplementation increased plasma n-3 PUFA concentration (P < 0.05) and reduced n-6: n-3 PUFA ratio (P < 0.05). Plasma IGF-1 was higher for n-3 PUFA relative to the CON (P < 0.05) and for High compared with Low plane of nutrition post-AI (P < 0.05) groups. A low plane of nutrition post-AI increased plasma concentrations of progesterone from Days 7-16 after insemination (P < 0.001) but reduced embryo length (P < 0.001). Supplementation with n-3 PUFA reduced and tended to reduce hepatic expression of CYP2C (P = 0.01) and CYP3A (P = 0.08), respectively. However, while dietary n-3 PUFA supplementation and an abrupt reduction in nutrient status following insemination elevated plasma concentrations of n-3 PUFA and mid and late phase P4, respectively, there was no effect of either PUFA supplementation or post-insemination plane of nutrition on embryo survival.

Rational Clinical Pathology Assessment in the Intensive Care Unit.

Blood tests are ordered on a daily basis in intensive care units (ICU). There are no widely accepted guidelines for testing requirements. This study investigated the impact on ICU laboratory test costs of a multi-strategy change in practice involving routine blood testing. A single centre, prospective, interventional study using historical controls was undertaken to investigate the impact of ICU specialist authorisation of high-volume routine tests on ICU laboratory test costs. Prior to commencement of the study, ICU nursing and junior ICU doctors were able to order tests. During the six-month intervention period, the ICU specialists authorised routine blood tests. Adverse events related to not performing blood tests were also recorded. Overall ICU laboratory test costs decreased by 12.3% over the six months (P=0.0022 versus historical control) with a mean compliance of 51% with the test authorisation protocol. The costs of frequently ordered tests (classified as high-volume) decreased by 20% (P=0.0022 versus historical control). These accounted for an average of 54 ± 3% of the overall ICU blood test costs (blood gas analyses 17%, simple chemistry tests consisting of electrolytes, liver function, calcium, phosphate, magnesium 14%, coagulation 12% and full blood count 11%). Two protocol-related adverse events were recorded and judged as minor and were resolved by ordering tests during the day. No adverse patient outcomes resulted from these two events. Blood testing authorisation by an ICU specialist was associated with significant cost savings in ICU and no adverse patient outcomes.

Establishment of critical timing of progesterone supplementation on corpus luteum and embryo development in beef heifers.

Optimal concentrations of progesterone (P4) during early pregnancy are a major determinant of embryo survival in cattle. This study examined the effects of P4 supplementation, following a period of induced low P4, on corpus luteum (CL) development, circulating P4 concentrations and embryo development. A total of 107 beef heifers from one herd were used in the study. Following AI (Day 0) at a synchronized oestrus, heifers were randomly assigned to 1 of 5 treatment groups: (1) no subsequent treatment (control; n=15); (2) administration of a synthetic prostaglandin F2α analogue (PG) on Days 3, 3.5 and 4 to induce low P4 and no further treatment (LP4; n=23); administration of PG as above followed by P4 supplementation via insertion of a Controlled Internal Drug Release device from (3) Day 4-7 (P4 d4-7; n=22); (4) Day 4-10 (P4 d4-10; n=23); or (5) Day 7-10 (P4 d7-10; n=24). Embryo and CL characteristics were determined following slaughter on Day 16. Embryo size was greater in heifers administered P4 d4-7 and P4 d4-10 in comparison with LP4. Corpus luteum area was significantly reduced in heifers administered P4 d4-7 and P4 d4-10 up to Day 7, and Day 10 (P4 d4-10), in comparison with LP4 heifers. Initiation of supplemental P4 on Day 7 had no effect on CL area or embryo size in comparison with LP4 heifers. Concentrations of P4 were less in P4 d4-7, P4 d7-10 and P4 d4-10 on Day 15 in comparison with LP4 heifers. The results of the study indicate an initiation and duration effect of supplemental P4 on circulating P4, embryo and CL development following induction of LP4 on Day 3 and 4.

Zero-valent Fe confined mesoporous silica nanocarriers (Fe(0) @ MCM-41) for targeting experimental orthotopic glioma in rats.

Mesoporous silica nanoparticles (MSNs) impregnated with zero-valent Fe (Fe(0) @ MCM-41) represent an attractive nanocarrier system for drug delivery into tumor cells. The major goal of this work was to assess whether MSNs can penetrate the blood-brain barrier in a glioblastoma rat model. Synthesized MSNs nanomaterials were characterized by energy dispersive X-ray spectroscopy, measurements of X-ray diffraction, scanning electron microscopy and Mössbauer spectroscopy. For the detection of the MSNs by MR and for biodistribution studies MSNs were labeled with zero-valent Fe. Subsequent magnetometry and nonlinear-longitudinal-response-M2 (NLR-M2) measurements confirmed the MR negative contrast enhancement properties of the nanoparticles. After incubation of different tumor (C6 glioma, U87 glioma, K562 erythroleukemia, HeLa cervix carcinoma) and normal cells such as fibroblasts and peripheral blood mononuclear cells (PBMCs) MSNs rapidly get internalized into the cytosol. Intracellular residing MSNs result in an enhanced cytotoxicity as Fe(0) @ MCM-41 promote the reactive oxygen species production. MRI and histological studies indicated an accumulation of intravenously injected Fe(0) @ MCM-41 MSNs in orthotopic C6 glioma model. Biodistribution studies with measurements of second harmonic of magnetization demonstrated an increased and dose-dependent retention of MSNs in tumor tissues. Taken together, this study demonstrates that MSNs can enter the blood-brain barrier and accumulate in tumorous tissues.

