The fully activated open state of KCNQ1 controls the cardiac "fight-or-flight" response.

The cardiac KCNQ1 + KCNE1 (IKs) channel regulates heart rhythm under both normal and stress conditions. Under stress, the ß-adrenergic stimulation elevates the intracellular cyclic adenosine monophosphate (cAMP) level, leading to KCNQ1 phosphorylation by protein kinase A and increased IKs, which shortens action potentials to adapt to accelerated heart rate. An impaired response to the ß-adrenergic stimulation due to KCNQ1 mutations is associated with the occurrence of a lethal congenital long QT syndrome (type 1, also known as LQT1). However, the underlying mechanism of ß-adrenergic stimulation of IKs remains unclear, impeding the development of new therapeutics. Here, we find that the unique properties of KCNQ1 channel gating with two distinct open states are key to this mechanism. KCNQ1's fully activated open (AO) state is more sensitive to cAMP than its intermediate open state. By enhancing the AO state occupancy, the small molecules ML277 and C28 are found to effectively enhance the cAMP sensitivity of the KCNQ1 channel, independent of KCNE1 association. This finding of enhancing AO state occupancy leads to a potential novel strategy to rescue the response of IKs to ß-adrenergic stimulation in LQT1 mutants. The success of this approach is demonstrated in cardiac myocytes and also in a high-risk LQT1 mutation. In conclusion, the present study not only uncovers the key role of the AO state in IKs channel phosphorylation, but also provides a target for antiarrhythmic strategy.

The fully activated open state of KCNQ1 controls the cardiac "fight-or-flight" response.

The cardiac KCNQ1+KCNE1 (I Ks ) channel regulates heart rhythm in both normal and stress conditions. Under stress, the ß-adrenergic stimulation elevates the intracellular cAMP level, leading to KCNQ1 phosphorylation by protein kinase A and increased I Ks , which shortens action potentials to adapt to accelerated heart rate. An impaired response to the ß-adrenergic stimulation due to KCNQ1 mutations is associated with the occurrence of a lethal congenital long QT syndrome (type 1, also known as LQT1). However, the underlying mechanism of ß-adrenergic stimulation of I Ks remains unclear, impeding the development of new therapeutics. Here we find that the unique properties of KCNQ1 channel gating with two distinct open states are key to this mechanism. KCNQ1's fully activated open (AO) state is more sensitive to cAMP than its' intermediate open (IO) state. By enhancing the AO state occupancy, the small molecules ML277 and C28 are found to effectively enhance the cAMP sensitivity of the KCNQ1 channel, independent of KCNE1 association. This finding of enhancing AO state occupancy leads to a potential novel strategy to rescue the response of I Ks to ß-adrenergic stimulation in LQT1 mutants. The success of this approach is demonstrated in cardiac myocytes and also in a high-risk LQT1 mutation. In conclusion the present study not only uncovers the key role of the AO state in I Ks channel phosphorylation, but also provides a new target for anti-arrhythmic strategy. Significance statement: The increase of I Ks potassium currents with adrenalin stimulation is important for "fight-or-flight" responses. Mutations of the IKs channel reducing adrenalin responses are associated with more lethal form of the type-1 long-QT syndrome (LQT). The alpha subunit of the IKs channel, KCNQ1 opens in two distinct open states, the intermediate-open (IO) and activated-open (AO) states, following a two-step voltage sensing domain (VSD) activation process. We found that the AO state, but not the IO state, is responsible for the adrenalin response. Modulators that specifically enhance the AO state occupancy can enhance adrenalin responses of the WT and LQT-associated mutant channels. These results reveal a mechanism of state dependent modulation of ion channels and provide an anti-arrhythmic strategy.

Effect of hydrogen peroxide on lens transparency, intracellular pH, gap junction coupling, hydrostatic pressure and membrane water permeability.

