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Abstract The concept of Targeted Protein Degradation (TPD) has been introduced as an attractive alternative to the development of classical inhibitors. TPD can extend the range of proteins that can be pharmacologically targeted beyond the classical targets for small molecule inhibitors, as a binding pocket is required but its occupancy does not need to lead to inhibition. The method is based on either small molecules that simultaneously bind to a protein of interest and to a cellular E3 ligase and bring them in close proximity (molecular glue) or a bi-functional molecule synthesized from the chemical linkage of a target protein-specific small molecule and one that binds to an E3 ligase (Proteolysis Targeting Chimeras (PROTAC)). The further extension of this approach to bioPROTACs, in which a small protein-based binding module is fused directly to an E3 ligase or an E3 ligase adaptor protein, makes virtually all proteins amenable to targeted degradation, as this method eliminates the requirement for binding pockets for small molecules. Designed Ankyrin Repeat Proteins (DARPins) represent a very attractive class of small protein-based binding modules that can be used for the development of bioPTOTACS. Here we describe the characterization of two DARPins generated against the oligomerization domain and the SAM domain of the transcription factor p73, a member of the p53 protein family. The DARPins can be used for (isoform-)selective pulldown experiments both in cell culture as well as primary tissue lysates. We also demonstrate that they can be used for staining in cell culture experiments. Fusing them to the speckle type POZ protein (SPOP), an adaptor protein for cullin-3 E3 ligase complexes, yields highly selective and effective degraders. We demonstrate that selective degradation of the ΔNp73α isoform reactivates p53.

Abstract Background Quadriceps strength deficits are known for patients with knee osteoarthritis (OA), whereas findings on hamstrings are less clear. The Adaptive Force (AF) as a special neuromuscular function has never been investigated in OA before. The maximal adaptive holding capacity (max. isometric AF; AFisomax) has been considered to be especially vulnerable to disruptive stimuli (e.g., nociception). It was hypothesized that affected limbs of OA patients would show clear deficits in AFisomax. Methods AF parameters and the maximal voluntary isometric contraction (MVIC) of hamstrings were assessed bilaterally comparing 20 patients with knee OA (ART) vs. controls (CON). AF was measured by a pneumatically driven device. Participants were instructed to maintain a static position despite an increasing load of the device. After reaching AFisomax, the hamstrings merged into eccentric action whereby the force increased further to the maximum (AFmax). MVIC was recorded before and after AF trials. Mixed ANOVA was used to identify differences between and within ART and CON (comparing 1st and 2nd measured sides). Results AFisomax and the torque development per degree of yielding were significantly lower only for the more affected side of ART vs. CON (p ≤ 0.001). The percentage difference of AFisomax amounted to − 40%. For the less affected side it was − 24% (p = 0.219). MVIC and AFmax were significantly lower for ART vs. CON for both sides (p ≤ 0.001). Differences of MVIC between ART vs. CON amounted to − 27% for the more, and − 30% for the less affected side; for AFmax it was − 34% and − 32%, respectively. Conclusion The results suggest that strength deficits of hamstrings are present in patients with knee OA possibly attributable to nociception, generally lower physical activity/relief of lower extremities or fear-avoidance. However, the more affected side of OA patients seems to show further specific impairments regarding neuromuscular control reflected by the significantly reduced adaptive holding capacity and torque development during adaptive eccentric action. It is assumed that those parameters could reflect possible inhibitory nociceptive effects more sensitive than maximal strengths as MVIC and AFmax. Their role should be further investigated to get more specific insights into these aspects of neuromuscular control in OA patients. The approach is relevant for diagnostics also in terms of severity and prevention.

Abstract In this study, we characterize Designed Ankyrin Repeat Proteins (DARPins) as investigative tools to probe botulinum neurotoxin A1 (BoNT/A1) structure and function. We identify DARPin-F5 that completely blocks SNAP25 substrate cleavage by BoNT/A1 in vitro. X-ray crystallography reveals that DARPin-F5 inhibits BoNT/A1 activity by interacting with a substrate-binding region between the α- and β-exosite. This DARPin does not block substrate cleavage of BoNT/A3, indicating that DARPin-F5 is a subtype-specific inhibitor. BoNT/A1 Glu-171 plays a critical role in the interaction with DARPin-F5 and its mutation to Asp, the residue found in BoNT/A3, results in a loss of inhibition of substrate cleavage. In contrast to the in vitro results, DARPin-F5 promotes faster substrate cleavage of BoNT/A1 in primary neurons and muscle tissue by increasing toxin translocation. Our findings could have important implications for the application of BoNT/A1 in therapeutic areas requiring faster onset of toxin action combined with long persistence.

Abstract The two p53 homologues p63 and p73 regulate transcriptional programs in epithelial tissues and several cell types in these tissues express both proteins. All members of the p53 family form tetramers in their active state through a dedicated oligomerization domain that structurally assembles as a dimer of dimers. The oligomerization domain of p63 and p73 share a high sequence identity, but the p53 oligomerization domain is more divergent and it lacks a functionally important C-terminal helix present in the other two family members. Based on these structural differences, p53 does not hetero-oligomerize with p63 or p73. In contrast, p63 and p73 form hetero-oligomers of all possible stoichiometries, with the hetero-tetramer built from a p63 dimer and a p73 dimer being thermodynamically more stable than the two homo-tetramers. This predicts that in cells expressing both proteins a p632/p732 hetero-tetramer is formed. So far, the tools to investigate the biological function of this hetero-tetramer have been missing. Here we report the generation and characterization of Designed Ankyrin Repeat Proteins (DARPins) that bind with high affinity and selectivity to the p632/p732 hetero-tetramer. Using these DARPins we were able to confirm experimentally the existence of this hetero-tetramer in epithelial mouse and human tissues and show that its level increases in squamous cell carcinoma.

