Local Calcium Elevation and Cell Elongation Initiate Guided Motility in Electrically Stimulated Osteoblast-Like Cells

BACKGROUND: Investigation of the mechanisms of guided cell migration can contribute to our understanding of many crucial biological processes, such as development and regeneration. Endogenous and exogenous direct current electric fields (dcEF) are known to induce directional cell migration, however the initial cellular responses to electrical stimulation are poorly understood. Ion fluxes, besides regulating intracellular homeostasis, have been implicated in many biological events, including regeneration. Therefore understanding intracellular ion kinetics during EF-directed cell migration can provide useful information for development and regeneration. METHODOLOGY/PRINCIPAL FINDINGS: We analyzed the initial events during migration of two osteogenic cell types, rat calvarial and human SaOS-2 cells, exposed to strong (10-15 V/cm) and weak (< or = 5 V/cm) dcEFs. Cell elongation and perpendicular orientation to the EF vector occurred in a time- and voltage-dependent manner. Calvarial osteoblasts migrated to the cathode as they formed new filopodia or lamellipodia and reorganized their cytoskeleton on the cathodal side. SaOS-2 cells showed similar responses except towards the anode. Strong dcEFs triggered a rapid increase in intracellular calcium levels, whereas a steady state level of intracellular calcium was observed in weaker fields. Interestingly, we found that dcEF-induced intracellular calcium elevation was initiated with a local rise on opposite sides in calvarial and SaOS-2 cells, which may explain their preferred directionality. In calcium-free conditions, dcEFs induced neither intracellular calcium elevation nor directed migration, indicating an important role for calcium ions. Blocking studies using cadmium chloride revealed that voltage-gated calcium channels (VGCCs) are involved in dcEF-induced intracellular calcium elevation. CONCLUSION/SIGNIFICANCE: Taken together, these data form a time scale of the morphological and physiological rearrangements underlying EF-guided migration of osteoblast-like cell types and reveal a requirement for calcium in these reactions. We show for the first time here that dcEFs trigger different patterns of intracellular calcium elevation and positional shifting in osteogenic cell types that migrate in opposite directions

Maturation of the functional mouse CRES amyloid from globular form

The epididymal lumen contains a complex cystatin-rich nonpathological amyloid matrix with putative roles in sperm maturation and sperm protection. Given our growing understanding for the biological function of this and other functional amyloids, the problem still remains: how functional amyloids assemble including their initial transition to early oligomeric forms. To examine this, we developed a protocol for the purification of nondenatured mouse CRES, a component of the epididymal amyloid matrix, allowing us to examine its assembly to amyloid under conditions that may mimic those in vivo. Herein we use X-ray crystallography, solution-state NMR, and solid-state NMR to follow at the atomic level the assembly of the CRES amyloidogenic precursor as it progressed from monomeric folded protein to an advanced amyloid. We show the CRES monomer has a typical cystatin fold that assembles into highly branched amyloid matrices, comparable to those in vivo, by forming β-sheet assemblies that our data suggest occur via two distinct mechanisms: a unique conformational switch of a highly flexible disulfide-anchored loop to a rigid β-strand and by traditional cystatin domain swapping. Our results provide key insight into our understanding of functional amyloid assembly by revealing the earliest structural transitions from monomer to oligomer and by showing that some functional amyloid structures may be built by multiple and distinctive assembly mechanisms

Quantifying antimicrobial access and usage for paediatric diarrhoeal disease in an urban community setting in Asia.

OBJECTIVES: Antimicrobial-resistant infections are a major global health issue. Ease of antimicrobial access in developing countries is proposed to be a key driver of the antimicrobial resistance (AMR) epidemic despite a lack of community antimicrobial usage data. METHODS: Using a mixed-methods approach (geospatial mapping, simulated clients, healthcare utilization, longitudinal cohort) we assessed antimicrobial access in the community and quantified antimicrobial usage for childhood diarrhoea in an urban Vietnamese setting. RESULTS: The study area had a pharmacy density of 15.7 pharmacies/km2 (a pharmacy for every 1316 people). Using a simulated client method at pharmacies within the area, we found that 8% (3/37) and 22% (8/37) of outlets sold antimicrobials for paediatric watery and mucoid diarrhoea, respectively. However, despite ease of pharmacy access, the majority of caregivers would choose to take their child to a healthcare facility, with 81% (319/396) and 88% (347/396) of responders selecting a specialized hospital as one of their top three preferences when seeking treatment for watery and mucoid diarrhoea, respectively. We calculated that at least 19% (2688/14427) of diarrhoea episodes in those aged 1 to <5 years would receive an antimicrobial annually; however, antimicrobial usage was almost 10 times greater in hospitals than in the community. CONCLUSIONS: Our data question the impact of community antimicrobial usage on AMR and highlight the need for better education and guidelines for all professionals with the authority to prescribe antimicrobials

