Myosin-I's motor and actin assembly activation activities are modular and separable in budding yeast clathrin-mediated endocytosis.

The myosin-Is, Myo3 and Myo5 in budding yeast, are implicated in force generation and actin assembly during clathrin-mediated endocytosis (CME). The myosin-Is have motor activity, bind the plasma membrane, and activate the Arp2/3 complex to promote branched actin assembly. We reveal that Myo5 's force-generating motor activity and nucleation-promoting factor (NPF) activity each must be coupled to membrane binding for successful CME. However, the motor and NPF activities are modular and separable, showing that these activities function independently rather than in an obligatorily integrated manner to provide myosin-I's essential functions in actin network assembly and force generation during budding yeast CME.

A Role for Cross-linking Proteins in Actin Filament Network Organization and Force Generation.

The high turgor pressure across the plasma membrane of yeasts creates a requirement for substantial force production by actin polymerization and myosin motor activity for clathrin-mediated endocytosis (CME). Endocytic internalization is severely impeded in the absence of fimbrin, an actin filament crosslinking protein called Sac6 in budding yeast. Here, we combine live-cell imaging and mathematical modeling to gain new insights into the role of actin filament crosslinking proteins in force generation. Genetic manipulation showed that CME sites with more crosslinking proteins are more effective at internalization under high load. Simulations of an experimentally constrained, agent-based mathematical model recapitulate the result that endocytic networks with more double-bound fimbrin molecules internalize the plasma membrane against elevated turgor pressure more effectively. Networks with large numbers of crosslinks also have more growing actin filament barbed ends at the plasma membrane, where the addition of new actin monomers contributes to force generation and vesicle internalization. Our results provide a richer understanding of the crucial role played by actin filament crosslinking proteins during actin network force generation, highlighting the contribution of these proteins to the self-organization of the actin filament network and force generation under increased load.

The Evaluation of an Integrated Tobacco Treatment Specialist in Primary Care.

Background: Primary care providers play a key role in screening for tobacco use and assessing desire to quit. Tobacco treatment specialists (TTS) are certified in helping patients who desire tobacco cessation. A primary care nurse practitioner within one Midwestern healthcare organization obtained TTS certification and integrated specialized tobacco cessation visits within a primary care clinic from February 2021 to February 2022. Purpose: To determine the efficiency and effectiveness of an integrated TTS-certified nurse practitioner (TTS-NP) in a primary care setting 1-year postimplementation. Method: This program evaluation utilized retrospective electronic health record review and included thirty-three patients. The logic model served as a framework to define efficiency and effectiveness. Results: Patients were referred by a provider (57.6%), nurse (15.2%), or self (27.3). Patients opted for in-person initial visits (81.8%) more than virtual (18.2%). Of a total of 73 scheduled visits, 8 (11%) were no-showed. Patients who self-referred had the lowest no-show rate (5.6%) compared to those referred by a provider (12.8%) or nurse (12.5%). Of the patients included, 87.9% set a goal quit date. Average time until first and second follow-up was 34.6 and 130.4 days after goal quit date. Follow-up was defined as the date of the patient's first message reply to the TTS-NP, or first visit following the goal quit date. A total of 51.9% (n = 14) and 63% (n = 17) reported cessation at the first and second follow-up. TTS-NP visit's cost, independent of any other coverage, was less than other specialty visits in primary care. Conclusion: TTS-NP visits in primary care enabled patients to benefit from lower cost and longitudinal follow-up within a familiar setting. Over half of patients achieved cessation. Results of this program evaluation suggest support for TTS-certified providers in primary care.

Structural basis for assembly and lipid-mediated gating of LRRC8A:C volume-regulated anion channels.

Leucine-rich repeat-containing protein 8 (LRRC8) family members form volume-regulated anion channels activated by hypoosmotic cell swelling. LRRC8 channels are ubiquitously expressed in vertebrate cells as heteromeric assemblies of LRRC8A (SWELL1) and LRRC8B-E subunits. Channels of different subunit composition have distinct properties that explain the functional diversity of LRRC8 currents across cell types. However, the basis for heteromeric LRRC8 channel assembly and function is unknown. Here we leverage a fiducial-tagging strategy to determine single-particle cryo-EM structures of heterohexameric LRRC8A:C channels in multiple conformations. Compared to homomers, LRRC8A:C channels show pronounced differences in architecture due to heterotypic LRR interactions that displace subunits away from the conduction axis and poise the channel for activation. Structures and functional studies further reveal that lipids embedded in the channel pore block ion conduction in the closed state. These results provide insight into determinants for heteromeric LRRC8 channel assembly, activity and gating by lipids.

