Cell separation by velocity sedimentation of postnatal mouse cerebellum

Trypsinized cells of newborn mouse cerebellum have been separated by velocity sedimentation at unit gravity in shallow gradients of Ficoll. The two main technical difficulties were formation of gels around the dissociated cells and clumping of cells before and during the sedimentation procedure. These were solved by adding DNase to the dissociation medium and with holding serum, respectively. Proliferating cells of the external granular layer separated according to size differences in the cell generation cycle. Identification of Purkinje or other early-forming neurons was made by labeling them with ³H-thymidine on their birthdays. Many of the fractions contain viable cells capable of aggregating in culture.     

Repercussions of the COVID-19 pandemic on athletes: a cross-sectional study

The COVID-19 pandemic has presented significant challenges and implications for the sports community. Thus, this study aimed to describe the prevalence of COVID-19 in Brazilian athletes and identify the epidemiological, clinical, athletic, life and health factors associated with the disease in these individuals. A cross-sectional study was performed involving 414 athletes from 22 different sports using an online questionnaire from August to November 2020. The association between the athletes’ characteristics and COVID-19 was evaluated using a logistic regression model. The prevalence of COVID-19 was 8.5%, although only 40% of athletes reported having been tested. Being under 27 years of age (3-fold), having children (~5-fold), having a teammate test positive for COVID-19 (2.5-fold), and smoking (14-fold) were associated with a possible higher risk of disease. Almost 20% of athletes self-reported musculoskeletal injuries during the period of the pandemic that was studied. Athletes with a university education (P = 0.02), a profession other than sports (P < 0.001), those from a low-income family (P = 0.01), and public health system users (P = 0.04) were significantly less frequently tested for COVID-19, whereas international competitors, athletes who received a wage, and athletes who had a teammate who tested positive for COVID-19 were 2-, 3-, and 15-fold more likely to be tested for COVID-19, respectively. Approximately 26% of the athletes who tested negative or were untested reported more than three characteristic COVID-19 symptoms, and 11% of athletes who tested positive for COVID-19 were asymptomatic. The identification of modifiable (have children, smoking, and teammates positively tested) and non-modifiable (age under 27 years) factors related to COVID-19 in athletes can contribute to implementing surveillance programmes to decrease the incidence of COVID-19 in athletes and its negative impacts in sports.     

Migration patterns and navigation cues of Atlantic salmon post-smolts migrating from 12 rivers through the coastal zones around the Irish Sea

The freshwater phase of the first seaward migration of juvenile Atlantic salmon (Salmo salar) is relatively well understood when compared with our understanding of the marine phase of their migration. In 2021, 1008 wild and 60 ranched Atlantic salmon smolts were tagged with acoustic transmitters in 12 rivers in England, Scotland, Northern Ireland and Ireland. Large marine receiver arrays were deployed in the Irish Sea at two locations: at the transition of the Irish Sea into the North Atlantic between Ireland and Scotland, and between southern Scotland and Northern Ireland, to examine the early phase of the marine migration of Atlantic salmon smolts. After leaving their natal rivers' post-smolt migration through the Irish Sea was rapid with minimum speeds ranging from 14.03 to 38.56 km.day⁻¹ for Atlantic salmon smolts that entered the Irish Sea directly from their natal river, to 9.69–39.94 km.day⁻¹ for Atlantic salmon smolts that entered the Irish Sea directly from their natal estuary. Population minimum migration success through the study area was strongly correlated with the distance of travel, populations further away from the point of entry to the open North Atlantic exhibited lower migration success. Post-smolts from different populations experienced different water temperatures on entering the North Atlantic. This was largely driven by the timing of their migration and may have significant consequences for feeding and ultimately survivorship. The influence of water currents on post-smolt movement was investigated using data from previously constructed numerical hydrodynamic models. Modeled water current data in the northern Irish Sea showed that post-smolts had a strong preference for migrating when the current direction was at around 283° (west-north-west) but did not migrate when exposed to strong currents in other directions. This is the most favorable direction for onward passage from the Irish Sea to the continental shelf edge current, a known accumulation point for migrating post-smolts. These results strongly indicate that post-smolts migrating through the coastal marine environment are: (1) not simply migrating by current following (2) engage in active directional swimming (3) have an intrinsic sense of their migration direction and (4) can use cues other than water current direction to orientate during this part of their migration.     

Interactions between cognitive and sensory load while planning and controlling complex gait adaptations in Parkinson’s disease

Background

Recent research has argued that removal of relevant sensory information during the planning and control of simple, self-paced walking can result in increased demand on central processing resources in Parkinson’s disease (PD). However, little is known about more complex gait tasks that require planning of gait adaptations to cross over an obstacle in PD.

