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We estimate global terrestrial gross primary production (GPP) based on models that use satellite data within a simplified light-use efficiency framework that does not rely upon other meteorological inputs. Satellite-based geometry-adjusted reflectances are from theMODerate-resolution Imaging Spectroradiometer (MODIS) and provide information about vegetation structure and chlorophyll content at both high temporal (daily to monthly) and spatial (~1 km) resolution. We use satellite-derived solar-induced fluorescence (SIF) to identify regions of high productivity crops and also evaluate the use of downscaled SIF to estimate GPP. We calibrate a set of our satellite-based models with GPP estimates from a subset of distributed eddy covariance flux towers (FLUXNET 2015). The results of the trained models are evaluated using an independent subset of FLUXNET 2015 GPP data. We show that variations in light-use efficiency (LUE) with incident PAR are important and can be easily incorporated into the models. Unlike many LUE-based models, our satellite-based GPP estimates do not use an explicit parameterization of LUE that reduces its value fromthe potentialmaximum under limiting conditions such as temperature and water stress. Even without the parameterized downward regulation, our simplified models are shown to perform as well as or better than state-of-the-art satellite data-driven products that incorporate such parameterizations. A significant fraction of both spatial and temporal variability in GPP across plant functional types can be accounted for using our satellite-based models. Our results provide an annual GPP value of ~140 PgCyear-1 for 2007 that is within the range of a compilation of observation-based, model, and hybrid results, but is higher than some previous satellite observation-based estimates.

Increased aridity and human population have reduced tree cover in parts of the African Sahel and degraded resources for local people. Yet, tree cover trends and the relative importance of climate and population remain unresolved. From field measurements, aerial photos, and Ikonos satellite images, we detected significant 1954–2002 tree density declines in the western Sahel of 18 ± 14% (P = 0.014, n = 204) and 17 ± 13% (P = 0.0009, n = 187). From field observations, we detected a significant 1960–2000 species richness decline of 21 ± 11% (P = 0.0028, n = 14) across the Sahel and a southward shift of the Sahel, Sudan, and Guinea zones. Multivariate analyses of climate, soil, and population showed that temperature most significantly (P

Fire is a major disturbance in the boreal forest, and has been shown to release significant amounts of carbon (C) to the atmosphere through combustion. However, less is known about the effects on ecosystems following fire, which include reduced productivity and changes in decomposition in the decade immediately following the disturbance. In this study, we assessed the impact of fire on net primary productivity (NPP) in the North American boreal forest using a 17-year record of satellite NDVI observations at 8-km spatial resolution together with a light-use efficiency model. We identified 61 fire scars in the satellite observations using digitized fire burn perimeters from a database of large fires. We studied the postfire response of NPP by analyzing the most impacted pixel within each burned area. NPP decreased in the year following the fire by 60-260g CM-2 yr-1 (30-80%). By comparing pre- and postfire observations, we estimated a mean NPP recovery period for boreal forests of about 9 years, with substantial variability among fires. We incorporated this behavior into a carbon cycle model simulation to demonstrate these effects on net ecosystem production. The disturbance resulted in a release of C to the atmosphere during the first 8 years, followed by a small, but long-lived, sink lasting 150 years. Postfire net emissions were three times as large as from a model run without changing NPP. However, only small differences in the C cycle occurred between runs after 8 years due to the rapid recovery of NPP. We conclude by discussing the effects of fire on the long-term continental trends in satellite NDVI observed across boreal North America during the 1980s and 1990s.

The Sea-viewing Wide Field-of-view Sensor (SeaWiFS) provides global monthly measurements of both oceanic phytoplankton chlorophyll biomass and light harvesting by land plants. These measurements allowed the comparison of simultaneous ocean and land net primary production (NPP) responses to a major El Niño to La Niña transition. Between September 1997 and August 2000, biospheric NPP varied by 6 petagrams of carbon per year (from 111 to 117 petagrams of carbon per year). Increases in ocean NPP were pronounced in tropical regions where El Niño-Southern Oscillation (ENSO) impacts on upwelling and nutrient availability were greatest. Globally, land NPP did not exhibit a clear ENSO response, although regional changes were substantial.

T cell receptor (TCR) engagement triggers T cell responses, yet how TCR-mediated activation is regulated at the plasma membrane remains unclear. Here, we report that deleting the membrane scaffolding protein Flotillin-2 (Flot2) increases T cell antigen-sensitivity, resulting in enhanced TCR signaling and effector function to weak TCR stimulation. T cell-specific Flot2-deficient mice exhibited reduced tumor growth and enhanced immunity to infection. Flot2-null CD4+ T cells exhibited increased T helper 1 polarization, proliferation, Nur77 induction, and phosphorylation of ZAP70 and ERK1/2 upon weak TCR stimulation, indicating a sensitized TCR-triggering threshold. Single cell-RNA sequencing suggested that Flot2-null CD4+ T cells follow a similar route of activation as wild-type CD4+ T cells but exhibit higher occupancy of a discrete activation state under weak TCR stimulation. Given prior reports that TCR clustering influences sensitivity of T cells to stimuli, we evaluated TCR distribution with super-resolution microscopy. Flot2 ablation increased the number of surface TCR nanoclusters on naïve CD4+ T cells. Collectively, we posit that Flot2 modulates T cell functionality to weak TCR stimulation, at least in part, by regulating surface TCR clustering. Our findings have implications for improving T cell reactivity in diseases with poor antigenicity, such as cancer and chronic infections.

Phosphate (Pi) serves countless metabolic pathways and is involved in macromolecule synthesis, energy storage, cellular signaling, and bone maintenance. Herein, we describe the coordination of Pi uptake and efflux pathways to maintain mammalian cell Pi homeostasis. We discover that XPR1, the presumed Pi efflux transporter, separately supervises rates of Pi uptake. This direct, regulatory interplay arises from XPR1 being a binding partner for the Pi uptake transporter PiT1, involving a predicted transmembrane helix/extramembrane loop in XPR1, and its hitherto unknown localization in a subset of intracellular LAMP1-positive puncta (named "XLPVs"). A pharmacological mimic of Pi homeostatic challenge is sensed by the inositol pyrophosphate IP 8 , which functionalizes XPR1 to respond in a temporally hierarchal manner, initially adjusting the rate of Pi efflux, followed subsequently by independent modulation of PiT1 turnover to reset the rate of Pi uptake. These observations generate a unifying model of mammalian cellular Pi homeostasis, expanding opportunities for therapeutic intervention., Competing Interests: Declaration of interests The authors declare no competing interests., (Published by Elsevier Inc.)