Abstract Diversity-oriented synthesis (DOS) is a powerful strategy to prepare molecules with underrepresented features in commercial screening collections, resulting in the elucidation of novel biological mechanisms. In parallel to the development of DOS, DNA-encoded libraries (DELs) have emerged as an effective, efficient screening strategy to identify protein binders. Despite recent advancements in this field, most DEL syntheses are limited by the presence of sensitive DNA-based constructs. Here, we describe the design, synthesis, and validation experiments performed for a 3.7 million-member DEL, generated using diverse skeleton architectures with varying exit vectors and derived from DOS, to achieve structural diversity beyond what is possible by varying appendages alone. We also show screening results for three diverse protein targets. We will make this DEL available to the academic scientific community to increase access to novel structural features and accelerate early-phase drug discovery.

Abstract The actin cytoskeleton is of fundamental importance for cellular structure and plasticity. However, abundance and function of filamentous actin in the nucleus are still controversial. Here we show that the actin-based molecular motor myosin VI contributes to the stabilization of stalled or reversed replication forks. In response to DNA replication stress, myosin VI associates with stalled replication intermediates and cooperates with the AAA ATPase Werner helicase interacting protein 1 (WRNIP1) in protecting these structures from DNA2-mediated nucleolytic attack. Using functionalized affinity probes to manipulate myosin VI levels in a compartment-specific manner, we provide evidence for the direct involvement of myosin VI in the nucleus and against a contribution of the abundant cytoplasmic pool during the replication stress response.

Drying-rewetting cycles are ubiquitous across natural and managed ecosystems. These cycles are known to mobilize carbon (C) in soils producing dramatic pulses in microbial respiration. While many factors contribute to these pulses, the drying-rewetting history of soils affecting carbon emissions remains unclear, especially in irrigated soils where soil moisture fluctuations are more repetitive and/or frequent than natural, seasonally influenced soils. To understand the controls of repeated wet-up and dry down effects on agricultural soils, we used a systems approach to examine the cross section of a furrow irrigated orchard to delineate soil C dynamics. Specifically, we compared two contrasting water regimes, (1) soils temporarily but repeatedly inundated during water delivery (i.e., furrows) and (2) soils at the base of trees (i.e., berms) that only receive water during precipitation events in a semi-arid Mediterranean climate. Overall, our findings show that the heterogeneous landscape of a furrow irrigated field results in two separate systems within the field scale in gaseous release of C as CO2, microbial selectivity of substrates, and mechanisms for C stabilization. By monitoring soil moisture as a function of depth for over two years, our results reveal that furrow soils undergo dramatic wet-dry cycles, while moisture within the berm is relatively constant. We were able to capture the distinct heterogeneity of soil moisture changes within the furrow and berm soils by continuously monitoring CO2 flux throughout water input events in both the wet and dry season. Soil CO2 efflux is suppressed upon irrigation within furrows, while carbon oxidation in berm soils exhibits pore-connectivity limitations that result in lower fluxes when dry. Solid phase soil C speciation determined by C 1s NEXAFS demonstrated C of higher aromaticity remained in furrow soil compared to berm soils. Microbial community analysis shows significantly different communities reside within berm and furrow soils, where furrow soils support more anaerobic metabolisms and spore-formers while berm soils have relatively higher abundance of aerobic microbes capable of degrading larger, more complex C compounds. Our findings show that water regime (periodic inundation vs episodic rainfall) controlling rewetting history can greatly differentiate C respiration within managed soils.

Abstract Background Isometric muscle actions can be performed either by initiating the action, e.g., pulling on an immovable resistance (PIMA), or by reacting to an external load, e.g., holding a weight (HIMA). In the present study, it was mainly examined if these modalities could be differentiated by oxygenation variables as well as by time to task failure (TTF). Furthermore, it was analyzed if variables are changed by intermittent voluntary muscle twitches during weight holding (Twitch). It was assumed that twitches during a weight holding task change the character of the isometric muscle action from reacting (≙ HIMA) to acting (≙ PIMA). Methods Twelve subjects (two drop outs) randomly performed two tasks (HIMA vs. PIMA or HIMA vs. Twitch, n = 5 each) with the elbow flexors at 60% of maximal torque maintained until muscle failure with each arm. Local capillary venous oxygen saturation (SvO2) and relative hemoglobin amount (rHb) were measured by light spectrometry. Results Within subjects, no significant differences were found between tasks regarding the behavior of SvO2 and rHb, the slope and extent of deoxygenation (max. SvO2 decrease), SvO2 level at global rHb minimum, and time to SvO2 steady states. The TTF was significantly longer during Twitch and PIMA (incl. Twitch) compared to HIMA (p = 0.043 and 0.047, respectively). There was no substantial correlation between TTF and maximal deoxygenation independently of the task (r = − 0.13). Conclusions HIMA and PIMA seem to have a similar microvascular oxygen and blood supply. The supply might be sufficient, which is expressed by homeostatic steady states of SvO2 in all trials and increases in rHb in most of the trials. Intermittent voluntary muscle twitches might not serve as a further support but extend the TTF. A changed neuromuscular control is discussed as possible explanation.

Adenylyl cyclases (ACs) generate the second messenger cAMP and play an important role in cellular signaling. Here, the authors use cryo-EM to trace the conformational changes resulting from binding to partial and full activators to one of these enzymes, AC9.

