Fibroblast growth factor-induced lens fiber cell elongation is driven by the stepwise activity of Rho and Rac.

The spheroidal shape of the eye lens is crucial for precise light focusing onto the retina. This shape is determined by concentrically aligned, convexly elongated lens fiber cells along the anterior and posterior axis of the lens. Upon differentiation at the lens equator, the fiber cells increase in height as their apical and basal tips migrate towards the anterior and posterior poles, respectively. The forces driving this elongation and migration remain unclear. We found that, in the mouse lens, membrane protrusions or lamellipodia are observed only in the maturing fibers undergoing cell curve conversion, indicating that lamellipodium formation is not the primary driver of earlier fiber migration. We demonstrated that elevated levels of fibroblast growth factor (FGF) suppressed the extension of Rac-dependent protrusions, suggesting changes in the activity of FGF controlling Rac activity, switching to lamellipodium-driven migration. Inhibitors of ROCK, myosin and actin reduced the height of both early and later fibers, indicating that elongation of these fibers relies on actomyosin contractility. Consistent with this, active RhoA was detected throughout these fibers. Given that FGF promotes fiber elongation, we propose that it does so through regulation of Rho activity.

Types of anomalies in two-dimensional video-based gait analysis in uncontrolled environments.

Two-dimensional video-based pose estimation is a technique that can be used to estimate human skeletal coordinates from video data alone. It is also being applied to gait analysis and in particularly, due to its simplicity of measurement, it has the potential to be applied to gait analysis of large populations. However, it is considered difficult to completely homogenize the environment and settings during the measurement of large populations. Therefore, it is necessary to appropriately deal with technical errors that are not related to the biological factors of interest. In this study, by analyzing a large cohort database, we have identified four major types of anomalies that occur during gait analysis using OpenPose in uncontrolled environments: anatomical, biomechanical, and physical anomalies and errors due to estimation. We have also developed a workflow for identifying and correcting these anomalies and confirmed that this workflow is reproducible through simulation experiments. Our results will help obtain a comprehensive understanding of the anomalies to be addressed during pre-processing for 2D video-based gait analysis of large populations.

Mentalis nerve branches supplying the lower lip revisited: a study of human fetuses and donated elderly cadavers.

PURPOSE: Little information is known about the mentalis nerve course from the lower lip approximation margin (free margin) to the upper lip. Likewise, no difference in nerve distribution has been observed between the cutaneous and mucosal parts of the lip. Therefore, this study reexamined mentalis nerve morphology. METHODS: For macroscopic observations, three fresh cadavers were dissected (one male and two females; aged 78-93). We also evaluated histological sections obtained from five donated elderly cadavers (two males and three females, aged 82-96 years) and 15 human fetuses (11-40 weeks or crown-rump length 80-372 mm). Immunohistochemical analysis for S100 protein and tyrosine hydroxylase was performed. RESULTS: In both fetuses and adult cadavers, one to three nerve branches ran upward in the submucosal tissue from the mental foramen. Near the free margin of the lip, some branches passed through the orbicularis oris muscle layer toward the lip skin, whereas others followed a reversed J-shaped course along the free margin. Nerve twigs ran in parallel beneath the mucosa, whereas wavy nerve twigs attached to the basal lamina of the lip epidermis. The difference in nerve endings abruptly occurred at the skin-mucosal junction. Tyrosine hydroxylase-positive sympathetic nerve twigs surrounded arteries and formed a branch composed of S100-negative unmyelinated fibers. CONCLUSION: The lower lip skin was innervated by a perforating branch passing through the orbicularis oris muscle, that was different from the lip mucosa. A sudden change in the nerve ending configuration at the mucocutaneous junction seemed to develop postnatally.

Exploratory Study of Growth of Circumference of Mandibular Fossa Adjacent to Petrous Portion of Temporal Bone Using Dried Skulls.

