[Survey of occupational health-related activities conducted at hospitals in the Kanto region (2020)].

OBJECTIVE: To survey occupational health-related activities conducted at hospitals certified by the Japan Council for Quality Health Care in the Kanto region of Japan. METHODS: The survey tool was sent to 470 hospitals and comprised the following items: hospital size, occupational health system, infection control practices, mental health services, promotion of work system reforms, and priorities in achieving occupational health. RESULTS: A total of 140 hospitals completed the survey. A monthly workplace inspection was conducted in approximately 60% of the hospitals. Testing of new employees for hepatitis and four other viruses was conducted in approximately 65% of the hospitals, and influenza vaccination was administered to the employees in all the hospitals. Most hospitals provided mental health services to their workers, which included consultation with an occupational physician. Work system reforms for changing conference time and task shifting or sharing were adopted in approximately 50% of the hospitals. Prevention of blood-borne pathogens, respiratory infections, and healthcare coverage for healthcare workers was identified as areas of improvement in several hospitals. CONCLUSIONS: Legally required infection control and occupational health-related practices were conducted in most hospitals. Additionally, several hospitals undertook work system reforms, including the management of changes in conference time and task shifting or sharing.

The extracellular loop of pendrin and prestin modulates their voltage-sensing property.

Pendrin and prestin belong to the solute carrier 26 (SLC26) family of anion transporters. Prestin is unique among the SLC26 family members in that it displays voltage-driven motor activity (electromotility) and concurrent gating currents that manifest as nonlinear cell membrane electrical capacitance (nonlinear capacitance (NLC)). Although the anion transport mechanism of the SLC26 proteins has begun to be elucidated, the molecular mechanism of electromotility, which is thought to have evolved from an ancestral ion transport mechanism, still remains largely elusive. Here, we demonstrate that pendrin also exhibits large NLC and that charged residues present in one of the extracellular loops of pendrin and prestin play significant roles in setting the voltage-operating points of NLC. Our results suggest that the molecular mechanism responsible for sensing voltage is not unique to prestin among the members of the SLC26 family and that this voltage-sensing mechanism works independently of the anion transport mechanism.

Real-time observation of flexible domain movements in CRISPR-Cas9.

The CRISPR-associated protein Cas9 is widely used for genome editing because it cleaves target DNA through the assistance of a single-guide RNA (sgRNA). Structural studies have revealed the multi-domain architecture of Cas9 and suggested sequential domain movements of Cas9 upon binding to the sgRNA and the target DNA These studies also hinted at the flexibility between domains; however, it remains unclear whether these flexible movements occur in solution. Here, we directly observed dynamic fluctuations of multiple Cas9 domains, using single-molecule FRET We found that the flexible domain movements allow Cas9 to adopt transient conformations beyond those captured in the crystal structures. Importantly, the HNH nuclease domain only accessed the DNA cleavage position during such flexible movements, suggesting the importance of this flexibility in the DNA cleavage process. Our FRET data also revealed the conformational flexibility of apo-Cas9, which may play a role in the assembly with the sgRNA Collectively, our results highlight the potential role of domain fluctuations in driving Cas9-catalyzed DNA cleavage.

Crystal structures of the TRIC trimeric intracellular cation channel orthologues.

Ca2+ release from the sarcoplasmic reticulum (SR) and endoplasmic reticulum (ER) is crucial for muscle contraction, cell growth, apoptosis, learning and memory. The trimeric intracellular cation (TRIC) channels were recently identified as cation channels balancing the SR and ER membrane potentials, and are implicated in Ca2+ signaling and homeostasis. Here we present the crystal structures of prokaryotic TRIC channels in the closed state and structure-based functional analyses of prokaryotic and eukaryotic TRIC channels. Each trimer subunit consists of seven transmembrane (TM) helices with two inverted repeated regions. The electrophysiological, biochemical and biophysical analyses revealed that TRIC channels possess an ion-conducting pore within each subunit, and that the trimer formation contributes to the stability of the protein. The symmetrically related TM2 and TM5 helices are kinked at the conserved glycine clusters, and these kinks are important for the channel activity. Furthermore, the kinks of the TM2 and TM5 helices generate lateral fenestrations at each subunit interface. Unexpectedly, these lateral fenestrations are occupied with lipid molecules. This study provides the structural and functional framework for the molecular mechanism of this ion channel superfamily.

