Mast cells promote choroidal neovascularization in a model of age-related macular degeneration.

'Wet' age-related macular degeneration (AMD) is characterized by pathologic choroidal neovascularization (CNV) that destroys central vision. Abundant evidence points to inflammation and immune cell dysfunction in the progression of CNV in AMD. Mast cells are resident immune cells that control the inflammatory response. Mast cells accumulate and degranulate in the choroid of patients with AMD, suggesting they play a role in CNV. Activated mast cells secrete various biologically active mediators, including inflammatory cytokines and proteolytic enzymes such as tryptase. We investigated the role of mast cells in AMD using a model of CNV. Conditioned media from activated mast cells exerts proangiogenic effects on choroidal endothelial cells and choroidal explants. Laser-induced CNV in vivo was markedly attenuated in mice genetically depleted of mast cells (KitW-sh/W-sh) and in wild-type mice treated with mast cell stabilizer, ketotifen fumarate. Tryptase was found to elicit pronounced choroidal endothelial cell sprouting, migration and tubulogenesis; while tryptase inhibition diminished CNV. Transcriptomic analysis of laser-treated RPE/choroid complex revealed collagen catabolism and extracellular matrix (ECM) reorganization as significant events correlated in clusters of mast cell activation. Consistent with these analyses, compared to wildtype mice choroids of laser-treated mast cell-deficient mice displayed less ECM remodelling evaluated using collagen hybridizing peptide tissue binding. Findings herein provide strong support for mast cells as key players in the progression of pathologic choroidal angiogenesis and as potential therapeutic targets to prevent pathological neovascularization in 'wet' AMD.

Results of Lucia Keratoprosthesis Implantation in Severe Corneal Disease.: Results of Lucia KPro.

PURPOSE: Describe complications, functional success, and retention of the Lucia Keratoprosthesis (KPro). DESIGN: Retrospective interventional case series METHODS: The demographic data, baseline characteristics, complications, functional success, and retention were analyzed for Lucia KPro at Instituto de Oftalmologia Conde de Valenciana in Mexico City from 2021-2023. Multivariate regression analysis and Kaplan-Meier plots were performed to identify associations with functional failure. The main outcome measures were postoperative complications; functional success defined as best-corrected visual acuity (BCVA) ≥ 20/200 (LogMAR 1.0); and device retention rate. RESULTS: Forty-eight eyes of 48 patients (56.88% male) were studied. The mean age at implantation was 57.29 ± 15.63 years (range 27-91) with a mean follow-up of 20.5 ± 8.83 months (range: 3-40). Diagnostic indications for implantation included recurrent graft rejection (54.17%), autoimmune disease (20.83%), chemical injury (12.5%), and other (12.5%). At least one postoperative complication occurred in 75% of patients leading to 65 additional interventions (mean number of subsequent procedures: 1.35 ± 1.3, range 0-5). The preoperative BCVA was 2.40 ± 0.36 LogMAR. At the final follow-up, 62.5% had achieved functional success and 22.92% had a BCVA of 20/40 (LogMAR 0.3) or better. Glaucoma (adjusted OR: 469.74, 95% CI 5.02 - 43939.14, p=0.007) and retinal pathology before KPro (adjusted OR: 372.38, 95% CI 4.18 - 33162.11, p=0.009) were associated with functional failure. The device remained in place in 95.83% of recipients over the follow-up period. CONCLUSION: The Lucia KPro offers functional success in severe corneal diseases and excellent retention in the short to intermediate term.

Erratum to 'Illustrated State-of-the-Art Capsules of the ISTH 2024 Congress' [Research and Practice in Thrombosis and Haemostasis Volume 8, Issue 4, May 2024, 102432].

[This corrects the article DOI: 10.1016/j.rpth.2024.102432.].

Endoplasmic Reticulum Stress Delays Choroid Development in the HCAR1 Knockout Mouse.