Sodium-glucose cotransporter 2 inhibitors for treatment of diabetes mellitus after kidney transplantation.

INTRODUCTION: Diabetes mellitus in kidney transplant recipients is a risk factor for cardiovascular events and premature death. Sodium-glucose cotransporter 2 inhibitors (SGLT2i) are increasingly used in nontransplant populations to improve diabetes control and cardiovascular and renal benefits. Limited literature exists regarding the safety and efficacy of these agents in renal transplant recipients. METHODS: We retrospectively reviewed all kidney transplant recipients within our health system who were prescribed a SGLT2i after transplantation for diabetes. The safety, tolerability, and effectiveness of SGLT2i were analyzed. RESULTS: Thirty-nine kidney transplant recipients were initiated on SGLT2i therapy, twenty-seven of which remained on therapy for at least 1 year. Ten (25%) patients experienced an adverse event while on a SGLT2i, with urinary tract infections (UTI) being the most common. Seventeen patients (43%) discontinued the SGLT2i at the time of chart review, most commonly due to cost and kidney function decline. The median [IQR] hemoglobin A1c (HbA1c) at SGLT2i initiation of 8.4% [7.8-9.2] decreased to 7.5% [6.8-8.0%] after 3 months and 7.5% [6.5-7.9] after 12 months. No meaningful change in kidney function or tacrolimus exposure was observed. CONCLUSION: SGLT2i may be a safe and effective treatment for diabetes in kidney transplant recipients. Cost is a barrier to SGLT2i therapy, and UTIs were the most frequently encountered adverse events in this cohort. More studies are needed to understand the safety profile and determine the effect of SGLT2i on diabetes-related comorbidities among kidney transplant recipients.

Mechanistic insights into actin force generation during vesicle formation from cryo-electron tomography.

Actin assembly provides force for a multitude of cellular processes. Compared to actin-assembly-based force production during cell migration, relatively little is understood about how actin assembly generates pulling forces for vesicle formation. Here, cryo-electron tomography identified actin filament number, organization, and orientation during clathrin-mediated endocytosis in human SK-MEL-2 cells, showing that force generation is robust despite variance in network organization. Actin dynamics simulations incorporating a measured branch angle indicate that sufficient force to drive membrane internalization is generated through polymerization and that assembly is triggered from ∼4 founding "mother" filaments, consistent with tomography data. Hip1R actin filament anchoring points are present along the entire endocytic invagination, where simulations show that it is key to pulling force generation, and along the neck, where it targets filament growth and makes internalization more robust. Actin organization described here allowed direct translation of structure to mechanism with broad implications for other actin-driven processes.

Accuracy of fetal anatomy survey in the diagnosis of velamentous cord insertion: a case-control study.

OBJECTIVE: Our objective was to determine the accuracy of ultrasound at the time of the fetal anatomy survey in the diagnosis of velamentous cord insertion (VCI). STUDY DESIGN: This retrospective case-control study identified placentas with VCI (cases) and randomly selected placentas with normal placental cord insertion (PCI) (controls) as documented by placental pathology for mothers delivered from 2002 through 2015. Archived ultrasound images for PCI at the time of the fetal anatomy survey were reviewed. Data analysis was by calculation of sensitivity, specificity, and accuracy and their 95% CI for the ultrasound diagnosis of VCI. RESULTS: The prevalence of VCI was 1.6% of placentas submitted for pathologic examination. There were 122 cases of VCI and 347 controls with normal PCI. The performance criteria calculated for the diagnosis of VCI at the time of fetal anatomy survey were as follows: sensitivity 33.6%; 95% CI: 25.3, 42.7; specificity 99.7%; 95% CI: 98.4, 99.9 and accuracy 82.5; 95% CI: 80.5, 82.9. CONCLUSION: The identification of a VCI at the time of fetal anatomy survey is highly specific for the presence of a VCI as documented by placental pathology. The sensitivity in this study was less than expected. Sensitivity could be improved by reducing the number of nonvisualized PCIs, creating an awareness of risk factors for VCI, and obtaining more detailed images in the case of an apparent marginal PCI.