The morphogenetic process of development of the circumference of the mandibular fossa during tooth eruption, which involves the replacement of deciduous teeth with permanent teeth, is strongly affected by occlusion. To the best of our knowledge, no studies have investigated the effect of occlusion on this process. This study investigated the morphogenetic process of development during tooth eruption using dried skulls harvested from Indian donors. The average distance between the ala-major-squamosa suture and the foramen ovale according to age group was as follows: 3.24 mm in the 8-month-old group and 8.92 mm in the adult group. The average distance between the ala-major-squamosa suture and the apex of the articular tubercle according to age groups was as follows: 10.38 mm in the 8-month-old group and 19.34 mm in the adult group. The average distance between the point of intersection of the petrosquamous fissure and petrotympanic fissure located on the perpendicular line drawn posteriorly from the shortest distance of the medio-lateral axis between the ala-major-squamosa suture and the apex of the articular tubercle according to age group was as follows: 9.68 mm in the 8-month-old group and 14.3 mm in the adult group. These results suggest that the mandibular fossa is strongly affected by load due to occlusion, unlike the growth of the neurocranium. This indicates that the effect of occlusion is a secondary element in the morphogenetic process of development of the circumference of the mandibular fossa.

7,8-Dihydroxy-3-(4'-hydroxyphenyl)coumarin inhibits invasion and migration of osteosarcoma cells.

Advances in pharmacy and medicine have led to the development of many anti-cancer and molecular targeted agents; however, there are few agents capable of suppressing metastasis. To prevent cancer recurrence, it is essential to develop novel agents for inhibiting metastasis. Coumarin-based compounds have multiple pharmacological activities including anti-cancer effects. We screened a compound library constructed at Kyoto Pharmaceutical University and showed that 7,8-dihydroxy-3-(4'-hydroxyphenyl)coumarin (DHC) inhibited invasion and migration of LM8 mouse osteosarcoma cells and 143B human osteosarcoma cells in a concentration-dependent manner. DHC decreased intracellular actin filament formation by downregulating Rho small GTP-binding proteins such as RHOA, RAC1, and CDC42, which regulate actin reorganization. However, DHC did not downregulate the corresponding mRNA transcripts, whereas it downregulated Rho small GTP-binding proteins in the presence of cycloheximide, suggesting that DHC enhances the degradation of these proteins. DHC treatment inhibited metastasis and prolonged overall survival in a spontaneous metastasis mouse model. These results indicate that DHC has the potential to suppress metastasis of osteosarcoma cells by downregulating Rho small GTP-binding proteins.

The Japanese Epidemiologic Study for Perioperative Anaphylaxis, a prospective nationwide study: clinical signs, severity, and therapeutic agents.

BACKGROUND: Diagnosis of perioperative anaphylaxis is difficult because of its non-specific and variable signs and symptoms. Therapeutic agents used to treat anaphylaxis and anaesthesiologist responses also vary depending on the case, which might affect outcomes; however, only a few studies have focused on these factors. METHODS: This prospective study of perioperative anaphylaxis, a part of the Japanese Epidemiologic Study for Perioperative Anaphylaxis, investigated the clinical signs, its severity, therapeutic drugs, epinephrine administration, and anaesthesiologist responses in cases of perioperative anaphylaxis to assess trends and variability. Shock index was used to assess severity of cardiovascular collapse. RESULTS: In 43 patients analysed in this study, cardiovascular signs (88.4%) were the most frequent, followed by skin (81.4%) and respiratory signs (60.5%). The presence of signs increased during the clinical course. The median time from the first signs to diagnosis of anaphylaxis was 10 (5.0-17.8) min. The rates of epinephrine use were 30.2% (unused), 48.8% (i.v.), and 20.9% (i.m.). The median time from diagnosis of anaphylaxis to epinephrine administration was 7 (inter-quartile range: 1.5-8.0) min. Antihistamines and corticosteroids were each used in 69.8% of cases. The worst shock index was higher in patients who received i.v. epinephrine (2.77 [0.90] mean [standard deviation]) than in both no epinephrine use cases (1.35 [0.41]) and i.m. epinephrine cases (1.89 [0.77] (P<0.001]). CONCLUSIONS: The clinical signs and treatments of perioperative anaphylaxis are variable, and the choice regarding epinephrine administration is based on symptom severity. CLINICAL TRIAL REGISTRATION: UMIN000035350.