The subretina, composed of the choroid and the retinal pigment epithelium (RPE), bears a critical role in proper vision. In addition to phagocytosis of photoreceptor debris, the RPE shuttles oxygen and nutrients to the neuroretina. For their own energy production, RPE cells mainly rely on lactate, a major by-product of glycolysis. Lactate, in turn, is believed to convey most of its biological effects via the hydroxycarboxylic acid receptor 1 (HCAR1). Here, the lactate-specific receptor, HCAR1, is found to be exclusively expressed in the RPE cells within the subretina, and Hcar1-/- mice exhibit a substantially thinner choroidal vasculature during development. Notably, the angiogenic properties of lactate on the choroid are impacted by the absence of Hcar1. HCAR1-deficient mice exhibit elevated endoplasmic reticulum stress along with eukaryotic initiation factor 2α phosphorylation, a significant decrease in the global protein translation rate, and a lower proliferation rate of choroidal vasculature. Strikingly, inhibition of the integrated stress response using an inhibitor that reverses the effect of eukaryotic initiation factor 2α phosphorylation restores protein translation and rescues choroidal thinning. These results provide evidence that lactate signalling via HCAR1 is important for choroidal development/angiogenesis and highlight the importance of this receptor in establishing mature vision.

A phase 3, randomized, double-blind, sham-controlled trial of SI-6603 (condoliase) in patients with radicular leg pain associated with lumbar disc herniation.

BACKGROUND CONTEXT: SI-6603 (condoliase) is a chemonucleolytic agent approved in Japan in 2018 for the treatment of lumbar disc herniation (LDH) associated with radicular leg pain. Condoliase, a mucopolysaccharidase with high substrate specificity for glycosaminoglycans (GAGs), offers a unique mechanism of action through the degradation of GAGs in the nucleus pulposus. As LDH management is currently limited to conservative approaches and surgical intervention, condoliase could offer a less invasive treatment option than surgery for patients with LDH. PURPOSE: The Discover 6603 study (NCT03607838) evaluated the efficacy and safety of a single-dose injection of SI-6603 (condoliase) vs sham for the treatment of radicular leg pain associated with LDH. STUDY DESIGN/SETTING: A randomized, double-blind, sham-controlled, phase 3 study conducted across 41 sites in the United States. PATIENT SAMPLE: Male and female participants (N=352; aged 30-70 years) with contained posterolateral LDH and unilateral radiculopathy/radicular leg pain for greater than 6 weeks. OUTCOME MEASURES: The primary endpoint was the change from baseline (CFB) in average worst leg pain score at 13 weeks, assessed using the 100-mm visual analogue scale. Key secondary endpoints were CFB in average worst leg pain score at 52 weeks, herniation volume at 13 weeks, and Oswestry Disability Index (ODI) score at 13 weeks. Safety evaluations included adverse events (AEs) and imaging findings. METHODS: Participants were randomized 1:1 to receive a single intradiscal injection of condoliase (1.25 units) or sham injection followed by 52 weeks of observation. The primary and key secondary endpoints were assessed using a mixed model for repeated measures (MMRM) analysis and a protocol-specified multiple imputation (MI) sensitivity analysis on the modified intention-to-treat (mITT) population. A prespecified serial gatekeeping algorithm was used for multiple comparisons. Safety endpoints included AEs, laboratory tests, vital signs, imaging (by X-ray and magnetic resonance imaging [MRI]), and occurrence of posttreatment lumbar surgery. RESULTS: Of the 352 randomized participants, 341 constituted the mITT population (condoliase n=169; sham n=172) and the safety population (condoliase n=167; sham n=174). For the primary endpoint, the condoliase group showed significantly greater improvement in CFB in worst leg pain at Week 13 (least squares mean [LSM] CFB: -41.7) compared with sham injection (-34.2; LSM difference: -7.5; 95% confidence interval [CI]: -14.1, -0.9; p=.0263) based on the MMRM analysis. CFB in worst leg pain at Week 52 favored condoliase vs sham, but the difference was not statistically significant (p=.0558), which halted the serial gatekeeping testing algorithm and dictated that the CFB in herniation volume and ODI scores at Week 13 would be considered nonsignificant, regardless of their p-values. Treatment group differences in CFB in herniation volume and ODI score favored the condoliase group vs sham at all timepoints. The MI sensitivity analysis showed differences in CFB in worst leg pain at Week 13 (p=.0223) and Week 52 (p=.0433) in favor of the condoliase group. Treatment-emergent AEs (TEAEs) were more common in the condoliase group (≥1 TEAE: 71.9%; ≥1 treatment-related TEAE: 28.1%) compared with the sham group (≥1 TEAE: 60.3%; ≥1 treatment-related TEAE: 10.3%). Of the TEAEs, spinal MRI abnormalities and back pain occurred most frequently. No treatment-related serious AEs occurred. CONCLUSIONS: Condoliase met its primary endpoint of significantly improving radicular leg pain at Week 13 and was generally well tolerated in patients with LDH. Chemonucleolysis with condoliase has the potential to provide a less invasive treatment option than surgery for those unresponsive to conservative treatment strategies.

