Modular Multiwell Viscoelastic Hydrogel Platform for Two- and Three-Dimensional Cell Culture Applications.

Hydrogels have gained significant popularity as model platforms to study reciprocal interactions between cells and their microenvironment. While hydrogel tools to probe many characteristics of the extracellular space have been developed, fabrication approaches remain challenging and time-consuming, limiting multiplexing or widespread adoption. Thus, we have developed a modular fabrication approach to generate distinct hydrogel microenvironments within the same 96-well plate for increased throughput of fabrication as well as integration with existing high-throughput assay technologies. This approach enables in situ hydrogel mechanical characterization and is used to generate both elastic and viscoelastic hydrogels across a range of stiffnesses. Additionally, this fabrication method enabled a 3-fold reduction in polymer and up to an 8-fold reduction in fabrication time required per hydrogel replicate. The feasibility of this platform for two-dimensional (2D) cell culture applications was demonstrated by measuring both population-level and single-cell-level metrics via microplate reader and high-content imaging. Finally, a 96-well hydrogel array was utilized for three-dimensional (3D) cell culture, demonstrating the ability to support high cell viability. Together, this work demonstrates a versatile and easily adaptable fabrication approach that can support the ever-expanding tool kit of hydrogel technologies for cell culture applications.

Modular multiwell viscoelastic hydrogel platform for two- and three-dimensional cell culture applications.

Hydrogels have gained significant popularity as model platforms to study the reciprocal interactions between cells and their microenvironment. While hydrogel tools to probe many characteristics of the extracellular space have been developed, fabrication approaches remain challenging and time-consuming, limiting multiplexing or widespread adoption. Thus, we have developed a modular fabrication approach to generate distinct hydrogel microenvironments within 96-well plates for increased throughput of fabrication as well as integration with existing high-throughput assay technologies. This approach enables in situ hydrogel mechanical characterization and was used to generate both elastic and viscoelastic hydrogels across a range of stiffnesses. Additionally, this fabrication method enabled a 3-fold reduction in polymer and up to an 8-fold reduction in fabrication time required per hydrogel replicate. The feasibility of this platform for cell culture applications was demonstrated by measuring both population-level and single cell-level metrics via microplate reader and high-content imaging. Finally, the 96-well hydrogel array was utilized for 3D cell culture, demonstrating the ability to support high cell viability. Together, this work demonstrates a versatile and easily adoptable fabrication approach that can support the ever-expanding tool kit of hydrogel technologies for cell culture applications.

Using Direct Laboratory Measurements of Electron Temperature Anisotropy to Identify the Heating Mechanism in Electron-Only Guide Field Magnetic Reconnection.

Anisotropic electron heating during electron-only magnetic reconnection with a large guide magnetic field is directly measured in a laboratory plasma through in situ measurements of electron velocity distribution functions. Electron heating preferentially parallel to the magnetic field is localized to one separatrix, and anisotropies of 1.5 are measured. The mechanism for electron energization is identified as the parallel reconnection electric field because of the anisotropic nature of the heating and spatial localization. These characteristics are reproduced in a 2D particle-in-cell simulation and are also consistent with numerous magnetosheath observations. A measured increase in the perpendicular temperature along both separatrices is not reproduced by our 2D simulations. This work has implications for energy partition studies in magnetosheath and laboratory reconnection.

The role of puberty on physical and brain development: A longitudinal study in male Rhesus Macaques.

This study examined the role of male pubertal maturation on physical growth and development of neurocircuits that regulate stress, emotional and cognitive control using a translational nonhuman primate model. We collected longitudinal data from male macaques between pre- and peri-puberty, including measures of physical growth, pubertal maturation (testicular volume, blood testosterone -T- concentrations) and brain structural and resting-state functional MRI scans to examine developmental changes in amygdala (AMY), hippocampus (HIPPO), prefrontal cortex (PFC), as well as functional connectivity (FC) between those regions. Physical growth and pubertal measures increased from pre- to peri-puberty. The indexes of pubertal maturation -testicular size and T- were correlated at peri-puberty, but not at pre-puberty (23 months). Our findings also showed ICV, AMY, HIPPO and total PFC volumetric growth, but with region-specific changes in PFC. Surprisingly, FC in these neural circuits only showed developmental changes from pre- to peri-puberty for HIPPO-orbitofrontal FC. Finally, testicular size was a better predictor of brain structural maturation than T levels -suggesting gonadal hormones-independent mechanisms-, whereas T was a strong predictor of functional connectivity development. We expect that these neural circuits will show more drastic pubertal-dependent maturation, including stronger associations with pubertal measures later, during and after male puberty.