The Japanese Epidemiologic Study for Perioperative Anaphylaxis, a prospective nationwide study: allergen exposure, epidemiology, and diagnosis of anaphylaxis during general anaesthesia.

BACKGROUND: Diagnosis of perioperative anaphylaxis is often challenging. This study describes the utility of a newly developed tool for identifying patients with a high possibility of anaphylaxis, and aimed to investigate the frequency of anaphylaxis with each drug during the perioperative period in Japan. METHODS: This study included patients with anaphylaxis of Grade 2 or higher severity during general anaesthesia at 42 facilities across Japan in 2019 and 2020. We developed and adopted a unique objective evaluation tool yielding a composite score for diagnosing anaphylaxis, which includes the results of skin tests and basophil activation tests, and clinical scores for perioperative anaphylaxis. The number of cases using each drug and the total number of anaphylaxis cases were investigated to calculate the frequency of anaphylaxis. RESULTS: General anaesthesia was performed in 218 936 cases, which included 55 patients with suspected perioperative anaphylaxis. The developed composite score diagnosed 43 of them with a high probability of anaphylaxis. The causative agent was identified in 32 cases. Plasma histamine levels showed high diagnostic accuracy for anaphylaxis. The top causative agents were rocuronium (10 cases in 210 852 patients, 0.005%), sugammadex (7 cases in 150 629 patients, 0.005%), and cefazolin (7 cases in 106 005 patients, 0.007%). CONCLUSIONS: We developed a composite tool to diagnose anaphylaxis, and found that the combination of tryptase levels, skin testing, and basophil activation testing results and clinical score improved the certainty of anaphylaxis diagnosis. The incidence of perioperative anaphylaxis in our study was 1 in about 5000 general anaesthesia cases. CLINICAL TRIAL REGISTRATION: UMIN000035350.

Catalytically inactive Cas9 impairs DNA replication fork progression to induce focal genomic instability.

Catalytically inactive Cas9 (dCas9) has become an increasingly popular tool for targeted gene activation/inactivation, live-cell imaging, and base editing. While dCas9 was reported to induce base substitutions and indels, it has not been associated with structural variations. Here, we show that dCas9 impedes replication fork progression to destabilize tandem repeats in budding yeast. When targeted to the CUP1 array comprising ∼16 repeat units, dCas9 induced its contraction in most cells, especially in the presence of nicotinamide. Replication intermediate analysis demonstrated replication fork stalling in the vicinity of dCas9-bound sites. Genetic analysis indicated that while destabilization is counteracted by the replisome progression complex components Ctf4 and Mrc1 and the accessory helicase Rrm3, it involves single-strand annealing by the recombination proteins Rad52 and Rad59. Although dCas9-mediated replication fork stalling is a potential risk in conventional applications, it may serve as a novel tool for both mechanistic studies and manipulation of genomic instability.

Chronological Changes in the Expression and Localization of Sox9 between Achilles Tendon Injury and Functional Recovery in Mice.

Tendons help transmit forces from the skeletal muscles and bones. However, tendons have inferior regenerative ability compared to muscles. Despite studies on the regeneration of muscles and bone tissue, only a few have focused on tendinous tissue regeneration, especially tendon regeneration. Sex-determining region Y-box transcription factor 9 (Sox9) is an SRY-related transcription factor with a DNA-binding domain and is an important control factor for cartilage formation. Sox9 is critical to the early-to-middle stages of tendon development. However, how Sox9 participates in the healing process after tendon injury is unclear. We hypothesized that Sox9 is expressed in damaged tendons and is crucially involved in restoring tendon functions. We constructed a mouse model of an Achilles tendon injury by performing a 0.3 mm wide partial excision in the Achilles tendon of mice, and chronologically evaluated the function restoration and localization of the Sox9 expressed in the damaged sites. The results reveal that Sox9 was expressed simultaneously with the formation of the pre-structure of the epitenon, an essential part of the tendinous tissue, indicating that its expression is linked to the functional restoration of tendons. Lineage tracing for Sox9 expressed during tendon restoration revealed the tendon restoration involvement of cells that switched into Sox9-expressing cells after tendon injury. The stem cells involved in tendon regeneration may begin to express Sox9 after injury.