Methods

In order to understand the interaction between availability of visual information relevant for self-motion and cognitive load, the current study evaluated PD participants and healthy controls while walking toward and stepping over an obstacle in three visual feedback conditions: (i) no visual restrictions; (ii) vision of the obstacle and their lower limbs while in complete darkness; (iii) vision of the obstacle only while in complete darkness; as well as two conditions including a cognitive load (with a dual task versus without a dual task). Each walk trial was divided into an early and late phase to examine changes associated with planning of step adjustments when approaching the obstacle.

Results

Interactions between visual feedback and dual task conditions during the obstacle approach were not significant. Patients with PD had greater deceleration and step time variability in the late phase of the obstacle approach phase while walking in both dark conditions compared to control participants. Additionally, participants with PD had a greater number of obstacle contacts when vision of their lower limbs was not available specifically during the dual task condition. Dual task performance was worse in PD compared to healthy control participants, but notably only while walking in the dark regardless of visual feedback.

Conclusions

These results suggest that reducing visual feedback while approaching an obstacle shifts processing to somatosensory feedback to guide movement which imposes a greater demand on planning resources. These results are key to fully understanding why trips and falls occur in those with PD.

     

Multivalent nanobodies targeting death receptor 5 elicit superior tumor cell killing through efficient caspase induction

Multiple therapeutic agonists of death receptor 5 (DR5) have been developed and are under clinical evaluation. Although these agonists demonstrate significant anti-tumor activity in preclinical models, the clinical efficacy in human cancer patients has been notably disappointing. One possible explanation might be that the current classes of therapeutic molecules are not sufficiently potent to elicit significant response in patients, particularly for dimeric antibody agonists that require secondary cross-linking via Fcγ receptors expressed on immune cells to achieve optimal clustering of DR5. To overcome this limitation, a novel multivalent Nanobody approach was taken with the goal of generating a significantly more potent DR5 agonist. In the present study, we show that trivalent DR5 targeting Nanobodies mimic the activity of natural ligand, and furthermore, increasing the valency of domains to tetramer and pentamer markedly increased potency of cell killing on tumor cells, with pentamers being more potent than tetramers in vitro. Increased potency was attributed to faster kinetics of death-inducing signaling complex assembly and caspase-8 and caspase-3 activation. In vivo, multivalent Nanobody molecules elicited superior anti-tumor activity compared to a conventional DR5 agonist antibody, including the ability to induce tumor regression in an insensitive patient-derived primary pancreatic tumor model. Furthermore, complete responses to Nanobody treatment were obtained in up to 50% of patient-derived primary pancreatic and colon tumor models, suggesting that multivalent DR5 Nanobodies may represent a significant new therapeutic modality for targeting death receptor signaling.     

Similar hypotensive responses to resistance exercise with and without blood flow restriction

Low intensity resistance exercise (RE) with blood flow restriction (BFR) has gained attention in the literature due to the beneficial effects on functional and morphological variables, similar to those observed during traditional RE without BFR, while the effects of BFR on post-exercise hypotension remain unclear. The aim of the present study was to compare the blood pressure (BP) response of trained normotensive individuals to RE with and without BFR. In this cross-over randomized trial, eight male subjects (23.8 ± 4 years, 74 ± 3 kg, 174 ± 4 cm) completed two exercise protocols: traditional RE (3 x 10 repetitions at 70% one-repetition maximum [1-RM]) and low intensity RE (3 x 15 repetitions at 20% 1-RM) with BFR. Blood pressure measurements were performed after 15 min of seated rest (0), immediately after and 10 min, 20 min, 30 min, 40 min, 50 min and 60 min after the experimental sessions. Similar hypotensive effects for systolic BP (SBP) were observed for both protocols (P < 0.05) after exercise, with no differences between groups (P > 0.05) and no statistically significant difference for diastolic BP (P > 0.05). These results suggest that in normotensive trained individuals, both traditional RE and RE with BFR induce hypotension for SBP, which is important to prevent cardiovascular disturbances.     