Pregnancy losses in cattle: potential for improvement.

For heifers, beef and moderate-yielding dairy cows, it appears that the fertilisation rate generally lies between 90% and 100%. For high-producing dairy cows, there is a less substantive body of literature, but it would appear that the fertilisation rate is somewhat lower and possibly more variable. In cattle, the major component of embryo loss occurs in the first 16 days following breeding (Day 0), with emerging evidence of greater losses before Day 8 in high-producing dairy cows. In cattle, late embryo mortality causes serious economic losses because it is often recognised too late to rebreed females. Systemic concentrations of progesterone during both the cycle preceding and following insemination affect embryo survival, with evidence of either excessive or insufficient concentrations being negatively associated with survival rate. The application of direct progesterone supplementation or treatments to increase endogenous output of progesterone to increase embryo survival cannot be recommended at this time. Energy balance and dry matter intake during the first 4 weeks after calving are critically important in determining pregnancies per AI when cows are inseminated at 70-100 days after calving. Level of concentrate supplementation of cows at pasture during the breeding period has minimal effects on conception rates, although sudden reductions in dietary intake should be avoided. For all systems of milk production, more balanced breeding strategies with greater emphasis on fertility and feed intake and/or energy must be developed. There is genetic variability within the Holstein breed for fertility traits, which can be exploited. Genomic technology will not only provide scientists with an improved understanding of the underlying biological processes involved in fertilisation and the establishment of pregnancy, but also, in the future, could identify genes responsible for improved embryo survival. Such information could be incorporated into breeding objectives in order to increase the rate of genetic progress for embryo survival. In addition, there is a range of easily adoptable management factors, under producer control, that can either directly increase embryo survival or ameliorate the consequences of low embryo survival rates. The correction of minor deficits in several areas can have a substantial cumulative positive effect on herd reproductive performance.

Ecdysteroids: A novel class of anabolic agents?

Increasing numbers of dietary supplements with ecdysteroids are marketed as "natural anabolic agents". Results of recent studies suggested that their anabolic effect is mediated by estrogen receptor (ER) binding. Within this study the anabolic potency of ecdysterone was compared to well characterized anabolic substances. Effects on the fiber sizes of the soleus muscle in rats as well the diameter of C2C12 derived myotubes were used as biological readouts. Ecdysterone exhibited a strong hypertrophic effect on the fiber size of rat soleus muscle that was found even stronger compared to the test compounds metandienone (dianabol), estradienedione (trenbolox), and SARM S 1, all administered in the same dose (5 mg/kg body weight, for 21 days). In C2C12 myotubes ecdysterone (1 µM) induced a significant increase of the diameter comparable to dihydrotestosterone (1 µM) and IGF 1 (1.3 nM). Molecular docking experiments supported the ERß mediated action of ecdysterone. To clarify its status in sports, ecdysterone should be considered to be included in the class "S1.2 Other Anabolic Agents" of the list of prohibited substances of the World Anti-Doping Agency.

The concurrent and carry over effects of long term changes in energy intake before insemination on pregnancy per artificial insemination in heifers.

Follicle development in a period of negative energy balance (NEB), as experienced by the postpartum dairy cow, could be affected by undesirable metabolic changes, and may contain a developmentally incompetent oocyte with an impaired potential to establish a pregnancy. A differential feeding model in heifers was developed to evaluate the concurrent and carryover effects of reduced energy intake before insemination on pregnancy per artificial insemination (P/AI). Heifers were randomly assigned to either (i) control feed intake group (CF), n=68, 1.3 times estimated maintenance energy (M) requirements for 50 days and 2.0 M for 83 days or (ii) restricted feed intake (RF), n=88, 0.65 M for 50 days and 2.0 M for 83 days. Pregnancy per AI was determined by transrectal ultrasonography at day 30 following AI. Despite significant loss of live weight (LW; 5.8±2.1 vs 70.5±2.8 kg, respectively) and body condition score (BCS; 0.05±0.03 vs 0.45±0.03) and a significant elevation in systemic concentrations of non-esterified fatty acids in RF heifers, there was no concurrent effect on P/AI (69 vs 72%) following AI at day 50. However, there was a carryover effect on P/AI as there was an 18 percentage point difference (64 vs 82%) between CF and RF heifers following AI on day 93. The results of the study indicate that a reduction in energy intake for a 50-day period pre-insemination had no concurrent effect but had a positive carryover effect on P/AI.