Program Evaluation of Pharmacist-Performed Medication Adherence Assessments in Candidates for Living Donor Kidney Transplant.

Introduction: Medication education and adherence assessments are integral to kidney transplant success. This program evaluation aimed to describe candidate-reported findings using a standardized medication adherence assessment in candidates undergoing living-donor kidney transplantation. Design: This was a single-center retrospective description of medication adherence on adult HIV-negative living-donor candidates from July 1, 2018 to December 1, 2018 who had ≥6 months post-operative follow-up. Medication adherence assessments were performed by a pharmacist at the pre-operative visit within 2 weeks prior to transplant. Candidates were considered to (a) have adherence concerns if they reported missed/late medications within 2 weeks of assessment or ever stopped a medication without medical advice and (b) considered using adherence strategies if they reported active use of pill box, method to keep track of refills/auto-refill use, medication list, or medication reminder(s). Missed medication data were collected at 3- and 6-months posttransplant. Results: Among 181 candidates included, 81 (45%) had adherence concerns and 169 (93%) reported using adherence strategies. There were no significant differences with adherence concerns by age ≤ 29 years, sex, race, prior transplant/dialysis, or less than a high school education. More candidates with greater than a high school education used adherence strategies (96% vs 86%, P = .002). Too few candidates had documentation on missing medications at 3 and 6 months. Conclusions: Over 40% of candidates reported characteristics concerning medication nonadherence despite over 90% reporting adherence strategies used. Medication adherence assessments can assist with identification of medication nonadherence and education individualization.

Therapeutic Potential of Brassica oleracea and Raphanus sativus Aqueous Extracts on a Prediabetic Rat Model.

Type 2 diabetes (T2D) is a serious health problem, and its prevalence is expected to increase worldwide in the years ahead. Cruciferous vegetables such as Brassica oleracea var. capitata L. (green cabbage) and Raphanus sativus L. (radish) have therapeutic properties that can be used to support the treatment of T2D. This study evaluated the effect of B. oleracea (BAE) and R. sativus (RAE) aqueous extracts on zoometric parameters, glycemic profiles, and pancreas and liver in prediabetic rats induced by a high-sucrose diet (HSD). BAE and RAE were administered to male HSD-induced Wistar rats (n = 35) at 5 and 10 mg/kg doses for 5 weeks. Zoometric and biochemical changes were measured, and then the pancreas and liver histological preparations were analyzed to observe the protective effect. BAE decreased feed intake and weight gain. Both extracts decreased fasting glucose and insulin levels compared with control (not treated), although not significantly (P > .05). The extracts significantly (P < .05) reduced homeostatic model assessment for insulin resistance, homeostasis model assessment of ß-cell function, and glucose intolerance, similar to metformin control. In addition, minor damage occurred in the pancreas and liver. The results indicated that BAE and RAE decreased weight gain, improved glucose regulation, and protected the pancreas and liver in HSD rats. Therefore, they have multiple therapeutical properties and may be helpful in the prevention of T2D.

Niche modelling and landscape genetics of the yellow-legged hornet (Vespa velutina): An integrative approach for evaluating central-marginal population dynamics in Europe.

Genetic diversity is an important biological trait for a successful invasion. During the expansion across a new territory, an invasive species may face unprecedented ecological conditions that will determine its demography and genetic diversity. The first record of the yellow-legged hornet (Vespa velutina) in Europe dates back to 2004 in France, from where it has successfully spread through a large territory in the continent, including Italy, Spain and Portugal. Integrative approaches offer a powerful strategy to detect and understand patterns of genetic variation in central and marginal populations. Here, we have analysed the relationship between genetic diversity parameters inferred from 15 V. velutina nuclear DNA microsatellite loci, and geographical and environmental drivers, such as the distance to the introduction focus, environmental suitability and distance to native and invasive niche centroids. Our results revealed a central-marginal dynamic, where allelic richness decreased towards the edge of the expansion range. The low environmental suitability of the territories invaded by marginal populations could prevent a diverse population from establishing and reducing the genetic diversity in populations at the expansion edge. Moreover, Markov chain Monte Carlo analysis showed both geographical and environmental distances were influencing population genetic differentiation. This study highlights the importance of combining genetic analysis with geographical and environmental drivers to understand genetic trends of invasive species to new environment.