Development and Regeneration of Muscle, Tendon, and Myotendinous Junctions in Striated Skeletal Muscle.

Owing to a rapid increase in aging population in recent years, the deterioration of motor function in older adults has become an important social problem, and several studies have aimed to investigate the mechanisms underlying muscle function decline. Furthermore, structural maintenance of the muscle-tendon-bone complexes in the muscle attachment sites is important for motor function, particularly for joints; however, the development and regeneration of these complexes have not been studied thoroughly and require further elucidation. Recent studies have provided insights into the roles of mesenchymal progenitors in the development and regeneration of muscles and myotendinous junctions. In particular, studies on muscles and myotendinous junctions have-through the use of the recently developed scRNA-seq-reported the presence of syncytia, thereby suggesting that fibroblasts may be transformed into myoblasts in a BMP-dependent manner. In addition, the high mobility group box 1-a DNA-binding protein found in nuclei-is reportedly involved in muscle regeneration. Furthermore, studies have identified several factors required for the formation of locomotor apparatuses, e.g., tenomodulin (Tnmd) and mohawk (Mkx), which are essential for tendon maturation.

Preoperative and postoperative complications of cerebrospinal fluid drainage in descending thoracic and thoraco-abdominal aortic aneurysm surgery: a single-center retrospective study.

PURPOSE: Cerebrospinal-fluid drainage (CSFD) has been performed to prevent paraplegia in descending thoracic or thoraco-abdominal aortic aneurysm (DTA/TAAA) surgery; however, CSFD itself has a risk of severe complications. We retrospectively investigated the incidence rates of CSFD-related preoperative and postoperative complications. METHODS: Patients who underwent DTA/TAAA surgery with a CSFD catheter that was inserted on the day before surgery were enrolled. The incidence rates of complications from spinal puncture until DTA/TAAA surgery were investigated as preoperative CSFD complications, and the incidence rates from DTA/TAAA surgery to postoperative day 7 were investigated as CSFD-related postoperative complications. RESULTS: Preoperative CSFD complications were analyzed in 123 cases. DTA/TAAA surgery was postponed due to bloody cerebrospinal fluid (2.5%) and due to meningitis (1.7%). The incidence rate of mild preoperative complications was 32.4%. Postoperative CSFD complications were analyzed in 108 cases. Intracranial hemorrhage occurred in 3.9% of cases in open surgery and other postoperative severe CSFD complications did not occur. The incidence rates of moderate/mild complications in open surgery were 2.6%/14.3% and those in TEVAR were 3.2%/19.4%. CONCLUSION: Bloody cerebrospinal fluid and meningitis, which are severe complications associated with spinal puncture, occurred within 1 day after spinal puncture. The incidence rates of moderate/mild complications were high in both the preoperative and postoperative periods. These results showed that CSFD catheter insertion and management should be performed carefully with consideration given to the risks and benefits of CSFD.

Capsaicin attenuates TGFß2-induced epithelial-mesenchymal-transition in lens epithelial cells in vivo and in vitro.

Posterior capsule opacification (PCO), the most common complication of cataract surgery occurring in 20-50% of patients after 2-5 years of cataract surgery, is a major problem in the aging society. The epithelial-mesenchymal transition (EMT) of lens epithelial cells after cataract surgery has been proposed as a major cause of PCO. Capsaicin, widely used as a food additive and analgesic agent, is a major pungent ingredient in red pepper. Although the effect of capsaicin on EMT has been reported in cancer cells, the biological reaction of capsaicin was unique in each cell type, and there have been no reports describing its effects on EMT earlier. In this study, we demonstrated that treatment with capsaicin inhibited TGFß2-induced EMT in vitro lens epithelial cells and ex vivo explant lens epithelial cells. Furthermore, eye drops of capsaicin inhibited the PCO model mice in vivo. Finally, we showed that capsaicin inhibited non-canonically induced Smad2/3 activation via suppression of EGFR activation and ERK phosphorylation. Our findings indicate that capsaicin and its derivatives are good candidate compounds for preventing PCO after cataract surgery.