Meniscal injury is common, and despite modern techniques, the failure rate following repair remains high. While there are recent treatment advances in the form of biologics, there is limited evidence and agreement on these emerging therapies and their role in meniscal healing. Amniotic tissue (umbilical cord allograft) is a biologic augmentation therapy that has been utilized in other musculoskeletal applications but has not been reported for use in meniscal repair. We describe a technique to deliver an allograft amniotic membrane into a meniscus tear repair site, potentially optimizing healing., Competing Interests: The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: J.M.T. is a board member of the 10.13039/100008542Arthroscopy Association of North America, Orthopedics Today, and Journal of Shoulder and Elbow Surgery; is a consultant or advisor for 10.13039/100004331Johnson & Johnson and Arthrex; receives speaking and lecture fees from Arthrex; and receives funding grants and nonfinancial support from the Journal of Shoulder and Elbow Surgery. C.J.T. is a board member of the Society of Military Orthopaedic Surgeons and is Podcast Editor for Arthroscopy Journal. All other authors (S.M.F., M.W.F., C.F.M., D.F.C.) declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

High-risk human papillomavirus (HR-HPV; HPV-16) and cigarette smoking are associated with cervical cancer (CC); however, the underlying mechanism(s) remain unclear. Additionally, the carcinogenic components of tobacco have been found in the cervical mucus of women smokers. Here, we determined the effects of cigarette smoke condensate (CSC; 3R4F) on human ectocervical cells (HPV-16 Ect/E6E7) exposed to CSC at various concentrations (10 -6 -100 μg/mL). We found CSC (10 -3 or 10 μg/mL)-induced proliferation, enhanced migration, and histologic and electron microscopic changes consistent with EMT in ectocervical cells with a significant reduction in E-cadherin and an increase in the vimentin expression compared to controls at 72 h. There was increased phosphorylation of receptor tyrosine kinases (RTKs), including Eph receptors, FGFR, PDGFRA/B, and DDR2, with downstream Ras/MAPK/ERK1/2 activation and upregulation of common EMT-related genes, TGFB SNAI2 , PDGFRB , and SMAD2. Our study demonstrated that CSC induces EMT in ectocervical cells with the upregulation of EMT-related genes, expression of protein biomarkers, and activation of RTKs that regulate TGFB expression, and other EMT-related genes. Understanding the molecular pathways and environmental factors that initiate EMT in ectocervical cells will help delineate molecular targets for intervention and define the role of EMT in the initiation and progression of cervical intraepithelial neoplasia and CC.

Purpose: To determine whether activity on Twitter was correlated with increasing impact factor (IF) among 6 orthopaedic sports medicine journals., Methods: Twitonomy software was used to collect account activity for the American Journal of Sports Medicine ; Arthroscopy: The Journal of Arthroscopic and Related Surgery; Knee Surgery, Sports Traumatology, Arthroscopy ; Journal of Shoulder and Elbow Surgery ; Orthopaedic Journal of Sports Medicine ; and Sports Health . Data from 2000 to 2020 were collected. Each journal's annual IF score was collected via scijournal.org. A multivariate regression model was used to predict the influence of different Twitter metrics on IF from 2012 to 2019. The journal name, number of tweets, and interaction of the two were used to predict IF. Additionally, Pearson correlation was used to assess correlations between Twitter account metrics and IF., Results: Over the study period, all IFs increased, with the exception of that for American Journal of Sports Medicine . The effect size between number of tweets and IF was not the same for each journal. For every additional tweet, American Journal of Sports Medicine increased its IF by 0.001 ( P  = .18). Sports Health and Orthopaedic Journal of Sports Medicine increased their IF by 0.01 ( P  = .002) and 0.022 ( P < .001), respectively. Knee Surgery, Sports Traumatology, Arthroscopy would expect a decrease in its IF by 0.004 ( P  = .55) and Journal of Shoulder and Elbow Surgery and Arthroscopy would increase its IF by 0.002 ( P  = .71) and 0.001 ( P  = .99), but this was not significant. There was a statistically significant positive correlation between annual tweets and IF across all journals., Conclusions: Markers of Twitter account activity, specifically the number of annual tweets, were predictive of an increase in IF among the orthopedic sports medicine journals included in this study., Clinical relevance: The findings of this study may allow orthopaedic sports medicine journals to make more effective, targeted, and productive use of their social media accounts to reach a broader audience, increase their influence, and increase the IF of their journal., Competing Interests: The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: M.K.M. reports a consulting or advisory role with Arthrex. M.K.M. is also a board or committee member of the American Academy of Orthopaedic Surgeons; American Orthopaedic Association; American Orthopaedic Society for Sports Medicine; Arthroscopy Association of North America; Association of Bone and Joint Surgeons; International Society of Arthroscopy, Knee Surgery, and Orthopaedic Sports Medicine; and Ruth Jackson Orthopaedic Society. M.K.M. is also a member of the editorial or governing board of American Journal of Sports Medicine Electronic Media, Arthroscopy, Journal of Bone and Joint Surgery (American), and OrthoInfo. C.J.T. is a board member of the Society of Military Orthopaedic Surgeons. C.J.T. also reports travel reimbursement from the Arthroscopy Association of North America. C.J.T. is also a podcast editor for Arthroscopy. All other authors (K.K., T.R.L., S.M.S.) declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Full ICMJE author disclosure forms are available for this article online, as supplementary material.

Aberrant fibroblast function plays a key role in the pathogenesis of idiopathic pulmonary fibrosis, a devastating disease of unrelenting extracellular matrix deposition in response to lung injury. Platelet-derived growth factor α-positive (Pdgfra+) lipofibroblasts (LipoFBs) are essential for lung injury response and maintenance of a functional alveolar stem cell niche. Little is known about the effects of lung injury on LipoFB function. Here, we used single-cell RNA-Seq (scRNA-Seq) technology and PdgfraGFP lineage tracing to generate a transcriptomic profile of Pdgfra+ fibroblasts in normal and injured mouse lungs 14 days after bleomycin exposure, generating 11 unique transcriptomic clusters that segregated according to treatment. While normal and injured LipoFBs shared a common gene signature, injured LipoFBs acquired fibrogenic pathway activity with an attenuation of lipogenic pathways. In a 3D organoid model, injured Pdgfra+ fibroblast-supported organoids were morphologically distinct from those cultured with normal fibroblasts, and scRNA-Seq analysis suggested distinct transcriptomic changes in alveolar epithelia supported by injured Pdgfra+ fibroblasts. In summary, while LipoFBs in injured lung have not migrated from their niche and retain their lipogenic identity, they acquire a potentially reversible fibrogenic profile, which may alter the kinetics of epithelial regeneration and potentially contribute to dysregulated repair, leading to fibrosis.

