BenzoHTag, a fluorogenic self-labeling protein developed using molecular evolution.

Self-labeling proteins are powerful tools in chemical biology as they enable the precise cellular localization of a synthetic molecule, often a fluorescent dye, with the genetic specificity of a protein fusion. HaloTag7 is the most popular self-labeling protein due to its fast labeling kinetics and the simplicity of its chloroalkane ligand. Reaction rates of HaloTag7 with different chloroalkane-containing substrates is highly variable and rates are only very fast for rhodamine-based dyes. This is a major limitation for the HaloTag system because fast labeling rates are critical for live-cell assays. Here, we report a molecular evolution system for HaloTag using yeast surface display that enables the screening of libraries up to 108 variants to improve reaction rates with any substrate of interest. We applied this method to produce a HaloTag variant, BenzoHTag, which has improved performance with a fluorogenic benzothiadiazole dye. The resulting system has improved brightness and conjugation kinetics, allowing for robust, no-wash fluorescent labeling in live cells. The new BenzoHTag-benzothiadiazole system has improved performance in live-cell assays compared to the existing HaloTag7-silicon rhodamine system, including saturation of intracellular enzyme in under 100 seconds and robust labeling at dye concentrations as low as 7 nM. It was also found to be orthogonal to the silicon HaloTag7-rhodamine system, enabling multiplexed no-wash labeling in live cells. The BenzoHTag system, and the ability to optimize HaloTag for a broader collection of substrates using molecular evolution, will be very useful for the development of cell-based assays for chemical biology and drug development.

Sex- and species-associated differences in complement-mediated immunity in humans and rhesus macaques.

The complement system can be viewed as a "moderator" of innate immunity, "instructor" of humoral immunity, and "regulator" of adaptive immunity. While sex is known to affect humoral and cellular immune systems, its impact on complement in humans and rhesus macaques, a commonly used non-human primate model system, has not been well studied. To address this knowledge gap, we analyzed serum samples from 90 humans and 72 rhesus macaques for the abundance and activity of the complement system components. While sequences of cascade proteins were highly conserved, dramatically different levels were observed between species. Whereas the low levels detected in rhesus samples raised questions about the suitability of the test for use with macaque samples, differences in levels of complement proteins were observed in male and female humans. Levels of total and antibody-dependent deposition of C1q and C3b on a glycosylated antigen differed between humans and rhesus, suggesting differential recognition of glycans and balance between classical and alternative activation pathways. Functional differences in complement-mediated lysis of antibody-sensitized cells were observed in multiple assays and showed that human females frequently exhibited higher lytic activity than human males or rhesus macaques, which typically did not exhibit such sex-associated differences. Other differences between species and sexes were observed in more narrow contexts-for only certain antibodies, antigens, or assays. Collectively, these results expand knowledge of sex-associated differences in the complement system in humans, identifying differences absent from rhesus macaques.IMPORTANCEThe complement system is a critical part of host defense to many bacterial, fungal, and viral infections. In parallel, rich epidemiological, clinical, and biomedical research evidence demonstrates that sex is an important biological variable in immunity, and many sex-specific differences in immune system are intimately tied with disease outcomes. This study focuses on the intersection of these two factors to define the impact of sex on complement pathway components and activities. This work expands our knowledge of sex-associated differences in the complement system in humans and also identifies the differences that appear to be absent in rhesus macaques, a popular non-human primate model. Whereas differences between species suggest potential limitations in the ability of macaque model to recapitulate human biology, knowledge of sex-based differences in humans has the potential to inform clinical research and practice.

The Association Between Black vs. White Race and 30-Day Hospitalization Among People Diagnosed with COVID-19 Within an Integrated Care Setting: a Cohort Study.