The Adaptive Force (AF) reflects the neuromuscular capacity to adapt to external loads during holding muscle actions and is similar to motions in real life and sports. The maximal isometric AF (AFisomax) was considered to be the most relevant parameter and was assumed to have major importance regarding injury mechanisms and the development of musculoskeletal pain. The aim of this study was to investigate the behavior of different torque parameters over the course of 30 repeated maximal AF trials. In addition, maximal holding vs. maximal pushing isometric muscle actions were compared. A side consideration was the behavior of torques in the course of repeated AF actions when comparing strength and endurance athletes. The elbow flexors of n = 12 males (six strength/six endurance athletes, non-professionals) were measured 30 times (120 s rest) using a pneumatic device. Maximal voluntary isometric contraction (MVIC) was measured pre and post. MVIC, AFisomax, and AFmax (maximal torque of one AF measurement) were evaluated regarding different considerations and statistical tests. AFmax and AFisomax declined in the course of 30 trials [slope regression (mean ± standard deviation): AFmax = −0.323 ± 0.263; AFisomax = −0.45 ± 0.45]. The decline from start to end amounted to −12.8% ± 8.3% (p < 0.001) for AFmax and −25.41% ± 26.40% (p < 0.001) for AFisomax. AF parameters declined more in strength vs. endurance athletes. Thereby, strength athletes showed a rather stable decline for AFmax and a plateau formation for AFisomax after 15 trials. In contrast, endurance athletes reduced their AFmax, especially after the first five trials, and remained on a rather similar level for AFisomax. The maximum of AFisomax of all 30 trials amounted 67.67% ± 13.60% of MVIC (p < 0.001, n = 12), supporting the hypothesis of two types of isometric muscle action (holding vs. pushing). The findings provided the first data on the behavior of torque parameters after repeated isometric–eccentric actions and revealed further insights into neuromuscular control strategies. Additionally, they highlight the importance of investigating AF parameters in athletes based on the different behaviors compared to MVIC. This is assumed to be especially relevant regarding injury mechanisms.

The increasing prevalence of Long COVID is an imminent public health disaster, and established approaches have not provided adequate diagnostics or treatments. Recently, anesthetic blockade of the stellate ganglion was reported to improve Long COVID symptoms in a small case series, purportedly by “rebooting” the autonomic nervous system. Here, we present a novel diagnostic approach based on the Adaptive Force (AF), and report sustained positive outcome for one severely affected Long COVID patient using individualized pulsed electromagnetic field (PEMF) at the area C7/T1. AF reflects the capacity of the neuromuscular system to adapt adequately to external forces in an isometric holding manner. In case, maximal isometric AF (AFisomax) is exceeded, the muscle merges into eccentric muscle action. Thereby, the force usually increases further until maximal AF (AFmax) is reached. In case adaptation is optimal, AFisomax is ~99–100% of AFmax. This holding capacity (AFisomax) was found to be vulnerable to disruption by unpleasant stimulus and, hence, was regarded as functional parameter. AF was assessed by an objectified manual muscle test using a handheld device. Prior to treatment, AFisomax was considerably lower than AFmax for hip flexors (62 N = ~28% AFmax) and elbow flexors (71 N = ~44% AFmax); i.e., maximal holding capacity was significantly reduced, indicating dysfunctional motor control. We tested PEMF at C7/T1, identified a frequency that improved neuromuscular function, and applied it for ~15 min. Immediately post-treatment, AFisomax increased to ~210 N (~100% AFmax) at hip and 184 N (~100% AFmax) at elbow. Subjective Long COVID symptoms resolved the following day. At 4 weeks post-treatment, maximal holding capacity was still on a similarly high level as for immediately post-treatment (~100% AFmax) and patient was symptom-free. At 6 months the patient's Long COVID symptoms have not returned. This case report suggests (1) AF could be a promising diagnostic for post-infectious illness, (2) AF can be used to test effective treatments for post-infectious illness, and (3) individualized PEMF may resolve post-infectious symptoms.

The V3-crown of the HIV-1 envelope protein largely elicits non-neutralizing antibodies. Here, the authors show that the V3-crown can be targeted by broadly neutralizing designed ankyrin repeat proteins recognizing two conformations one of which resembles CCR5- bound V3.

Morphogens disperse to pattern tissues and control their growth during development, allowing for the specification of multiple fates across space. Here the authors block dispersal of a morphogen Dpp (BMP2/4) and show that the requirement for Dpp dispersal is much lower than previously thought.

Abstract The olfactomotor system is especially investigated by examining the sniffing in reaction to olfactory stimuli. The motor output of respiratory-independent muscles was seldomly considered regarding possible influences of smells. The Adaptive Force (AF) characterizes the capability of the neuromuscular system to adapt to external forces in a holding manner and was suggested to be more vulnerable to possible interfering stimuli due to the underlying complex control processes. The aim of this pilot study was to measure the effects of olfactory inputs on the AF of the hip and elbow flexors, respectively. The AF of 10 subjects was examined manually by experienced testers while smelling at sniffing sticks with neutral, pleasant or disgusting odours. The reaction force and the limb position were recorded by a handheld device. The results show, inter alia, a significantly lower maximal isometric AF and a significantly higher AF at the onset of oscillations by perceiving disgusting odours compared to pleasant or neutral odours (p

Sensorimotor control can be impaired by slacked muscle spindles. This was shown for reflex responses and, recently, also for muscular stability in the sense of Adaptive Force (AF). The slack in muscle spindles was generated by contracting the lengthened muscle followed by passive shortening. AF was suggested to specifically reflect sensorimotor control since it requires tension-length control in adaptation to an increasing load. This study investigated AF parameters in reaction to another, manually performed slack procedure in a preselected sample (n = 13). The AF of 11 elbow and 12 hip flexors was assessed by an objectified manual muscle test (MMT) using a handheld device. Maximal isometric AF was significantly reduced after manual spindle technique vs. regular MMT. Muscle lengthening started at 64.93 ± 12.46% of maximal voluntary isometric contraction (MVIC). During regular MMT, muscle length could be maintained stable until 92.53 ± 10.12% of MVIC. Hence, muscular stability measured by AF was impaired after spindle manipulation. Force oscillations arose at a significantly lower level for regular vs. spindle. This supports the assumption that they are a prerequisite for stable adaptation. Reduced muscular stability in reaction to slack procedures is considered physiological since sensory information is misled. It is proposed to use slack procedures to test the functionality of the neuromuscular system, which is relevant for clinical practice.

