Deletion of a conserved genomic region associated with adolescent idiopathic scoliosis leads to vertebral rotation in mice.

Adolescent idiopathic scoliosis (AIS) is the most common form of scoliosis, in which spinal curvature develops in adolescence, and 90% of patients are female. Scoliosis is a debilitating disease that often requires bracing or surgery in severe cases. AIS affects 2%-5.2% of the population; however, the biological origin of the disease remains poorly understood. In this study, we aimed to determine the function of a highly conserved genomic region previously linked to AIS using a mouse model generated by CRISPR-CAS9 gene editing to knockout this area of the genome to understand better its contribution to AIS, which we named AIS_CRMΔ. We also investigated the upstream factors that regulate the activity of this enhancer in vivo, whether the spatial expression of the LBX1 protein would change with the loss of AIS-CRM function, and whether any phenotype would arise after deletion of this region. We found a significant increase in mRNA expression in the developing neural tube at E10.5, and E12.5, for not only Lbx1 but also other neighboring genes. Adult knockout mice showed vertebral rotation and proprioceptive deficits, also observed in human AIS patients. In conclusion, our study sheds light on the elusive biological origins of AIS, by targeting and investigating a highly conserved genomic region linked to AIS in humans. These findings provide valuable insights into the function of the investigated region and contribute to our understanding of the underlying causes of this debilitating disease.

Ichthyosis linked to sphingosine 1-phosphate lyase insufficiency is due to aberrant sphingolipid and calcium regulation.

Sphingosine 1-phosphate lyase (SGPL1) insufficiency (SPLIS) is a syndrome which presents with adrenal insufficiency, steroid-resistant nephrotic syndrome, hypothyroidism, neurological disease, and ichthyosis. Where a skin phenotype is reported, 94% had abnormalities such as ichthyosis, acanthosis, and hyperpigmentation. To elucidate the disease mechanism and the role SGPL1 plays in the skin barrier we established clustered regularly interspaced short palindromic repeats-Cas9 SGPL1 KO and a lentiviral-induced SGPL1 overexpression (OE) in telomerase reverse-transcriptase immortalised human keratinocytes (N/TERT-1) and thereafter organotypic skin equivalents. Loss of SGPL1 caused an accumulation of S1P, sphingosine, and ceramides, while its overexpression caused a reduction of these species. RNAseq analysis showed perturbations in sphingolipid pathway genes, particularly in SGPL1_KO, and our gene set enrichment analysis revealed polar opposite differential gene expression between SGPL1_KO and _OE in keratinocyte differentiation and Ca2+ signaling genesets. SGPL1_KO upregulated differentiation markers, while SGPL1_OE upregulated basal and proliferative markers. The advanced differentiation of SGPL1_KO was confirmed by 3D organotypic models that also presented with a thickened and retained stratum corneum and a breakdown of E-cadherin junctions. We conclude that SPLIS associated ichthyosis is a multifaceted disease caused possibly by sphingolipid imbalance and excessive S1P signaling, leading to increased differentiation and an imbalance of the lipid lamellae throughout the epidermis.

C-C Bond Activation and Demethylenation of Epoxides by Amine Radical Dications.

In this note, we explore a unique reactivity pattern that involves a rare radical-based C-C bond scission of epoxides followed by demethylenation. The reaction is accomplished by Selecfluor and its radical dication working in tandem; a mechanism supported by experiment and DFT calculations is proposed that involves the generation and identification of a key reactive intermediate. The reaction seems to be fairly general for 1,1-disubstituted epoxides.

Photoactivated Pyridine Directed Fluorination through Hydrogen Atom Transfer.

We have established hydrogen atom transfer (HAT) as the key player in a directed, photopromoted fluorination of pyridylic groups. The Lewis basic pyridyl nitrogen directs amine radical dication propagated HAT and Selectfluor fluorination of various ortho substituents in a highly regioselective manner with little to no side product formation. A variety of pyridines and quinolines were employed to showcase the directing capability of the nitrogen atom. Additionally, both experimental and computational data are provided that illuminate how this mechanism differs from and complements prior work in the area.

VirA+EmiC project: Evaluating real-world effectiveness and sustainability of integrated routine opportunistic hepatitis B and C testing in a large urban emergency department.

