Trichoderma harzianum) reduces cropping surface and damages basidiomes, limiting production and harvest quality. The goal of the current study was to evaluate new generation fungicides, to devise suitable management strategies against the green mold disease under prevailing agro-climatic conditions. Six non-systemic and five systemic fungitoxicants were evaluated for their efficacy against pathogen, T. harzianum, and host, Agaricus bisporus, under in vitro conditions. Among non-systemic fungicides, chlorothalonil and prochloraz manganese with mean mycelium inhibition of 76.87 and 93.40 percent, respectively, were highly inhibitory against the pathogen. The least inhibition percentage of 7.16 of A. bisporus was exhibited by chlorothalonil. Under in vivo conditions, use of captan 50 WP resulted in a maximum yield of button mushroom of 14.96 kg/qt. So far, systemic fungicides were concerned, carbendazim proved extremely inhibitory to the pathogen (89.22%), with least inhibitory effect on host mycelium (1.56%). However, application of non-systemic fungitoxicants further revealed that fungicide prochloraz manganese 50 WP at 0.1–0.2 percent or chlorothalonil 50 WP at 0.2 percent, exhibited maximum disease control of 89.06–96.30 percent. Moreover, the results of systemic fungitoxicants showed that carbendazim 50 WP or thiophanate methyl 70 WP at 0.1 percent reduced disease to 2.29–3.69 percent, hence exhibiting the disease control of 80.11–87.66 percent. Under in vivo conditions, fungicide myclobutanil at 0.1 percent concentration produced the maximum button mushroom production of 12.87 kg/q.Mycoparasites cause serious losses in profitable mushroom farms worldwide. The negative impact of green mold ( Trichoderma spp. . T−1 concentrations and five systemic fungitoxicants, namely myclobutanil, carbendazim, hexaconazole, thiophanate methyl and difenoconazole at concentrations of 10, 25, 50, 75 and 100 μg mL−1 were evaluated against the mycelial growth of green mold pathogen and host by using the poisoned food technique . TT. harzianum) and host (A. bisporus) mycelium at 50, 75, 100, 200 and 500 g mL−1 doses using the poisoned food technique  against the test pathogen . There w−1, respectively. The development of the test pathogen was fully inhibited by prochloraz manganese at 100 μg mL−1 and higher concentrations. After prochloraz manganese chemical, the next best fungicide showing 94.73 and 90.56 percent growth inhibition was chlorothalonil at a concentration of 500 μg mL−1 and 200 μg mL−1, respectively. However, fungitoxicants mancozeb proved to be least effective at 50 μg mL−1 with minimal growth inhibition of just 11.57 percent.The maximum and minimum inhibition percentages of all evaluated non-systemic fungicides were achieved at concentration 500 and 50 μg mL−1 whereas the highest (25.12%) was attained at 500 μg mL−1. At concentration 50 μg mL−1, prochloraz manganese, chlorothalonil and captan showed no growth inhibition of the host mycelium. However, at concentration 50 μg mL−1 fungicide zineb, propineb and mancozeb exhibited inhibition percentage of 2.84, 3.86 and 4.69, respectively. The next least inhibitory fungicides with respective concentration were chlorothalonil, captan, prochloraz manganese at 75 μg mL−1 with inhibition percentage of 2.88, 3.27 and 3.32, followed by chlorothalonil, zineb prochloraz manganese, captan with inhibition percentage 4.94, 5.28, 5.80, 6.66 at concentration 100, 75, 100 and 100 μg mL−1, respectively. Highest inhibition percentage of 33.52 and 31.34 of test fungal mycelium growth was displayed by mancozeb and propineb, respectively, at 500 μg mL−1.Results  showed tWhen compared to pathogen-infested and untreated check-I, the findings mentioned in Among different non-systemic fungicides that were used for study, captan showed a minimum number (90.08) of fruit bodies per kg of mushrooms at a concentration of 0.2%, trailed by prochloraz manganese and chlorothalonil at a concentration of 0.2% and captan at a concentration of 0.1% yielding 90.46–91.68 fruit bodies per kg of mushroom, whereas, 94.48 and 91.73 fruit bodies per kg of mushroom were recorded in infested-untreated and uninfested untreated, respectively, as illustrated in By the application of non-systemic fungitoxicants, it was observed that mushroom crop yield from per quintal compost also grew dramatically. Highest button yield for treatments receiving captan (0.10–0.20%) and prochloraz manganese (0.20%) was 14.18–14.96 kg/qt compost, compared to 7.46 and 13.78 kg/qt compost obtained from controls that were infested-untreated and uninfested-untreated. The next best treatments were chlorothalonil (0.10%) or captan (0.05%) with mean yield of (12.33–13.52) kg/qt compost. The least successful fungicides at a concentration of 0.05 percent were both prochloraz manganese and chlorothalonil. From the table, it is evident, with an increase in concentration of particular fungicides, button yield also increased, e.g., prochloraz manganese at 0.2% concentration give 14.88 kg/qt compost.viz., weight and diameter of pileus and stipe were also significantly affected by non-systemic fungicidal applications on infested casings . The next best treatment was captan followed by prochloraz manganese at concentration 0.20 and 0.10% with pileus weight of 7.35 and 7.14 g, respectively.Pileus diameter: Among the various fungicidal treatments, the largest pileus diameter of 3.81 cm was measured from