BACKGROUND: Black Americans are more likely to experience hospitalization from COVID-19 compared with White Americans. Whether this excess risk differs by age, sex, obesity, or diabetes, key risk factors for COVID hospitalization, among an integrated population with uniform healthcare access, are less clear. METHODS: We identified all adult members (≥ 18 years) of Kaiser Permanente Georgia (KPGA) diagnosed with COVID-19 between January 1, 2020, and September 30, 2021 (N = 24,564). We restricted the analysis to members of Black or White race identified from electronic medical records. Our primary outcome was first hospitalization within 30 days of COVID-19 diagnosis. To assess the association between race and 30-day hospitalization, we performed multivariable logistic regression adjusting for several member and neighborhood-level characteristics, and tested for interactions of race with age, sex, diabetes, and obesity. A regression-based decomposition method was then used to estimate how much of the observed race disparity in 30-day hospitalization could be explained by member and neighborhood-level factors. RESULTS: Overall, 11.27% of Black KPGA members were hospitalized within 30 days of a COVID diagnosis, as compared with 9.44% of White KPGA members. Black (vs. White) KPGA members had a 34% (aOR: 1.32 [95% CI: 1.19-1.47]) higher odds of 30-day hospitalization following COVID-19 after accounting for clinical differences. The odds of 30-day hospitalization in Black vs. White KPGA members did not differ significantly by sex (men: 1.46 [1.25-1.70]; women: 1.24 [1.07-1.43]), by age (18-29 years: 1.33 [0. 841-2.10]; 30-49 years: 1.26 [1.02-1.56]; ≥ 50 years: 1.24 [1.10-1.41]); by diabetes status (with diabetes: 1.38 [1.16-1.66]; without diabetes: 1.26 [1.11-1.44]), or by obesity (with obesity: 1.31 [1.15-1.50]; without obesity: 1.28 [1.06-1.53]). Factors that, if Black and White KPGA members had the same level of exposure, would be most likely to reduce the Black-White disparity in 30-day hospitalization from COVID-19 were obesity, history of flu vaccine, and neighborhood-level income and social vulnerability. CONCLUSIONS: Early in the pandemic, Black (vs. White) members of an integrated health system had higher odds of being hospitalized within 30 days of COVID-19 diagnosis and this excess risk was similar by sex, age, and comorbidities. Factors that explained the largest proportions of race-based disparities were obesity, receipt of flu vaccine, and neighborhood-level social determinants of health. These findings suggest that social determinants of health, or other unmeasured factors, may be drivers of racial disparities in COVID-19 outcomes.

Sex and species associated differences in Complement-mediated immunity in Humans and Rhesus macaques.

The complement system can be viewed as a 'moderator' of innate immunity, 'instructor' of humoral immunity, and 'regulator' of adaptive immunity. While sex and aging are known to affect humoral and cellular immune systems, their impact on the complement pathway in humans and rhesus macaques, a commonly used non-human primate model system, have not been well-studied. To address this knowledge gap, we analyzed serum samples from 90 humans and 75 rhesus macaques for the abundance and activity of the complement system components. While sequences of cascade proteins were highly conserved, dramatically different levels were observed between species. Whereas the low levels detected in rhesus samples raised questions about the suitability of the test, differences in levels of complement proteins were observed in male and female humans. Levels of total and antibody-dependent deposition of C1q and C3b on a glycosylated antigen differed between human and rhesus, suggesting differential recognition of glycans. Functional differences in complement-mediated lysis of antibody-sensitized cells were observed in multiple assays and showed that human females frequently exhibited higher lytic activity than human males or rhesus macaques, which typically did not exhibit such sexual dimorphism. Other differences between species and sexes were observed in more narrow contexts-for only certain antibodies, antigens, or assays. Collectively, these results expand our knowledge of sexual dimorphism in the complement system in humans, identifying differences that appear to be absent from rhesus macaques.

Silk fibroin, gelatin, and human placenta extracellular matrix-based composite hydrogels for 3D bioprinting and soft tissue engineering.

BACKGROUND: There is a great clinical need and it remains a challenge to develop artificial soft tissue constructs that can mimic the biomechanical properties and bioactivity of natural tissue. This is partly due to the lack of suitable biomaterials. Hydrogels made from human placenta offer high bioactivity and represent a potential solution to create animal-free 3D bioprinting systems that are both sustainable and acceptable, as placenta is widely considered medical waste. A combination with silk and gelatin polymers can bridge the biomechanical limitations of human placenta chorion extracellular matrix hydrogels (hpcECM) while maintaining their excellent bioactivity. METHOD: In this study, silk fibroin (SF) and tyramine-substituted gelatin (G-TA) were enzymatically crosslinked with human placental extracellular matrix (hpcECM) to produce silk-gelatin-ECM composite hydrogels (SGE) with tunable mechanical properties, preserved elasticity, and bioactive functions. The SGE composite hydrogels were characterized in terms of gelation kinetics, protein folding, and bioactivity. The cyto- and biocompatibility of the SGE composite was determined by in vitro cell culture and subcutaneous implantation in a rat model, respectively. The most cell-supportive SGE formulation was then used for 3-dimensional (3D) bioprinting that induced chemical crosslinking during extrusion. CONCLUSION: Addition of G-TA improved the mechanical properties of the SGE composite hydrogels and inhibited crystallization and subsequent stiffening of SF for up to one month. SGE hydrogels exhibit improved and tunable biomechanical properties and high bioactivity for encapsulated cells. In addition, its use as a bioink for 3D bioprinting with free reversible embedding of suspended hydrogels (FRESH) has been validated, opening the possibility to fabricate highly complex scaffolds for artificial soft tissue constructs with natural biomechanics in future.