Regulation of the Membrane Trafficking of the Mechanosensitive Ion Channels TRPV1 and TRPV4 by Zonular Tension, Osmotic Stress and Activators in the Mouse Lens.

Lens water transport generates a hydrostatic pressure gradient that is regulated by a dual-feedback system that utilizes the mechanosensitive transient receptor potential vanilloid (TRPV) channels, TRPV1 and TRPV4, to sense changes in mechanical tension and extracellular osmolarity. Here, we investigate whether the modulation of TRPV1 or TRPV4 activity dynamically affects their membrane trafficking. Mouse lenses were incubated in either pilocarpine or tropicamide to alter zonular tension, exposed to osmotic stress, or the TRPV1 and TRPV4 activators capsaicin andGSK1016790A (GSK101), and the effect on the TRPV1 and TRPV4 membrane trafficking in peripheral fiber cells visualized using confocal microscopy. Decreases in zonular tension caused the removal of TRPV4 from the membrane of peripheral fiber cells. Hypotonic challenge had no effect on TRPV1, but increased the membrane localization of TRPV4. Hypertonic challenge caused the insertion of TRPV1 and the removal of TRPV4 from the membranes of peripheral fiber cells. Capsaicin caused an increase in TRPV4 membrane localization, but had no effect on TRPV1; while GSK101 decreased the membrane localization of TRPV4 and increased the membrane localization of TRPV1. These reciprocal changes in TRPV1/4 membrane localization are consistent with the channels acting as mechanosensitive transducers of a dual-feedback pathway that regulates lens water transport.

Effects of isomaltulose ingestion on postexercise hydration state and heat loss responses in young men.

NEW FINDINGS: What is the central question of this study? What are the effects of isomaltulose, an ingredient in carbohydrate-electrolyte beverages to maintain glycaemia and attenuate the risk of dehydration during exercise heat stress, on postexercise rehydration and physiological heat loss responses? What is the main finding and its importance? Consumption of a 6.5% isomaltulose-electrolyte beverage following exercise heat stress restored hydration following a 2 h recovery as compared to a 2% solution or water only. While the 6.5% isomaltulose-electrolytes increased plasma volume and plasma osmolality, which are known to modulate postexercise heat loss, sweating and cutaneous vascular responses did not differ between conditions. Consequently, ingestion beverages containing 6.5% isomaltulose-electrolytes enhanced postexercise rehydration without affecting heat loss responses. ABSTRACT: Isomaltulose is a disaccharide carbohydrate widely used during exercise to maintain glycaemia and hydration. We investigated the effects of ingesting a beverage containing isomaltulose and electrolytes on postexercise hydration state and physiological heat loss responses. In a randomized, single-blind cross-over design, 10 young healthy men were hypohydrated by performing up to three 30 min successive moderate-intensity (50% heart rate reserve) bouts of cycling, each separated by 10 min, while wearing a water-perfusion suit heated to 45°C. The protocol continued until a 2% reduction in body mass was achieved. Thereafter, participants performed a final 15 min moderate-intensity exercise bout followed by a 2 h recovery. Following cessation of exercise, participants ingested a beverage consisting of (i) water only (Water), (ii) 2% isomaltulose (CHO-2%), or (iii) 6.5% isomaltulose (CHO-6.5%) equal to the volume of 2% body mass loss within the first 30 min of the recovery. Changes in plasma volume (ΔPV) after fluid ingestion were greater for CHO-6.5% compared with CHO-2% (120 min postexercise) and Water (90 and 120 min) (all P ≤ 0.040). Plasma osmolality remained elevated with CHO-6.5% compared with consumption of the other beverages at 30 and 90 min postexercise (all P ≤ 0.050). Urine output tended to be reduced with CHO-6.5% compared to other fluid conditions (main effect, P = 0.069). Rectal and mean skin temperatures, chest sweat rate and cutaneous perfusion did not differ between conditions (all P > 0.05). In conclusion, compared with CHO-2% and Water, consuming a beverage consisting of CHO-6.5% and electrolytes during recovery under heat stress enhances PV recovery without modulating physiological heat loss responses.