Abstract The mechanical muscular oscillations are rarely the objective of investigations regarding the identification of a biomarker for Parkinson’s disease (PD). Therefore, the aim of this study was to investigate whether or not this specific motor output differs between PD patients and controls. The novelty is that patients without tremor are investigated performing a unilateral isometric motor task. The force of armflexors and the forearm acceleration (ACC) were recorded as well as the mechanomyography of the biceps brachii (MMGbi), brachioradialis (MMGbra) and pectoralis major (MMGpect) muscles using a piezoelectric-sensor-based system during a unilateral motor task at 70% of the MVIC. The frequency, a power-frequency-ratio, the amplitude variation, the slope of amplitudes and their interlimb asymmetries were analysed. The results indicate that the oscillatory behavior of muscular output in PD without tremor deviates from controls in some parameters: Significant differences appeared for the power-frequency-ratio (p = 0.001, r = 0.43) and for the amplitude variation (p = 0.003, r = 0.34) of MMGpect. The interlimb asymmetries differed significantly concerning the power-frequency-ratio of MMGbi (p = 0.013, r = 0.42) and MMGbra (p = 0.048, r = 0.39) as well as regarding the mean frequency (p = 0.004, r = 0.48) and amplitude variation of MMGpect (p = 0.033, r = 0.37). The mean (M) and variation coefficient (CV) of slope of ACC differed significantly (M: p = 0.022, r = 0.33; CV: p = 0.004, r = 0.43). All other parameters showed no significant differences between PD and controls. It remains open, if this altered mechanical muscular output is reproducible and specific for PD.

Adaptation to external forces relies on a well-functioning proprioceptive system including muscle spindle afferents. Muscle length and tension control in reaction to external forces is most important regarding the Adaptive Force (AF). This study investigated the effect of different procedures, which are assumed to influence the function of muscle spindles, on the AF. Elbow flexors of 12 healthy participants (n = 19 limbs) were assessed by an objectified manual muscle test (MMT) with different procedures: regular MMT, MMT after precontraction (self-estimated 20% MVIC) in lengthened position with passive return to test position (CL), and MMT after CL with a second precontraction in test position (CL-CT). During regular MMTs, muscles maintained their length up to 99.7% ± 1.0% of the maximal AF (AFmax). After CL, muscles started to lengthen at 53.0% ± 22.5% of AFmax. For CL-CT, muscles were again able to maintain the static position up to 98.3% ± 5.5% of AFmax. AFisomax differed highly significantly between CL vs. CL-CT and regular MMT. CL was assumed to generate a slack of muscle spindles, which led to a substantial reduction of the holding capacity. This was immediately erased by a precontraction in the test position. The results substantiate that muscle spindle sensitivity seems to play an important role for neuromuscular functioning and musculoskeletal stability.

Long COVID patients show symptoms, such as fatigue, muscle weakness and pain. Adequate diagnostics are still lacking. Investigating muscle function might be a beneficial approach. The holding capacity (maximal isometric Adaptive Force; AFisomax) was previously suggested to be especially sensitive for impairments. This longitudinal, non-clinical study aimed to investigate the AF in long COVID patients and their recovery process. AF parameters of elbow and hip flexors were assessed in 17 patients at three time points (pre: long COVID state, post: immediately after first treatment, end: recovery) by an objectified manual muscle test. The tester applied an increasing force on the limb of the patient, who had to resist isometrically for as long as possible. The intensity of 13 common symptoms were queried. At pre, patients started to lengthen their muscles at ~50% of the maximal AF (AFmax), which was then reached during eccentric motion, indicating unstable adaptation. At post and end, AFisomax increased significantly to ~99% and 100% of AFmax, respectively, reflecting stable adaptation. AFmax was statistically similar for all three time points. Symptom intensity decreased significantly from pre to end. The findings revealed a substantially impaired maximal holding capacity in long COVID patients, which returned to normal function with substantial health improvement. AFisomax might be a suitable sensitive functional parameter to assess long COVID patients and to support therapy process.

The link between emotions and motor function has been known for decades but is still not clarified. The Adaptive Force (AF) describes the neuromuscular capability to adapt to increasing forces and was suggested to be especially vulnerable to interfering inputs. This study investigated the influence of pleasant and unpleasant food imagery on the manually assessed AF of elbow and hip flexors objectified by a handheld device in 12 healthy women. The maximal isometric AF was significantly reduced during unpleasant vs. pleasant imagery and baseline (p < 0.001, dz = 0.98–1.61). During unpleasant imagery, muscle lengthening started at 59.00 ± 22.50% of maximal AF, in contrast to baseline and pleasant imagery, during which the isometric position could be maintained mostly during the entire force increase up to ~97.90 ± 5.00% of maximal AF. Healthy participants showed an immediately impaired holding function triggered by unpleasant imagery, presumably related to negative emotions. Hence, AF seems to be suitable to test instantaneously the effect of emotions on motor function. Since musculoskeletal complaints can result from muscular instability, the findings provide insights into the understanding of the causal chain of linked musculoskeletal pain and mental stress. A case example (current stress vs. positive imagery) suggests that the approach presented in this study might have future implications for psychomotor diagnostics and therapeutics.