polybags with treatment of prochloraz at concentration 0.20%, followed by prochloraz manganese (3.67 cm) at concentration 0.10 which was statistically equal to Check II (uninfested-untreated). The next best treatments were captan and chlorothalonil at concentration 0.20% with pileus diameter of 3.62 and 3.59 cm, respectively.Stipe weight: Treatment with captan at concentration 0.20% produced stripes with maximum weight of 4.79 g compared to that in infested-untreated control (4.29 g). The subsequent efficient treatments for producing buttons with maximum stripe weight are, chlorothalonil, prochloraz manganese 50 WP and captan 50 WP at concentration 0.20, 0.20 and 0.10, respectively. For uninfested-untreated control, the mean stipe weight was 4.17 cm.Stipe diameter: Prochloraz manganese (0.20%) treatment produced mushrooms with highest stipe diameter of 1.35 cm followed by prochloraz manganese at concentration 0.10% with a stipe diameter of 1.31 cm compared to 1.22 cm obtained in infested-untreated control.T. harzianum) and host (A. bisporus) mycelium at different concentrations of 10, 25, 50, 75 and 100 μg mL−1 by poisoned food technique  in overall comparison. Myclobutanil was the next least inhibitory (18.21%) to the A. bisporus mycelium, whereas difenoconazole and hexaconazole showed highest growth inhibition (21.48–39.99%). The mycelial inhibition was observed to rise continuously when the fungicide concentration was increased. Carbendazim showed no inhibition of the host fungus at up to 25 μg mL−1 and thiophanate methyl at 10 μg mL−1; however, at concentrations 50–100 and 25–50 µg mL−1 these were least inhibitory by displaying inhibitory percentages of 1.72–3.57, respectively. Fungitoxicants including hexaconazole, difenoconazole and myclobutanil were highly suppressive against A. bisporus. At a concentration of 100 µg ml−1 fungicides, viz., hexaconazole, difenoconazole and myclobutanil, exhibited inhibitory percentages of 24.13, 31.19 and 61.90 to mycelial growth of mushroom.The inhibitory action of all the fungicides on host mycelium when evaluated differed substantially . CarbendSignificant impacts on yield and yield parameters such as weight and number of fruiting bodies were seen in the fungicidal application on pathogen infested casings. From T. harzianum-infested casings, the average weight of fruit-bodies was significantly affected. Thiophanate methyl (0.1% concentration) and carbendazim (0.025% concentration) produced 10.44–11.26 g of average fruiting body weight, which increased from 10.33 g obtained from infested-untreated polybags. Fruiting bodies weighing 10.56 g were obtained in uninfested-untreated control. The next best fungicide treatments were carbendazim 50 WP (0.1%) and myclobutanil (0.1%) producing fruit bodies of 10.96–11.21 g weight.Applying systemic fungicides to −1 compost at 0.1 percent concentration that was as good as that obtained in the uninfested control (11.08 kg q−1 compost). The same was found in the systemic fungitoxicants, yield increase with an increase in fungitoxicant concentration of particular fungitoxicant, e.g., in carbendazim the highest yield was achieved at 0.1% concentration, which gave 12.63 kg q−1 compost.Button mushroom yield  has been increased significantly by application of systemic fungicide. Application of fungicides, myclobutanil and carbendazim both displayed a maximum yield of 12.63 and 12.87 kg qThe fungitoxicant impact on the infested casing has greatly afflicted the quality parameters of sporophore, such as diameter of pileus and weight of stipe, although there was no substantial change in the pileus weight and stipe diameter .Weight of pileus: The pileus weight of A. bisporus sporophores did not change considerably after fungicidal treatments in pathogen-infested casing.Pileus diameter: The maximum pileus diameter of 3.82 cm was recorded in polybags receiving myclobutanil (0.1%) fungitoxicant treatment. Carbendazim (0.1%) or myclobutanil (0.05%) or thiophanate methyl (0.05%) applications were the next best treatments that produced 3.67–3.73 cm of pileus diameter, parallel to those attained in infested-untreated controls. However, none of the fungitoxicant applications developed a pileus diameter of 4.12 cm like that of the uninfested-untreated control.Stipe weight: Weight of stripe enlarged significantly to 4.91–5.01 g at 0.1 percent in treatments receiving carbendazim or myclobutanil, compared to 4.37 g in infested-untreated control and 4.69 g in uninfested-untreated control. Thiophanate methyl (0.1%), myclobutanil (0.05%) or carbendazim (0.05%), yielding a mean stipe weight of 4.83–4.87 g, were the next best treatments.Stipe diameter: The fungicidal treatment had no discernible effect on the stipe diameter of A. bisporus sporophores.T. harzianum with no or minimal impact on host mycelium (A. bisporus). Under in vitro and in vivo conditions, different fungicides were tested against both T. harzianum and A. bisporus in order to choose the most efficient ones regarding the disease’s restraint. Maximum reticence under in vitro conditions against T. harzianum was exhibited by prochloraz manganese, followed by chlorothalonil, and were least effective against A. bisporus growth. Among six in vitro fungitoxicants tested, chlorothalonil, prochloraz manganese and captan were further assessed in a mushroom house for in vivo efficacy. In contrast to other fungicides, the selected fungitoxicants expressed