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Despite effective countermeasures, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) persists worldwide because of its ability to diversify and evade human immunity. This evasion stems from amino acid substitutions, particularly in the receptor binding domain (RBD) of the spike protein that confers resistance to vaccine-induced antibodies and antibody therapeutics. To constrain viral escape through resistance mutations, we combined antibody variable regions that recognize different RBD sites into multispecific antibodies. Here, we describe multispecific antibodies, including a trivalent trispecific antibody that potently neutralized diverse SARS-CoV-2 variants and prevented virus escape more effectively than single antibodies or mixtures of the parental antibodies. Despite being generated before the appearance of Omicron, this trispecific antibody neutralized all major Omicron variants through BA.4/BA.5 at nanomolar concentrations. Negative stain electron microscopy suggested that synergistic neutralization was achieved by engaging different epitopes in specific orientations that facilitated binding across more than one spike protein. Moreover, a tetravalent trispecific antibody containing the same variable regions as the trivalent trispecific antibody also protected Syrian hamsters against Omicron variants BA.1, BA.2, and BA.5 challenge, each of which uses different amino acid substitutions to mediate escape from therapeutic antibodies. These results demonstrated that multispecific antibodies have the potential to provide broad SARS-CoV-2 coverage, decrease the likelihood of escape, simplify treatment, and provide a strategy for antibody therapies that could help eliminate pandemic spread for this and other pathogens.

Background: Anterior cruciate ligament injury and anterior cruciate ligament reconstruction (ACLR) are risk factors for symptomatic posttraumatic osteoarthritis (PTOA). After ACLR, individuals demonstrate altered joint tissue metabolism indicative of increased inflammation and cartilage breakdown. Serum biomarker changes have been associated with tibiofemoral cartilage composition indicative of worse knee joint health but not with PTOA-related symptoms., Purpose/hypothesis: The purpose of this study was to determine associations between changes in serum biomarker profiles from the preoperative sample collection to 6 months after ACLR and clinically relevant knee PTOA symptoms at 12 months after ACLR. It was hypothesized that increases in biomarkers of inflammation, cartilage metabolism, and cartilage degradation would be associated with clinically relevant PTOA symptoms after ACLR., Study Design: Case-control study; Level of evidence, 3., Methods: Individuals undergoing primary ACLR were included (N = 30). Serum samples collected preoperatively and 6 months after ACLR were processed to measure markers indicative of changes in inflammation (ie, monocyte chemoattract protein 1 [MCP-1]) and cartilage breakdown (ie, cartilage oligomeric matrix protein [COMP], matrix metalloproteinase 3, ratio of type II collagen breakdown to type II collagen synthesis). Knee injury and Osteoarthritis Outcome Score surveys were completed at 12 months after ACLR and used to identify participants with and without clinically relevant PTOA-related symptoms. K-means cluster analyses were used to determine serum biomarker profiles. One-way analyses of variance and logistic regressions were used to assess differences in Knee injury and Osteoarthritis Outcome Score subscale scores and clinically relevant PTOA-related symptoms between biomarker profiles., Results: Two profiles were identified and characterized based on decreases (profile 1: 67% female; age, 21.4 ± 5.1 years; body mass index, 24.4 ± 2.4) and increases (profile 2: 33% female; age, 21.3 ± 3.2 years; body mass index, 23.4 ± 2.6) in sMCP-1 and sCOMP preoperatively to 6 months after ACLR. Participants with profile 2 did not demonstrate differences in knee pain, symptoms, activities of daily living, sports function, or quality of life at 12 months after ACLR compared to those with profile 1 ( P = .56-.81; η 2 = 0.002-0.012). No statistically significant associations were noted between biomarker profiles and clinically relevant PTOA-related symptoms (odds ratio, 1.30; 95% CI, 0.23-6.33)., Conclusion: Serum biomarker changes in MCP-1 and sCOMP within the first 6 months after ACLR were not associated with clinically relevant PTOA-related symptoms., Competing Interests: One or more of the authors has declared the following potential conflict of interest or source of funding: Research reported in this manuscript was supported by funding from the University of North Carolina's Department of Orthopaedics Laurence E. Dahners Research Grant, the National Institute of Arthritis and Musculoskeletal and Skin Disease of the National Institutes of Health (1R03 AR066840-01A1 and P30 AR072580), the North Carolina Translational and Clinical Sciences (TraCS) Institute, and the National Athletic Trainers’ Association Research and Education Foundation (14NewINV001). R.A.C. has received consulting fees from Arthrex and support for education from SouthTech Orthopedics. G.K. has received compensation for services other than consulting from Arthrex. J.S. has received support for education from SouthTech Orthopedics. AOSSM checks author disclosures against the Open Payments Database (OPD). AOSSM has not conducted an independent investigation on the OPD and disclaims any liability or responsibility relating thereto.