Transcranial motor-evoked potentials of laryngeal muscles for intraoperative neuromonitoring of the vagus nerve during thyroid surgery.

PURPOSE: The aim of this study was to elucidate normative features of vagal motor-evoked potentials (MEPs) induced by transcranial electrical stimulation (TES) and to determine the influence of functional decline of the recurrent laryngeal nerve (RLN) on vagal MEPs during thyroid surgery. METHODS: A total of 54 patients undergoing elective thyroid surgery under general anesthesia were enrolled in this study. Vagal MEPs induced by TES were measured from the vocal cord using one of two types of electrodes (wire type or wide and flat type) mounted on an endotracheal tube. We investigated the effects of stimulation intensity and train pulse number on vagal MEP amplitude, the time course of vagal MEP amplitude during surgery, and the effects of functional decline of the RLN on vagal MEPs. RESULTS: The success rate of vagal MEP monitoring with wide- and flat-type electrodes was significantly higher than that with wire-type electrodes. Reliable vagal MEPs were obtained at a stimulation intensity of approximately 300 V with 3 or more pulses in 91% of the patients without preoperative RLN palsy (RLNP), and the amplitude was augmented with increasing stimulation intensity and train pulse number. Vagal MEP amplitude decreased during thyroid surgery and then partially recovered at the end of surgery. Vagal MEP amplitude recorded from the electrode ipsilateral to preoperative RLNP was significantly lower than that on the contralateral intact side. CONCLUSION: Vagal MEPs induced by TES can be obtained with a high success rate during thyroid surgery and would reflect functional status of the RLN.

Various Arsenic Network Structures in 112-Type Ca1-xLaxFe1-yPdyAs2 Revealed by Synchrotron X-ray Diffraction Experiments.

Two novel 112-type palladium-doped iron arsenides were synthesized and identified using comprehensive studies involving synchrotron X-ray diffraction and X-ray absorption near-edge structure (XANES) experiments. Whereas in-plane arsenic zigzag chains were found in the 112-type superconducting iron arsenide Ca1-xLaxFeAs2 with maximum Tc = 34 K, deformed arsenic network structures appeared in other 112-type materials, such as longitudinal arsenic zigzag chains in CaFe1-yPdyAs2 (y ∼ 0.51) and arsenic square sheets constructed via hypervalent bonding in Ca1-xLaxFe1-yPdyAs2 (x ∼ 0.31, y ∼ 0.30). As K-edge XANES spectra clarified the similar oxidization states around FeAs4 tetrahedrons, alluding to possible parents for high-Tc 112-type iron arsenide superconductors.

Novel coiled-coil proteins regulate exocyst association with cortical microtubules in xylem cells via the conserved oligomeric golgi-complex 2 protein.

Xylem vessel cells develop secondary cell walls in distinct patterns. Cortical microtubules are rearranged into distinct patterns and regulate secondary cell wall deposition; however, it is unclear how exocytotic membrane trafficking is linked to cortical microtubules. Here, we show that the novel coiled-coil proteins vesicle tethering 1 (VETH1) and VETH2 recruit EXO70A1, an exocyst subunit essential for correct patterning of secondary cell wall deposition, to cortical microtubules via the conserved oligomeric Golgi complex (COG) 2 protein. VETH1 and VETH2 encode an uncharacterized domain of an unknown function designated DUF869, and were preferentially up-regulated in xylem cells. VETH1-green fluorescent protein (GFP) and VETH2-GFP co-localized at novel vesicle-like small compartments, which exhibited microtubule plus-end-directed and end-tracking dynamics. VETH1 and VETH2 interacted with COG2, and this interaction promoted the association between cortical microtubules and EXO70A1 These results suggest that the VETH-COG2 complex ensures the correct secondary cell wall deposition pattern by recruiting exocyst components to cortical microtubules.

[Airway Management Using McGrath MAC in a Pediatric Patient with a Laryngeal Saccular Cyst].