Features of functional and dysfunctional CD8+ T cells to guide HIV vaccine development.

PURPOSE OF REVIEW: CD8+ T cell responses are a key component of the host immune response to human immunodeficiency virus (HIV) but vary significantly across individuals with distinct clinical outcomes. These differences help inform the qualitative features of HIV-specific CD8+ T cells that we should aim to induce by vaccination. RECENT FINDINGS: We review previous and more recent findings on the features of dysfunctional and functional CD8+ T cell responses that develop in individuals with uncontrolled and controlled HIV infection, with particular emphasis on proliferation, cytotoxic effector function, epitope specificity, and responses in lymph nodes. We also discuss the implications of these findings for both prophylactic and therapeutic T cell vaccine development within the context of T cell vaccine trials. SUMMARY: The induction of HIV specific CD8+ T cell responses is an important goal of ongoing vaccine efforts. Emerging data on the key features of CD8+ T cell responses that distinguish individuals who spontaneously control from those with progressive disease continues to provide key guidance.

Complement contributes to antibody-mediated protection against repeated SHIV challenge.

The first clinical efficacy trials of a broadly neutralizing antibody (bNAb) resulted in less benefit than expected and suggested that improvements are needed to prevent HIV infection. While considerable effort has focused on optimizing neutralization breadth and potency, it remains unclear whether augmenting the effector functions elicited by broadly neutralizing antibodies (bNAbs) may also improve their clinical potential. Among these effector functions, complement-mediated activities, which can culminate in the lysis of virions or infected cells, have been the least well studied. Here, functionally modified variants of the second-generation bNAb 10-1074 with ablated and enhanced complement activation profiles were used to examine the role of complement-associated effector functions. When administered prophylactically against simian-HIV challenge in rhesus macaques, more bNAb was required to prevent plasma viremia when complement activity was eliminated. Conversely, less bNAb was required to protect animals from plasma viremia when complement activity was enhanced. These results suggest that complement-mediated effector functions contribute to in vivo antiviral activity, and that their engineering may contribute to the further improvements in the efficacy of antibody-mediated prevention strategies.

Evidence for the Role of a Second Fc-Binding Receptor in Placental IgG Transfer in Nonhuman Primates.

Transplacental transfer of maternal antibodies provides the fetus and newborn with passive protection against infectious diseases. While the role of the highly conserved neonatal Fc receptor (FcRn) in transfer of IgG in mammals is undisputed, recent reports have suggested that a second receptor may contribute to transport in humans. We report poor transfer efficiency of plant-expressed recombinant HIV-specific antibodies, including engineered variants with high FcRn affinity, following subcutaneous infusion into rhesus macaques close to parturition. Unexpectedly, unlike those derived from mammalian tissue culture, plant-derived antibodies were essentially unable to cross macaque placentas. This defect was associated with poor Fcγ receptor binding and altered Fc glycans and was not recapitulated in mice. These results suggest that maternal-fetal transfer of IgG across the three-layer primate placenta may require a second receptor and suggest a means of providing maternal antibody treatments during pregnancy while avoiding fetal harm. IMPORTANCE This study compared the ability of several human HIV envelope-directed monoclonal antibodies produced in plants with the same antibodies produced in mammalian cells for their ability to cross monkey and mouse placentas. We found that the two types of antibodies have comparable transfer efficiencies in mice, but they are differentially transferred across macaque placentas, consistent with a two-receptor IgG transport model in primates. Importantly, plant-produced monoclonal antibodies have excellent binding characteristics for human FcRn receptors, permitting desirable pharmacokinetics in humans. The lack of efficient transfer across the primate placenta suggests that therapeutic plant-based antibody treatments against autoimmune diseases and cancer could be provided to the mother while avoiding transfer and preventing harm to the fetus.