Clouding of the eye lens or cataract is an age-related anomaly that affects middle-aged humans. Exploration of the etiology points to a great extent to oxidative stress due to different forms of reactive oxygen species/metabolites such as Hydrogen peroxide (H2O2) that are generated due to intracellular metabolism and environmental factors like radiation. If accumulated and left unchecked, the imbalance between the production and degradation of H2O2 in the lens could lead to cataracts. Our objective was to explore ex vivo the effects of H2O2 on lens physiology. We investigated transparency, intracellular pH (pHi), intercellular gap junction coupling (GJC), hydrostatic pressure (HP) and membrane water permeability after subjecting two-month-old C57 wild-type (WT) mouse lenses for 3 h or 8 h in lens saline containing 50 µM H2O2; the results were compared with control lenses incubated in the saline without H2O2. There was a significant decrease in lens transparency in H2O2-treated lenses. In control lenses, pHi decreases from ∼7.34 in the surface fiber cells to 6.64 in the center. Experimental lenses exposed to H2O2 for 8 h showed a significant decrease in surface pH (from 7.34 to 6.86) and central pH (from 6.64 to 6.56), compared to the controls. There was a significant increase in GJC resistance in the differentiating (12-fold) and mature (1.4-fold) fiber cells compared to the control. Experimental lenses also showed a significant increase in HP which was ∼2-fold higher at the junction between the differentiating and mature fiber cells and ∼1.5-fold higher at the center compared to these locations in control lenses; HP at the surface was 0 mm Hg in either type lens. Fiber cell membrane water permeability significantly increased in H2O2-exposed lenses compared to controls. Our data demonstrate that elevated levels of lens intracellular H2O2 caused a decrease in intracellular pH and led to acidosis which most likely uncoupled GJs, and increased AQP0-dependent membrane water permeability causing a consequent rise in HP. We infer that an abnormal increase in intracellular H2O2 could induce acidosis, cause oxidative stress, alter lens microcirculation, and lead to the development of accelerated lens opacity and age-related cataracts.

Mechanisms of antiviral action and toxicities of ipecac alkaloids: Emetine and dehydroemetine exhibit anti-coronaviral activities at non-cardiotoxic concentrations.

The emergence of highly infectious pathogens with their potential for triggering global pandemics necessitate the development of effective treatment strategies, including broad-spectrum antiviral therapies to safeguard human health. This study investigates the antiviral activity of emetine, dehydroemetine (DHE), and congeneric compounds against SARS-CoV-2 and HCoV-OC43, and evaluates their impact on the host cell. Concurrently, we assess the potential cardiotoxicity of these ipecac alkaloids. Significantly, our data reveal that emetine and the (-)-R,S isomer of 2,3-dehydroemetine (designated in this paper as DHE4) reduce viral growth at nanomolar concentrations (i.e., IC50 ∼ 50-100 nM), paralleling those required for inhibition of protein synthesis, while calcium channel blocking activity occurs at elevated concentrations (i.e., IC50 ∼ 40-60 µM). Our findings suggest that the antiviral mechanisms primarily involve disruption of host cell protein synthesis and is demonstrably stereoisomer specific. The prospect of a therapeutic window in which emetine or DHE4 inhibit viral propagation without cardiotoxicity renders these alkaloids viable candidates in strategies worthy of clinical investigation.

A crystallin mutant cataract with mineral deposits.

Connexin mutant mice develop cataracts containing calcium precipitates. To test whether pathologic mineralization is a general mechanism contributing to the disease, we characterized the lenses from a nonconnexin mutant mouse cataract model. By cosegregation of the phenotype with a satellite marker and genomic sequencing, we identified the mutant as a 5-bp duplication in the γC-crystallin gene (Crygcdup). Homozygous mice developed severe cataracts early, and heterozygous animals developed small cataracts later in life. Immunoblotting studies showed that the mutant lenses contained decreased levels of crystallins, connexin46, and connexin50 but increased levels of resident proteins of the nucleus, endoplasmic reticulum, and mitochondria. The reductions in fiber cell connexins were associated with a scarcity of gap junction punctae as detected by immunofluorescence and significant reductions in gap junction-mediated coupling between fiber cells in Crygcdup lenses. Particles that stained with the calcium deposit dye, Alizarin red, were abundant in the insoluble fraction from homozygous lenses but nearly absent in wild-type and heterozygous lens preparations. Whole-mount homozygous lenses were stained with Alizarin red in the cataract region. Mineralized material with a regional distribution similar to the cataract was detected in homozygous lenses (but not wild-type lenses) by micro-computed tomography. Attenuated total internal reflection Fourier-transform infrared microspectroscopy identified the mineral as apatite. These results are consistent with previous findings that loss of lens fiber cell gap junctional coupling leads to the formation of calcium precipitates. They also support the hypothesis that pathologic mineralization contributes to the formation of cataracts of different etiologies.