Purpose: Strong observational evidence has linked changes in limb loading during walking following anterior cruciate ligament reconstruction (ACLR) to posttraumatic osteoarthritis (PTOA). It remains unknown if manipulating peak loading influences joint tissue biochemistry. Thus, the purpose of this study is to determine whether manipulating peak vertical ground reaction force (vGRF) during gait influences changes in serum cartilage oligomeric matrix protein (sCOMP) concentrations in ACLR participants., Methods: Forty ACLR individuals participated in this randomized crossover study (48% female, age = 21.0 ± 4.4 years, BMI = 24.6 ± 3.1). Participants attended four sessions, wherein they completed one of four biofeedback conditions (habitual loading (no biofeedback), high loading (5% increase in vGRF), low loading (5% decrease in vGRF), and symmetrical loading (between-limb symmetry in vGRF)) while walking on a treadmill for 3000 steps. Serum was collected before (baseline), immediately (acute post), 1 h (1 h post), and 3.5 h (3.5 h post) following each condition. A comprehensive general linear mixed model was constructed to address the differences in sCOMP across all conditions and timepoints in all participants and a subgroup of sCOMP Increasers., Results: No sCOMP differences were found across the entire cohort. In the sCOMP Increasers, a significant time × condition interaction was found (F 9,206  = 2.6, p = 0.009). sCOMP was lower during high loading than low loading (p = 0.009) acutely (acute post). At 3.5 h post, sCOMP was higher during habitual loading than symmetrical loading (p = 0.001)., Conclusion: These data suggest that manipulating lower limb loading in ACLR patients who habitually exhibit an acute increase in sCOMP following walking results in improved biochemical changes linked to cartilage health. Key Points • This study assesses the mechanistic link between lower limb load modification and joint tissue biochemistry at acute and delayed timepoints. • Real-time biofeedback provides a paradigm to experimentally assess the mechanistic link between loading and serum biomarkers. • Manipulating peak loading during gait resulted in a metabolic effect of lower sCOMP concentrations in a subgroup of ACLR individuals. • Peak loading modifications may provide an intervention strategy to mitigate the development of PTOA following ACLR., (© 2024. The Author(s), under exclusive licence to International League of Associations for Rheumatology (ILAR).)

Purpose: Less physical activity has been associated with systemic biomarkers of cartilage breakdown after anterior cruciate ligament reconstruction (ACLR). However, previous research lacks analysis of deleterious cartilage compositional changes and objective physical activity after ACLR. The purpose of this study was to determine the association between physical activity quantified via accelerometer-based measures of daily steps and time in moderate-to-vigorous physical activity (MVPA), and T1rho magnetic resonance imaging (MRI) of the femoral articular cartilage, a marker of proteoglycan density in individuals with ACLR., Methods: Daily steps and MVPA were assessed over 7 d using an accelerometer worn on the hip in 26 individuals between 6 and 12 months after primary unilateral ACLR. Resting T1rho MRI was collected bilaterally, and T1rho MRI interlimb ratios (ILR: ACLR limb/contralateral limb) were calculated for lateral and medial femoral condyle regions of interest. We conducted univariate linear regression analyses to determine associations between T1rho MRI ILRs and daily steps and MVPA with and without controlling for sex., Results: Greater T1rho MRI ILR of the central lateral femoral condyle, indicative of less proteoglycan density in the ACLR limb, was associated with greater time in MVPA ( R2 = 0.178, P = 0.032). Sex-adjusted models showed significant interaction terms between daily steps and sex in the anterior ( P = 0.025), central ( P = 0.002), and posterior ( P = 0.002) medial femoral condyle., Conclusions: Lesser physical activity may be a risk factor for maintaining cartilage health after ACLR; additionally, the relationship between physical activity and cartilage health may be different between males and females., (Copyright © 2023 by the American College of Sports Medicine.)