Community-engaged Research Partnerships as Healing Spaces for Health Professionals and Researchers.

Drawing from collective experiences in our capacity building project: Health Equity Activation Research Team for Inclusion Health, we argue that while community-engaged partnerships tend to focus on understanding health inequities and developing solutions, they can be healing spaces for health professionals and researchers. Data were obtained from a 15-month participatory ethnography, including focus groups and interviews. Ethnographic notes and transcripts were coded and analyzed using both deductive and inductive coding. Practices of radical welcome, vulnerability, valuing the whole person, acknowledging how partnerships can cause harm, and centering lived experience expertise in knowledge creation processes were identified as key characteristics of healing spaces. Ultimately, health professionals and researchers work within the same social, political and economic contexts of populations with the worst health outcomes. Their own healing is critical for tackling larger systemic changes aimed at improving the well-being of communities harmed by legacies of exclusion.

Efficacy and safety of bempedoic acid in acute coronary syndrome. Design of the clinical trial ES-BempeDACS.

INTRODUCTION AND OBJECTIVES: Only about 1 out of every 3 patients with acute myocardial infarction (AMI) achieve low-density lipoprotein cholesterol (LDL-C) values <55mg/dL in the first year. The present study aims to evaluate the impact of early intensive therapy on lipid control after an AMI. METHODS: An independent, prospective, pragmatic, controlled, randomized, open-label, evaluator-blinded clinical trial (PROBE design) will analyze the efficacy and safety of an oral lipid-lowering triple therapy: high-potency statin+bempedoic acid (BA) 180mg+ezetimibe (EZ) 10mg versus current European-based guidelines (high-potency statin±EZ 10mg), in AMI patients. LDL-C will be determined within the first 48hours. Patients with LDL-C ≥ 115mg/dL (without previous statin therapy), ≥ 100mg/dL (with previous low-potency or high-potency statin therapy at submaximal dose), or ≥ 70mg/dL (with previous high-potency statin therapy at high dose) will be randomly assigned 1:1 between 24 and 72hours post-AMI to the BA/EZ combination or to statin±EZ, without BA. The primary endpoint is the proportion of patients reaching LDL-C <55mg/dL at 8 weeks after treatment. RESULTS: The results of this study will provide novel information for post-AMI LDL-C control by evaluating the usefulness of an early intensive lipid-lowering strategy based on triple oral therapy. CONCLUSIONS: Early intensive lipid-lowering triple oral therapy vs the treatment recommended by current clinical practice guidelines could facilitate the achievement of optimal LDL-C levels in the first 2 months after AMI (a high-risk period). IDENTIFICATION NUMBER: EudraCT 2021-006550-31.

A randomized, placebo-controlled trial of long-acting dexamethasone viscous gel delivered by transforaminal injection for lumbosacral radicular pain.

TRIAL REGISTRATION: ClinicalTrials.gov Identifier: NCT03372161.

Improvement of digital Gabor holographic microscopy using a lens in plankton studies.

This work utilizes a Gabor Holographic Optical Scheme integrated with a microscope objective and a thin convex plane lens. This bi-telecentric lens system corrects spherical aberration from the objective, maintains consistent magnification across various reconstruction distances, and ensures a plane incidence on CMOS. Depending on the focal lengths of the objective and lens, the final image can be enlarged or reduced compared to the classic Gabor system, resulting in high-quality reconstructed phase images without spherical aberration. This setup was employed to capture phase distribution and intensity images of planktonic objects, such as copepods, achieving superior image quality.

Health Care Access and COVID-19 Vaccination in the United States: A Cross-Sectional Analysis.