GPX1 knockout, not catalase knockout, causes accelerated abnormal optical aberrations and cataract in the aging lens.

Purpose: Glutathione peroxidase 1 (GPX1) and catalase are expressed in the lens epithelial cells and cortical fiber cells, where they detoxify H2O2 to reduce oxidative stress, which is a major cause for cataractogenesis. We sought to find out, between these two enzymes, which is critical for transparency and homeostasis in the aging lens by investigating alterations in the lens's refractive property, transparency, and gap junction coupling (GJC) resistance. Methods: Wild-type (C57BL/6J), GPX1 knockout (GPX1-/-) and catalase knockout (CAT-/-) mice were used. Lens transparency was quantified using dark-field images and ImageJ software. For optical aberration evaluation, each lens was placed over a copper electron microscopy specimen grid; the grid image was captured through the lens using a digital camera attached to a dark-field binocular microscope. Optical aberrations were assessed by the quality of the magnified gridlines. Microelectrode-based intact lens intracellular impedance was measured to determine GJC resistance. Results: In contrast to wild-type (WT) and CAT-/- lenses, GPX1-/- lenses developed accelerated age-related cataracts. While two-month-old lenses were normal, at nine months of age, GPX1-/- mice started to show the development of abnormal optical distortion aberrations and loss of transparency. At 12 months of age, GPX1-/- lenses developed significant opacity and abnormal optical distortion aberrations compared to CAT-/- and WT (p<0.001); these aberrations gradually increased with age and matured into cataracts by 24 months of age. There was also a significant increase (p<0.001) in GJC resistance in the differentiating and mature fiber cells of GPX1-/- lenses at 12 months of age compared to that in similar areas of age-matched CAT-/- and WT lenses. Conclusions: Changes in the refractive and physiological properties of the lens occurred before cataract formation in GPX1-/- lenses but not in CAT-/- lenses. GPX1 is more critical than catalase for lens transparency, optical quality, and homeostasis in the aging lens under normal physiological conditions. GPX1 could be a promising therapeutic target for developing potential strategies to reduce adverse oxidative stress and delay/treat/prevent age-related cataracts.

Eph-ephrin Signaling Affects Eye Lens Fiber Cell Intracellular Voltage and Membrane Conductance.

The avascular eye lens generates its own microcirculation that is required for maintaining lifelong lens transparency. The microcirculation relies on sodium ion flux, an extensive network of gap junction (GJ) plaques between lens fiber cells and transmembrane water channels. Disruption of connexin proteins, the building blocks of GJs, or aquaporins, which make up water and adhesion channels, lead to lens opacification or cataracts. Recent studies have revealed that disruption of Eph-ephrin signaling, in particular the receptor EphA2 and the ligand ephrin-A5, in humans and mice lead to congenital and age-related cataracts. We investigated whether changes in lens transparency in EphA2 or ephrin-A5 knockout (-/-) mice is related to changes in GJ coupling and lens fluid and ion homeostasis. Immunostaining revealed changes in connexin 50 (Cx50) subcellular localization in EphA2 -/- peripheral lens fibers and alteration in aquaporin 0 (Aqp0) staining patterns in ephrin-A5 -/- and EphA2 -/- inner mature fiber cells. Surprisingly, there was no obvious change in GJ coupling in knockout lenses. However, there were changes in fiber cell membrane conductance and intracellular voltage in knockout lenses from 3-month-old mice. These knockout lenses displayed decreased conductance of mature fiber membranes and were hyperpolarized compared to control lenses. This is the first demonstration that the membrane conductance of lens fibers can be regulated. Together these data suggest that EphA2 may be needed for normal Cx50 localization to the cell membrane and that conductance of lens fiber cells requires normal Eph-ephrin signaling and water channel localization.

