Does Higher Intensity Increase the Rate of Responders to Endurance Training When Total Energy Expenditure Remains Constant? A Randomized Controlled Trial.

BACKGROUND: Standardized training prescriptions often result in large variation in training response with a substantial number of individuals that show little or no response at all. The present study examined whether the response in markers of cardiorespiratory fitness (CRF) to moderate intensity endurance training can be elevated by an increase in training intensity. METHODS: Thirty-one healthy, untrained participants (46 ± 8 years, BMI 25.4 ± 3.3 kg m-2 and [Formula: see text]O2max 34 ± 4 mL min-1 kg-1) trained for 10 weeks with moderate intensity (3 day week-1 for 50 min per session at 55% HRreserve). Hereafter, the allocation into two groups was performed by stratified randomization for age, gender and VO2max response. CON (continuous moderate intensity) trained for another 16 weeks at moderate intensity, INC (increased intensity) trained energy-equivalent for 8 weeks at 70% HRreserve and then performed high-intensity interval training (4 × 4) for another 8 weeks. Responders were identified as participants with VO2max increase above the technical measurement error. RESULTS: There was a significant difference in [Formula: see text]O2max response between INC (3.4 ± 2.7 mL kg-1 min-1) and CON (0.4 ± 2.9 mL kg-1 min-1) after 26 weeks of training (P = 0.020). After 10 weeks of moderate training, in total 16 of 31 participants were classified as VO2max responders (52%). After another 16 weeks continuous moderate intensity training, no further increase of responders was observed in CON. In contrast, the energy equivalent training with increasing training intensity in INC significantly (P = 0.031) increased the number of responders to 13 of 15 (87%). The energy equivalent higher training intensities increased the rate of responders more effectively than continued moderate training intensities (P = 0.012). CONCLUSION: High-intensity interval training increases the rate of response in VO2max to endurance training even when the total energy expenditure is held constant. Maintaining moderate endurance training intensities might not be the best choice to optimize training gains. Trial Registration German Clinical Trials Register, DRKS00031445, Registered 08 March 2023-Retrospectively registered, https://www.drks.de/DRKS00031445.

[Pandemic relief volunteers-increasing resilience for disasters]. / Pandemienotfallhelfer ­ gestärkte Resilienz für den Katastrophenfall.

Background: The corona pandemic demands new solutions from our health care system in order to expand treatment capacities in a resilient manner within a short period of time. The last stage of expansion is disaster protection, the resilience of which can also be improved by volunteers. However, these spontaneous volunteers require training in order to be integrated into the disaster relief structures. Methods: In a step-by-step process, an ad hoc expert panel developed a curriculum for pandemic relief volunteer (PRV) training. Results: The goal of PRV is to assist fully trained responders during transport and care in a makeshift hospital. The curriculum for training as a PRV comprises 16 instructional units of 45 min each on the topics of deployment, self-protection, protection of others, and direct patient care. The focus is on practical skills for which the participants can take responsibility for execution. Conclusion: The concept of the PRV is the first structured training and integration of spontaneous responders in German civil protection. It is not a substitute for fully trained full-time and voluntary staff, but can provide useful support.

Effects on cardiorespiratory fitness of moderate-intensity training vs. energy-matched training with increasing intensity.