The consistent monitoring of trees both inside and outside of forests is key to sustainable land management. Current monitoring systems either ignore trees outside forests or are too expensive to be applied consistently across countries on a repeated basis. Here we use the PlanetScope nanosatellite constellation, which delivers global very high-resolution daily imagery, to map both forest and non-forest tree cover for continental Africa using images from a single year. Our prototype map of 2019 (RMSE = 9.57%, bias = -6.9%). demonstrates that a precise assessment of all tree-based ecosystems is possible at continental scale, and reveals that 29% of tree cover is found outside areas previously classified as tree cover in state-of-the-art maps, such as in croplands and grassland. Such accurate mapping of tree cover down to the level of individual trees and consistent among countries has the potential to redefine land use impacts in non-forest landscapes, move beyond the need for forest definitions, and build the basis for natural climate solutions and tree-related studies., (© 2023. The Author(s).)

A-kinase anchoring protein 79 (AKAP79) is a human scaffolding protein that organizes Ca 2+ /calmodulin-dependent protein phosphatase calcineurin, calmodulin, cAMP-dependent protein kinase, protein kinase C, and the transcription factor nuclear factor of activated T cells (NFAT1) into a signalosome at the plasma membrane. Upon Ca 2+ store depletion, AKAP79 interacts with the N-terminus of STIM1-gated Orai1 Ca 2+ channels, enabling Ca 2+ nanodomains to stimulate calcineurin. Calcineurin then dephosphorylates and activates NFAT1, which then translocates to the nucleus. A fundamental question is how signalosomes maintain long-term signaling when key effectors are released and therefore removed beyond the reach of the activating signal. Here, we show that the AKAP79-Orai1 interaction is considerably more transient than that of STIM1-Orai1. Free AKAP79, with calcineurin and NFAT1 in tow, is able to replace rapidly AKAP79 devoid of NFAT1 on Orai1, in the presence of continuous Ca 2+ entry. We also show that Ca 2+ nanodomains near Orai1 channels activate almost the entire cytosolic pool of NFAT1. Recycling of inactive NFAT1 from the cytoplasm to AKAP79 in the plasma membrane, coupled with the relatively weak interaction between AKAP79 and Orai1, maintain excitation-transcription coupling. By measuring rates for AKAP79-NFAT interaction, we formulate a mathematical model that simulates NFAT dynamics at the plasma membrane.

Purpose: To evaluate the maximal load at failure, cyclic displacement, and stiffness of onlay subpectoral biceps tenodesis (BT) with an intramedullary unicortical metal button (MB) versus a unicortical all-suture button (ASB)., Methods: Eighteen matched paired human cadaveric proximal humeri were randomly allocated for subpectoral BT with either ASB or MB using a high-strength suture. Specimens were tested on a servohydraulic mechanical testing apparatus under cyclic load for 1,000 cycles and then loaded to failure. The clamp was then adjusted to isolate the suture-anchor point interface and loaded to failure. Maximal load to failure, displacement, and stiffness were compared., Results: There was no significant difference between groups in stiffness, displacement, or yield load. The maximal load to failure for the MB was greater than the ASB (347.6 ± 74.1N vs 266.5 ± 69.3N, P  = .047). Eight specimens in each group failed by suture pull-through on the tendon. When the suture-anchor point interface was isolated, there was no significant difference in maximal load at failure (MB 586.5 ± 215.8N vs ASB 579.6 ± 255.9N, P  = .957)., Conclusions: This study demonstrates that the MB and ASB have similar biomechanical performance when used in subpectoral BT. Although the MB showed statistically significant greater maximal load to failure, there was no difference between the MB and ASB when the suture-tendon interface was eliminated. Suture pull-through was the most common mode of failure for both implants, underscoring the importance of the suture-tendon interface., Clinical relevance: Fixation techniques for the treatment of long head of the biceps brachii tenodesis continue to evolve. The use of an all-suture suspensory button has advantages, but it is important to understand if this implant is a biomechanically suitable alternative to a metal suspensory button.

In many cell types, the rise in cytosolic Ca 2+ due to opening of Ca 2+ release-activated Ca 2+ (CRAC) channels drives a plethora of responses, including secretion, motility, energy production, and gene expression. The amplitude and time course of the cytosolic Ca 2+ rise is shaped by the rates of Ca 2+ entry into and removal from the cytosol. However, an extended bulk Ca 2+ rise is toxic to cells. Here, we show that the plasma membrane Ca 2+ ATPase (PMCA) pump plays a major role in preventing a prolonged cytosolic Ca 2+ signal following CRAC channel activation. Ca 2+ entry through CRAC channels leads to a sustained sub-plasmalemmal Ca 2+ rise but bulk Ca 2+ is kept low by the activity of PMCA4b. Despite the low cytosolic Ca 2+ , membrane permeability to Ca 2+ is still elevated and Ca 2+ continues to enter through CRAC channels. Ca 2+ -dependent NFAT activation, driven by Ca 2+ nanodomains near the open channels, is maintained despite the return of bulk Ca 2+ to near pre-stimulation levels. Our data reveal a central role for PMCA4b in determining the pattern of a functional Ca 2+ signal and in sharpening local Ca 2+ gradients near CRAC channels, whilst protecting cells from a toxic Ca 2+ overload., Competing Interests: A.B.P. holds the position of Executive Editor for Function and is blinded from reviewing or making decisions for the manuscript. The authors declare they have no conflict of interest., (© The Author(s) 2022. Published by Oxford University Press on behalf of American Physiological Society.)

During 2019, an infrared camera, the compact thermal imager (CTI), recorded 15 million images of the Earth from the International Space Station. CTI is based on strained-layer superlattice (SLS) detector technology. The camera covered the spectral range from 3 to 11 µm in two spectral channels, 3.3-5.4 and 7.8-10.7 µm. Individual image frames were 26×21 k m 2 projected on the ground, with 82 m pixel resolution. A frame time of 2.54 s created continuous image swaths with a 13% along-track image overlap. Upper limits determined on the ground and in flight for the electronic offset, read noise, and dark current demonstrated the stability of the SLS detector and camera over many months. Temperature calibration was established using a combination of preflight and in-flight measurements. A narrowband approximation of temperature as a function of photon counts produced an analytic relationship covering a temperature range of 0°-400°C. Examples of CTI images illustrate temperature retrievals over sea ice, urban and agricultural areas, desert, and wildfires.