Innovative testing approaches and care pathways are required to meet global hepatitis B virus (HBV) and hepatitis C virus (HCV) elimination goals. Routine blood-borne virus (BBV) testing in emergency departments (EDs) in high-prevalence areas is suggested by the European Centre for Disease Prevention and Control (ECDC) but there is limited evidence for this. Universal HIV testing in our ED according to UK guidance has been operational since 2015. We conducted a real-world service evaluation of a modified electronic patient record (EPR) system to include opportunistic opt-out HBV/reflex-HCV tests for any routine blood test orders for ED attendees aged ≥16 years. Reactive laboratory results were communicated directly to specialist clinical teams. Our model for contacting patients requiring linkage to care (new diagnoses/known but disengaged) evolved from initially primarily hospital-led to collaborating with regional health and community service networks. Over 11 months, 81,088 patients attended the ED; 36,865 (45.5%) had a blood test. Overall uptake for both HBV and HCV testing was 75%. Seroprevalence was 0.9% for hepatitis B surface antigen (HBsAg) and 0.9% for HCV antigen (HCV-Ag). 79% of 140 successfully contacted HBsAg+patients required linkage to care, of which 87% engaged. 76% of 130 contactable HCV-Ag+patients required linkage, 52% engaged. Our results demonstrate effectiveness and sustainability of universal ED EPR opt-out HBV/HCV testing combined with comprehensive linkage to care pathways, allowing care provision particularly for marginalized at-risk groups with limited healthcare access. The findings support the ECDC BBV testing guidance and may inform future UK hepatitis testing guidance.

Universal testing for hepatitis B and hepatitis C in the emergency department: a cost-effectiveness and budget impact analysis of two urban hospitals in the United Kingdom.

BACKGROUND: Numerous studies have shown the effectiveness of testing for hepatitis B (HBV) and hepatitis C (HCV) in emergency departments (ED), due to the elevated prevalence amongst attendees. The aim of this study was to conduct a cost-effectiveness analysis of universal opt-out HBV and HCV testing in EDs based on 2 long-term studies of the real-world effectiveness of testing in 2 large ED's in the UK. METHODS: A Markov model was used to evaluate ED-based HBV and HCV testing versus no ED testing, in addition to current testing practice. The two EDs had a HBV HBsAg prevalence of 0.5-0.9% and an HCV RNA prevalence of 0.9-1.0%. The analysis was performed from a UK health service perspective, over a lifetime time horizon. Costs are reported in British pounds (GBP), and outcomes as quality adjusted life years (QALYs), with both discounted at 3.5% per year. Incremental cost-effectiveness ratios (ICER) are calculated as costs per QALY gained. A willingness-to-pay threshold of £20,000/QALY was used. The cost-effectiveness was estimated for both infections, in both ED's. RESULTS: HBV and HCV testing were highly cost-effective in both settings, with ICERs ranging from £7,177 to £12,387 per QALY gained. In probabilistic analyses, HBV testing was 89-94% likely to be cost-effective at the threshold, while HCV testing was 94-100% likely to be cost-effective, across both settings. In deterministic sensitivity analyses, testing remained cost-effective in both locations at ≥ 0.25% HBsAg prevalence, and ≥ 0.49% HCV RNA prevalence. This is much lower than the prevalence observed in the two EDs included in this study. CONCLUSIONS: HBV and HCV testing in urban EDs is highly cost-effective in the UK, and can be cost-effective at relatively low prevalence. These results should be reflected in UK and European hepatitis testing guidelines.

Brain Activity Fluctuations Propagate as Waves Traversing the Cortical Hierarchy.

The brain exhibits highly organized patterns of spontaneous activity as measured by resting-state functional magnetic resonance imaging (fMRI) fluctuations that are being widely used to assess the brain's functional connectivity. Some evidence suggests that spatiotemporally coherent waves are a core feature of spontaneous activity that shapes functional connectivity, although this has been difficult to establish using fMRI given the temporal constraints of the hemodynamic signal. Here, we investigated the structure of spontaneous waves in human fMRI and monkey electrocorticography. In both species, we found clear, repeatable, and directionally constrained activity waves coursed along a spatial axis approximately representing cortical hierarchical organization. These cortical propagations were closely associated with activity changes in distinct subcortical structures, particularly those related to arousal regulation, and modulated across different states of vigilance. The findings demonstrate a neural origin of spatiotemporal fMRI wave propagation at rest and link it to the principal gradient of resting-state fMRI connectivity.