Modulating the voltage sensor of a cardiac potassium channel shows antiarrhythmic effects.

Cardiac arrhythmias are the most common cause of sudden cardiac death worldwide. Lengthening the ventricular action potential duration (APD), either congenitally or via pathologic or pharmacologic means, predisposes to a life-threatening ventricular arrhythmia, Torsade de Pointes. IKs (KCNQ1+KCNE1), a slowly activating K+ current, plays a role in action potential repolarization. In this study, we screened a chemical library in silico by docking compounds to the voltage-sensing domain (VSD) of the IKs channel. Here, we show that C28 specifically shifted IKs VSD activation in ventricle to more negative voltages and reversed the drug-induced lengthening of APD. At the same dosage, C28 did not cause significant changes of the normal APD in either ventricle or atrium. This study provides evidence in support of a computational prediction of IKs VSD activation as a potential therapeutic approach for all forms of APD prolongation. This outcome could expand the therapeutic efficacy of a myriad of currently approved drugs that may trigger arrhythmias.

Absence of S100A4 in the mouse lens induces an aberrant retina-specific differentiation program and cataract.

S100A4, a member of the S100 family of multifunctional calcium-binding proteins, participates in several physiological and pathological processes. In this study, we demonstrate that S100A4 expression is robustly induced in differentiating fiber cells of the ocular lens and that S100A4 (-/-) knockout mice develop late-onset cortical cataracts. Transcriptome profiling of lenses from S100A4 (-/-) mice revealed a robust increase in the expression of multiple photoreceptor- and Müller glia-specific genes, as well as the olfactory sensory neuron-specific gene, S100A5. This aberrant transcriptional profile is characterized by corresponding increases in the levels of proteins encoded by the aberrantly upregulated genes. Ingenuity pathway network and curated pathway analyses of differentially expressed genes in S100A4 (-/-) lenses identified Crx and Nrl transcription factors as the most significant upstream regulators, and revealed that many of the upregulated genes possess promoters containing a high-density of CpG islands bearing trimethylation marks at histone H3K27 and/or H3K4, respectively. In support of this finding, we further documented that S100A4 (-/-) knockout lenses have altered levels of trimethylated H3K27 and H3K4. Taken together, our findings suggest that S100A4 suppresses the expression of retinal genes during lens differentiation plausibly via a mechanism involving changes in histone methylation.

Connexin Mutants Compromise the Lens Circulation and Cause Cataracts through Biomineralization.

Gap junction-mediated intercellular communication facilitates the circulation of ions, small molecules, and metabolites in the avascular eye lens. Mutants of the lens fiber cell gap junction proteins, connexin46 (Cx46) and connexin50 (Cx50), cause cataracts in people and in mice. Studies in mouse models have begun to elucidate the mechanisms by which these mutants lead to cataracts. The expression of the dominant mutants causes severe decreases in connexin levels, reducing the gap junctional communication between lens fiber cells and compromising the lens circulation. The impairment of the lens circulation results in several changes, including the accumulation of Ca2+ in central lens regions, leading to the formation of precipitates that stain with Alizarin red. The cataract morphology and the distribution of Alizarin red-stained material are similar, suggesting that the cataracts result from biomineralization within the organ. In this review, we suggest that this may be a general process for the formation of cataracts of different etiologies.

A PIP2 substitute mediates voltage sensor-pore coupling in KCNQ activation.

KCNQ family K+ channels (KCNQ1-5) in the heart, nerve, epithelium and ear require phosphatidylinositol 4,5-bisphosphate (PIP2) for voltage dependent activation. While membrane lipids are known to regulate voltage sensor domain (VSD) activation and pore opening in voltage dependent gating, PIP2 was found to interact with KCNQ1 and mediate VSD-pore coupling. Here, we show that a compound CP1, identified in silico based on the structures of both KCNQ1 and PIP2, can substitute for PIP2 to mediate VSD-pore coupling. Both PIP2 and CP1 interact with residues amongst a cluster of amino acids critical for VSD-pore coupling. CP1 alters KCNQ channel function due to different interactions with KCNQ compared with PIP2. We also found that CP1 returned drug-induced action potential prolongation in ventricular myocytes to normal durations. These results reveal the structural basis of PIP2 regulation of KCNQ channels and indicate a potential approach for the development of anti-arrhythmic therapy.