Phosphorylation by the stress-activated MAPK Slt2 down-regulates the yeast TOR complex 2.

Saccharomyces cerevisiae target of rapamycin (TOR) complex 2 (TORC2) is an essential regulator of plasma membrane lipid and protein homeostasis. How TORC2 activity is modulated in response to changes in the status of the cell envelope is unclear. Here we document that TORC2 subunit Avo2 is a direct target of Slt2, the mitogen-activated protein kinase (MAPK) of the cell wall integrity pathway. Activation of Slt2 by overexpression of a constitutively active allele of an upstream Slt2 activator (Pkc1) or by auxin-induced degradation of a negative Slt2 regulator (Sln1) caused hyperphosphorylation of Avo2 at its MAPK phosphoacceptor sites in a Slt2-dependent manner and diminished TORC2-mediated phosphorylation of its major downstream effector, protein kinase Ypk1. Deletion of Avo2 or expression of a phosphomimetic Avo2 allele rendered cells sensitive to two stresses (myriocin treatment and elevated exogenous acetic acid) that the cell requires Ypk1 activation by TORC2 to survive. Thus, Avo2 is necessary for optimal TORC2 activity, and Slt2-mediated phosphorylation of Avo2 down-regulates TORC2 signaling. Compared with wild-type Avo2, phosphomimetic Avo2 shows significant displacement from the plasma membrane, suggesting that Slt2 inhibits TORC2 by promoting Avo2 dissociation. Our findings are the first demonstration that TORC2 function is regulated by MAPK-mediated phosphorylation.

The Stress-Sensing TORC2 Complex Activates Yeast AGC-Family Protein Kinase Ypk1 at Multiple Novel Sites.

Yeast (Saccharomyces cerevisiae) target of rapamycin (TOR) complex 2 (TORC2) is a multi-subunit plasma membrane-associated protein kinase and vital growth regulator. Its essential functions are exerted via phosphorylation and stimulation of downstream protein kinase Ypk1 (and its paralog Ypk2). Ypk1 phosphorylates multiple substrates to regulate plasma membrane lipid and protein composition. Ypk1 function requires phosphorylation of Thr504 in its activation loop by eisosome-associated Pkh1 (and its paralog Pkh2). For cell survival under certain stresses, however, Ypk1 activity requires further stimulation by TORC2-mediated phosphorylation at C-terminal sites, dubbed the "turn" (Ser644) and "hydrophobic" (Thr662) motifs. Here we show that four additional C-terminal sites are phosphorylated in a TORC2-dependent manner, collectively defining a minimal consensus. We found that the newly identified sites are as important for Ypk1 activity, stability, and biological function as Ser644 and Thr662. Ala substitutions at the four new sites abrogated the ability of Ypk1 to rescue the phenotypes of Ypk1 deficiency, whereas Glu substitutions had no ill effect. Combining the Ala substitutions with an N-terminal mutation (D242A), which has been demonstrated to bypass the need for TORC2-mediated phosphorylation, restored the ability to complement a Ypk1-deficient cell. These findings provide new insights about the molecular basis for TORC2-dependent activation of Ypk1.

The Mini C-arm Adds Quality and Efficiency to the Pediatric Orthopedic Outpatient Clinic.

The mini C-arm has become increasingly popular in the practice of orthopedics. To the authors' knowledge, its use in the pediatric orthopedic outpatient clinic has not been reported. The purpose of this study was to evaluate the practice efficiency and radiation exposure to the patient when the mini C-arm was used in the pediatric orthopedic outpatient clinic. One hundred consecutive midshaft and distal forearm fractures were evaluated by one orthopedic surgeon in follow-up using a mini C-arm. For each case, the radiation physicist calculated the amount of skin exposure in milligray (mGy). The average skin exposure to the patient from the mini C-arm was 0.58 mGy, compared with 0.2 mGy for anteroposterior view and lateral view radiographs. Use of the mini C-arm, in place of plain radiographs obtained in the radiology department, decreased time waiting during clinic visits by 23 minutes. This study reports 2 important findings. First, surprisingly, the mini C-arm used a slightly higher radiation dose than standard imaging with plain radiographs. Second, use of the mini C-arm saved time and improved the efficiency of the clinic visit. Overall, the mini C-arm improves quality and efficiency in the pediatric orthopedic outpatient clinic. [Orthopedics. 2016; 39(6):e1097-e1099.].