Infraspinatus Muscle Fiber Moment Arms During Abduction: A Biomechanical Comparison of Values for Intact Rotator Cuff, Supraspinatus Tear, Superior Capsular Reconstruction, and Reverse Total Shoulder Arthroplasty.

Background: Lines of action of the superior, middle, and inferior infraspinatus muscle fibers work together to produce moment arms that change throughout abduction in an intact shoulder, after a supraspinatus tear, and after superior capsular reconstruction (SCR) and reverse total shoulder arthroplasty (rTSA). Purpose: To use moment arm values to indicate the efficacy of SCR and rTSA to restore infraspinatus function during shoulder abduction. Study Design: Descriptive laboratory study. Methods: A total of 5 human cadaveric shoulders placed in a testing apparatus were each actively abducted (0°-90°) under the following 4 conditions: intact, complete supraspinatus tear, SCR, and rTSA. The 3-dimensional coordinates of points were tracked along the origin and insertion of the superior, middle, and inferior infraspinatus fibers during abduction. Moment arm values were calculated using the origin-insertion method to determine abduction contribution of infraspinatus fiber sections. Analysis of variance and post hoc Tukey testing were used to compare differences in moment arms between the 4 conditions and between fiber sections. Results: In the intact condition, the superior infraspinatus fibers had an abduction moment that decreased with elevation until shifting to adduction. Conversely, the middle and inferior fibers had an adduction moment that turned to abduction (mean moment arm values from 0° to 90°: inferior, from -5.9 to 19.4 mm; middle, from -4.7 to 15.9 mm; superior, from 5.6 to -5.1 mm; P < .05). After a supraspinatus tear, superior fibers lacked any torque, and inferior and middle fibers lost adduction potential (inferior, from 4.8 to 14.0 mm; middle, from -0.2 to 9.6 mm; superior, from 1.0 to 0.7 mm; P < .05). SCR restored the initial superior fiber abduction moment (5.6 mm at 0°; P < .05); middle and inferior fibers had some restoration but were weaker than intact fibers. Loss of abduction moment in all fibers was seen with rTSA (inferior, from -9.6 to -1.6 mm; middle, from -10.5 to -3.6 mm; superior, from -1.7 to -4.6 mm; P < .05). Conclusion: Infraspinatus fiber groups had different and inverse moment arms during scapular plane elevation. SCR most closely resembled the intact shoulder, whereas rTSA transformed the infraspinatus into an adductor. Clinical Relevance: These results support the efficacy of SCR at restoring biomechanical muscle function and suggest that the changes in moment arms for each fiber group be considered when choosing treatment modalities and rehabilitation protocols after rotator cuff tear.

Phagocytosis by an HIV antibody is associated with reduced viremia irrespective of enhanced complement lysis.

Increasingly, antibodies are being used to treat and prevent viral infections. In the context of HIV, efficacy is primarily attributed to dose-dependent neutralization potency and to a lesser extent Fc-mediated effector functions. It remains unclear whether augmenting effector functions of broadly neutralizing antibodies (bNAbs) may improve their clinical potential. Here, we use bNAb 10E8v4 targeting the membrane external proximal region (MPER) to examine the role of antibody-mediated effector and complement (C') activity when administered prophylactically against SHIV challenge in rhesus macaques. With sub-protective dosing, we find a 78-88% reduction in post-acute viremia that is associated with 10E8v4-mediated phagocytosis acting at the time of challenge. Neither plasma nor tissue viremic outcomes in vivo is improved with an Fc-modified variant of 10E8v4 enhanced for C' functions as determined in vitro. These results suggest that effector functions inherent to unmodified 10E8v4 contribute to efficacy against SHIVSF162P3 in the absence of plasma neutralizing titers, while C' functions are dispensable in this setting, informing design of bNAb modifications for improving protective efficacy.

Revisiting an IgG Fc Loss-of-Function Experiment: the Role of Complement in HIV Broadly Neutralizing Antibody b12 Activity.