Orthopaedic surgery has revolutionized the expectations for restoration of physical function after musculoskeletal injury and, along with physical therapy, has transformed the limits of recovery. Many orthopaedic procedures have a high success rate for improving quality of life and patient-reported outcomes, yet these procedures carry some level of risk, including postoperative complications. The stepped-care model of health care delivery, when applied to musculoskeletal care, recommends implementing less-intense and lower-risk treatments with known efficacy, such as promotion and education of self-management strategies and physical therapy, before more-invasive and higher-risk treatments such as surgery. This model of managing musculoskeletal disability can improve efficiency of care delivery and reduce medical costs at the health system level. Unfortunately, there is a documented lack of implementing an appropriate course of conservative care, especially physical therapy, prior to surgery across multiple orthopaedic disciplines including sports, spine, and trauma medicine and joint arthroplasty. Failure to respond to nonsurgical treatment has been suggested as a requisite component of the surgical appropriateness criteria, yet practical application can be elusive. Multiple barriers to adequate utilization of conservative treatment exist, including U.S. payment models that increase out-of-pocket expense for patients, negative patient perception of therapy, unreasonable patient expectations from therapy versus surgery, and communication barriers between patient, surgeon, and therapist. Surgeons should ensure that high-quality guideline-appropriate care is delivered early and adequately to their patients. Rehabilitation professionals have a responsibility to deliver high-value care, properly documenting the type and extent of treatment to improve surgical decision-making between surgeons and patients. Criteria to determine appropriateness for surgery should include a standardized and extensive assessment of failed therapies prior to certain elective surgeries. Improved collaboration between surgeons and rehabilitation professionals can result in improved outcomes for patients with musculoskeletal disorders., Level of Evidence: V, expert opinion., (© 2021 The Authors.)

Perioperative pain control for hip arthroscopy procedures represents a significant challenge for both surgeons and anesthetists. In light of the opioid crisis, greater emphasis has been placed on multimodal pain control techniques. There is considerable debate regarding regional nerve blockade for hip arthroscopy. Although regional anesthesia has a significant role in perioperative pain management strategies, many of the most common techniques present their own risks and limitations. In particular, the less desirable effects of postoperative weakness in the lower extremity and difficulty with ambulation are commonly produced with standard regional blockades. We present a technique for performing a targeted, sensory nerve block that can be done efficiently and safely in the immediate preoperative period. This block can potentially lower the intraoperative and postoperative opioid requirements without the risks of muscle blockade and falls from other regional anesthesia modalities., (© 2021 Published by Elsevier on behalf of the Arthroscopy Association of North America.)

To avoid conflicting and deleterious outcomes, eukaryotic cells often confine second messengers to spatially restricted subcompartments. The smallest signaling unit is the Ca 2+ nanodomain, which forms when Ca 2+ channels open. Ca 2+ nanodomains arising from store-operated Orai1 Ca 2+ channels stimulate the protein phosphatase calcineurin to activate the transcription factor nuclear factor of activated T cells (NFAT). Here, we show that NFAT1 tethered directly to the scaffolding protein AKAP79 (A-kinase anchoring protein 79) is activated by local Ca 2+ entry, providing a mechanism to selectively recruit a transcription factor. We identify the region on the N terminus of Orai1 that interacts with AKAP79 and demonstrate that this site is essential for physiological excitation-transcription coupling. NMR structural analysis of the AKAP binding domain reveals a compact shape with several proline-driven turns. Orai2 and Orai3, isoforms of Orai1, lack this region and therefore are less able to engage AKAP79 and activate NFAT. A shorter, naturally occurring Orai1 protein that arises from alternative translation initiation also lacks the AKAP79-interaction site and fails to activate NFAT1. Interfering with Orai1-AKAP79 interaction suppresses cytokine production, leaving other Ca 2+ channel functions intact. Our results reveal the mechanistic basis for how a subtype of a widely expressed Ca 2+ channel is able to activate a vital transcription pathway and identify an approach for generation of immunosuppressant drugs., Competing Interests: The authors declare no competing interest., (Copyright © 2021 the Author(s). Published by PNAS.)

The first documented Rift Valley hemorrhagic fever outbreak in the Arabian Peninsula occurred in northwestern Yemen and southwestern Saudi Arabia from August 2000 to September 2001. This Rift Valley fever outbreak is unique because the virus was introduced into Arabia during or after the 1997-1998 East African outbreak and before August 2000, either by wind-blown infected mosquitos or by infected animals, both from East Africa. A wet period from August 2000 into 2001 resulted in a large number of amplification vector mosquitoes, these mosquitos fed on infected animals, and the outbreak occurred. More than 1,500 people were diagnosed with the disease, at least 215 died, and widespread losses of domestic animals were reported. Using a combination of satellite data products, including 2 x 2 m digital elevation images derived from commercial satellite data, we show rainfall and potential areas of inundation or water impoundment were favorable for the 2000 outbreak. However, favorable conditions for subsequent outbreaks were present in 2007 and 2013, and very favorable conditions were also present in 2016-2018. The lack of subsequent Rift Valley fever outbreaks in this area suggests that Rift Valley fever has not been established in mosquito species in Southwest Arabia, or that strict animal import inspection and quarantine procedures, medical and veterinary surveillance, and mosquito control efforts put in place in Saudi Arabia following the 2000 outbreak have been successful. Any area with Rift Valley fever amplification vector mosquitos present is a potential outbreak area unless strict animal import inspection and quarantine proceedures are in place., Competing Interests: The authors have declared that no competing interests exist.

A large proportion of dryland trees and shrubs (hereafter referred to collectively as trees) grow in isolation, without canopy closure. These non-forest trees have a crucial role in biodiversity, and provide ecosystem services such as carbon storage, food resources and shelter for humans and animals 1,2 . However, most public interest relating to trees is devoted to forests, and trees outside of forests are not well-documented 3 . Here we map the crown size of each tree more than 3 m 2 in size over a land area that spans 1.3 million km 2 in the West African Sahara, Sahel and sub-humid zone, using submetre-resolution satellite imagery and deep learning 4 . We detected over 1.8 billion individual trees (13.4 trees per hectare), with a median crown size of 12 m 2 , along a rainfall gradient from 0 to 1,000 mm per year. The canopy cover increases from 0.1% (0.7 trees per hectare) in hyper-arid areas, through 1.6% (9.9 trees per hectare) in arid and 5.6% (30.1 trees per hectare) in semi-arid zones, to 13.3% (47 trees per hectare) in sub-humid areas. Although the overall canopy cover is low, the relatively high density of isolated trees challenges prevailing narratives about dryland desertification 5-7 , and even the desert shows a surprisingly high tree density. Our assessment suggests a way to monitor trees outside of forests globally, and to explore their role in mitigating degradation, climate change and poverty.