Multi-dimensional incoherent Thomson scattering system in PHAse Space MApping (PHASMA) facility.

A multi-dimensional incoherent Thomson scattering diagnostic system capable of measuring electron temperature anisotropies at the level of the electron velocity distribution function (EVDF) is implemented on the PHAse Space MApping facility to investigate electron energization mechanisms during magnetic reconnection. This system incorporates two injection paths (perpendicular and parallel to the axial magnetic field) and two collection paths, providing four independent EVDF measurements along four velocity space directions. For strongly magnetized electrons, a 3D EVDF comprised of two characteristic electron temperatures perpendicular and parallel to the local magnetic field line is reconstructed from the four measured EVDFs. Validation of isotropic electrons in a single magnetic flux rope and a steady-state helicon plasma is presented.

Laboratory Observations of Electron Heating and Non-Maxwellian Distributions at the Kinetic Scale during Electron-Only Magnetic Reconnection.

Non-Maxwellian electron velocity distribution functions composed of a warm bulk population and a cold beam are directly measured during electron-only reconnection with a strong out-of-plane (guide) magnetic field in a laboratory plasma. Electron heating is localized to the separatrix, and the electron temperature increases continuously along the separatrix. The measured gain in enthalpy flux is 70% of the incoming Poynting flux. The electron beams are oppositely directed on either side of the X point, and their velocities are comparable to, and scale with, the electron Alfvén speed. Particle-in-cell simulations are consistent with the measurements. The experimental results are consistent with, and go beyond, recent observations in the magnetosheath.

Incoherent Thomson scattering system for PHAse space MApping (PHASMA) experiment.

A new incoherent Thomson scattering system measures the evolution of electron velocity distribution functions perpendicular and parallel to the ambient magnetic field during kinking of a single flux rope and merging of two flux ropes through magnetic reconnection. The Thomson scattering system provides sub-millimeter spatial resolution, sufficient to diagnose the several millimeters sized magnetic reconnection electron diffusion region in the PHAse Space MAppgin experiment. Due to the relatively modest plasma density ∼1019 m-3 and electron temperature ∼1 eV, stray light suppression is critical for these measurements. Two volume Bragg gratings are used in series as a notch filter with a spectral bandwidth <0.1 nm in the collection branch. A CCD with a Gen III intensifier with peak quantum efficiency >47% is used as the detector in a 1.3 m spectrometer. Preliminary results of gun plasma electron temperature will be reported and compared with measurements obtained from a triple Langmuir probe.

Optimizing humeral stem fixation in revision reverse shoulder arthroplasty with the cement-within-cement technique.

BACKGROUND: The purpose of this study was to report on the clinical outcomes of patients undergoing revision reverse shoulder arthroplasty (RSA) by the cement-within-cement technique, as well as to identify whether surgical technique can affect subsequent humeral loosening. METHODS: In 98 patients, cemented humeral components that were revised to RSA using the cement-within-cement technique were identified and included in this study. We compared 8 patients in whom humeral stem loosening developed with 90 patients whose stem remained fixed. Preoperative and postoperative radiographs of each patient were downloaded in DICOM (Digital Imaging and Communications in Medicine) format and analyzed in Mimics. The total area of the cement mantle (in square millimeters) and of the stem (in square millimeters), as visualized on 2-dimensional plain films, was measured in each subject on both preoperative and postoperative radiographs. Outcomes at a minimum of 2 years of follow-up were analyzed. RESULTS: Clinical outcomes were available in 57 patients, with a mean follow-up period of 54 months (range, 21-156 months). Patients demonstrated significantly improved functional outcome scores and shoulder range of motion. In the group without loosening, the mean increase in the cement mantle area was 4380 ± 12701 mm2 (P < .0001). In the group with loosening, the mean increase in the cement mantle area was only 811 ± 4014 mm2 (P = .484). CONCLUSIONS: Use of the cement-within-cement technique for fixation of the humeral component in revision RSA is effective in improving functional outcome scores and shoulder range of motion. Furthermore, these findings suggest that efforts to maximize the cement volume during reimplantation may lessen the chance of humeral stem loosening requiring additional revision.

Defining the younger patient: age as a predictive factor for outcomes in shoulder arthroplasty.