Lever arm extension of myosin VI is unnecessary for the adjacent binding state.

Myosin VI is a processive myosin that has a unique stepping motion, which includes three kinds of steps: a large forward step, a small forward step and a backward step. Recently, we proposed the parallel lever arms model to explain the adjacent binding state, which is necessary for the unique motion. In this model, both lever arms are directed the same direction. However, experimental evidence has not refuted the possibility that the adjacent binding state emerges from myosin VI folding its lever arm extension (LAE). To clarify this issue, we constructed a myosin VI/V chimera that replaces the myosin VI LAE with the IQ3-6 domains of the myosin V lever arm, which cannot fold, and performed single molecule imaging. Our chimera showed the same stepping patterns as myosin VI, indicating the LAE is not responsible for the adjacent binding state.

Spontaneous detachment of the leading head contributes to myosin VI backward steps.

Myosin VI is an ATP driven molecular motor that normally takes forward and processive steps on actin filaments, but also on occasion stochastic backward steps. While a number of models have attempted to explain the backwards steps, none offer an acceptable mechanism for their existence. We therefore performed single molecule imaging of myosin VI and calculated the stepping rates of forward and backward steps at the single molecule level. The forward stepping rate was proportional to the ATP concentration, whereas the backward stepping rate was independent. Using these data, we proposed that spontaneous detachment of the leading head is uncoupled from ATP binding and is responsible for the backward steps of myosin VI.

Simultaneous observation of the lever arm and head explains myosin VI dual function.

Myosin VI is an adenosine triphosphate (ATP)-driven dimeric molecular motor that has dual function as a vesicle transporter and a cytoskeletal anchor. Recently, it was reported that myosin VI generates three types of steps by taking either a distant binding or adjacent binding state (noncanonical hand-over-hand step pathway). The adjacent binding state, in which both heads bind to an actin filament near one another, is unique to myosin VI and therefore may help explain its distinct features. However, detailed information of the adjacent binding state remains unclear. Here simultaneous observations of the head and tail domain during stepping are presented. These observations show that the lever arms tilt forward in the adjacent binding state. Furthermore, it is revealed that either head could take the subsequent step with equal probability from this state. Together with previous results, a comprehensive stepping scheme is proposed; it includes the tail domain motion to explain how myosin VI achieves its dual function.

G146V mutation at the hinge region of actin reveals a myosin class-specific requirement of actin conformations for motility.

The G146V mutation in actin is dominant lethal in yeast. G146V actin filaments bind cofilin only minimally, presumably because cofilin binding requires the large and small actin domains to twist with respect to one another around the hinge region containing Gly-146, and the mutation inhibits that twisting motion. A number of studies have suggested that force generation by myosin also requires actin filaments to undergo conformational changes. This prompted us to examine the effects of the G146V mutation on myosin motility. When compared with wild-type actin filaments, G146V filaments showed a 78% slower gliding velocity and a 70% smaller stall force on surfaces coated with skeletal heavy meromyosin. In contrast, the G146V mutation had no effect on either gliding velocity or stall force on myosin V surfaces. Kinetic analyses of actin-myosin binding and ATPase activity indicated that the weaker affinity of actin filaments for myosin heads carrying ADP, as well as reduced actin-activated ATPase activity, are the cause of the diminished motility seen with skeletal myosin. Interestingly, the G146V mutation disrupted cooperative binding of myosin II heads to actin filaments. These data suggest that myosin-induced conformational changes in the actin filaments, presumably around the hinge region, are involved in mediating the motility of skeletal myosin but not myosin V and that the specific structural requirements for the actin subunits, and thus the mechanism of motility, differ among myosin classes.

Switch between large hand-over-hand and small inchworm-like steps in myosin VI.