Membrane Potential and Catecholamine Secretion by Bovine Adrenal Chromaffin Cells: Use of Tetraphenylphosphonium Distribution and Carbocyanine Dye Fluorescence

Changes in plasma membrane potential of isolated bovine adrenal chromaffin cells were measured independently by two chemical probe methods and related to corresponding effects on catecholamine secretion. The lipophilic cation tetraphenylphosphonium (TPP+) and the carbocyanine dye 3,3'-dipropylthiadicarbocyanine [DiS-C3-(5)] were used. The necessity of evaluating the subcellular distribution of TPP+ among cytoplasmic, mitochondrial, secretory granule, and bound compartments was demonstrated and the resting plasma membrane potential determined to be -55 mV. The relationship between membrane potential and catecholamine secretion was determined in response to variations in extracellular K+ and to the presence of several secretagogues including cholinergic receptor ligands, veratridine, and ionophores for Na+ and K+. The dependence of potential on K+ concentration fit the Goldman constant field equation with a Na/K permeability ratio of 0.1. The dependence of both K+- and veratridine-evoked catecholamine secretion on membrane potential exhibited a potential threshold of about -40 mV before a significant rise in secretion occurred. This is likely related to the threshold for opening of voltage-sensitive Ca2+ channels. Acetylcholine and nicotine evoked a large secretory response without a sufficiently sustained depolarization to be detectable by the relatively slow potential sensitive chemical probes. Decamethonium induced a detectable depolarization of the chromaffin cells. Veratridine and gramicidin evoked both membrane depolarization and catecholamine release. By contrast the K ionophore valinomycin evoked significant levels of secretion without any depolarization. This is consistent with its utilization of an intracellular source of Ca2+ and the independence of its measured secretory response on extracellular Ca2+.     

Three dimensional typological studies using scanning electron microscopy for characterization of Termitomyces pellets obtained from submerged growth conditions

There are gaps in existing understanding of fungal pellet growth dynamics. We used scanning electron microscopy (SEM) for morphological characterization of the biomass organization of Termitomyces pellets for seven species: T. microcarpus (TMI1), T. albuminosus (TAL1, TAL2), T. striatus (TSTR), T. aurantiacus (TAUR), T. heimii (THE1, THE2), T. globulus (TGLO) and T. clypeatus (TCL1, TCL2, TCL3, TCL4, TCL5). We assessed the utility of SEM for morphological and structural characterization of Termitomyces spp. in three dimensional (3D) pellet form to identify ideal pellet morphology for industrial use. Typological classification of Termitomyces species was based on furrows, isotropy, total motifs and fractal dimensions. The pellets formed were entangled and exhibited highly compacted mycelial mass with microheterogeneity and microporosity. The mean density of furrows of Termitomyces species was between 10,000 and 11,300 cm/cm², percentage isotropy was 30?80 and total motifs varied from 300 to 2500. TGLO exhibited the highest furrow mean density, 11243 cm/cm², which indicated a compact, cerebroid structure with complex ridges and furrows, whereas TAL2 exhibited the lowest furrow density. TMI1a exhibited a high percentage isotropic value, 74.6, TSTR exhibited the lowest, 30.9. Total motif number also was used as a typological classification parameter. Fractal values were 2.64?2.78 for various submerged conditions of Termitomyces species. TAL1 exhibited the highest fractal dimension and TAL2 the lowest, which indicates the complexity of branching patterns. Three-dimensional SEM image analysis can provide insight into pellet micromorphology and is a powerful tool for exploring topographical details of pellets.     

Time-resolved fluorescence studies of tomaymycin bonding to synthetic DNAs.

Tomaymycin reacts covalently with guanine in the DNA minor groove, exhibiting considerable specificity for the flanking bases. The sequence dependence of tomaymycin bonding to DNA was investigated in synthetic DNA oligomers and polymers. The maximum extent of bonding to DNA is greater for homopurine and natural DNA sequences than for alternating purine-pyrimidine sequences. Saturation of DNA with tomaymycin has little effect on the melting temperature in the absence of unbound drug. Fluorescence lifetimes were measured for DNA adducts at seven of the ten unique trinucleotide bonding sites. Most of the adducts had two fluorescence lifetimes, representing two of the four possible binding modes. The lifetimes cluster around 2-3 ns and 5-7 ns; the longer lifetime is the major component for most bonding sites. The two lifetime classes were assigned to R and S diastereomeric adducts by comparison with previous NMR results for oligomer adducts. The lifetime difference between binding modes is interpreted in terms of an anomeric effect on the excited-state proton transfer reaction that quenches tomaymycin fluorescence. Bonding kinetics of polymer adducts were monitored by fluorescence lifetime measurements. Rates of adduct formation vary by two orders of magnitude with poly(dA-dG).poly(dC-dT), reacting the fastest at 4 x 10(-2) M-1 s-1. The sequence specificity of tomaymycin is discussed in light of these findings and other reports in the literature.