An 18-month-old female infant was scheduled for resection of a laryngeal saccular cyst inducing narrow airway. Since the cyst protruded from the left side of the epiglottis and the vocal cord was compressed to the right side, a difficult airway was anticipated. In addition, there was a risk of tracheal occlusion by rupture of the cyst Awake intubation was considered to be dangerous. Slow induction with sevoflurane and neuromusclar blockade was attempted, preparing reversal of the neuromusclar blockade. Fortunately, mask ventilation was achieved without difficulty. It was necessary to insert a tracheal tube avoiding the cyst We used McGrath MAC (Aircraft Medical Co., UK), which enabled us to manipulate the tracheal tube. A tracheal tube was successfully inserted under McGrath monitor guidance.

Sfrp1 and Sfrp2 are not involved in Wnt/ß-catenin signal silencing during lens induction but are required for maintenance of Wnt/ß-catenin signaling in lens epithelial cells.

During eye lens development, regulation of Wnt/ß-catenin signaling is critical for two major processes: initially it must be silent in the lens placode for lens development to proceed, but subsequently it is required for maintenance of the lens epithelium. It is not known how these different phases of Wnt/ß-catenin activity/inactivity are regulated. Secreted frizzled related protein-2 (Sfrp2), a putative Wnt-Fz antagonist, is expressed in lens placode and in lens epithelial cells and has been put forward as a candidate for regional Wnt/ß-catenin pathway regulation. Here we show its closely-related isoform, Sfrp1, has a complimentary pattern of expression in the lens, being absent from the placode and epithelium but expressed in the fibers. As mice with single knockouts of Sfrp1 or Sfrp2 had no defects in lens formation, we examined lenses of Sfrp1 and Sfrp2 double knockout (DKO) mice and showed that they formed lens placode and subsequent lens structures. Consistent with this we did not observe ectopic TCF/Lef activity in lens placode of DKOs. This indicates that Sfrp1 and Sfrp2 individually, or together, do not constitute the putative negative regulator that blocks Wnt/ß-catenin signaling during lens induction. In contrast, Sfrp1 and Sfrp2 appear to have a positive regulatory function because Wnt/ß-catenin signaling in lens epithelial cells was reduced in Sfrp1 and Sfrp2 DKO mice. Lenses that formed in DKO mice were smaller than controls and exhibited a deficient epithelium. Thus Sfrps play a role in lens development, at least in part, by regulating aspects of Wnt/ß-catenin signaling in lens epithelial cells.

Smad2 and Smad3 are redundantly essential for the suppression of iNOS synthesis in macrophages by regulating IRF3 and STAT1 pathways.

Although transforming growth factor (TGF)-ß1 is a well-known immunosuppressive cytokine, little is known about the role of its downstream transcription factors, Smad2 and Smad3, in the suppression of macrophage activation. Previous studies have demonstrated that Smad3 is critical for the suppression of LPS-mediated inducible nitric oxide (NO) synthase (iNOS) induction, although the role of Smad2 remains to be investigated. In this study, we found that iNOS induction was enhanced in Smad2-deficient bone marrow-derived macrophages (BMDMs) and peritoneal macrophages in vitro and tumor-associated macrophages in vivo, compared with wild-type (WT) macrophages. However, TGF-ß1 still suppressed iNOS induction in Smad2-deficient macrophages. In Smad2/3 double knockout (KO) (Smad2/3 DKO) BMDMs, LPS-mediated NO/iNOS induction was more strongly elevated than in Smad2 or Smad3 single KO BMDMs, and its suppression by exogenous TGF-ß1 was severely impaired. These data suggest that Smad2 and Smad3 redundantly regulate iNOS induction. Similarly, the production of IL-6 and TNFα, but not IL-10 was augmented in Smad2/3 DKO BMDMs, suggesting that Smad2 and Smad3 also redundantly suppressed some cytokines production. In Smad2/3 DKO macrophages, TLR3- as well as TLR4-mediated IRF3 activation and IFN-ß production were strongly augmented, which resulted in hyper STAT1 phosphorylation. Furthermore, IFN-ß- and IFN-γ-induced iNOS induction in the absence of TLR signaling and STAT1 transcriptional activity were augmented in Smad2/3 DKO BMDMs. These results suggest that Smad2 and Smad3 negatively regulate iNOS induction in macrophages by suppressing multiple steps in the IRF3-IFN-ß-STAT1 pathway.