Purpose: To determine associations between immediate and delayed response of serum cartilage oligomeric matrix protein (sCOMP) to loading (i.e., 3000 walking steps) and femoral cartilage interlimb T1ρ relaxation times in individual's post-anterior cruciate ligament reconstruction (ACLR)., Methods: This cross-sectional study included 20 individuals 6-12 months following primary ACLR (65% female, 20.5 ± 4.0 years old, 24.9 ± 3.0 kg/m 2 , 7.3 ± 1.5 months post-ACLR). Serum samples were collected prior to, immediately following, and 3.5 h following walking 3000 steps on a treadmill at habitual walking speed. sCOMP concentrations were processed using enzyme-linked immunosorbent assays. Immediate and delayed absolute sCOMP responses to loading were evaluated immediately and 3.5 h post-walking, respectively. Participants underwent bilateral magnetic resonance imaging with T1ρ sequences to calculate resting femoral cartilage interlimb T1ρ relaxation time ratios between limbs (i.e., ACLR/Uninjured limb). Linear regression models were fitted to determine associations between sCOMP response to loading and femoral cartilage T1ρ outcomes controlling for pre-loading sCOMP concentrations., Results: Greater increases in delayed sCOMP response to loading were associated with greater lateral (∆R 2  = 0.29, p = 0.02) but not medial (∆R 2  < 0.01, p = 0.99) femoral cartilage interlimb T1ρ ratios. Associations between immediate sCOMP response to loading with femoral cartilage interlimb T1ρ ratios were weak and non-significant (∆R 2 range = 0.02-0.09, p range = 0.21-0.58)., Conclusion: Greater delayed sCOMP response to loading, a biomarker of cartilage breakdown, is associated with worse lateral femoral cartilage composition in the ACLR limb compared to the uninjured limb. Delayed sCOMP response to loading may be a more indicative metabolic indicator linked to deleterious changes in composition than immediate sCOMP response., (© 2023. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

In osteoarthritis (OA), bone changes are radiological hallmarks and are considered important for disease progression. The C-C chemokine receptor-2 (CCR2) has been shown to play an important role in bone physiology. In this study, we investigated whether Ccr2 osteoblast-specific inactivation at different times during post-traumatic OA (PTOA) progression improves joint structures, bone parameters, and pain. We used a tamoxifen-inducible Ccr2 inactivation in Collagen1α-expressing cells to obtain osteoblasts lacking Ccr2 ( CCR2 - Col1αKO ). We stimulated PTOA changes in CCR2 - Col1αKO and CCR2 +/+ mice using the destabilization of the meniscus model (DMM), inducing recombination before or after DMM (early- vs. late-inactivation). Joint damage was evaluated at two, four, eight, and twelve weeks post-DMM using multiple scores: articular-cartilage structure (ACS), Safranin-O, histomorphometry, osteophyte size/maturity, subchondral bone thickness and synovial hyperplasia. Spontaneous and evoked pain were assessed for up to 20 weeks. We found that early osteoblast- Ccr2 inactivation delayed articular cartilage damage and matrix degeneration compared to CCR2 +/+, as well as DMM-induced bone thickness. Osteophyte formation and maturation were only minimally affected. Late Collagen1α- Ccr2 deletion led to less evident improvements. Osteoblast- Ccr2 deletion also improved static measures of pain, while evoked pain did not change. Our study demonstrates that Ccr2 expression in osteoblasts contributes to PTOA disease progression and pain by affecting both cartilage and bone tissues.