The role of the complement system in HIV-1 immunity and pathogenesis is multifaceted, and an improved understanding of complement activities mediated by HIV-1-specific antibodies has the potential to inform and advance clinical development efforts. A seminal nonhuman primate challenge experiment suggested that complement was dispensable for the protective effect of the early broadly neutralizing antibody (bnAb) b12, but recent experiments have raised questions about the breadth of circumstances under which this conclusion may hold. Here, we reassess the original observation using Fc variants of IgG1 b12 that enhance complement activity and report that complement fixation on recombinant antigen, virions, and cells and complement-dependent viral and cellular lysis in vitro vary among bnAbs. Specifically, while the clinically significant V3 glycan-specific bnAb 10-1074 demonstrates activity, we found that b12 does not meaningfully activate the classical complement cascade. Consistent with avid engagement by C1q and its complex system of regulatory factors, these results suggest that complement-mediated antibody activities demonstrate a high degree of context dependence and motivate revisiting the role of complement in antibody-mediated prevention of HIV-1 infection by next-generation bnAbs in new translational studies in animal models. IMPORTANCE Given the suboptimal outcome of VRC01 antibody-mediated prevention of HIV-1 infection in its first field trial, means to improve diverse antiviral activities in vivo have renewed importance. This work revisits a loss-of-function experiment that investigated the mechanism of action of b12, a similar antibody, and finds that the reason why complement-mediated antiviral activities were not observed to contribute to protection may be the inherent lack of activity of wild-type b12, raising the prospect that this mechanism may contribute in the context of other HIV-specific antibodies.

Evaluation of rotator cuff abduction moment arms for superior capsular reconstruction and reverse total shoulder arthroplasty.

PURPOSE: The rotator cuff (RC) muscles contribute to dynamic stability and rotational actions of the glenohumeral joint. Moment arm can be used to demonstrate the potential work a muscle contributes to a musculoskeletal joint rotation. This study aimed to understand the moment arm contributions of the RC muscles and explore changes following a complete supraspinatus tear treated with either superior capsular reconstruction (SCR) or reverse total shoulder arthroplasty (rTSA). METHODS: Five fresh-frozen cadaveric specimens were prepared and mounted in an apparatus where each intact RC muscle was held in tension with a line of action toward its origin on the scapula. Mean moment arms for each muscle were determined experimentally based on Optotrak data collected during cadaveric shoulder arm abduction. RESULTS: Using ANOVA testing, our analysis demonstrated significant differences (p < 0.001) in infraspinatus and teres minor moment arms after rTSA compared to the intact shoulder model. After SCR, significant differences (p < 0.001) were seen in teres minor, with these differences being statistically similar to the changes seen in teres minor after rTSA. Subscapularis showed no significant difference in moment arm values between the models (p = 0.148). CONCLUSION: Our results illustrate that mean moment arms were preserved in the RC muscles after complete supraspinatus tear. This study also shows evidence that subscapularis function may be maintained after SCR or rTSA. After SCR, infraspinatus may maintain similar abduction ability compared to the anatomical shoulder, while teres minor ability may increase. Infraspinatus may have decreased abduction ability after rTSA while teres minor may have increased ability.

Effects of implant rotational malposition on contact surface area after implantation of the augmented glenoid baseplate in the setting of glenoid bone loss.

AIM OF THE STUDY: Augmented glenoid baseplates are utilized in reverse total shoulder arthroplasty in the setting of glenoid bone loss. These implants permit lateralization of the joint line and correction of bony version abnormalities. To allow bone preservation in the setting of abnormal bony version or deficiency, the backside of the augmented glenoid baseplate is not perpendicular to the axis of the central post/screw. Thus, if the baseplate is implanted with any rotational malposition, this could affect the backside contact area available for ingrowth. The purpose of this study was to assess if rotational malpositioning of a full-wedge augmented baseplate alongside the axis of the central screw significantly affects the glenoid implant backside contact area. METHODS: Seven synthetic scapulas (Sawbones, Vashon, WA) were used to implant a 15° full-wedge glenoid baseplate (Wright Medical, Memphis, TN) according to the manufacturer's technique. The contact pressure between the baseplate and the glenoid surface at rotational positions 5°, 10°, and 15° clockwise (CW) and counterclockwise (CCW) from the central axis was measured with Extreme Low Fujifilm Prescale (Tekscan, Boston, MA). The data was analyzed digitally to obtain a percentage of contact surface area. To evaluate gross contact, a computed tomography (CT) scan was performed and manual measurements of contact between the glenoid and the baseplate were conducted using a standardized axial CT slice. RESULTS: The average contact area at zero degrees of malrotation was 37.26 ± 3.27%. Average contact areas for the simulated malposition cases were 13.99 ± 9.39% at 15° CCW, 24.89 ± 5.11% at 10° CW, and 19.32 ± 3.13% at 15° CW. Each of these results was significant (p < 0.003). On computed tomography, at 15° CCW, the contact area decreased by 39%; at 15° CW, the contact area decreased by 38%. DISCUSSION: The use of augmented glenoid baseplates presents a technical challenge. It is difficult to avoid implant malrotation along the axis of the central peg/screw, because the final rotation of the baseplate must be chosen while the implant is several centimeters away from the bone. This study found that 10° and 15° malrotation about the glenoid baseplate's central axis leads to significant decreases in the implant-bone contact area. CONCLUSIONS: When implanting an augmented baseplate for total shoulder arthroplasty, it is important to minimize baseplate malrotation to decrease the risk of baseplate loosening.