Background: The rates of return to full activity, persistent disability, complications, and surgical revisions after operative management of posterior cruciate ligament (PCL) tears in a physically active population have not been reported., Purpose: To evaluate the clinical outcomes of active military patients with symptomatic PCL tears who underwent surgical reconstruction and compare outcomes between isolated PCL and multiligament injuries., Study Design: Cohort study; Level of evidence, 3., Methods: Individuals undergoing surgical reconstruction of the PCL (Current Procedural Terminology code 29889) were isolated from the Military Health System Management Analysis and Reporting Tool between fiscal years 2005 and 2010. Demographic variables and rates of postoperative complications, activity limitations, rates of revision surgery, physical disability ratings, and ultimate medical discharge were recorded from the electronic medical record and US Army Physical Disability Agency database., Results: A total of 182 patients underwent 193 surgeries, including 118 isolated PCL reconstructions and 75 multiligament knee reconstructions, with an average follow-up of 19.5 months. There were 174 primary procedures and 19 revision reconstructions. The mean ± SD patient age was 28.4 ± 7.2 years, with males comprising 96.2% of patients. The overall surgical complication rate was 12.4%, with a significantly higher rate in multiligament knee reconstructions compared with isolated PCL reconstructions (18.7% vs 8.5%; P = .045). Overall, 35.1% of patients were discharged from military service due to disability. Rates of discharge were significantly higher in those undergoing surgery at lower volume institutions (those that performed <2 PCL reconstructions per year during the study period) than those at higher volume institutions (41.1% vs 26%; P = .040). The overall revision rate was 10.9%, with no significant difference between the isolated PCL and multiligament knee reconstructions. Of the 103 patients with primary isolated PCL reconstructions, 35% underwent medical discharge for persistent knee complaints, and 12.6% required revision PCL reconstruction. The overall failure rate for primary isolated PCL reconstructions, which includes both revision surgery and knee-related medical discharge from military service, was 42.7%., Conclusion: In a physically active, military population, nearly one-third of patients were unable to return to previous level of military function, and 12.6% required revision at short-term follow-up due to persistent instability. Perioperative complication rates were significantly higher among patients with multiligament knee reconstructions., Competing Interests: One or more of the authors has declared the following potential conflict of interest or source of funding: B.R.W. has received publishing royalties from Elsevier, speaking fees from Genzyme, and honoraria from Vericel; has grants/grants pending from Encore Medical; has received educational support from Arthrex, Desert Mountain Medical, Medwest, and Smith & Nephew; and has received hospitality payments from Arthrex, DePuy Synthes, Desert Mountain Medical, Encore Medical, Vericel, and Wright Medical. K.L.C. has received consulting fees from Novartis and publishing royalties from Springer. B.D.O. is a paid associate editor for the American Journal of Sports Medicine and has received consulting fees from ConMed Linvatec, DePuy Synthes, Flexion Therapeutics, Mitek, the Musculoskeletal Transplant Foundation, and Vericel; research support from Histogenics; and publishing royalties from Saunders/Mosby-Elsevier, SLACK Inc, and Springer. 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., (© The Author(s) 2019.)

Dryland ecosystems comprise a balance between woody and herbaceous vegetation. Climate change impacts rainfall timing, which may alter the respective contributions of woody and herbaceous plants on the total vegetation production. Here, we apply 30 years of field-measured woody foliage and herbaceous mass from Senegal and document a faster increase in woody foliage mass (+17 kg ha -1 yr -1 ) as compared to herbaceous mass (+3 kg ha -1 yr -1 ). Annual rainfall trends were partitioned into core wet-season rains (+0.7 mm yr -1 ), supporting a weak but periodic (5-year cycles) increase in herbaceous mass, and early/late rains (+2.1 mm yr -1 ), explaining the strongly increased woody foliage mass. Satellite observations confirm these findings for the majority of the Sahel, with total herbaceous/woody foliage mass increases by 6%/20%. We conclude that the rainfall recovery in the Sahel does not benefit herbaceous vegetation to the same extent as woody vegetation, presumably favoured by increased early/late rains., Competing Interests: The authors declare no competing interests.

Interannual climate variability patterns associated with the El Niño-Southern Oscillation phenomenon result in climate and environmental anomaly conditions in specific regions worldwide that directly favor outbreaks and/or amplification of variety of diseases of public health concern including chikungunya, hantavirus, Rift Valley fever, cholera, plague, and Zika. We analyzed patterns of some disease outbreaks during the strong 2015-2016 El Niño event in relation to climate anomalies derived from satellite measurements. Disease outbreaks in multiple El Niño-connected regions worldwide (including Southeast Asia, Tanzania, western US, and Brazil) followed shifts in rainfall, temperature, and vegetation in which both drought and flooding occurred in excess (14-81% precipitation departures from normal). These shifts favored ecological conditions appropriate for pathogens and their vectors to emerge and propagate clusters of diseases activity in these regions. Our analysis indicates that intensity of disease activity in some ENSO-teleconnected regions were approximately 2.5-28% higher during years with El Niño events than those without. Plague in Colorado and New Mexico as well as cholera in Tanzania were significantly associated with above normal rainfall (p < 0.05); while dengue in Brazil and southeast Asia were significantly associated with above normal land surface temperature (p < 0.05). Routine and ongoing global satellite monitoring of key climate variable anomalies calibrated to specific regions could identify regions at risk for emergence and propagation of disease vectors. Such information can provide sufficient lead-time for outbreak prevention and potentially reduce the burden and spread of ecologically coupled diseases.

Visualization and quantification of fluorescently labeled axonal fibers are widely employed in studies of neuronal connectivity in the brain. However, accurate analysis of axon density is often confounded by autofluorescence and other fluorescent artifacts. By the time these problems are detected in labeled tissue sections, significant time and resources have been invested, and the tissue may not be easy to replace. In response to these difficulties, we have developed Digital Enhancement of Fibers with Noise Elimination (DEFiNE), a method for eliminating fluorescent artifacts from digital images based on their morphology and fluorescence spectrum, thus permitting enhanced visualization and quantification of axonal fibers. Application of this method is facilitated by a DEFiNE macro, written using ImageJ Macro Language (IJM), which includes an automated and customizable procedure for image processing and a semi-automated quantification method that accounts for any remaining local variation in background intensity. The DEFiNE macro is open-source and used with the widely available FIJI software for maximum accessibility.