Introduction: The present study investigated the role of training intensity in the dose-response relationship between endurance training and cardiorespiratory fitness (CRF). The hypothesis was that beginners would benefit from an increase in training intensity after an initial training phase, even if the energy expenditure was not altered. For this purpose, 26 weeks of continuous moderate training (control group, CON) was compared to training with gradually increasing intensity (intervention group, INC) but constant energy expenditure. Methods: Thirty-one healthy, untrained subjects (13 men, 18 women; 46 ± 8 years; body mass index 25.4 ± 3.3 kg m-2; maximum oxygen uptake, VO2max 34 ± 4 ml min-1 kg-1) trained for 10 weeks with moderate intensity [3 days/week for 50 min/session at 55% heart rate reserve (HRreserve)] before allocation to one of two groups. A minimization technique was used to ensure homogeneous groups. While group CON continued with moderate intensity for 16 weeks, the INC group trained at 70% HRreserve for 8 weeks and thereafter participated in a 4 × 4 training program (high-intensity interval training, HIIT) for 8 weeks. Constant energy expenditure was ensured by indirect calorimetry and corresponding adjustment of the training volume. Treadmill tests were performed at baseline and after 10, 18, and 26 weeks. Results: The INC group showed improved VO2max (3.4 ± 2.7 ml kg-1 min-1) to a significantly greater degree than the CON group (0.4 ± 2.9 ml kg-1 min-1) (P = 0.020). In addition, the INC group exhibited improved Vmax (1.7 ± 0.7 km h-1) to a significantly greater degree than the CON group (1.0 ± 0.5 km h-1) (P = 0.001). The reduction of resting HR was significantly larger in the INC group (7 ± 4 bpm) than in the CON group (2 ± 6 bpm) (P = 0.001). The mean heart rate in the submaximal exercise test was reduced significantly in the CON group (5 ± 6 bpm; P = 0.007) and in the INC group (8 ± 7 bpm; P = 0.001), without a significant interaction between group and time point. Conclusion: Increasing intensity leads to greater adaptations in CRF than continuing with moderate intensity, even without increased energy expenditure. After 26 weeks of training in the moderate- and higher-intensity domain, energy-matched HIIT elicited further adaptations in cardiorespiratory fitness. Thus, training intensity plays a crucial role in the dose-response relationship between endurance training and fitness in untrained but healthy individuals. Clinical Trial Registration: https://www.drks.de/DRKS00031445, identifier DRKS00031445.

Changes in Key Biomechanical Parameters According to the Expertise Level in Runners at Different Running Speeds.

Running has become increasingly popular worldwide. Among runners, there exists a wide range of expertise levels. Investigating the differences between runners at two extreme levels, that is novices and experts, is crucial to understand the changes that occur as a result of multiple years of training. Vertical oscillation of center of mass (CoM), stride frequency normalized to the leg length, and duty factor, which describes the step time relative to the flight time, are key biomechanical parameters that have been shown to be closely related to the running economy and are used to characterize the running style. The variability characteristics of these parameters may reveal valuable information concerning the control of human locomotion. However, how the expertise level and running speed affect the variability of these key biomechanical parameters has not yet been investigated. The aim of this study was to analyze the effects of expertise level (novice vs. expert) and running speed (10 km/h vs. 15 km/h) on these parameters and their variability. It was hypothesized that expert runners would have lower vertical oscillation of CoM, normalized stride frequency, and duty factor and show less variability in these parameters. The parameters' variability was operationalized by the coefficient of variation. The mean values and variability of these key biomechanical parameters according to expertise level and running speed were compared with rmANOVAs. The results showed that the experts had a lower duty factor and less variable vertical oscillation of CoM and normalized stride frequency, independently of the running speed. At a higher running speed, the variability of vertical oscillation of CoM was higher, whereas that of normalized stride frequency and duty factor did not change significantly. To the best of our knowledge, this is the first study analyzing the effects of expertise level and running speed on the variability of key biomechanical parameters.

HSF2BP Interacts with a Conserved Domain of BRCA2 and Is Required for Mouse Spermatogenesis.

The tumor suppressor BRCA2 is essential for homologous recombination (HR), replication fork stability, and DNA interstrand crosslink repair in vertebrates. We identify HSF2BP, a protein previously described as testis specific and not characterized functionally, as an interactor of BRCA2 in mouse embryonic stem cells, where the 2 proteins form a constitutive complex. HSF2BP is transcribed in all cultured human cancer cell lines tested and elevated in some tumor samples. Inactivation of the mouse Hsf2bp gene results in male infertility due to a severe HR defect during spermatogenesis. The BRCA2-HSF2BP interaction is highly evolutionarily conserved and maps to armadillo repeats in HSF2BP and a 68-amino acid region between the BRC repeats and the DNA binding domain of human BRCA2 (Gly2270-Thr2337) encoded by exons 12 and 13. This region of BRCA2 does not harbor known cancer-associated missense mutations and may be involved in the reproductive rather than the tumor-suppressing function of BRCA2.

BRCA2 diffuses as oligomeric clusters with RAD51 and changes mobility after DNA damage in live cells.

Genome maintenance by homologous recombination depends on coordinating many proteins in time and space to assemble at DNA break sites. To understand this process, we followed the mobility of BRCA2, a critical recombination mediator, in live cells at the single-molecule level using both single-particle tracking and fluorescence correlation spectroscopy. BRCA2-GFP and -YFP were compared to distinguish diffusion from fluorophore behavior. Diffusive behavior of fluorescent RAD51 and RAD54 was determined for comparison. All fluorescent proteins were expressed from endogenous loci. We found that nuclear BRCA2 existed in oligomeric clusters, and exhibited heterogeneous mobility. DNA damage increased BRCA2 transient binding, presumably including binding to damaged sites. Despite its very different size, RAD51 displayed mobility similar to BRCA2, which indicates physical interaction between these proteins both before and after induction of DNA damage. We propose that BRCA2-mediated sequestration of nuclear RAD51 serves to prevent inappropriate DNA interactions and that all RAD51 is delivered to DNA damage sites in association with BRCA2.