BACKGROUND: The purpose of this study was to define an age cutoff at which clinical outcomes and revision rates differ for patients undergoing primary anatomic total shoulder arthroplasty (TSA) and patients undergoing primary reverse shoulder arthroplasty (RSA). METHODS: This retrospective cohort study included 1250 primary shoulder arthroplasties (1131 patients) with minimum 2-year clinical follow-up (mean, 50 months [range, 24-146 months]). TSA (n = 518; mean age, 68.1 years [range, 28-90 years]) was performed for osteoarthritis in most cases (99%), whereas the primary diagnoses for RSA (n = 732; mean age, 70.8 years [range, 22-91 years]) included rotator cuff arthropathy (35%), massive cuff tear without osteoarthritis (29.8%), and osteoarthritis (20.5%). Outcomes included range of motion, the American Shoulder and Elbow Surgeons (ASES) score, and the revision rate. The relationship between age at the time of surgery in 5-year increments (46-50 years, 51-55 years, and so on) and the revision rate was examined to identify the age cutoff; this was then used to assess clinical outcomes. RESULTS: In patients younger than 65 years, TSA was associated with a 3.4-fold increased risk of revision (P = .01). RSA performed in patients younger than 60 years was associated with a 4.8-fold increased risk of revision (P < .001). TSA patients aged 65 years or older and RSA patients aged 60 years or older had better total ASES scores (82 vs. 77 [P = .03] and 72 vs. 62 [P = .002], respectively) and better internal rotation (interquartile range, TSA 5-6 vs. 4-5 [P = .002] and RSA 4-5 vs 3-4 [P = .04])-where 6 represents T4 to T6 and 4 represents T11 to L1-than their younger counterparts. CONCLUSION: Age at index arthroplasty affects outcomes and the risk of revision. Primary TSA patients younger than 65 years and RSA patients younger than 60 years have a significantly increased revision risk. These age cutoffs are also correlated with differences in ASES scores and internal rotation.

Use of a modified Outerbridge-Kashiwagi procedure for the treatment of posttraumatic elbow sequelae.

BACKGROUND: To review our 10-year experience treating posttraumatic sequelae of the elbow using a modified Outerbridge-Kashiwagi (O-K) procedure. METHODS: Twenty-one patients with posttraumatic sequelae of the elbow treated using the technique were evaluated clinically using the Mayo Elbow Performance Score, range of motion testing, and pain level. We noted the presence of preoperative and postoperative ulnar nerve symptoms, complications, and reoperations. Open contracture release was selected to address either removal of hardware or ulnar nerve pathology. RESULTS: At a mean of 39 months (range, 12-116 months), the Mayo Elbow Performance Score improved from 52 to 84 (P < .0001) and the mean arc of motion improved from 44° to 98° (P < .0001). At the final follow-up, 90% of patients reported no pain or mild pain, and 81% of patients had a satisfactory objective result. In 15 of 21 cases (71%), it was necessary to mobilize the ulnar nerve. After contracture release, 1 patient developed new onset ulnar nerve symptoms. Three patients underwent reoperation: 2 for recalcitrant contracture and 1 for new onset ulnar nerve symptoms. CONCLUSIONS: The mini-open O-K procedure is safe and effective in restoring function in patients with retained hardware and posttraumatic contracture. Posttraumatic arthritic patients often require both removal of hardware and neurolysis of the ulnar nerve. The mini-open O-K procedure allows complete access to the elbow joint, which facilitates release for both intrinsic and extrinsic contracture.

Early Developmental Trajectories of Functional Connectivity Along the Visual Pathways in Rhesus Monkeys.

Early social interactions shape the development of social behavior, although the critical periods or the underlying neurodevelopmental processes are not completely understood. Here, we studied the developmental changes in neural pathways underlying visual social engagement in the translational rhesus monkey model. Changes in functional connectivity (FC) along the ventral object and motion pathways and the dorsal attention/visuo-spatial pathways were studied longitudinally using resting-state functional MRI in infant rhesus monkeys, from birth through early weaning (3 months), given the socioemotional changes experienced during this period. Our results revealed that (1) maturation along the visual pathways proceeds in a caudo-rostral progression with primary visual areas (V1-V3) showing strong FC as early as 2 weeks of age, whereas higher-order visual and attentional areas (e.g., MT-AST, LIP-FEF) show weak FC; (2) functional changes were pathway-specific (e.g., robust FC increases detected in the most anterior aspect of the object pathway (TE-AMY), but FC remained weak in the other pathways (e.g., AST-AMY)); (3) FC matures similarly in both right and left hemispheres. Our findings suggest that visual pathways in infant macaques undergo selective remodeling during the first 3 months of life, likely regulated by early social interactions and supporting the transition to independence from the mother.