Polyfunctional Tier 2-Neutralizing Antibodies Cloned following HIV-1 Env Macaque Immunization Mirror Native Antibodies in a Human Donor.

Vaccine efforts to combat HIV are challenged by the global diversity of viral strains and shielding of neutralization epitopes on the viral envelope glycoprotein trimer. Even so, the isolation of broadly neutralizing Abs from infected individuals suggests the potential for eliciting protective Abs through vaccination. This study reports a panel of 58 mAbs cloned from a rhesus macaque (Macaca mulatta) immunized with envelope glycoprotein immunogens curated from an HIV-1 clade C-infected volunteer. Twenty mAbs showed neutralizing activity, and the strongest neutralizer displayed 92% breadth with a median IC50 of 1.35 µg/ml against a 13-virus panel. Neutralizing mAbs predominantly targeted linear epitopes in the V3 region in the cradle orientation (V3C) with others targeting the V3 ladle orientation (V3L), the CD4 binding site (CD4bs), C1, C4, or gp41. Nonneutralizing mAbs bound C1, C5, or undetermined conformational epitopes. Neutralization potency strongly correlated with the magnitude of binding to infected primary macaque splenocytes and to the level of Ab-dependent cellular cytotoxicity, but did not predict the degree of Ab-dependent cellular phagocytosis. Using an individualized germline gene database, mAbs were traced to 23 of 72 functional IgHV alleles. Neutralizing V3C Abs displayed minimal nucleotide somatic hypermutation in the H chain V region (3.77%), indicating that relatively little affinity maturation was needed to achieve in-clade neutralization breadth. Overall, this study underscores the polyfunctional nature of vaccine-elicited tier 2-neutralizing V3 Abs and demonstrates partial reproduction of the human donor's humoral immune response through nonhuman primate vaccination.

Biomechanics in an Incomplete Versus Complete Supraspinatus Tear: A Cadaveric Study.

BACKGROUND: Degenerative and traumatic changes to the rotator cuff can result in massive and irreparable rotator cuff tears (RCTs). PURPOSE/HYPOTHESIS: The study objective was to conduct a biomechanical comparison between a small, incomplete RCT and a large, complete RCT. We hypothesized that the incomplete supraspinatus (SS) tear would lead to an incremental loss of abduction force and preserve vertical position of the humeral head, while a complete SS tear would cause superior humeral migration, decrease functional deltoid abduction force, and increase passive range of motion (ROM). STUDY DESIGN: Controlled laboratory study. METHODS: Six cadaveric shoulders were evaluated using a custom testing apparatus. Each shoulder was subjected to 3 conditions: (1) intact/control, (2) 50%, full-thickness, incomplete SS tear, and (3) 100%, complete SS tear. Deltoid abduction force, superior humeral head migration, and passive ROM were measured in static conditions at 0°, 30°, and 60° of glenohumeral abduction, respectively. RESULTS: The intact SS resulted in a mean deltoid abduction force of 2.5, 3.3, and 3.8 N at 0°, 30°, and 60° of abduction, respectively. Compared with the intact shoulder, there was no significant difference in mean abduction force seen in the incomplete tear, while the force was significantly decreased by 52% at 30° of abduction in the complete tear (P = .009). Compared with the incomplete tear, there were significant decreases in abduction force seen in the complete tear, by 33% and 48% (0.9 N and 1.1 N) at 0° and 30° of abduction, respectively (P = .04 and .004). The intact configuration experienced a mean superior humeral head migration of 1.5, 1.4, and 1.1 mm at 0°, 30°, and 60° of abduction, respectively. The complete tear resulted in a superior migration of 3.0 and 4.4 mm greater than the intact configuration at 0° and 30° of abduction, respectively (P = .001). There was a 5° and 10° increase in abduction ROM with 50% and 100% tears, respectively (P = .003 and .03). CONCLUSION: An incomplete SS tear does not significantly alter the biomechanics of the shoulder, while a large, complete SS tear leads to a significant superior humeral migration, a decreased deltoid abduction force, and a mild increase in passive ROM. CLINICAL RELEVANCE: Our findings demonstrate the effects of large SS tears on key biomechanical parameters, as they progress from partial tears.