The link between emotions and motor control has been discussed for years. The measurement of the Adaptive Force (AF) provides the possibility to get insights into the adaptive control of the neuromuscular system in reaction to external forces. It was hypothesized that the holding isometric AF is especially vulnerable to disturbing inputs. Here, the behavior of the AF under the influence of positive (tasty) vs. negative (disgusting) food imaginations was investigated. The AF was examined in n = 12 cases using an objectified manual muscle test of the hip flexors, elbow flexors or pectoralis major muscle, performed by one of two experienced testers while the participants imagined their most tasty or most disgusting food. The reaction force and the limb position were measured by a handheld device. While the slope of force rises and the maximal AF did not differ significantly between tasty and disgusting imaginations (p > 0.05), the maximal isometric AF was significantly lower and the AF at the onset of oscillations was significantly higher under disgusting vs. tasty imaginations (both p = 0.001). A proper length tension control of muscles seems to be a crucial functional parameter of the neuromuscular system which can be impaired instantaneously by emotionally related negative imaginations. This might be a potential approach to evaluate somatic reactions to emotions.

The mechanotendography (MTG) is a method for analyzing the mechanical oscillations of tendons during muscular actions. The aim of this investigation was to evaluate the technical reliability of a piezo-based measurement system used for MTG. The reliability measurements were performed by using audio samples played by a subwoofer. The thereby generated pressure waves were recorded by a piezo-based measurement system. An audio of 40 Hz sine oscillations and four different formerly in vivo recorded MTG-signals were converted into audio files and were used as test signals. Five trials with each audio were performed and one audio was used for repetition trials on another day. The signals’ correlation was estimated by Spearman (MCC) and intraclass correlation coefficients (ICC(3,1)), Cronbach’s alpha (CA) and by mean distances (MD). All parameters were compared between repetition and randomized matched signals. The repetition trials show high correlations (MCC: 0.86 ± 0.13, ICC: 0.89 ± 0.12, CA: 0.98 ± 0.03), low MD (0.03 ± 0.03V) and differ significantly from the randomized matched signals (MCC: 0.15 ± 0.10, ICC: 0.17 ± 0.09, CA: 0.37 ± 0.16, MD: 0.19 ± 0.01V) (p = 0.001 – 0.043). This speaks for an excellent reliability of the measurement system. Presuming the skin above superficial tendons oscillates adequately, we estimate this tool as valid for the application in musculoskeletal system.

In sports and movement sciences isometric muscle function is usually measured by pushing against a stable resistance. However, subjectively one can hold or push isometrically. Several investigations suggest a distinction of those forms. The aim of this study was to investigate whether these two forms of isometric muscle action can be distinguished by objective parameters in an interpersonal setting. 20 subjects were grouped in 10 same sex pairs, in which one partner should perform the pushing isometric muscle action (PIMA) and the other partner executed the holding isometric muscle action (HIMA). The partners had contact at the distal forearms via an interface, which included a strain gauge and an acceleration sensor. The mechanical oscillations of the triceps brachii (MMGtri) muscle, its tendon (MTGtri) and the abdominal muscle (MMGobl) were recorded by a piezoelectric-sensor-based measurement system. Each partner performed three 15s (80% MVIC) and two fatiguing trials (90% MVIC) during PIMA and HIMA, respectively. Parameters to compare PIMA and HIMA were the mean frequency, the normalized mean amplitude, the amplitude variation, the power in the frequency range of 8 to 15 Hz, a special power-frequency ratio and the number of task failures during HIMA or PIMA (partner who quit the task). A "HIMA failure" occurred in 85% of trials (p < 0.001). No significant differences between PIMA and HIMA were found for the mean frequency and normalized amplitude. The MMGobl showed significantly higher values of amplitude variation (15s: p = 0.013; fatiguing: p = 0.007) and of power-frequency-ratio (15s: p = 0.040; fatiguing: p = 0.002) during HIMA and a higher power in the range of 8 to 15 Hz during PIMA (15s: p = 0.001; fatiguing: p = 0.011). MMGtri and MTGtri showed no significant differences. Based on the findings it is suggested that a holding and a pushing isometric muscle action can be distinguished objectively, whereby a more complex neural control is assumed for HIMA.

Inter-brain synchronization is primarily investigated during social interactions but had not been examined during coupled muscle action between two persons until now. It was previously shown that mechanical muscle oscillations can develop coherent behavior between two isometrically interacting persons. This case study investigated if inter-brain synchronization appears thereby, and if differences of inter- and intrapersonal muscle and brain coherence exist regarding two different types of isometric muscle action. Electroencephalography (EEG) and mechanomyography/mechanotendography (MMG/MTG) of right elbow extensors were recorded during six fatiguing trials of two coupled isometrically interacting participants (70% MVIC). One partner performed holding and one pushing isometric muscle action (HIMA/PIMA; tasks changed). The wavelet coherence of all signals (EEG, MMG/MTG, force, ACC) were analyzed intra- and interpersonally. The five longest coherence patches in 8–15 Hz and their weighted frequency were compared between real vs. random pairs and between HIMA vs. PIMA. Real vs. random pairs showed significantly higher coherence for intra-muscle, intra-brain, and inter-muscle-brain activity (p < 0.001 to 0.019). Inter-brain coherence was significantly higher for real vs. random pairs for EEG of right and central areas and for sub-regions of EEG left (p = 0.002 to 0.025). Interpersonal muscle-brain synchronization was significantly higher than intrapersonal one, whereby it was significantly higher for HIMA vs. PIMA. These preliminary findings indicate that inter-brain synchronization can arise during muscular interaction. It is hypothesized both partners merge into one oscillating neuromuscular system. The results reinforce the hypothesis that HIMA is characterized by more complex control strategies than PIMA. The pilot study suggests investigating the topic further to verify these results on a larger sample size. Findings could contribute to the basic understanding of motor control and is relevant for functional diagnostics such as the manual muscle test which is applied in several disciplines, e.g., neurology, physiotherapy.