BACKGROUND: Although federal legislation made COVID-19 vaccines free, inequities in access to medical care may affect vaccine uptake. OBJECTIVE: To assess whether health care access was associated with uptake and timeliness of COVID-19 vaccination in the United States. DESIGN: A cross-sectional study. SETTING: 2021 National Health Interview Survey (Q2-Q4). SUBJECTS: In all, 21,532 adults aged≥18 were included in the study. MEASURES: Exposures included 4 metrics of health care access: health insurance, having an established place for medical care, having a physician visit within the past year, and medical care affordability. Outcomes included receipt of 1 or more COVID-19 vaccines and receipt of a first vaccine within 6 months of vaccine availability. We examined the association between each health care access metric and outcome using logistic regression, unadjusted and adjusted for demographic, geographic, and socioeconomic covariates. RESULTS: In unadjusted analyses, each metric of health care access was associated with the uptake of COVID-19 vaccination and (among those vaccinated) early vaccination. In adjusted analyses, having health coverage (adjusted odds ratio [AOR] 1.60; 95% CI: 1.39, 1.84), a usual place of care (AOR 1.58; 95% CI: 1.42, 1.75), and a doctor visit within the past year (AOR 1.45, 95% CI: 1.31, 1.62) remained associated with higher rates of COVID-19 vaccination. Only having a usual place of care was associated with early vaccine uptake in adjusted analyses. LIMITATIONS: Receipt of COVID-19 vaccination was self-reported. CONCLUSIONS: Several metrics of health care access are associated with the uptake of COVID-19 vaccines. Policies that achieve universal coverage, and facilitate long-term relationships with trusted providers, may be an important component of pandemic responses.

Effect of Water Absorption and Stacking Sequences on the Tensile Properties and Damage Mechanisms of Hybrid Polyester/Glass/Jute Composites.

The aim of this work is to analyze the effect of water absorption on the mechanical properties and damage mechanisms of polyester/glass fiber/jute fiber hybrid composites obtained using the compression molding and vacuum-assisted resin transfer molding (VARTM) techniques with different stacking sequences. For this purpose, the mechanical behavior under tensile stress of the samples was evaluated before and after hygrothermal aging at different temperatures: TA, 50 °C, and 70 °C for a period of 696 h. The damage mechanism after the mechanical tests was evaluated using SEM analysis. The results showed a tendency for the mechanical properties of the composites to decrease with exposure to an aqueous ambient, regardless of the molding technique used to conform the composites. It was also observed that the stacking sequence had no significant influence on the dry composites. However, exposure to the aqueous ambient led to a reduction in mechanical properties, both for the molding technique and the stacking sequence. Damage such as delamination, fiber pull-out, fiber/matrix detachment, voids, and matrix removal were observed in the composites in the SEM analyses.

Clinical Presentation, Risk Factors, and Comparison of Laboratory Diagnostics for Seasonal Influenza Virus Among Cambodians From 2007 to 2020.

Background: Despite its global significance, challenges associated with understanding the epidemiology and accurately detecting, measuring, and characterizing the true burden of seasonal influenza remain in many resource-poor settings. Methods: A prospective observational study was conducted in Cambodia at 28 health facilities between 2007 and 2020 utilizing passive surveillance data of patients presenting with acute undifferentiated febrile illness (AUFI) to describe the prevalence of influenza A and B and characterize associated risk factors and symptoms using a questionnaire. A comparison of rapid influenza diagnostic tests (RIDTs) and real-time reverse transcription polymerase chain reaction (rRT-PCR) results was also conducted. Results: Of 30 586 total participants, 5634 (18.4%) tested positive for either influenza A or B, with 3557 (11.6%) positive for influenza A and 2288 (7.5%) positive for influenza B during the study. Influenza A and B were strongly associated with the rainy season (odds ratio [OR], 2.30; P < .001) and being from an urban area (OR, 1.45; P < .001). Analysis of individual symptoms identified cough (OR, 2.8; P < .001), chills (OR, 1.4; P < .001), and sore throat (OR, 1.4; P < .001) as having the strongest positive associations with influenza among patients with AUFI. Analysis comparing RIDTs and rRT-PCR calculated the overall sensitivity of rapid tests to be 0.492 (95% CI, 0.479-0.505) and specificity to be 0.993 (95% CI, 0.992-0.994) for both influenza type A and B. Conclusions: Findings from this 14-year study include describing the epidemiology of seasonal influenza over a prolonged time period and identifying key risk factors and clinical symptoms associated with infection; we also demonstrate the poor sensitivity of RIDTs in Cambodia.