Taking it one step at a time in homologous recombination repair.

The individual steps in the process of homologous recombination are particularly amenable to analysis by single-molecule imaging and manipulation experiments. Over the past 20 years these have provided a wealth of new information on the DNA transactions that make up this vital process. Exciting progress in developing new tools and techniques to analyze more complex components, dynamic reaction steps and molecular coordination continues at a rapid pace. Here we highlight recent results and indicate some emerging techniques likely to produce the next stage of advanced insight into homologous recombination. In this and related fields the future is bright.

Mechanosensitive channels and bacterial cell wall integrity: does life end with a bang or a whimper?

Mechanogated channels are fundamental components of bacterial cells that enable retention of physical integrity during extreme increases in cell turgor. Optical tweezers combined with microfluidics have been used to study the fate of individual Escherichia coli cells lacking such channels when subjected to a bursting stress caused by increased turgor. Fluorescence-activated cell sorting and electron microscopy complement these studies. These analyses show that lysis occurs with a high probability, but the precise path differs between individual cells. By monitoring the loss of cytoplasmic green fluorescent protein, we have determined that some cells release this protein but remain phase dark (granular) consistent with the retention of the majority of large proteins. By contrast, most cells suffer cataclysmic wall failure leading to loss of granularity but with the retention of DNA and overall cell shape (protein-depleted ghosts). The time span of these events induced by hypo-osmotic shock varies but is of the order of milliseconds. The data are interpreted in terms of the timing of mechanosensitive channel gating relative to osmotically induced water influx.

ArdA proteins from different mobile genetic elements can bind to the EcoKI Type I DNA methyltransferase of E. coli K12.

Anti-restriction and anti-modification (anti-RM) is the ability to prevent cleavage by DNA restriction-modification (RM) systems of foreign DNA entering a new bacterial host. The evolutionary consequence of anti-RM is the enhanced dissemination of mobile genetic elements. Homologues of ArdA anti-RM proteins are encoded by genes present in many mobile genetic elements such as conjugative plasmids and transposons within bacterial genomes. The ArdA proteins cause anti-RM by mimicking the DNA structure bound by Type I RM enzymes. We have investigated ArdA proteins from the genomes of Enterococcus faecalis V583, Staphylococcus aureus Mu50 and Bacteroides fragilis NCTC 9343, and compared them to the ArdA protein expressed by the conjugative transposon Tn916. We find that despite having very different structural stability and secondary structure content, they can all bind to the EcoKI methyltransferase, a core component of the EcoKI Type I RM system. This finding indicates that the less structured ArdA proteins become fully folded upon binding. The ability of ArdA from diverse mobile elements to inhibit Type I RM systems from other bacteria suggests that they are an advantage for transfer not only between closely-related bacteria but also between more distantly related bacterial species.

The kinetics of YOYO-1 intercalation into single molecules of double-stranded DNA.

The cyanine dye, YOYO-1, has frequently been used in single DNA molecule imaging work to stain double-stranded DNA as it fluoresces strongly when bound. The binding of YOYO-1 lengthens the DNA due to bis-intercalation. We have investigated the kinetics of binding, via this increase in DNA length, for single, hydrodynamically-stretched molecules of lambda DNA observed via Total Internal Reflection Fluorescence (TIRF) microscopy. The rate and degree of lengthening in 40mM NaHCO(3) (pH 8.0) buffer depend upon the free dye concentration with the reaction taking several minutes to reach completion even in relatively high, 40nM, concentrations of YOYO-1. In the absence of overstretching of the DNA molecule, we determine the second order rate constant to be 3.8±0.7×10(5)s(-1)M(-1), the dissociation constant to be 12.1±3.4nM and the maximum DNA molecule extension to be 36±4%. The intercalation time constant (inverse of the pseudo-first order rate constant), τ, decreased from 309 to 62s as YOYO-1 levels increased from 10 to 40nM. The kinetics of binding help with interpretation of the behavior of DNA-YOYO-1 complexes when overstretched and establish defined conditions for the preparation of DNA-YOYO-1 complexes.