Aim. The aim of the research was to study the features of mild cervical dysplasia (CIN I) and determine the management of patients with such abnormal cytological results found in cervical screening against bacterial vaginosis and HPV infection. Materials and methods. The study included 252 patients aged 19 to 35. Ill-defined cells were detected during cytological screening. All patients were divided into 2 groups. The first group consisted of 93 patients with ASC-US cytological Bethesda classification results. The second group included 159 women with LSIL cytological Bethesda classification results. PCRdiagnostics was performed to determine qualitative and quantitative composition of genital tract microflora. The presence of 16th, 18th, 31st, 33rd, 35th, 39th, 45th, 51st, 52nd, 56th, 58th, 59th types HPV was detected. Patients with HPV-infection underwent widened colposcopy with evaluation of the grade of abnormal colposcopy findings.Results. In the 1st group the bacterial vaginosis was diagnosed in 83,9% of women, HPV – in 39,7%, trichomoniasis – in 7,5% of patients. In the 2nd group bacterial vaginosis was diagnosed in 64,8% of patients, HPV – in 78,0%, trichomoniasis – in 2,5%. In the 2nd group the incidence of abnormal colposcopy findings was twice higher than in 1st group. Patients underwent uterine cervix biopsy. Biopsy material was subjected to histological investigation. In the 1st group (n=22) 50,0% of patients had mild cervical dysplasia (CIN I), 9,1% – had moderate cervical dysplasia (CIN II). In the second group (n=61) 68,8% of patients had mild cervical dysplasia (CIN I), 9,83% – had moderate cervical dysplasia (CIN II). Patients of both groups were treated for 3-6 months. They underwent control cytological and microbiological studies after the treatment. The recurrence of bacterial vaginosis was 4,3% in 1st group and 14,0% in 2nd group. In 1st group 16,43% of patients had HPV positive test; it was positive in 19,5% of women of 2nd group. LSIL cytologicalBethesda classification results were in 1 (1,1%) patient in the 1st and in 5 (3,1%) patients in the 2nd group.Conclusion. Bacterial vaginosis is an additional factor of progression of dysplasia in patients with HPV positive test. Patients with LSIL and ASC-US cytological Bethesda classification results need correct antibiotic therapy which should be followed by control cytological studies and HPV test. These are the main ways of cervical cancer prevention.

The regulation of oxygen and blood supply during isometric muscle actions is still unclear. Recently, two behavioral types of oxygen saturation (SvO2) and relative hemoglobin amount (rHb) in venous microvessels were described during a fatiguing holding isometric muscle action (HIMA) (type I: nearly parallel behavior of SvO2 and rHb; type II: partly inverse behavior). The study aimed to ascertain an explanation of these two regulative behaviors. Twelve subjects performed one fatiguing HIMA trial with each arm by weight holding at 60% of the maximal voluntary isometric contraction (MVIC) in a 90° elbow flexion. Six subjects additionally executed one fatiguing PIMA trial by pulling on an immovable resistance with 60% of the MVIC with each side and same position. Both regulative types mentioned were found during HIMA (I: n = 7, II: n = 17) and PIMA (I: n = 3, II: n = 9). During the fatiguing measurements, rHb decreased initially and started to increase in type II at an average SvO2-level of 58.75 ± 2.14%. In type I, SvO2 never reached that specific value during loading. This might indicate the existence of a threshold around 59% which seems to trigger the increase in rHb and could explain the two behavioral types. An approach is discussed to meet the apparent incompatibility of an increased capillary blood filling (rHb) despite high intramuscular pressures which were found by other research groups during isometric muscle actions.

Abstract Iron (Fe) and manganese (Mn) are the two most common redox-active elements in the Earth’s crust and are well known to influence mineral formation and dissolution, trace metal sequestration, and contaminant transformations in soils and sediments. Here, we characterized the reaction of aqueous Fe(II) with pyrolusite (β-MnO2) using electron microscopy, X-ray diffraction, aqueous Fe and Mn analyses, and 57Fe Mössbauer spectroscopy. We reacted pyrolusite solids repeatedly with 3 mM Fe(II) at pH 7.5 to evaluate whether electron transfer occurs and to track the evolving reactivity of the Mn/Fe solids. We used Fe isotopes (56 and 57) in conjunction with 57Fe Mössbauer spectroscopy to isolate oxidation of Fe(II) by Fe(III) precipitates or pyrolusite. Using these complementary techniques, we determined that Fe(II) is initially oxidized by pyrolusite and that lepidocrocite is the dominant Fe oxidation product. Additional Fe(II) exposures result in an increasing proportion of magnetite on the pyrolusite surface. Over a series of nine 3 mM Fe(II) additions, Fe(II) continued to be oxidized by the Mn/Fe particles suggesting that Mn/Fe phases are not fully passivated and remain redox active even after extensive surface coverage by Fe(III) oxides. Interestingly, the initial Fe(III) oxide precipitates became further reduced as Fe(II) was added and additional Mn was released into solution suggesting that both the Fe oxide coating and underlying Mn phase continue to participate in redox reactions when freshly exposed to Fe(II). Our findings indicate that Fe and Mn chemistry is influenced by sustained reactions of Fe(II) with Mn/Fe oxides.