Many biological motor molecules move within cells using stepsizes predictable from their structures. Myosin VI, however, has much larger and more broadly distributed stepsizes than those predicted from its short lever arms. We explain the discrepancy by monitoring Qdots and gold nanoparticles attached to the myosin-VI motor domains using high-sensitivity nanoimaging. The large stepsizes were attributed to an extended and relatively rigid lever arm; their variability to two stepsizes, one large (72 nm) and one small (44 nm). These results suggest that there exist two tilt angles during myosin-VI stepping, which correspond to the pre- and postpowerstroke states and regulate the leading head. The large steps are consistent with the previously reported hand-over-hand mechanism, while the small steps follow an inchworm-like mechanism and increase in frequency with ADP. Switching between these two mechanisms in a strain-sensitive, ADP-dependent manner allows myosin VI to fulfill its multiple cellular tasks including vesicle transport and membrane anchoring.

Simultaneous measurement of nucleotide occupancy and mechanical displacement in myosin-V, a processive molecular motor.

Adenosine triphosphate (ATP) turnover drives various processive molecular motors and adenosine diphosphate (ADP) release is a principal transition in this cycle. Biochemical and single molecule mechanical studies have led to a model in which a slow ADP release step contributes to the processivity of myosin-V. To test the relationship between force generation and ADP release, we utilized optical trapping nanometry and single molecule total internal reflection fluorescence imaging for simultaneous and direct observation of both processes in myosin-V. We found that ADP was released 69 +/- 5.3 ms after force generation and displacement of actin, providing direct evidence for slow ADP release. As proposed by several previous studies, this slow ADP release probably ensures processivity by prolonging the strong actomyosin state in the ATP turnover cycle.

Measurement system for simultaneous observation of myosin V chemical and mechanical events.

Myosin V is an actin-based processive molecular motor driven by the chemical energy of ATP hydrolysis. Although the chemo-mechanical coupling in processive movement has been postulated by separate structural, mechanical and biochemical studies, no experiment has been able to directly test these conclusions. Therefore the relationship between ATP-turnover and force generation remains unclear. Currently, the most direct method to measure the chemo-mechanical coupling in processive motors is to simultaneously observe ATP-turnover cycles and displacement at the single molecule level. In this study, we developed a simultaneous measurement system suitable for mechanical and chemical assays of myosin V in order to directly elucidate its chemo-mechanical coupling.

Site-directed spin labeling electron paramagnetic resonance study of the calcium-induced structural transition in the N-domain of human cardiac troponin C complexed with troponin I.

Calcium-induced structural transition in the amino-terminal domain of troponin C (TnC) triggers skeletal and cardiac muscle contraction. The salient feature of this structural transition is the movement of the B and C helices, which is termed the "opening" of the N-domain. This movement exposes a hydrophobic region, allowing interaction with the regulatory domain of troponin I (TnI) as can be seen in the crystal structure of the troponin ternary complex [Takeda, S., Yamashita, A., Maeda, K., and Maeda, Y. (2003) Nature 424, 35-41]. In contrast to skeletal TnC, Ca(2+)-binding site I (an EF-hand motif that consists of an A helix-loop-B helix motif) is inactive in cardiac TnC. The question arising from comparisons with skeletal TnC is how both helices move according to Ca(2+) binding or interact with TnI in cardiac TnC. In this study, we examined the Ca(2+)-induced movement of the B and C helices relative to the D helix in a cardiac TnC monomer state and TnC-TnI binary complex by means of site-directed spin labeling electron paramagnetic resonance (EPR). Doubly spin-labeled TnC mutants were prepared, and the spin-spin distances were estimated by analyzing dipolar interactions with the Fourier deconvolution method. An interspin distance of 18.4 A was estimated for mutants spin labeled at G42C on the B helix and C84 on the D helix in a Mg(2+)-saturated monomer state. The interspin distance between Q58C on the C helix and C84 on the D helix was estimated to be 18.3 A under the same conditions. Distance changes were observed by the addition of Ca(2+) ions and the formation of a complex with TnI. Our data indicated that the C helix moved away from the D helix in a distinct Ca(2+)-dependent manner, while the B helix did not. A movement of the B helix by interaction with TnI was observed. Both Ca(2+) and TnI were also shown to be essential for the full opening of the N-domain in cardiac TnC.