1-D imaging of rotation-vibration non-equilibrium from pure rotational ultrafast coherent anti-Stokes Raman scattering.

We present one-dimensional (1-D) imaging of rotation-vibration non-equilibrium measured by two-beam pure rotational hybrid femtosecond/picosecond coherent anti-Stokes Raman scattering (fs/ps CARS). Simultaneous measurements of the spatial distribution of molecular rotation-vibration non-equilibrium are critical for understanding molecular energy transfer in low temperature plasmas and hypersonic flows. However, non-equilibrium CARS thermometry until now was limited to point measurements. The red shift of rotational energy levels by vibrational excitation was used to determine the rotational and vibrational temperatures from 1-D images of the pure rotational spectrum. Vibrational temperatures up to 5500 K were detected in a CH4/N2 nanosecond-pulsed pin-to-pin plasma within 2 mm near the cathode. This approach enables study of non-equilibrium systems with 40 µm spatial resolution.

Biomechanical effects of superior capsular reconstruction in a rotator cuff-deficient shoulder: a cadaveric study.

BACKGROUND: Superior capsular reconstruction (SCR) has been gaining popularity as a treatment for irreparable rotator cuff tears (RCTs), especially in younger patients. This biomechanical study aimed to investigate how SCR affects functional abduction force, humeral head migration, and passive range of motion following an irreparable RCT. We hypothesized that SCR will restore these parameters to nearly intact shoulder levels. METHODS: Six fresh-frozen cadaveric shoulders were evaluated using a custom biomechanical testing apparatus. Each shoulder was taken through 3 conditions: (1) intact (control); (2) irreparable, complete supraspinatus (SS) tear; and (3) SCR. Functional abduction force, superior humeral head migration, and passive range of motion, including axial shoulder rotation, were measured in static condition at 0°, 30°, and 60° of glenohumeral abduction. Data were analyzed using the paired Student t test or Wilcoxon signed rank test, depending on the results of normality testing. RESULTS: The irreparable SS tear resulted in significantly lower functional abduction force at 30° of abduction (P = .01) and a trend toward a decrease (P = .17) at 60° compared with the intact configuration. SCR shoulders produced greater functional force at 0° compared with the tear configuration (P = .046). Humeral head migration was significantly increased by 4.4 and 3.0 mm at 0° and 30° of abduction, respectively, when comparing the intact vs. SS tear configurations (P = .001). SCR decreased superior migration down to levels of intact shoulders at 0° and 30° of abduction (P = .008 and P = .013, respectively) and was not significantly different from the intact configuration at any angle. SCR decreased passive shoulder extension compared with the tear configuration and increased abduction compared with the intact configuration (P = .007 and P = .03, respectively). The overall arc of axial rotation was not significantly different between SCR and the intact configuration at any angle. CONCLUSIONS: In the setting of an irreparable SS tear, SCR restores key biomechanical parameters of the shoulder to intact levels. SCR should be considered for qualifying patients with irreparable RCTs.

The biomechanics of the supraspinatus-deficient shoulder treated with superior capsular reconstruction vs. reverse total shoulder arthroplasty-experimental study.