Purpose of Review: The goal of this paper is to review the biomechanical and clinical rationale for single-bundle versus double-bundle posterior cruciate ligament (PCL) reconstruction. The primary question is whether there has been demonstrated any clear biomechanical or clinical superiority of a double-bundle reconstruction over a single-bundle reconstruction., Recent Findings: There is some recent evidence demonstrating biomechanical superiority of double-bundle versus single-bundle reconstruction; however, this is not definitive. Clinical superiority has not been clearly demonstrated as of yet. The primary question which served as the basis of this review remains unanswered. There is recent biomechanical data to suggest a potential benefit of double-bundle versus single-bundle reconstruction, but not all studies are in agreement. Furthermore, the possible biomechanical advantages have not yet been borne out in clinical studies. At this point, we cannot clearly recommend one technique versus another and the decision should be left to the treating surgeon.

GATA3 is frequently mutated in breast cancer; these mutations are widely presumed to be loss-of function despite a dearth of information regarding their effect on disease course or their mechanistic impact on the breast cancer transcriptional network. Here, we address molecular and clinical features associated with GATA3 mutations. A novel classification scheme defines distinct clinical features for patients bearing breast tumors with mutations in the second GATA3 zinc-finger (ZnFn2). An engineered ZnFn2 mutant cell line by CRISPR-Cas9 reveals that mutation of one allele of the GATA3 second zinc finger (ZnFn2) leads to loss of binding and decreased expression at a subset of genes, including Progesterone Receptor. At other loci, associated with epithelial to mesenchymal transition, gain of binding correlates with increased gene expression. These results demonstrate that not all GATA3 mutations are equivalent and that ZnFn2 mutations impact breast cancer through gain and loss-of function.

Introduction: Acromioclavicular (AC) joint separation is a common entity in athletic patient populations. The surgical treatment of these injuries varies based upon extent of injury, with numerous imaging modalities recommended to differentiate injury severity and treatment options. The use of weighted stress radiographs is controversial in the diagnostic evaluation of AC separation with previous consensus recommending against their use. No study to date has investigated the clinical utilization of diagnostic studies in the evaluation of AC joint separations in a military surgeon population., Methods: Thirty-eight shoulder or sports medicine sub-specialty certified orthopaedic surgeons on active service in the Army, Air Force, and Navy were surveyed on their evaluation and treatment protocols for AC joint injuries. Specifically analyzed were imaging choice including the use of weighted stress radiographs as well as treatment recommendations based upon Rockwood grade. Responses were recorded in addition to surgeon descriptive data. Responses were analyzed with descriptive statistics., Results: Thirty-seven of the identified thirty eight surgeons responded to the survey, for a 97% response rate. Of the group, 70% of surgeons were within 10 years of completing fellowship with an estimated average of 15 AC joint separations treated annually. Plain radiographic examination was relied upon by 48% of surgeons for treatment of AC joint separation with 13% using weighted stress radiographs. Overall, 10% of surgeon stated that their treatment plan would vary based upon results from a weighted stress view. 51% of surgeons included magnetic resonance imaging in their diagnostic approach of these injuries. Treatment recommendation varied according to injury severity with 78% preferring nonoperative treatment for acute Grade III injuries with 86% waiting a minimum of 3 months before proceeding with operative treatment. For Grade V injuries, 81% of surgeon preferred operative treatment, with 59% incorporating a soft-tissue graft in their repair or reconstructive procedure., Discussion: This study identified substantial practice variation amongst military surgeons treating a relatively homogenous population with AC joint separations, reflective of a lack of definitive evidence to guide diagnosis and treatment. Overall, nonoperative management is the preferred initial approach for Type III injuries and operative treatment is the preferred initial approach for Type V injuries. The diagnostic evaluation varied across the surgeon cohort, but 87% elected against the use of weighted stress radiographs for the evaluation of AC joint separations, with only 10% relying upon them to dictate their recommended treatment. Future research identifying optimal diagnosis and treatment of AC joint separations is needed.

Background: Multi-walled carbon nanotubes (MWCNTs) are engineered nanomaterials used for a variety of industrial and consumer products. Their high tensile strength, hydrophobicity, and semi-conductive properties have enabled many novel applications, increasing the possibility of accidental nanotube inhalation by either consumers or factory workers. While MWCNT inhalation has been previously shown to cause inflammation and pulmonary fibrosis at high doses, the susceptibility of differentiating bronchial epithelia to MWCNT exposure remains unexplored. In this study, we investigate the effect of MWCNT exposure on cilia development in a differentiating air-liquid interface (ALI) model. Primary bronchial epithelial cells (BECs) were isolated from human donors via bronchoscopy and treated with non-cytotoxic doses of MWCNTs in submerged culture for 24 h. Cultures were then allowed to differentiate in ALI for 28 days in the absence of further MWCNT exposure. At 28 days, mucociliary differentiation endpoints were assessed, including whole-mount immunofluorescent staining, histological, immunohistochemical and ultrastructural analysis, gene expression, and cilia beating analysis., Results: We found a reduction in the prevalence and beating of ciliated cells in MWCNT-treated cultures, which appeared to be caused by a disruption of cellular microtubules and cytoskeleton during ciliogenesis and basal body docking. Expression of gene markers of mucociliary differentiation, such as FOXJ1 and MUC5AC/B, were not affected by treatment. Colocalization of basal body marker CEP164 with γ-tubulin during days 1-3 of ciliogenesis, as well as abundance of basal bodies up to day 14, were attenuated by treatment with MWCNTs., Conclusions: Our results suggest that a single exposure of bronchial cells to MWCNT during a vulnerable period before differentiation may impair their ability to develop into fully functional ciliated cells.

Central noradrenergic neurons, collectively defined by synthesis of the neurotransmitter norepinephrine, are a diverse collection of cells in the hindbrain, differing in their anatomy, physiological and behavioral functions, and susceptibility to disease and environmental insult. To investigate the developmental basis of this heterogeneity, we have used an intersectional genetic fate mapping strategy in mice to study the dorsoventral origins of the En1 -derived locus coeruleus (LC) complex which encompasses virtually all of the anatomically defined LC proper, as well as a portion of the A7 and subcoeruleus (SubC) noradrenergic nuclei. We show that the noradrenergic neurons of the LC complex originate in two different territories of the En1 expression domain in the embryonic hindbrain. Consistent with prior studies, we confirm that the majority of the LC proper arises from the alar plate, the dorsal domain of the neural tube, as defined by expression of Pax7 Cre . In addition, our analysis shows that a large proportion of the En1 -derived A7 and SubC nuclei also originate in the Pax7 Cre -defined alar plate. Surprisingly, however, we identify a smaller subpopulation of the LC complex that arises from outside the Pax7 Cre expression domain. We characterize the distribution of these neurons within the LC complex, their cell morphology, and their axonal projection pattern. Compared to the broader LC complex, the newly identified Pax7 Cre -negative noradrenergic subpopulation has very sparse projections to thalamic nuclei, suggestive of distinct functions. This developmental genetic analysis opens new avenues of investigation into the functional diversity of the LC complex.