Zeeman splitting measurements of magnetic fields in iodine plasma.

Iodine is an attractive propellant for next generation ion thrusters. Laser induced fluorescence (LIF) is widely used with other propellant species as a non-perturbative technique for measuring flow for thruster prediction models. We apply LIF methods recently demonstrated for singly-ionized iodine to a magnetized plasma environment similar to those found in ion thrusters and in magnetically confined laboratory plasmas. We demonstrate the feasibility of remotely determining the local magnetic field from the Zeeman effect-split spectrum of I+.

Demonstration of confocal laser induced fluorescence at long focal lengths.

We add a collection path obscuration to a confocal telescope and confirm theoretical predictions of significant improvement in the longitudinal spatial localization. The improvements of spatial localization permitted an extension of the confocal telescope's focal length from 150 mm to 500 mm. At this longer focal length, millimeter-scale spatial localization is confirmed by comparing radial profiles of metastable state density obtained via confocal and conventional optical arrangements in a helicon source. The long focal length arrangement enables the measurement of argon neutral velocity distribution functions in the conventionally inaccessible region under a helicon source antenna.

Range position and climate sensitivity: The structure of among-population demographic responses to climatic variation.

Species' distributions will respond to climate change based on the relationship between local demographic processes and climate and how this relationship varies based on range position. A rarely tested demographic prediction is that populations at the extremes of a species' climate envelope (e.g., populations in areas with the highest mean annual temperature) will be most sensitive to local shifts in climate (i.e., warming). We tested this prediction using a dynamic species distribution model linking demographic rates to variation in temperature and precipitation for wood frogs (Lithobates sylvaticus) in North America. Using long-term monitoring data from 746 populations in 27 study areas, we determined how climatic variation affected population growth rates and how these relationships varied with respect to long-term climate. Some models supported the predicted pattern, with negative effects of extreme summer temperatures in hotter areas and positive effects on recruitment for summer water availability in drier areas. We also found evidence of interacting temperature and precipitation influencing population size, such as extreme heat having less of a negative effect in wetter areas. Other results were contrary to predictions, such as positive effects of summer water availability in wetter parts of the range and positive responses to winter warming especially in milder areas. In general, we found wood frogs were more sensitive to changes in temperature or temperature interacting with precipitation than to changes in precipitation alone. Our results suggest that sensitivity to changes in climate cannot be predicted simply by knowing locations within the species' climate envelope. Many climate processes did not affect population growth rates in the predicted direction based on range position. Processes such as species-interactions, local adaptation, and interactions with the physical landscape likely affect the responses we observed. Our work highlights the need to measure demographic responses to changing climate.

Hearing aid fitting and developmental outcomes of children fit according to either the NAL or DSL prescription: fit-to-target, audibility, speech and language abilities.

OBJECTIVE: This study examined the influence of prescription on hearing aid (HA) fitting characteristics and 5-year developmental outcomes of children. DESIGN: A randomised controlled trial implemented as part of a population-based study on Longitudinal Outcomes of Children with Hearing Impairment (LOCHI). STUDY SAMPLE: Two-hundred and thirty-two children that were fit according to either the National Acoustic Laboratories (NAL) or Desired Sensation Level (DSL) prescription. RESULTS: Deviation from targets and root-mean-square error in HA fitting revealed no significant difference between fitting prescriptions. Aided audibility quantified by using the Speech Intelligibility Index (SII) model showed that DSL provided higher audibility than NAL at low and medium input levels but not at high input level. After allowing for hearing loss desensitisation, differences in audibility between prescription groups were significant only at low input level. The randomised trial of prescription that was implemented for 163 children revealed no significant between-group differences in speech production, perception, and language; but parent-rated functional performance was higher for the DSL than for the NAL group. CONCLUSIONS: Proximity to prescriptive targets was similar between fitting prescriptions. The randomised trial revealed differences in aided audibility at low input level between prescription groups, but no significant differences in speech and language abilities.

Confocal laser induced fluorescence with comparable spatial localization to the conventional method.