AIM OF THE STUDY: Our objective was to compare biomechanical effects of superior capsular reconstruction (SCR) and reverse total shoulder arthroplasty (rTSA) on shoulder motion, in the setting of an irreparable supraspinatus (SS) tear. We hypothesized that rTSA would produce greater improvement in abduction force and shift the humerus inferiorly, while SCR would produce greater range of motion (ROM) and prevent superior migration of the humerus during abduction. METHODS: Six cadaveric shoulders were evaluated using a custom biomechanical apparatus. Each shoulder underwent four experimental conditions: (1) intact/control, (2) irreparable SS tear, (3) SCR using dermal allograft, and (4) rTSA without SCR. Deltoid abduction force, superior humeral head translation, and passive range of motion were measured in static tendon loading condition at 0, 30, and 60° of glenohumeral abduction. RESULTS: Both rTSA and SCR restored abduction force to intact levels at all abduction angles. rTSA significantly increased abduction force compared with the SS-deficient shoulder at 0, 30, 60° (p = 0.04), while SCR produced a significant increase at 0° (p = 0.05) abduction. rTSA inferiorly shifted the humeral head by 27 mm (p = 0.002). SCR restored superior humeral head translation to intact SS levels. Compared with SCR, rTSA resulted in 25° less passive abduction (p = 0.001) without significant differences in forward flexion/extension. Compared with SCR, rTSA achieved 10° less passive internal rotation at 0° abduction (p = 0.03) and 26° and 17° greater external rotation at 30° and 60° abduction, respectively (p = 0.03, p = 0.04). DISCUSSION: Our investigation found that abduction force was restored to intact cuff levels by both procedures, without significant differences between the two techniques. SCR restored superior humeral head migration and rTSA translated the humerus inferiorly. rTSA resulted in decreased passive abduction ROM and increased external rotation, compared with SCR. CONCLUSION: Both SCR and rTSA restore key biomechanical parameters following an irreparable SS tear, although SCR offers superior passive abduction ROM.

Electric field vector measurements via nanosecond electric-field-induced second-harmonic generation.

Electric-field-induced second-harmonic generation, or E-FISH, has received renewed interest as a nonintrusive tool for probing electric fields in gas discharges and plasmas using ultrashort laser pulses. An important contribution of this work lies in establishing that the E-FISH method works effectively in the nanosecond regime, yielding field sensitivities of about a kV/cm at atmospheric pressure from a 16 ns pulse. This is expected to broaden its applicability within the plasma community, given the wider access to conventional nanosecond laser sources. A Pockels-cell-based pulse-slicing scheme, which may be readily integrated with such nanosecond laser systems, is shown to be a complementary and cost-effective option for improving the time resolution of the electric field measurement. Using this scheme, a time resolution of ∼3 ns is achieved, without any detriment to the signal sensitivity. This could prove invaluable for nonequilibrium plasma applications, where time resolution of a few nanoseconds or less is often critical. Finally, we take advantage of the field vector sensitivity of the E-FISH signal to demonstrate simultaneous measurements of both the horizontal and vertical components of the electric field.

Antibody-mediated complement activation in pathology and protection.

Antibody-dependent complement activity is associated not only with autoimmune morbidity, but also with antitumor efficacy. In infectious disease, both recombinant monoclonal antibodies and polyclonal antibodies generated in natural adaptive responses can mediate complement activity to protective, therapeutic or disease-enhancing effect. Recent advances have contributed to the structural resolution of molecular complexes involved in antibody-mediated complement activation, defining the avid nature of participating interactions and pointing to how antibody isotype, subclass, hinge flexibility, glycosylation state, amino acid sequence and the contextual nature of the cognate antigen/epitope are all factors that can determine complement activity through impact on antibody multimerization and subsequent recruitment of complement component 1q. Beyond the efficiency of activation, complement activation products interact with various cell types that mediate immune adherence, trafficking, immune education and innate functions. Similarly, depending on the anatomical location and extent of activation, complement can support homeostatic restoration or be leveraged by pathogens or neoplasms to enhance infection or promote tumorigenic microenvironments, respectively. Advances in means to suppress complement activation by intravenous immunoglobulin (IVIG), IVIG mimetics and complement-intervening antibodies represent proven and promising exploratory therapeutic strategies, while antibody engineering has likewise offered frameworks to enhance, eliminate or isolate complement activation to interrogate in vivo mechanisms of action. Such strategies promise to support the optimization of antibody-based drugs that are able to tackle emerging and difficult-to-treat diseases by improving our understanding of the synergistic and antagonistic relationships between antibody mechanisms mediated by Fc receptors, direct binding and the products of complement activation.