Signaling Between TRPV1/TRPV4 and Intracellular Hydrostatic Pressure in the Mouse Lens.

Purpose: The lens uses feedback to maintain zero pressure in its surface cells. Positive pressures are detected by transient receptor potential vanilloid (TRPV4), which initiates a cascade that reduces surface cell osmolarity. The first step is opening of gap junction hemichannels. One purpose of the current study was to identify the connexin(s) in the hemichannels. Negative pressures are detected by TRPV1, which initiates a cascade that increases surface osmolarity. The increase in osmolarity was initially reported to be through inhibition of Na/K ATPase activity, but a recent study reported it was through stimulation of Na/K/2Cl (NKCC) cotransport. A second purpose of this study was to reconcile these two reports. Methods: Intracellular hydrostatic pressures were measured using a microelectrode/manometer system. Lenses from TRPV1 or Cx50 null mice were studied. Specific inhibitors of Cx50 gap junction channels, NKCC, and Akt were used. Results: Either knockout of Cx50 or blockade of Cx50 channels completely eliminated the response to positive surface pressures. Knockout of Cx50 also caused a positive drift in surface pressure. The short-term (∼20-minute) response to negative surface pressures was eliminated by blockade of NKCC, but a long-term (∼4-hour) response restored pressure to zero. Both short- and long-term responses were eliminated by knockout of TRPV1 or inhibition of Akt. Conclusions: Hemichannels made from Cx50 are required for the response to positive surface pressures. Negative surface pressures first activate NKCC, but a backup system is inhibition of Na/K ATPase activity. Both responses are initiated by TRPV1 and go through PI3K/Akt before branching.

TRPV1 activation stimulates NKCC1 and increases hydrostatic pressure in the mouse lens.

The porcine lens response to a hyperosmotic stimulus involves an increase in the activity of an ion cotransporter sodium-potassium/two-chloride cotransporter 1 (NKCC1). Recent studies with agonists and antagonists pointed to a mechanism that appears to depend on activation of transient receptor potential vanilloid 1 (TRPV1) ion channels. Here, we compare responses in lenses and cultured lens epithelium obtained from TRPV1-/- and wild type (WT) mice. Hydrostatic pressure (HP) in lens surface cells was determined using a manometer-coupled microelectrode approach. The TRPV1 agonist capsaicin (100 nM) caused a transient HP increase in WT lenses that peaked after ∼30 min and then returned toward baseline. Capsaicin did not cause a detectable change of HP in TRPV1-/- lenses. The NKCC inhibitor bumetanide prevented the HP response to capsaicin in WT lenses. Potassium transport was examined by measuring Rb+ uptake. Capsaicin increased Rb+ uptake in cultured WT lens epithelial cells but not in TRPV1-/- cells. Bumetanide, A889425, and the Akt inhibitor Akti prevented the Rb+ uptake response to capsaicin. The bumetanide-sensitive (NKCC-dependent) component of Rb+ uptake more than doubled in response to capsaicin. Capsaicin also elicited rapid (<2 min) NKCC1 phosphorylation in WT but not TRPV1-/- cells. HP recovery was shown to be absent in TRPV1-/- lenses exposed to hyperosmotic solution. Bumetanide and Akti prevented HP recovery in WT lenses exposed to hyperosmotic solution. Taken together, responses to capsaicin and hyperosmotic solution point to a functional role for TRPV1 channels in mouse lens. Lack of NKCC1 phosphorylation and Rb+ uptake responses in TRPV1-/- mouse epithelium reinforces the notion that a hyperosmotic challenge causes TRPV1-dependent NKCC1 activation. The results are consistent with a role for the TRPV1-activated signaling pathway leading to NKCC1 stimulation in lens osmotic homeostasis.

The Ciliary Muscle and Zonules of Zinn Modulate Lens Intracellular Hydrostatic Pressure Through Transient Receptor Potential Vanilloid Channels.