We present measurements of ion velocity distributions obtained by laser induced fluorescence (LIF) using a single viewport in an argon plasma. A patent pending design, which we refer to as the confocal fluorescence telescope, combines large objective lenses with a large central obscuration and a spatial filter to achieve high spatial localization along the laser injection direction. Models of the injection and collection optics of the two assemblies are used to provide a theoretical estimate of the spatial localization of the confocal arrangement, which is taken to be the full width at half maximum of the spatial optical response. The new design achieves approximately 1.4 mm localization at a focal length of 148.7 mm, improving on previously published designs by an order of magnitude and approaching the localization achieved by the conventional method. The confocal method, however, does so without requiring a pair of separated, perpendicular optical paths. The confocal technique therefore eases the two window access requirement of the conventional method, extending the application of LIF to experiments where conventional LIF measurements have been impossible or difficult, or where multiple viewports are scarce.

Safety limit warning levels for the avoidance of excessive sound amplification to protect against further hearing loss.

OBJECTIVE: To determine safe output sound pressure levels (SPL) for sound amplification devices to preserve hearing sensitivity after usage. DESIGN: A mathematical model consisting of the Modified Power Law (MPL) (Humes & Jesteadt, 1991 ) combined with equations for predicting temporary threshold shift (TTS) and subsequent permanent threshold shift (PTS) (Macrae, 1994b ) was used to determine safe output SPL. STUDY SAMPLE: The study involves no new human subject measurements of loudness tolerance or threshold shifts. PTS was determined by the MPL model for 234 audiograms and the SPL output recommended by four different validated prescription recommendations for hearing aids. RESULTS: PTS can, on rare occasion, occur as a result of SPL delivered by hearing aids at modern day prescription recommendations. The trading relationship of safe output SPL, decibel hearing level (dB HL) threshold, and PTS was captured with algebraic expressions. Better hearing thresholds lowered the safe output SPL and higher thresholds raised the safe output SPL. CONCLUSION: Safe output SPL can consider the magnitude of unaided hearing loss. For devices not set to prescriptive levels, limiting the output SPL below the safe levels identified should protect against threshold worsening as a result of long-term usage.

The distribution of echinostome parasites in ponds and implications for larval anuran survival.

Parasites can influence host population dynamics, community composition and evolution. Prediction of these effects, however, requires an understanding of the influence of ecological context on parasite distributions and the consequences of infection for host fitness. We address these issues with an amphibian - trematode (Digenea: Echinostomatidae) host-parasite system. We initially performed a field survey of trematode infection in first (snail) and second (larval green frog, Rana clamitans) intermediate hosts over 5 years across a landscape of 23 ponds in southeastern Michigan. We then combined this study with a tadpole enclosure experiment in eight ponds. We found echinostomes in all ponds during the survey, although infection levels in both snails and amphibians differed across ponds and years. Echinostome prevalence (proportion of hosts infected) in snails also changed seasonally depending on host species, and abundance (parasites per host) in tadpoles depended on host size and prevalence in snails. The enclosure experiment demonstrated that infection varied at sites within ponds, and tadpole survival was lower in enclosures with higher echinostome abundance. The observed effects enhance our ability to predict when and where host-parasite interactions will occur and the potential fitness consequences of infection, with implications for population and community dynamics, evolution and conservation.

Effect of acute and chronic autophagy deficiency on skeletal muscle apoptotic signaling, morphology, and function.

Autophagy is a catabolic process that targets and degrades cytoplasmic materials. In skeletal muscle, autophagy is required for the control of mass under catabolic conditions, but is also basally active in the maintenance of myofiber homeostasis. In this study, we found that some specific autophagic markers (LC3-I, LC3-II, SQSTM1) were basally lower in glycolytic muscle compared to oxidative muscle of autophagy competent mice. In contrast, basal autophagic flux was higher in glycolytic muscle. In addition, we used several skeletal muscle-specific Atg7 transgenic mouse models to investigate the effect of acute (iAtg7-/-) and chronic (cAtg7-/-) autophagy deficiency on skeletal muscle morphology, contractility, and apoptotic signaling. While acute autophagy ablation (iAtg7-/-) resulted in increased centralized nuclei in glycolytic muscle, it did not alter contractile properties or measures of apoptosis and proteolysis. In contrast, with chronic autophagy deficiency (cAtg7-/-) there was an increased proportion of centralized nuclei, as well as reduced force and altered twitch kinetics in glycolytic muscle. Glycolytic muscle of cAtg7-/- mice also displayed an increased level of the pro-apoptotic protein BAX, as well as calpain and proteasomal enzymatic activity. Collectively, our data demonstrate cumulative damage from chronic skeletal muscle-specific autophagy deficiency with associated apoptotic and proteasomal upregulation. These findings point towards the importance of investigating different muscle/fiber types when studying skeletal muscle autophagy, and the critical role of autophagy in the maintenance of myofiber function, integrity, and cellular health.