A three-step enantioselective synthesis of (+)- and (-)-α-thujone.

The stereocontrolled three-step synthesis of either enantiomer of α-thujone from commercially available 3-methyl-1-butyne is described. The enantioselectivity originates from a Brown crotylation which is then conferred to the all-carbon quaternary center via chirality transfer in a gold-catalyzed cycloisomerization. The route is highly atom economical and requires no protecting groups or redox manipulations.

Loss of Nnt Increases Expression of Oxidative Phosphorylation Complexes in C57BL/6J Hearts.

Nicotinamide nucleotide transhydrogenase (NNT) is a proton pump in the inner mitochondrial membrane that generates reducing equivalents in the form of NAPDH, which can be used for anabolic pathways or to remove reactive oxygen species (ROS). A number of studies have linked NNT dysfunction to cardiomyopathies and increased risk of atherosclerosis; however, biallelic mutations in humans commonly cause a phenotype of adrenal insufficiency, with rare occurrences of cardiac dysfunction and testicular tumours. Here, we compare the transcriptomes of the hearts, adrenals and testes from three mouse models: the C57BL/6N, which expresses NNT; the C57BL/6J, which lacks NNT; and a third mouse, expressing the wild-type NNT sequence on the C57BL/6J background. We saw enrichment of oxidative phosphorylation genes in the C57BL/B6J in the heart and adrenal, possibly indicative of an evolved response in this substrain to loss of Nnt. However, differential gene expression was mainly driven by mouse background with some changes seen in all three tissues, perhaps reflecting underlying genetic differences between the C57BL/B6J and -6N substrains.

Epithelial and stromal circadian clocks are inversely regulated by their mechano-matrix environment.

The circadian clock is an autonomous molecular feedback loop inside almost every cell in the body. We have shown that the mammary epithelial circadian clock is regulated by the cellular microenvironment. Moreover, a stiff extracellular matrix dampens the oscillations of the epithelial molecular clock. Here, we extend this analysis to other tissues and cell types, and identify an inverse relationship between circadian clocks in epithelia and fibroblasts. Epithelial cells from mammary gland, lung and skin have significantly stronger oscillations of clock genes in soft 3D microenvironments, compared to stiff 2D environments. Fibroblasts isolated from the same tissues show the opposite response, exhibiting stronger oscillations and more prolonged rhythmicity in stiff microenvironments. RNA analysis identified that a subset of mammary epithelial clock genes, and their regulators, are upregulated in 3D microenvironments in soft compared to stiff gels. Furthermore, the same genes are inversely regulated in fibroblasts isolated from the same tissues. Thus, our data reveal for the first time an intrinsic difference in the regulation of circadian genes in epithelia and fibroblasts.

In Situ Determination of Carbon Number Distributions of Mixtures of Linear Hydrocarbons Confined within Porous Media Using Pulsed Field Gradient NMR.

Pulsed field gradient (PFG) NMR measurements, combined with a novel optimization method, are used to determine the composition of hydrocarbon mixtures of linear alkanes (C7-C16) in both the bulk liquid state and when imbibed within a porous medium of mean pore diameter 28.6 nm. The method predicts the average carbon number of a given mixture to an accuracy of ±1 carbon number and the mole fraction of a mixture component to within an average root-mean-square error of ±0.036 with just three calibration mixtures. Given that the method can be applied at any conditions of temperature and pressure at which the PFG NMR measurements are made, the method has the potential for application in characterizing hydrocarbon liquid mixtures inside porous media and at the operating conditions relevant to, for example, hydrocarbon recovery and heterogeneous catalysis.

An Economic Evaluation of the Cost-Effectiveness of Opt-Out Hepatitis B and Hepatitis C Testing in an Emergency Department Setting in the United Kingdom.