Charcoal production for urban energy consumption is a main driver of forest degradation in sub Saharan Africa. Urban growth projections for the continent suggest that the relevance of this process will increase in the coming decades. Forest degradation associated to charcoal production is difficult to monitor and commonly overlooked and underrepresented in forest cover change and carbon emission estimates. We use a multitemporal dataset of very high-resolution remote sensing images to map kiln locations in a representative study area of tropical woodlands in central Mozambique. The resulting maps provided a characterization of the spatial extent and temporal dynamics of charcoal production. Using an indirect approach we combine kiln maps and field information on charcoal making to describe the magnitude and intensity of forest degradation linked to charcoal production, including aboveground biomass and carbon emissions. Our findings reveal that forest degradation associated to charcoal production in the study area is largely independent from deforestation driven by agricultural expansion and that its impact on forest cover change is in the same order of magnitude as deforestation. Our work illustrates the feasibility of using estimates of urban charcoal consumption to establish a link between urban energy demands and forest degradation. This kind of approach has potential to reduce uncertainties in forest cover change and carbon emission assessments in sub-Saharan Africa.

Background: The phytoestrogen, genistein at low doses nongenomically activates mitogen-activated protein kinase p44/42 (MAPKp44/42) via estrogen receptor alpha (ERα) leading to proliferation of human uterine leiomyoma cells. In this study, we evaluated if MAPKp44/42 could activate downstream effectors such as mitogen- and stress-activated protein kinase 1 (MSK1), which could then epigenetically modify histone H3 by phosphorylation following a low dose (1 μg/ml) of genistein., Results: Using hormone-responsive immortalized human uterine leiomyoma (ht-UtLM) cells, we found that genistein activated MAPKp44/42 and MSK1, and also increased phosphorylation of histone H3 at serine10 (H3S10ph) in ht-UtLM cells. Colocalization of phosphorylated MSK1 and H3S10ph was evident by confocal microscopy in ht-UtLM cells (r = 0.8533). Phosphorylation of both MSK1and H3S10ph was abrogated by PD98059 (PD), a MEK1 kinase inhibitor, thereby supporting genistein's activation of MSK1 and Histone H3 was downstream of MAPKp44/42. In proliferative (estrogenic) phase human uterine fibroid tissues, phosphorylated MSK1 and H3S10ph showed increased immunoexpression compared to normal myometrial tissues, similar to results observed in in vitro studies following low-dose genistein administration. Real-time RT-PCR arrays showed induction of growth-related transcription factor genes, EGR1, Elk1, ID1, and MYB (cMyb) with confirmation by western blot, downstream of MAPK in response to low-dose genistein in ht-UtLM cells. Additionally, genistein induced associations of promoter regions of the above transcription factors with H3S10ph as evidenced by Chromatin Immunoprecipitation (ChIP) assays, which were inhibited by PD. Therefore, genistein epigenetically modified histone H3 by phosphorylation of serine 10, which was regulated by MSK1 and MAPK activation., Conclusion: Histone H3 phosphorylation possibly represents a mechanism whereby increased transcriptional activation occurs following low-dose genistein exposure.

The ZFP36L3 protein is a rodent-specific, placenta- and yolk sac-specific member of the tristetraprolin (TTP) family of CCCH tandem zinc finger proteins. These proteins bind to AU-rich elements in target mRNAs, and promote their deadenylation and decay. We addressed the hypotheses that the absence of ZFP36L3 would result in the accumulation of target transcripts in placenta and/or yolk sac, and that some of these would be important for female reproductive physiology and overall fecundity. Mice deficient in ZFP36L3 exhibited decreased neonatal survival rates, but no apparent morphological changes in the placenta or surviving offspring. We found Zfp36l3 to be paternally imprinted, with profound parent-of-origin effects on gene expression. The protein was highly expressed in the syncytiotrophoblast cells of the labyrinth layer of the placenta, and the epithelial cells of the yolk sac. RNA-Seq of placental mRNA from Zfp36l3 knockout (KO) mice revealed many significantly upregulated transcripts, whereas there were few changes in KO yolk sacs. Many of the upregulated placental transcripts exhibited decreased decay rates in differentiated trophoblast stem cells derived from KO blastocysts. Several dozen transcripts were deemed high probability targets of ZFP36L3; these include proteins known to be involved in trophoblast and placenta physiology. Type 1 transferrin receptor mRNA was unexpectedly decreased in KO placentas, despite an increase in its stability in KO stem cells. This receptor is crucial for placental iron uptake, and its decrease was accompanied by decreased iron stores in the KO fetus, suggesting that this intrauterine deficiency might have deleterious consequences in later life., (© 2016. Published by The Company of Biologists Ltd.)

Investigating the developmental, structural and functional complexity of mammalian tissues and organs depends on identifying and gaining experimental access to diverse cell populations. Here, we describe a set of recombinase-responsive fluorescent indicator alleles in mice that significantly extends our ability to uncover cellular diversity by exploiting the intrinsic genetic signatures that uniquely define cell types. Using a recombinase-based intersectional strategy, these new alleles uniquely permit non-invasive labeling of cells defined by the overlap of up to three distinct gene expression domains. In response to different combinations of Cre, Flp and Dre recombinases, they express eGFP and/or tdTomato to allow the visualization of full cellular morphology. Here, we demonstrate the value of these features through a proof-of-principle analysis of the central noradrenergic system. We label previously inaccessible subpopulations of noradrenergic neurons to reveal details of their three-dimensional architecture and axon projection profiles. These new indicator alleles will provide experimental access to cell populations at unprecedented resolution, facilitating analysis of their developmental origin and anatomical, molecular and physiological properties., (© 2015. Published by The Company of Biologists Ltd.)

We show that the vegetation canopy of the Amazon rainforest is highly sensitive to changes in precipitation patterns and that reduction in rainfall since 2000 has diminished vegetation greenness across large parts of Amazonia. Large-scale directional declines in vegetation greenness may indicate decreases in carbon uptake and substantial changes in the energy balance of the Amazon. We use improved estimates of surface reflectance from satellite data to show a close link between reductions in annual precipitation, El Niño southern oscillation events, and photosynthetic activity across tropical and subtropical Amazonia. We report that, since the year 2000, precipitation has declined across 69% of the tropical evergreen forest (5.4 million km(2)) and across 80% of the subtropical grasslands (3.3 million km(2)). These reductions, which coincided with a decline in terrestrial water storage, account for about 55% of a satellite-observed widespread decline in the normalized difference vegetation index (NDVI). During El Niño events, NDVI was reduced about 16.6% across an area of up to 1.6 million km(2) compared with average conditions. Several global circulation models suggest that a rise in equatorial sea surface temperature and related displacement of the intertropical convergence zone could lead to considerable drying of tropical forests in the 21st century. Our results provide evidence that persistent drying could degrade Amazonian forest canopies, which would have cascading effects on global carbon and climate dynamics.