Single-molecule imaging of Bacteroides fragilis AddAB reveals the highly processive translocation of a single motor helicase.

The AddAB helicase and nuclease complex is used for repairing double-strand DNA breaks in the many bacteria that do not possess RecBCD. Here, we show that AddAB, from the Gram-negative opportunistic pathogen Bacteroides fragilis, can rescue the ultraviolet sensitivity of an Escherichia coli recBCD mutant and that addAB is required for survival of B. fragilis following DNA damage. Using single-molecule observations we demonstrate that AddAB can translocate along DNA at up to 250 bp per second and can unwind an average of 14,000 bp, with some complexes capable of unwinding 40,000 bp. These results demonstrate the importance of processivity for facilitating encounters with recognition sequences that modify enzyme function during homologous recombination.

Screening by imaging: scaling up single-DNA-molecule analysis with a novel parabolic VA-TIRF reflector and noise-reduction techniques.

Bacteriophage lambda-DNA molecules are frequently used as a scaffold to characterize the action of single proteins unwinding, translocating, digesting or repairing DNA. However, scaling up such single-DNA-molecule experiments under identical conditions to attain statistically relevant sample sizes remains challenging. Additionally the movies obtained are frequently noisy and difficult to analyse with any precision. We address these two problems here using, firstly, a novel variable-angle total internal reflection fluorescence (VA-TIRF) reflector composed of a minimal set of optical reflective elements, and secondly, using single value decomposition (SVD) to improve the signal-to-noise ratio prior to analysing time-lapse image stacks. As an example, we visualize under identical optical conditions hundreds of surface-tethered single lambda-DNA molecules, stained with the intercalating dye YOYO-1 iodide, and stretched out in a microcapillary flow. Another novelty of our approach is that we arrange on a mechanically driven stage several capillaries containing saline, calibration buffer and lambda-DNA, respectively, thus extending the approach to high-content, high-throughput screening of single molecules. Our length measurements of individual DNA molecules from noise-reduced kymograph images using SVD display a 6-fold enhanced precision compared to raw-data analysis, reaching approximately 1 kbp resolution. Combining these two methods, our approach provides a straightforward yet powerful way of collecting statistically relevant amounts of data in a semi-automated manner. We believe that our conceptually simple technique should be of interest for a broader range of single-molecule studies, well beyond the specific example of lambda-DNA shown here.

Poly-l-lysines and poly-l-arginines induce leakage of negatively charged phospholipid vesicles and translocate through the lipid bilayer upon electrostatic binding to the membrane.

Poly-l-lysines (PLL) and poly-l-arginines (PLA) of different polymer chain lengths interact strongly with negatively charged phospholipid vesicles mainly due to their different electrical charges. 1-Palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol (POPG), 1,2-dipalmitoyl-sn-glycero-3-phosphoglycerol (DPPG) and their mixtures (1/1 mol/mol) with the respective phosphatidylcholines of equivalent chain length were chosen as model membrane systems that form at room temperature either the fluid L(alpha) or the gel phase L(beta) lipid bilayer membranes, respectively. Leakage experiments revealed that the fluid POPG membranes are more perturbed compared to the gel phase DPPG membranes upon peptide binding. Furthermore, it was found that pure PG membranes are more prone to release the vesicle contents as a result of pore formation than the lipid mixtures POPG/POPC and DPPG/DPPC. For the longer polymers (>or=44 amino acids) maximal dye-release was observed when the molar ratio of the concentrations of amino acid residues to charged lipid molecules reached a value of R(P)=0.5, i.e. when the outer membrane layer was theoretically entirely covered by the polymer. At ratios lower or higher than 0.5 leakage dropped significantly. Furthermore, PLL and PLA insertions and/or translocations through lipid membranes were analyzed by using FITC-labeled polymers by monitoring their fluorescence intensity upon membrane binding. Short PLL molecules and PLA molecules of all lengths seemed to translocate through both fluid and gel phase lipid bilayers. Comparison of the PLL and PLA fluorescence assay results showed that PLA interacts stronger with phospholipid membranes compared to PLL. Isothermal titration calorimetry (ITC) measurements were performed to give further insight into these mechanisms and to support the findings obtained by fluorescence assays. Cryo-transmission electron microscopy (cryo-TEM) was used to visualize changes in the vesicles' morphology after addition of the polypeptides.