Posttraumatic osteoarthritis (PTOA) is mostly treated via corticosteroid administration, and total joint arthroplasty continues to be the sole effective intervention in severe conditions. To assess the therapeutic potential of CCR2 targeting in PTOA, we used biodegradable microplates (µPLs) to achieve a slow and sustained intraarticular release of the CCR2 inhibitor RS504393 into injured knees and followed joint damage during disease progression. RS504393-loaded µPLs (RS-µPLs) were fabricated via a template-replica molding technique. A mixture of poly(lactic-co-glycolic acid) (PLGA) and RS504393 was deposited into 20 × 10 μm (length × height) wells in a polyvinyl alcohol (PVA) square-patterned template. After physicochemical and toxicological characterizations, the RS504393 release profile from µPL was assessed in PBS buffer. C57BL/6 J male mice were subjected to destabilization of the medial meniscus (DMM)/sham surgery, and RS-µPLs (1 mg/kg) were administered intraarticularly 1 week postsurgery. Administrations were repeated at 4 and 7 weeks post-DMM. Drug free-µPLs (DF-µPLs) and saline injections were performed as controls. Mice were euthanized at 4 and 10 weeks post-DMM, corresponding to the early and severe PTOA stages, respectively. Knees were evaluated for cartilage structure score (ACS, H&E), matrix loss (safranin O score), osteophyte formation and maturation from cartilage to bone (cartilage quantification), and subchondral plate thickness. The RS-µPL architecture ensured the sustained release of CCR2 inhibitors over several weeks, with ~ 20% of RS504393 still available at 21 days. This prolonged release improved cartilage structure and reduced bone damage and synovial hyperplasia at both PTOA stages. Extracellular matrix loss was also attenuated, although with less efficacy. The results indicate that local sustained delivery is needed to optimize CCR2-targeted therapies., (© 2022. The Author(s).)

Despite effective countermeasures, SARS-CoV-2 persists worldwide due to its ability to diversify and evade human immunity 1 . This evasion stems from amino-acid substitutions, particularly in the receptor-binding domain of the spike, that confer resistance to vaccines and antibodies 2-16 . To constrain viral escape through resistance mutations, we combined antibody variable regions that recognize different receptor binding domain (RBD) sites 17,18 into multispecific antibodies. Here, we describe multispecific antibodies, including a trispecific that prevented virus escape >3000-fold more potently than the most effective clinical antibody or mixtures of the parental antibodies. Despite being generated before the evolution of Omicron, this trispecific antibody potently neutralized all previous variants of concern and major Omicron variants, including the most recent BA.4/BA.5 strains at nanomolar concentrations. Negative stain electron microscopy revealed that synergistic neutralization was achieved by engaging different epitopes in specific orientations that facilitated inter-spike binding. An optimized trispecific antibody also protected Syrian hamsters against Omicron variants BA.1, BA.2 and BA.5, each of which uses different amino acid substitutions to mediate escape from therapeutic antibodies. Such multispecific antibodies decrease the likelihood of SARS-CoV-2 escape, simplify treatment, and maximize coverage, providing a strategy for universal antibody therapies that could help eliminate pandemic spread for this and other pathogens.

The processes leading to the emergence of new species are poorly understood in marine plankton, where weak physical barriers and homogeneous environmental conditions limit spatial and ecological segregation. Here, we combine molecular and ecological information from a long-term time series and propose Pseudo-nitzschia allochrona , a new cryptic planktonic diatom, as a possible case of speciation by temporal segregation. The new species differs in several genetic markers (18S, 28S and ITS rDNA fragments and rbc L) from its closest relatives, which are morphologically very similar or identical, and is reproductively isolated from its sibling species P. arenysensis . Data from a long-term plankton time series show P. allochrona invariably occurring in summer-autumn in the Gulf of Naples, where its closely related species P. arenysensis , P. delicatissima , and P. dolorosa are instead found in winter-spring. Temperature and nutrients are the main factors associated with the occurrence of P. allochrona , which could have evolved in sympatry by switching its phenology and occupying a new ecological niche. This case of possible speciation by time shows the relevance of combining ecological time series with molecular information to shed light on the eco-evolutionary dynamics of marine microorganisms., Competing Interests: We declare no conflict of interest., (© 2022 The Authors. Ecology and Evolution published by John Wiley & Sons Ltd.)