Abstract Background In isometric muscle function, there are subjectively two different modes of performance: one can either hold isometrically – thus resist an impacting force – or push isometrically – therefore work against a stable resistance. The purpose of this study is to investigate whether or not two different isometric muscle actions – the holding vs. pushing one (HIMA vs PIMA) – can be distinguished by objective parameters. Methods Ten subjects performed two different measuring modes at 80% of MVC realized by a special pneumatic system. During HIMA the subject had to resist the defined impacting force of the pneumatic system in an isometric position, whereby the force of the cylinder works in direction of elbow flexion against the subject. During PIMA the subject worked isometrically in direction of elbow extension against a stable position of the system. The signals of pressure, force, acceleration and mechanomyography/-tendography (MMG/MTG) of the elbow extensor (MMGtri/MTGtri) and the abdominal muscle (MMGobl) were recorded and evaluated concerning the duration of maintaining the force level (force endurance) and the characteristics of MMG-/MTG-signals. Statistical group differences comparing HIMA vs. PIMA were estimated using SPSS. Results Significant differences between HIMA and PIMA were especially apparent regarding the force endurance: During HIMA the subjects showed a decisively shorter time of stable isometric position (19 ± 8 s) in comparison with PIMA (41 ± 24 s; p = .005). In addition, during PIMA the longest isometric plateau amounted to 59.4% of the overall duration time of isometric measuring, during HIMA it lasted 31.6% (p = .000). The frequency of MMG/MTG did not show significant differences. The power in the frequency ranges of 8–15 Hz and 10–29 Hz was significantly higher in the MTGtri performing HIMA compared to PIMA (but not for the MMGs). The amplitude of MMG/MTG did not show any significant difference considering the whole measurement. However, looking only at the last 10% of duration time (exhaustion), the MMGtri showed significantly higher amplitudes during PIMA. Conclusion The results suggest that under holding isometric conditions muscles exhaust earlier. That means that there are probably two forms of isometric muscle action. We hypothesize two potential reasons for faster yielding during HIMA: (1) earlier metabolic fatigue of the muscle fibers and (2) the complexity of neural control strategies.

The improvement of power is an objective in training of athletes. In order to detect effective methods of exercise, basic research is required regarding the mechanisms of muscular activity. The purpose of this study is to investigate whether or not a muscular pre-activation prior to an external impulse-like force impact has an effect on the maximal explosive eccentric Adaptive Force (xpAFeccmax). This power capability combines different probable power enhancing mechanisms. To measure the xpAFeccmax an innovative pneumatic device was used. During measuring, the subject tries to hold an isometric position as long as possible. In the moment in which the subjects’ maximal isometric holding strength is exceeded, it merges into eccentric muscle action. This process is very close to motions in sports, where an adaptation of the neuromuscular system is required, e.g., force impacts caused by uneven surfaces during skiing. For investigating the effect of pre-activation on the xpAFeccmax of the quadriceps femoris muscle, n = 20 subjects had to pass three different pre-activation levels in a randomized order (level 1: 0.4 bar, level 2: 0.8 bar, level 3: 1.2 bar). After adjusting the standardized pre-pressure by pushing against the interface, an impulse-like load impacted on the distal tibia of the subject. During this, the xpAFeccmax was detected. The maximal voluntary isometric contraction (MVIC) was also measured. The torque values of the xpAFeccmax were compared with regard to the pre-activation levels. The results show a significant positive relation between the pre-activation of the quadriceps femoris muscle and the xpAFeccmax (male: p = 0.000, η2= 0.683; female: p = 0.000, η2= 0.907). The average percentage increase of torque amounted +28.15 ± 25.4% between MVIC and xpAFeccmax with pre-pressure level 1, +12.09 ± 7.9% for the xpAFeccmax comparing pre-pressure levels 1 vs. 2 and +2.98 ± 4.2% comparing levels 2 and 3. A higher but not maximal muscular activation prior to a fast impacting eccentric load seems to produce an immediate increase of force outcome. Different possible physiological explanatory approaches and the use as a potential training method are discussed.

Adaptive Force (AF) reflects the capability of the neuromuscular system to adapt adequately to external forces with the intention of maintaining a position or motion. One specific approach to assessing AF is to measure force and limb position during a pneumatically applied increasing external force. Through this method, the highest (AFmax), the maximal isometric (AFisomax) and the maximal eccentric Adaptive Force (AFeccmax) can be determined. The main question of the study was whether the AFisomax is a specific and independent parameter of muscle function compared to other maximal forces. In 13 healthy subjects (9 male and 4 female), the maximal voluntary isometric contraction (pre- and post-MVIC), the three AF parameters and the MVIC with a prior concentric contraction (MVICpri-con) of the elbow extensors were measured 4 times on two days. Arithmetic mean (M) and maximal (Max) torques of all force types were analyzed. Regarding the reliability of the AF parameters between days, the mean changes were 0.31–1.98 Nm (0.61%–5.47%, p = 0.175–0.552), the standard errors of measurements (SEM) were 1.29–5.68 Nm (2.53%–15.70%) and the ICCs(3,1) = 0.896–0.996. M and Max of AFisomax, AFmax and pre-MVIC correlated highly (r = 0.85–0.98). The M and Max of AFisomax were significantly lower (6.12–14.93 Nm; p ≤ 0.001–0.009) and more variable between trials (coefficient of variation (CVs) ≥ 21.95%) compared to those of pre-MVIC and AFmax (CVs ≤ 5.4%). The results suggest the novel measuring procedure is suitable to reliably quantify the AF, whereby the presented measurement errors should be taken into consideration. The AFisomax seems to reflect its own strength capacity and should be detected separately. It is suggested its normalization to the MVIC or AFmax could serve as an indicator of a neuromuscular function.

Targeted therapy of ErbB2-dependent tumours often provokes an adaptive response leading to reactivation of the PI3K/AKT pathway. Here the authors identify an ErbB3-independent compensatory mechanism comprising Ras/PI3K activation directly by ErbB2, and develop biparatopic panErbB inhibitors to block this mode of resistance.