Purpose: Lenses have an intracellular hydrostatic pressure gradient to drive fluid from central fiber cells to surface epithelial cells. Pressure is regulated by a feedback control system that relies on transient receptor potential vanilloid (TRPV)1 and TRPV4 channels. The ciliary muscle transmits tension to the lens through the zonules of Zinn. Here, we have examined if ciliary muscle tension influenced the lens intracellular hydrostatic pressure gradient. Methods: We measured the ciliary body position and intracellular hydrostatic pressures in mouse lenses while pharmacologically causing relaxation or contraction of the ciliary muscle. We also used inhibitors of TRPV1 and TRPV4, in addition to phosphoinositide 3-kinase (PI3K) p110α knockout mice and immunostaining of phosphorylated protein kinase B (Akt), to determine how changes in ciliary muscle tension resulted in altered hydrostatic pressure. Results: Ciliary muscle relaxation increased the distance between the ciliary body and the lens and caused a decrease in intracellular hydrostatic pressure that was dependent on intact zonules and could be blocked by inhibition of TRPV4. Ciliary contraction moved the ciliary body toward the lens and caused an increase in intracellular hydrostatic pressure and Akt phosphorylation that required intact zonules and was blocked by either inhibition of TRPV1 or genetic deletion of the p110α catalytic subunit of PI3K. Conclusions: These results show that the hydrostatic pressure gradient within the lens was influenced by the tension exerted on the lens by the ciliary muscle through the zonules of Zinn. Modulation of the gradient of intracellular hydrostatic pressure in the lens could alter the water content, and the gradient of refractive index.

Regulation of heart rate and the pacemaker current by phosphoinositide 3-kinase signaling.

Heart rate in physiological conditions is set by the sinoatrial node (SN), the primary cardiac pacing tissue. Phosphoinositide 3-kinase (PI3K) signaling is a major regulatory pathway in all normal cells, and its dysregulation is prominent in diabetes, cancer, and heart failure. Here, we show that inhibition of PI3K slows the pacing rate of the SN in situ and in vitro and reduces the early slope of diastolic depolarization. Furthermore, inhibition of PI3K causes a negative shift in the voltage dependence of activation of the pacemaker current, I F, while addition of its second messenger, phosphatidylinositol 3,4,5-trisphosphate, induces a positive shift. These shifts in the activation of I F are independent of, and larger than, those induced by the autonomic nervous system. These results suggest that PI3K is an important regulator of heart rate, and perturbations in this signaling pathway may contribute to the development of arrhythmias.

C-Terminal End of Aquaporin 0 Regulates Lens Gap Junction Channel Function.

Purpose: We reported previously that aquaporin 0 (AQP0) modulates lens fiber cell gap junction (GJ) channel function. The present study was conducted to find out whether the C-terminal end of AQP0 is involved in this regulation. Methods: A mouse model, AQP0ΔC/ΔC, was genetically engineered to express AQP0 with 1-246 amino acids, without the normal intact AQP0 (1-263 amino acids) in the lens. Transparency and focusing of the lens were assessed. Intracellular impedance was measured to determine GJ coupling resistance. Intracellular hydrostatic pressure (HP) was also determined. Western blotting was performed to determine connexin (Cx46 and Cx50) expression levels. Results: At postnatal day 10, AQP0ΔC/ΔC mouse lenses relative to age-matched wild-type lenses showed loss of transparency and abnormal optical distortion; GJ coupling resistance increased in the differentiating (1.6-fold) and mature (8-fold) fiber cells; lens HP increased approximately 1.5-fold at the junction between the differentiating and mature fiber cells and approximately 2.0-fold in the center; there was no significant change (P > 0.05) in expression levels of Cx46 or Cx50. Conclusions: The increase in GJ coupling resistance was not associated with reduced connexin expression, suggesting either a reduction in the open probability or some physical change in plaque location. The increase in resistance was significantly greater than the increase in HP, suggesting less pressure-driven water flow through each open GJ channel. These changes may lead to a loss of transparency and abnormal optical distortion. Overall, our data demonstrate the C-terminal end of AQP0 is involved in modulating GJ coupling to maintain lens transparency and homeostasis.

The Connexin50D47A Mutant Causes Cataracts by Calcium Precipitation.