Context: Better knee function is linked to psychological readiness to return to sport after anterior cruciate ligament reconstruction (ACLR). Individuals with ACLR participate in less physical activity than matched uninjured control individuals, yet the association between knee function and physical activity post-ACLR remains unclear., Objective: To determine the associations between (1) patient-reported knee function measured using the Knee Injury and Osteoarthritis Outcome Score Knee-Related Quality of Life (KOOS-QOL), daily steps, and minutes spent in moderate-to-vigorous physical activity (MVPA) of individuals with ACLR and (2) KOOS-QOL and daily steps and MVPA in individuals with ACLR who presented with (ie, symptomatic) or without (ie, asymptomatic) clinically meaningful knee-related symptoms., Design: Cross-sectional study., Setting: Laboratory, free-living conditions., Patients or Other Participants: A total of 66 individuals with primary unilateral ACLR (36 women, 30 men; age = 22 ± 4 years, height = 1.71 ± 0.1 m, mass = 71.3 ± 12.6 kg, body mass index = 24.2 ± 2.9, time post-ACLR = 28 ± 33 months)., Main Outcome Measure(s): We collected KOOS data and retrospectively stratified participants into those with (symptomatic group, n = 30) or without (asymptomatic group, n = 36) clinically meaningful knee-related symptoms based on previously defined KOOS cutoffs. We assessed daily steps and MVPA using accelerometers that participants wore on the right hip for 7 days. We conducted linear regressions to determine associations between KOOS-QOL and daily steps and MVPA., Results: In the entire sample, no associations existed between KOOS-QOL and daily steps (ΔR2 = 0.01, P = .50) or MVPA (ΔR2 = 0.01, P = .36). In the symptomatic group, a greater KOOS-QOL was associated with more time in MVPA (ΔR2 = 0.12, P = .05). In the asymptomatic group, no associations were identified between the KOOS-QOL and daily steps and MVPA., Conclusions: Individuals with symptoms post-ACLR who spent more time in MVPA reported higher QOL., (© by the National Athletic Trainers' Association, Inc.)

Phytoplankton play a pivotal role in global biogeochemical and trophic processes and provide essential ecosystem services. However, there is still no broad consensus on how and to what extent their community composition responds to environmental variability. Here, high-frequency oceanographic and biological data collected over more than 25 years in a coastal Mediterranean site are used to shed light on the temporal patterns of phytoplankton species and assemblages in their environmental context. Because of the proximity to the coast and due to large-scale variations, environmental conditions showed variability on the short and long-term scales. Nonetheless, an impressive regularity characterised the annual occurrence of phytoplankton species and their assemblages, which translated into their remarkable stability over decades. Photoperiod was the dominant factor related to community turnover and replacement, which points at a possible endogenous regulation of biological processes associated with species-specific phenological patterns, in analogy with terrestrial plants. These results highlight the considerable stability and resistance of phytoplankton communities in response to different environmental pressures, which contrast the view of these organisms as passively undergoing changes that occur at different temporal scales in their habitat, and show how, under certain conditions, biological processes may prevail over environmental forcing., (© 2022. The Author(s).)

Objective: To test the hypothesis that an altered gut microbiota (dysbiosis) plays a role in obesity-associated osteoarthritis (OA)., Methods: Stool and blood samples were collected from 92 participants with a body mass index (BMI) ≥30 kg/m 2 , recruited from the Johnston County Osteoarthritis Project. OA patients (n = 50) had hand and knee OA (Kellgren/Lawrence [K/L] grade ≥2 or arthroplasty). Controls (n = 42) had no hand OA and a K/L grade of 0-1 for the knees. Compositional analysis of stool samples was carried out by 16S ribosomal RNA amplicon sequencing. Alpha- and beta-diversity and differences in taxa relative abundances were determined. Blood samples were used for multiplex cytokine analysis and measures of lipopolysaccharide (LPS) and LPS binding protein. Germ-free mice were gavaged with patient- or control-pooled fecal samples and fed a 40% fat, high-sucrose diet for 40 weeks. Knee OA was evaluated histologically., Results: On average, OA patients were slightly older than the controls, consisted of more women, and had a higher mean BMI, higher mean Western Ontario and McMaster Universities Osteoarthritis Index pain score, and higher mean K/L grade. There were no significant differences in α- or β-diversity or genus level composition between patients and controls. Patients had higher plasma levels of osteopontin (P = 0.01) and serum LPS (P < 0.0001) compared to controls. Mice transplanted with patient or control microbiota exhibited a significant difference in α-diversity (P = 0.02) and β-diversity, but no differences in OA severity were observed., Conclusion: The lack of differences in the gut microbiota, but increased serum LPS levels, suggest the possibility that increased intestinal permeability allowing for greater absorption of LPS, rather than a dysbiotic microbiota, may contribute to the development of OA associated with obesity., (© 2021, American College of Rheumatology.)