The role of temperature on the development of circadian rhythms in honey bee workers.

Circadian rhythms in honey bees are involved in various processes that impact colony survival. For example, young nurses take care of the brood constantly throughout the day and lack circadian rhythms. At the same time, foragers use the circadian clock to remember and predict food availability in subsequent days. Previous studies exploring the ontogeny of circadian rhythms of workers showed that the onset of rhythms is faster in the colony environment (~2 days) than if workers were immediately isolated after eclosion (7-9 days). However, which specific environmental factors influenced the early development of worker circadian rhythms remained unknown. We hypothesized that brood nest temperature plays a key role in the development of circadian rhythmicity in young workers. Our results show that young workers kept at brood nest-like temperatures (33-35 °C) in the laboratory develop circadian rhythms faster and in greater proportion than bees kept at lower temperatures (24-26 °C). In addition, we examined if the effect of colony temperature during the first 48 h after emergence is sufficient to increase the rate and proportion of development of circadian rhythmicity. We observed that twice as many individuals exposed to 35 °C during the first 48 h developed circadian rhythms compared to individuals kept at 25 °C, suggesting a critical developmental period where brood nest temperatures are important for the development of the circadian system. Together, our findings show that temperature, which is socially regulated inside the hive, is a key factor that influences the ontogeny of circadian rhythmicity of workers.

Impact of Frailty on Heart Failure Prognosis: Is Sex Relevant?

PURPOSE OF REVIEW: Heart failure (HF) is one of the most frequent causes of hospital admission in elderly patients, especially in women, who present a high prevalence of geriatric syndromes like frailty. Studies have suggested that frailty and its impact may also differ between males and females. Understanding how frailty may differently affect HF patients depending on sex is therefore imperative for providing personalized care. The aim of this review is to summarize the role of sex in the prognostic impact of frailty in HF patients. RECENT FINDINGS: Numerous studies have identified frailty as a significant predictor of all-cause mortality and hospital readmissions. A recent study of elderly HF out-patients demonstrated that while women had a higher prevalence of frailty, it was an independent predictor of mortality and readmission only in men. Moreover, another study revealed that physical frailty was associated with time to first clinical event among men but not among women. These results raise the question about why frailty affects differently HF prognosis in men and women. Women with HF present a higher prevalence of frailty, especially when it is considered as physical decline. Nevertheless, frailty affects differently HF prognosis in men and women. Women with HF present lower mortality than men and frailty is related with prognosis only in men. The different severity of HF between men and women and other hormonal, psychosocial, and clinical factors might be involved in this fact.

Cocaine sensitization in male rats requires activation of estrogen receptors.

Gonadal steroids play a modulatory role in cocaine use disorders, and are responsible for many sex differences observed in the behavioral response to cocaine. In females, it is well established that estradiol enhances the behavioral response to cocaine. In males, we have recently shown that testosterone enhances sensitization to cocaine but its mechanism of action remains to be elucidated. The current study investigated the contribution of DHT, a non-aromatizable androgen, and of estradiol, in regulating cocaine-induced sensitization in male rats. Gonadectomized (GDX) male rats treated with estradiol sensitized to repeated cocaine administration, while GDX rats treated with DHT did not, implicating estradiol in cocaine sensitization. Furthermore, intact male rats treated with the antiestrogen ICI 182,780 did not show sensitization to repeated cocaine. This study demonstrates the pivotal role of estradiol in cocaine-induced neuroplasticity and neuroadaptations in the rodent brain.

Epstein Barr virus-directed T-cell therapy for refractory EBV-PTLD in a toddler post Orthotopic heart transplantation.

Epstein-Barr Virus (EBV) is a ubiquitous herpes type virus that is associated with post-transplant lymphoproliferative disorder (PTLD). Usual management includes reduction or cessation of immunosuppression and in some cases chemotherapy including rituximab. However, limited therapies are available if PTLD is refractory to rituximab. Several clinical trials have investigated the use of EBV-directed T cells in rituximab-refractory patients; however, data regarding response is scarce and inconclusive. Herein, we describe a patient with EBV-PTLD refractory to rituximab after orthotopic heart transplantation (OHT) requiring EBV-directed T-cell therapy. This article aims to highlight the unique and aggressive clinical presentation and progression of PTLD with utilization of EBV-directed T-cell therapy for management and associated pitfalls.