Purpose: Mutations in connexin50 (Cx50) and connexin46 (Cx46) cause cataracts. Because the expression of Cx46fs380 leads to decreased gap junctional coupling and formation of calcium precipitates, we studied Cx50D47A lenses to test whether Cx50 mutants also cause cataracts due to calcium precipitation. Methods: Connexin levels were determined by immunoblotting. Gap junctional coupling conductance was calculated from intracellular impedance studies of intact lenses. Intracellular hydrostatic pressure was measured using a microelectrode/manometer system. Intracellular free calcium ion concentrations ([Ca2+]i) were measured using Fura-2 and fluorescence imaging. Calcium precipitation was assessed by Alizarin red staining and compared to the distribution of opacities in darkfield images. Results: In Cx50D47A lenses, Cx50 levels were 11% (heterozygotes) and 1.2% (homozygotes), and Cx46 levels were 52% (heterozygotes) and 30% (homozygotes) when compared to wild-type at 2.5 months. Gap junctional coupling in differentiating fibers of Cx50D47A lenses was 49% (heterozygotes) and 29% (homozygotes), and in mature fibers, it was 24% (heterozygotes) and 4% (homozygotes) compared to wild-type lenses. Hydrostatic pressure was significantly increased in Cx50D47A lenses. [Ca2+]i was significantly increased in Cx50D47A lenses. Alizarin red-stained calcium precipitates were present in homozygous Cx50D47A lenses with a similar distribution to the cataracts. Conclusions: Cx50D47A expression altered the lens internal circulation by decreasing connexin levels and gap junctional coupling. Reduced water and ion outflow through gap junctions increased the gradients of intracellular hydrostatic pressure and concentrations of free calcium ions. In these lenses, calcium ions accumulated, precipitated, and formed cataracts. These results suggest that mutant lens fiber connexins lead to calcium precipitates, which may cause cataracts.

A Vision-Based Counting and Recognition System for Flying Insects in Intelligent Agriculture.

Rapid and accurate counting and recognition of flying insects are of great importance, especially for pest control. Traditional manual identification and counting of flying insects is labor intensive and inefficient. In this study, a vision-based counting and classification system for flying insects is designed and implemented. The system is constructed as follows: firstly, a yellow sticky trap is installed in the surveillance area to trap flying insects and a camera is set up to collect real-time images. Then the detection and coarse counting method based on You Only Look Once (YOLO) object detection, the classification method and fine counting based on Support Vector Machines (SVM) using global features are designed. Finally, the insect counting and recognition system is implemented on Raspberry PI. Six species of flying insects including bee, fly, mosquito, moth, chafer and fruit fly are selected to assess the effectiveness of the system. Compared with the conventional methods, the test results show promising performance. The average counting accuracy is 92.50% and average classifying accuracy is 90.18% on Raspberry PI. The proposed system is easy-to-use and provides efficient and accurate recognition data, therefore, it can be used for intelligent agriculture applications.

Disruption of the lens circulation causes calcium accumulation and precipitates in connexin mutant mice.

The lens is an avascular organ whose function and survival depend on an internal circulation system. Cx46fs380 mice model a human autosomal dominant cataract caused by a mutant lens connexin. In these mice, fiber cell connexin levels and gap junction coupling are severely decreased. The present studies were conducted to examine components of the lens circulation system that might be altered and contribute to the pathogenesis of cataracts. Lenses from wild-type mice and Cx46fs380 heterozygotes and homozygotes were studied at 2 months of age. Cx46fs380-expressing lens fiber cells were depolarized. Cx46fs380 lenses had increased intracellular hydrostatic pressure and concentrations of Na+ and Ca2+. The activity of epithelial Na+-K+-ATPase was decreased in Cx46fs380 lenses. All of these changes were more severe in homozygous than in heterozygous Cx46fs380 lenses. Cx46fs380 cataracts were stained by Alizarin red, a dye used to detect insoluble Ca2+. These data suggest that the lens internal circulation was disrupted by expression of Cx46fs380, leading to several consequences including accumulation of Ca2+ to levels so high that precipitates formed. Similar Ca2+-containing precipitates may contribute to cataract formation due to other genetic or acquired etiologies.