OBJECTIVES: The prevalence of hepatitis is high in emergency department (ED) attendees in the United Kingdom, with a prevalence of up to 2% for hepatitis B (HBV) HBsAg, and 2.9% for hepatitis C (HCV) RNA. The aim of this paper is to perform an economic evaluation of opt-out ED-based HCV and HBV testing. METHODS: A Markov model was developed to analyze the cost-effectiveness of opt-out HCV and HBV testing in EDs in the UK. The model used data from UK studies of ED testing to parameterize the HCV and HBV prevalence (1.4% HCV RNA, 0.84% HBsAg), test costs, and intervention effects (contact rates and linkage to care). For HCV, we used an antibody test cost of £3.64 and RNA test cost of £68.38, and assumed direct-acting antiviral treatment costs of £10 000. For HBV, we used a combined HBsAg and confirmatory test cost of £5.79. We also modeled the minimum prevalence of HCV (RNA-positive) and HBV (HBsAg) required to make ED testing cost-effective at a £20 000 willingness to pay per quality-adjusted life-year threshold. RESULTS: In the base case, ED testing was highly cost-effective, with HCV and HBV testing costing £8019 and £9858 per quality-adjusted life-year gained, respectively. HCV and HBV ED testing remained cost-effective at 0.25% HCV RNA or HBsAg prevalence or higher. CONCLUSIONS: Emergency department testing for HCV and HBV is highly likely to be cost-effective in many areas across the UK depending on their prevalence. Ongoing studies will help evaluate ED testing across different regions to inform testing guidelines.

Cost-Effectiveness of One-Time Birth Cohort Screening for Hepatitis C as Part of the National Health Service Health Check Program in England.

BACKGROUND AND OBJECTIVES: Birth cohort screening for the hepatitis C virus (HCV) has been implemented in the US, but there is little evidence of its cost-effectiveness in England. We aim to evaluate the cost-effectiveness of one-time HCV screening for individuals born between 1950 and 1979 as part of the National Health Service health check in England, a health check for adults aged 40 to 74 years in primary care. METHODS: A Markov model was developed to analyze add-on HCV testing to the National Health Service health check for individuals in birth cohorts between 1950 and 1979, versus current background HCV testing only, over a lifetime horizon. The model used data from a back-calculation model of the burden of HCV in England, sentinel surveillance of HCV testing, and published literature. Results are presented from a health service perspective in pounds in 2017, as incremental cost-effectiveness ratios per quality-adjusted life years gained. RESULTS: The base-case incremental cost-effectiveness ratios ranged from £7648 to £24 434, and £18 681 to £46 024, across birth cohorts when considering 2 sources of HCV transition probabilities. The intervention is most likely to be cost-effective for those born in the 1970s, and potentially cost-effective for those born from 1955 to 1969. The model results were most sensitive to the source of HCV transition probabilities, the probability of referral and receiving treatment, and the HCV prevalence among testers. The maximum value of future research across all birth cohorts was £11.3 million at £20 000 per quality-adjusted life years gained. CONCLUSION: Birth cohort screening is likely to be cost-effective for younger birth cohorts, although considerable uncertainty exists for other birth cohorts. Further studies are warranted to reduce uncertainty in cost-effectiveness and consider the acceptability of the intervention.

Disrupted circadian clocks and altered tissue mechanics in primary human breast tumours.

BACKGROUND: Circadian rhythms maintain tissue homeostasis during the 24-h day-night cycle. Cell-autonomous circadian clocks play fundamental roles in cell division, DNA damage responses and metabolism. Circadian disruptions have been proposed as a contributing factor for cancer initiation and progression, although definitive evidence for altered molecular circadian clocks in cancer is still lacking. In this study, we looked at circadian clocks in breast cancer. METHODS: We isolated primary tumours and normal tissues from the same individuals who had developed breast cancer with no metastases. We assessed circadian clocks within primary cells of the patients by lentiviral expression of circadian reporters, and the levels of clock genes in tissues by qPCR. We histologically examined collagen organisation within the normal and tumour tissue areas, and probed the stiffness of the stroma adjacent to normal and tumour epithelium using atomic force microscopy. RESULTS: Epithelial ducts were disorganised within the tumour areas. Circadian clocks were altered in cultured tumour cells. Tumour regions were surrounded by stroma with an altered collagen organisation and increased stiffness. Levels of Bmal1 messenger RNA (mRNA) were significantly altered in the tumours in comparison to normal epithelia. CONCLUSION: Circadian rhythms are suppressed in breast tumour epithelia in comparison to the normal epithelia in paired patient samples. This correlates with increased tissue stiffness around the tumour region. We suggest possible involvement of altered circadian clocks in the development and progression of breast cancer.

The intervertebral disc contains intrinsic circadian clocks that are regulated by age and cytokines and linked to degeneration.