The manual muscle test (MMT) is a flexible diagnostic tool, which is used in many disciplines, applied in several ways. The main problem is the subjectivity of the test. The MMT in the version of a “break test” depends on the tester’s force rise and the patient’s ability to resist the applied force. As a first step, the investigation of the reproducibility of the testers’ force profile is required for valid application. The study examined the force profiles of n = 29 testers (n = 9 experiences (Exp), n = 8 little experienced (LitExp), n = 12 beginners (Beg)). The testers performed 10 MMTs according to the test of hip flexors, but against a fixed leg to exclude the patient’s reaction. A handheld device recorded the temporal course of the applied force. The results show significant differences between Exp and Beg concerning the starting force (padj = 0.029), the ratio of starting to maximum force (padj = 0.005) and the normalized mean Euclidean distances between the 10 trials (padj = 0.015). The slope is significantly higher in Exp vs. LitExp (p = 0.006) and Beg (p = 0.005). The results also indicate that experienced testers show inter-tester differences and partly even a low intra-tester reproducibility. This highlights the necessity of an objective MMT-assessment. Furthermore, an agreement on a standardized force profile is required. A suggestion for this is given.

Over the last few years, protein-based affinity reagents have proven very helpful in cell and developmental biology. While many of these versatile small proteins can be expressed both in the intracellular and extracellular milieu in cultured cells and in living organisms, they can also be functionalized by fusing them to different protein domains in order to regulate or modulate their target proteins in diverse manners. For example, protein binders have been employed to degrade, trap, localize or enzymatically modify specific target proteins. Whereas binders to many endogenous proteins or small protein tags have been generated, several affinity reagents against fluorescent proteins have also been created and used to manipulate target proteins tagged with the corresponding fluorescent protein. Both of these approaches have resulted in improved methods for cell biological and developmental studies. While binders against GFP and mCherry have been previously isolated and validated, we now report the generation and utilization of designed ankyrin repeat proteins (DARPins) against the monomeric teal fluorescent protein 1 (mTFP1). Here we use the generated DARPins to delocalize Rab proteins to the nuclear compartment, in which they cannot fulfil their regular functions anymore. In the future, such manipulations might enable the production of acute loss-of-function phenotypes in different cell types or in living organisms based on direct protein manipulation rather than on genetic loss-of-function analyses.

Recurring dry-wet cycles of soils, such as in rice paddies and on floodplains, have a dramatic impact on biogeochemical processes. The rates and trajectories of microbial metabolic functions during transition periods from drained to flooded conditions affect the transformation rates and phase partitioning of carbon, nutrients, and contaminants. However, the regulating mechanisms responsible for diverging functional metabolisms during such transitions are poorly resolved. The chemistry of organic carbon within the microbially available pool likely holds key information regarding carbon cycling and redox transformation rates. In this study, we used mesocosms to examine the influence of different carbon sources (glucose, straw, manure, char) on microbial energetics, respiration rates, and carbon balances in rice paddy soils during the transition from drained to flooded conditions following inundation. We found that variability in carbon solubility (1.6–400 mg g−1) and chemical composition of the amendments led to non-uniform stimulation of carbon dioxide production per unit carbon added (0.4–32.9 mmol CO2 mol−1 added C). However, there was a clear linear correlation between energy release and net CO2 production rate (R2 = 0.85), between CO2 and initial soluble C (R2 = 0.91, excluding glucose treatment) and between heat output and Gibbs free energy of initial soluble C (R2 = 0.78 and 0.69, with/without glucose respectively). Our results further indicated that the chemical composition of the soluble C from amendments initiated divergent anaerobic respiration behavior, impacting methane production and the partitioning of elements between soil solid phase and solution. This study shows the benefit of monitoring energy and element mass balances for elucidating the contribution of various microbial metabolic functions in complex systems. Further, our results highlight the importance of organic carbon composition within the water soluble pool as a key driver of microbially mediated redox transformations with major impacts on greenhouse gas emissions, contaminant fate, and nutrient cycling in paddy soils and similar ecosystems.

Increasing soil organic carbon (SOC) stocks in agricultural soils can contribute to stabilizing or even lowering atmospheric greenhouse gas (GHG) concentrations. Cover crop rotation has been shown to increase SOC and provide productivity benefits for agriculture. Here we used a split field design to evaluate the short-term effect of cover crop on SOC distribution and chemistry using a combination of bulk, isotopic, and spectroscopic analyses of size-and density-separated soil aggregates. Macroaggregates (>250 µm) incorporated additional plant material with cover crop as evidenced by more negative δ13C values (−25.4‱ with cover crop compared to −25.1‱ without cover crop) and increased phenolic (plant-like) resonance in carbon NEXAFS spectra. Iron EXAFS data showed that the Fe pool was composed of 17−21% Fe oxide with the remainder a mix of primary and secondary minerals. Comparison of oxalate and dithionite extractions suggests that cover crop may also increase Fe oxide crystallinity, especially in the dense (>2.4 g cm−3) soil fraction. Cover crop δ13C values were more negative across density fractions of bulk soil, indicating the presence of less processed organic carbon. Although no significant difference was observed in bulk SOC on a mass per mass basis between cover and no cover crop fields after one season, isotopic and spectroscopic data reveal enhanced carbon movement between aggregates in cover crop soil.

The scope of targets investigated in pharmaceutical research is continuously moving into uncharted territory. Consequently, finding suitable chemical matter with current compound collections is proving increasingly difficult. Encoded library technologies enable the rapid exploration of large chemical space for the identification of ligands for such targets. These binders facilitate drug discovery projects both as tools for target validation, structural elucidation and assay development as well as starting points for medicinal chemistry. Novartis internalized two complementing encoded library platforms to accelerate the initiation of its drug discovery programs. For the identification of low-molecular weight ligands, we apply DNA-encoded libraries. In addition, encoded peptide libraries are employed to identify cyclic peptides. This review discusses how we apply these two platforms in our research and why we consider it beneficial to run both pipelines in-house.