Effects of water scarcity awareness and climate change belief on recycled water usage willingness: Evidence from New Mexico, United States

The global water crisis is being exacerbated by climate change, even in the United States. Recycled water is a feasible alternative to alleviate the water shortage, but it is constrained by humans’ perceptions. The current study examines how residents’ water scarcity awareness and climate change belief influence their willingness to use recycled water directly and indirectly. Bayesian Mindsponge Framework (BMF) analytics was employed on a dataset of 1831 residents in Albuquerque, New Mexico, an arid inland region in the US. We discovered that residents’ willingness to use direct recycled potable water is positively affected by their awareness of water scarcity, but the effect is conditional on their belief in the impacts of climate change on the water cycle. Meanwhile, the willingness to use indirect recycled potable water is influenced by water scarcity awareness, and the belief in climate change further enhances this effect. These findings implicate that fighting climate change denialism and informing the public of the water scarcity situation in the region can contribute to the effectiveness and sustainability of long-term water conservation and climate change alleviation efforts

Safety and efficacy of fluoxetine on functional outcome after acute stroke (AFFINITY): a randomised, double-blind, placebo-controlled trial

Background Trials of fluoxetine for recovery after stroke report conflicting results. The Assessment oF FluoxetINe In sTroke recoverY (AFFINITY) trial aimed to show if daily oral fluoxetine for 6 months after stroke improves functional outcome in an ethnically diverse population. Methods AFFINITY was a randomised, parallel-group, double-blind, placebo-controlled trial done in 43 hospital stroke units in Australia (n=29), New Zealand (four), and Vietnam (ten). Eligible patients were adults (aged ≥18 years) with a clinical diagnosis of acute stroke in the previous 2–15 days, brain imaging consistent with ischaemic or haemorrhagic stroke, and a persisting neurological deficit that produced a modified Rankin Scale (mRS) score of 1 or more. Patients were randomly assigned 1:1 via a web-based system using a minimisation algorithm to once daily, oral fluoxetine 20 mg capsules or matching placebo for 6 months. Patients, carers, investigators, and outcome assessors were masked to the treatment allocation. The primary outcome was functional status, measured by the mRS, at 6 months. The primary analysis was an ordinal logistic regression of the mRS at 6 months, adjusted for minimisation variables. Primary and safety analyses were done according to the patient's treatment allocation. The trial is registered with the Australian New Zealand Clinical Trials Registry, ACTRN12611000774921. Findings Between Jan 11, 2013, and June 30, 2019, 1280 patients were recruited in Australia (n=532), New Zealand (n=42), and Vietnam (n=706), of whom 642 were randomly assigned to fluoxetine and 638 were randomly assigned to placebo. Mean duration of trial treatment was 167 days (SD 48·1). At 6 months, mRS data were available in 624 (97%) patients in the fluoxetine group and 632 (99%) in the placebo group. The distribution of mRS categories was similar in the fluoxetine and placebo groups (adjusted common odds ratio 0·94, 95% CI 0·76–1·15; p=0·53). Compared with patients in the placebo group, patients in the fluoxetine group had more falls (20 [3%] vs seven [1%]; p=0·018), bone fractures (19 [3%] vs six [1%]; p=0·014), and epileptic seizures (ten [2%] vs two [<1%]; p=0·038) at 6 months. Interpretation Oral fluoxetine 20 mg daily for 6 months after acute stroke did not improve functional outcome and increased the risk of falls, bone fractures, and epileptic seizures. These results do not support the use of fluoxetine to improve functional outcome after stroke

Full-length Vpu and human CD4(372–433) in phospholipid bilayers as seen by magic angle spinning NMR

HIV-1 Vpu and CD4(372-433), a peptide comprising the transmembrane and cytoplasmic domain of human CD4, were recombinantly expressed in Escherichia coli, uniformly labeled with 13C and 15N isotopes, and separately reconstituted into phospholipid bilayers. Highly resolved dipolar cross-polarization (CP)-based solid-state NMR spectra of the two transmembrane proteins were recorded under magic angle sample spinning. Partial assignment of 13C resonances was achieved. Site-specific assignments were obtained for 13 amino acid residues of CD4(372-433) and two Vpu residues. Additional amino acid type-specific assignments were achieved for 10 amino acid spin systems for both CD4(372-433) and Vpu. Further, structural flexibility was probed with different dipolar recoupling techniques, and the correct insertion of the transmembrane domains into the lipid bilayers was confirmed by proton spin diffusion experiments