OBJECTIVES: The circadian clocks are internal timing mechanisms that drive ∼24-hour rhythms in a tissue-specific manner. Many aspects of the physiology of the intervertebral disc (IVD) show clear diurnal rhythms. However, it is unknown whether IVD tissue contains functional circadian clocks and if so, how their dysregulation is implicated in IVD degeneration. METHODS: Clock gene dynamics in ex vivo IVD explants (from PER2:: luciferase (LUC) reporter mice) and human disc cells (transduced with lentivirus containing Per2::luc reporters) were monitored in real time by bioluminescence photon counting and imaging. Temporal gene expression changes were studied by RNAseq and quantitative reverse transcription (qRT)-PCR. IVD pathology was evaluated by histology in a mouse model with tissue-specific deletion of the core clock gene Bmal1. RESULTS: Here we show the existence of the circadian rhythm in mouse IVD tissue and human disc cells. This rhythm is dampened with ageing in mice and can be abolished by treatment with interleukin-1ß but not tumour necrosis factor α. Time-series RNAseq revealed 607 genes with 24-hour patterns of expression representing several essential pathways in IVD physiology. Mice with conditional knockout of Bmal1 in their disc cells demonstrated age-related degeneration of IVDs. CONCLUSIONS: We have established autonomous circadian clocks in mouse and human IVD cells which respond to age and cytokines, and control key pathways involved in the homeostasis of IVDs. Genetic disruption to the mouse IVD molecular clock predisposes to IVD degeneration. These results support the concept that disruptions to circadian rhythms may be a risk factor for degenerative IVD disease and low back pain.

Circadian clocks and breast cancer.

Circadian clocks respond to environmental time cues to coordinate 24-hour oscillations in almost every tissue of the body. In the breast, circadian clocks regulate the rhythmic expression of numerous genes. Disrupted expression of circadian genes can alter breast biology and may promote cancer. Here we overview circadian mechanisms, and the connection between the molecular clock and breast biology. We describe how disruption of circadian genes contributes to cancer via multiple mechanisms, and link this to increased tumour risk in women who work irregular shift patterns. Understanding the influence of circadian rhythms on breast cancer could lead to more efficacious therapies, reformed public health policy and improved patient outcome.

Flow regime, temperature, and biotic interactions drive differential declines of trout species under climate change.

Broad-scale studies of climate change effects on freshwater species have focused mainly on temperature, ignoring critical drivers such as flow regime and biotic interactions. We use downscaled outputs from general circulation models coupled with a hydrologic model to forecast the effects of altered flows and increased temperatures on four interacting species of trout across the interior western United States (1.01 million km(2)), based on empirical statistical models built from fish surveys at 9,890 sites. Projections under the 2080s A1B emissions scenario forecast a mean 47% decline in total suitable habitat for all trout, a group of fishes of major socioeconomic and ecological significance. We project that native cutthroat trout Oncorhynchus clarkii, already excluded from much of its potential range by nonnative species, will lose a further 58% of habitat due to an increase in temperatures beyond the species' physiological optima and continued negative biotic interactions. Habitat for nonnative brook trout Salvelinus fontinalis and brown trout Salmo trutta is predicted to decline by 77% and 48%, respectively, driven by increases in temperature and winter flood frequency caused by warmer, rainier winters. Habitat for rainbow trout, Oncorhynchus mykiss, is projected to decline the least (35%) because negative temperature effects are partly offset by flow regime shifts that benefit the species. These results illustrate how drivers other than temperature influence species response to climate change. Despite some uncertainty, large declines in trout habitat are likely, but our findings point to opportunities for strategic targeting of mitigation efforts to appropriate stressors and locations.

In vitro cytotoxicity of Artemisia vulgaris L. essential oil is mediated by a mitochondria-dependent apoptosis in HL-60 leukemic cell line.