We document significant worldwide weather anomalies that affected agriculture and vector-borne disease outbreaks during the 2010-2012 period. We utilized 2000-2012 vegetation index and land surface temperature data from NASA's satellite-based Moderate Resolution Imaging Spectroradiometer (MODIS) to map the magnitude and extent of these anomalies for diverse regions including the continental United States, Russia, East Africa, Southern Africa, and Australia. We demonstrate that shifts in temperature and/or precipitation have significant impacts on vegetation patterns with attendant consequences for agriculture and public health. Weather extremes resulted in excessive rainfall and flooding as well as severe drought, which caused ∼10 to 80% variation in major agricultural commodity production (including wheat, corn, cotton, sorghum) and created exceptional conditions for extensive mosquito-borne disease outbreaks of dengue, Rift Valley fever, Murray Valley encephalitis, and West Nile virus disease. Analysis of MODIS data provided a standardized method for quantifying the extreme weather anomalies observed during this period. Assessments of land surface conditions from satellite-based systems such as MODIS can be a valuable tool in national, regional, and global weather impact determinations.

Foam and emulsion stability has long been believed to correlate with the surface shear viscosity of the surfactant used to stabilize them. Many subtleties arise in interpreting surface shear viscosity measurements, however, and correlations do not necessarily indicate causation. Using a sensitive technique designed to excite purely surface shear deformations, we make the most sensitive and precise measurements to date of the surface shear viscosity of a variety of soluble surfactants, focusing on SDS in particular. Our measurements reveal the surface shear viscosity of SDS to be below the sensitivity limit of our technique, giving an upper bound of order 0.01 μN·s/m. This conflicts directly with almost all previous studies, which reported values up to 10(3)-10(4) times higher. Multiple control and complementary measurements confirm this result, including direct visualization of monolayer deformation, for SDS and a wide variety of soluble polymeric, ionic, and nonionic surfactants of high- and low-foaming character. No soluble, small-molecule surfactant was found to have a measurable surface shear viscosity, which seriously undermines most support for any correlation between foam stability and surface shear rheology of soluble surfactants.

Since the first isolation of Rift Valley fever virus (RVFV) in the 1930s, there have been multiple epizootics and epidemics in animals and humans in sub-Saharan Africa. Prospective climate-based models have recently been developed that flag areas at risk of RVFV transmission in endemic regions based on key environmental indicators that precede Rift Valley fever (RVF) epizootics and epidemics. Although the timing and locations of human case data from the 2006-2007 RVF outbreak in Kenya have been compared to risk zones flagged by the model, seroprevalence of RVF antibodies in wildlife has not yet been analyzed in light of temporal and spatial predictions of RVF activity. Primarily wild ungulate serum samples from periods before, during, and after the 2006-2007 RVF epizootic were analyzed for the presence of RVFV IgM and/or IgG antibody. Results show an increase in RVF seropositivity from samples collected in 2007 (31.8%), compared to antibody prevalence observed from 2000-2006 (3.3%). After the epizootic, average RVF seropositivity diminished to 5% in samples collected from 2008-2009. Overlaying maps of modeled RVF risk assessments with sampling locations indicated positive RVF serology in several species of wild ungulate in or near areas flagged as being at risk for RVF. Our results establish the need to continue and expand sero-surveillance of wildlife species Kenya and elsewhere in the Horn of Africa to further calibrate and improve the RVF risk model, and better understand the dynamics of RVFV transmission.

Previously, we reported that fenvalerate (Fen) promotes proliferation of human uterine leiomyoma (UtLM) cells by enhancing progression of cells from G(0)-G(1) to S phase through molecular mechanisms independent of estrogen receptor-α and -β. The cyclin-dependent kinase (CDK) inhibitor p27, which blocks G(1) to S phase transitions and is an important regulator of CDK2, is often decreased in hormonally regulated diseases, including uterine leiomyomas. Therefore, we were interested in whether Fen could regulate the expression of p27 and whether p27 might play a role in Fen-induced cell proliferation. Expression of p27 in Fen-treated UtLM and uterine smooth muscle cells (UtSMCs) was examined. We found that p27 mRNA was significantly downregulated and that protein levels were decreased in both cell types treated with 10 μM Fen for 24 h compared with respective controls. Overexpression of p27 in UtLM cells and UtSMCs using an adenovirus doxycycline (Dox)-regulated Tet-off system abrogated the proliferative effects of Fen, as evidenced by decreased total cell numbers and BrdU incorporation. Fen treatment increased CDK2 mRNA expression levels; however, overexpression of p27 also abolished this effect. In contrast, Dox treatment dramatically restored the above muted responses. Finally, we utilized siRNA to knock down p27 expression. After transfection, mRNA levels of p27 were downregulated in UtLM cells and UtSMCs and total cell numbers and BrdU incorporation increased significantly compared with nontransfected cells. Fen treatment in the presence of p27 silencing enhanced the increased cell counts and BrdU labeling in UtLM cells and UtSMCs. Taken together, these results indicate that p27 downregulation is critical for Fen-induced cell proliferation.

Previously, we found that high doses of genistein show an inhibitory effect on uterine leiomyoma (UtLM) cell proliferation. In this study, using microarray analysis and Ingenuity Pathways Analysis™, we identified genes (up- or down-regulated, ≥ 1.5 fold, P ≤ 0.001), functions and signaling pathways that were altered following treatment with an inhibitory concentration of genistein (50 μg/ml) in UtLM cells. Downregulation of TGF-β signaling pathway genes, activin A, activin B, Smad3, TGF-β2 and genes related to cell cycle regulation, with the exception of the upregulation of the CDK inhibitor P15, were identified and validated by real- time RT-PCR studies. Western blot analysis further demonstrated decreased protein expression of activin A and Smad3 in genistein-treated UtLM cells. Moreover, we found that activin A stimulated the growth of UtLM cells, and the inhibitory effect of genistein was partially abrogated in the presence of activin A. Overexpression of activin A and Smad3 were found in tissue samples of leiomyoma compared to matched myometrium, supporting the contribution of activin A and Smad3 in promoting the growth of UtLM cells. Taken together, these results suggest that downregulation of activin A and Smad3, both members of the TGF-β pathway, may offer a mechanistic explanation for the inhibitory effect of a high-dose of genistein on UtLM cells, and might be potential therapeutic targets for treatment of clinical cases of uterine leiomyomas.