Behavioral health integration: Transforming patient care, medical resident education, and physician effectiveness.

Integrating behavioral health care into the family medicine residency has had a dramatic impact on patient care, resident training, and physician effectiveness. With a cursory glance, it may seem that integrated behavioral health providers spend their days exclusively having 30-min office visits with patients. A full exploration, however, reveals that well-integrated behavioral health offers not only brief and effective visits with patients but also increased collaboration, better patient comanagement, and multiple opportunities for resident learning. Behavioral health integration transforms the way in which medical providers at all levels of training work, teach, and conceptualize their future practice. In an internal survey of 33 medical providers in an integrated family medicine residency, 97% of respondents report they value behavioral health integration to such a degree that they are more likely to accept a job in a setting that offers integrated behavioral health.

Comparison of soft toothbrush and new ultra-soft cleaner in ability to remove plaque from teeth.

In this single-blind, crossover study, the difference between a brushless tooth cleaner and a soft toothbrush was studied to compare plaque removal efficiency. The sample was composed of 15 human subjects who were categorized into two groups. Group 1 was composed of subjects randomly assigned to the brushless tooth cleaner for the first two weeks. Group 2 was composed of those randomly assigned to begin the study using the soft toothbrush. After two weeks of brushing with their assigned device, subjects returned to their normal modality to brush their teeth for one week. For the last two weeks of the study, subjects were told to brush with the opposite device they were originally assigned to at the beginning of the trial. Investigators recorded the subjects' gingival indices (based on probe depths) and Quigley scores (based on plaque indices using disclosing solution) at the beginning of week one, the end of week two, the end of week three and the end of week five. The main outcomes in this study were the Silness Loe Index (SLI) and the Quigley Hein Index (QHI). The SLI was assessed on the buccal, lingual, mesial and distal surfaces of six teeth, for a total of 24 surfaces. The QHI was assessed on the buccal and lingual surfaces of six teeth, for a total of 12 surfaces. Each index was measured at each visit by the sum total score divided by the total number of surfaces. The data were analyzed separately using a mixed-effects repeated measures analysis of variance (RMANOVA) for crossover designs. Results indicate that, according to the SLI, there is no significant difference between the two treatments after the first or second weeks. However, based on the QHI, statistically significant differences existed between the two treatments after week one and two. After week one, the soft toothbrush use had a higher QHI than the brushless tooth cleaner. After week two, the brushless tooth cleaner had a higher QHI than the soft toothbrush.

Predator biomass determines the magnitude of non-consumptive effects (NCEs) in both laboratory and field environments.

Predator body size often indicates predation risk, but its significance in non-consumptive effects (NCEs) and predator risk assessment has been largely understudied. Although studies often recognize that predator body size can cause differing cascading effects, few directly examine prey foraging behavior in response to individual predator sizes or investigate how predator size is discerned. These mechanisms are important since perception of the risk imposed by predators dictates behavioral responses to predators and subsequent NCEs. Here, we evaluate the role of predator body size and biomass on risk assessment and the magnitude of NCEs by investigating mud crab foraging behavior and oyster survival in response to differing biomasses of blue crab predators using both laboratory and field methods. Cues from high predator biomass treatments including large blue crab predators and multiple small blue crab predators decreased mud crab foraging and increased oyster survival, whereas mud crab foraging in response to a single small blue crab did not differ from controls. Mud crabs also increased refuge use in the presence of large and multiple small, but not single small, blue crab predators. Thus, both predator biomass and aggregation patterns may affect the expression of NCEs. Understanding the impact of predator biomass may therefore be necessary to successfully predict the role of NCEs in shaping community dynamics. Further, the results of our laboratory experiments were consistent with observed NCEs in the field, suggesting that data from mesocosm environments can provide insight into field situations where flow and turbulence levels are moderate.