BACKGROUND: The essential oil (EO) of Artemisia vulgaris L. has been traditionally used worldwide for treating a large number of diseases. Although major components in A. vulgaris EO have been shown to inhibit growth of different cancer cells, as pure compounds or part of other plants extracted oil, no information is known about its anti-proliferative activities. Therefore, the current investigation has evaluated the toxicity of the plant extracted oil from buds (AVO-b) and leaves (AVO-l) and characterized their growth inhibitory effects on cancer cells. METHODS: AVO-b and AVO-l from A. vulgaris L. were extracted by hydrodistillation, and their effect on the viability of human HL-60 promyelocytic leukemia and various other cancer cell lines was tested using MTT assay. Flow cytometric analysis of apoptosis, DNA fragmentation assay, caspases enzymatic activities and Western blotting were used to determine the apoptotic pathway triggered by their action on HL-60 cells. RESULTS: Low concentrations of AVO-b and AVO-l inhibited the growth of HL-60 cells in a dose- and time-dependent manner. Employing flow cytometric, DNA fragmentation and caspase activation analyses, demonstrated that the cytotoxic effect of the oils is mediated by a caspase-dependent apoptosis. Kinetic studies in the presence and absence specific caspase inhibitors showed that activation of caspase-8 was dependent and subsequent to the activation of caspases-9 and -3. In addition, the essential oil caused a disruption of the mitochondrial transmembrane potential (ΔΨm), increased the release of cytochrome c to the cytosol, and altered the expression of certain members of Bcl-2 family (Bcl-2, Bax and Bid), Apaf-1 and XIAP. Interestingly, low doses of AVO-b and AVO-1 also induced apoptosis in various cancer cell lines, but not in noncancerous cells. CONCLUSIONS: The results demonstrate that the EO-induced apoptosis in HL-60 cells is mediated by caspase-dependent pathways, involving caspases-3, -9, and -8, which are initiated by Bcl-2/Bax/Bid-dependent loss of ΔΨm leading to release of cytochrome c to the cytoplasm to activate the caspase cascade. The finding that AVO-b and AVO-l are more efficient to induce apoptosis in different cancer cell lines than noncancerous cells, suggests that A. vulgaris might be a promising source for new anticancer agents.

Inhaled methoxyflurane - an explorable alternative to nitrous oxide?

Nitrous oxide is a widely used and well-established form of inhalation sedation in dentistry. Its properties have a wide margin of safety and allow for anxious, paediatric and adult patients to receive dental treatment with minimal impact upon discharge. Nitrous oxide has drawbacks, however, including its environmental impact and need for specialist equipment. Methoxyflurane is another drug which could prove to be an alternative to nitrous oxide. Methoxyflurane's use has proved popular within emergency medicine in Australia and New Zealand for its potent analgesic effects and recognition of its anxiolytic effect. As a result, its use in invasive outpatient procedures has now become popular. Unfortunately, there is very limited evidence of its use within dentistry as a form of inhalation sedation and analgesic. A wider evidence base should be established, as methoxyflurane could prove to be an effective and environmentally friendly alternative to nitrous oxide.

Unbalanced Medial-to-Lateral Knee Muscle Co-Contractions are Associated with Medial Tibiofemoral Underloading during Gait Three Months after Anterior Cruciate Ligament Reconstruction.

Altered medial/lateral knee muscle co-contraction (measure by co-contraction indices, CCI) occurs during gait early after anterior cruciate ligament reconstruction (ACLR). Changes in peak medial compartment forces (pMCF) are also observed early after ACLR and are linked to the development of knee osteoarthritis. We do not know if imbalanced co-contraction is associated with these alterations in knee load. The purpose of this study was to evaluate the association between pMCF and the CCIs of medial/lateral knee muscle pairs during walking three months after ACLR. Bilateral knee gait mechanics and electromyography (EMG) data were collected from 44 participants 3 months following surgery. CCIs of six muscle pairs and medial-to-lateral (M:L) CCIs ratios were calculated during the weight acceptance interval. Bilateral pMCFs were calculated using a subject-based neuromusculoskeletal model. Based on interlimb pMCF symmetry, participants were divided into three groups: symmetric loaders, underloaders, and overloaders. A 2 × 3 (limb × group) ANOVA was used to compare CCIs between limbs in all groups. A partial Spearman's test was performed to examine the association between CCIs ratios and pMCF. The CCIs of the vastus lateralis-lateral gastrocnemius muscle pair was higher in the involved limb of underloaders (vs. the uninvolved limb and vs. the involved limb of symmetric loaders). The ratio of M:L CCIs was significantly lower (more lateral CCIs) in the involved limb, which was associated with lower pMCF. These results suggest that individuals early after ACLR who walk with higher CCIs of lateral knee musculature (vs. medial), have medial tibiofemoral underloading.