partial restraint of mycelium of mushrooms but maximum pathogen inhibition. Among systemic fungitoxicants, carbendazim, thiophante methyl and myclobutanil were highly inhibitory to T. harzianum and were less inhibitory to A. bisporus mycelium growth; whereas the least inhibitory fungicide against the pathogen was hexaconazole under in vitro conditions. Carbendazim displayed a lower degree of mushroom inhibition, suggesting that A. bisporus is marginally resistant to this fungitoxicant. Our results were in limited covenant with Thapa and Seth [Trichoderma without affecting mushroom growth. By application of carbendazim or thiophanate methyl at concentration 0.1 percent, the infection was reduced to 2.29–3.69 percent with disease control of 80.11–87.66 percent. Again, the influence of systemic fungitoxicants does not have much impact on sporophore quality parameter. Similarly, several other workers have also documented the inhibitory effects of these fungicides, thus these results were in agreement with the present findings [T. harzianum was observed in carbendazim followed by bitertanol, captan, hexaconzole and at least mancozeb, which strongly supports the present findings. Prochloraz manganese is the officially recommended fungicide in the mushroom industry. The aggressive growth of Trichoderma isolates has been found to be inhibited without affecting the growth of A. bisporus by prochloraz manganese, as it interferes with the demethylation step in ergosterol biosynthesis [Trichoderma spp. isolated from button mushroom were studied and carbendazim was proved most effective [Trichoderma growth was also inhibited by benomyl, prochloraz manganese and imazalil, different fungicides were considered by IC50 values > 2 ppm for both thermophilic fungi and A. bisporus. Prochloraz, flusilazol and benomyl had partial effects on A. bisporus growth and development between the concentration of 0 and 2 ppm but were found extra lethal for thermophilic fungi [Usage of non-chemical (fungicide) ingredients such as botanicals and bio-control agents are ideally necessary for the successful management of green mold disease in mushroom production houses. It is also possible to use certain fungicides to control the disease economically. All these applications and amendments are, however, intended to verify or inhibit the proliferation and growth of and Seth , and demfindings ,19,20,21findings . The finynthesis ,19. Seveffective . Trichodic fungi ,24.Pleurotus sp. and T. viride increased with an increase in concentration of various fungicides [Trichoderma sp. when added to an agar medium at 5 ppm and 10 ppm, respectively, and, hence, supports our present findings. Other researchers have also demonstrated the inclusion of efficient fungicides, bio control agents or botanicals for the control of green mold disease of button mushrooms [T. Harzianum edible fungi mold was also reported earlier by some workers [With the in vivo assessment of this pathogen, it would possible to determine the effectiveness of every aspect of disease management. The in vivo evaluation of fungitoxicants showed that by using prochloraz manganese at 0.10–0.2 percent or chlorothalonil at 0.2 percent, the disease was reduced to 0.70–2.07 percent, showing disease control of 89.06–96.30 percent. Due to the impact of non-systemic fungitoxicants, there is no influence on the quality parameters of sporophores, carbendazim and thiophanate methyl (systemic) or prochloraz manganese and chlorothalonil (non-systemic) were successful in controlling green mold disease with a concomitant increase in the yield of button mushrooms incorporated into pathogen-infested casing soil. Furthermore, growth inhibition of ngicides . The mosngicides , in addiushrooms ,28,29. T workers ,18,19,21−1 concentration, carbendazim and thiophante methyl fully inhibited the growth of the pathogen under in vitro and in vivo. Among non-systemic prochloraz, manganese was found to be highly effective in reducing the disease. Carbendazim at 0.1% and prochloraz manganese at 0.2% provided an efficient control of green mold infection of white button mushroom. However, biological control of disease management, an alternative to fungicides, is also highly recommended and encouraged.The present finding on evaluation of non-systemic and systemic fungicides revealed the promising effects in reducing the infection of green mold. At 100 μg mL


Immunoglobulin E (IgE) elevation is a hallmark of allergic conditions such as atopic dermatitis (AD). The pathogenesis of AD is typically associated with high levels of IL-4 and IL-13 produced by activated T helper 2 (Th2) cells. Psoriasis, on the other hand, is an inflammatory skin disease mainly driven by Th17 cells and their related cytokines. Although the immunopathologic reactions and clinical manifestations are often easily distinguished in the two skin conditions, patients with psoriasis may sometimes exhibit AD-like manifestations, such as elevated IgE and persistent pruritic lesions. Given the fact that the effective T cells have great plasticity to re-differentiate in response to innate and environmental factors, this unusual skin condition could be a consequence of a cross-reaction between distinct arms of T-cell and humoral immunity. Here we review the literature concerning the roles of IgE in the development of AD and psoriasis, showing that elevated IgE seems to be an important indicator for this non-typical psoriasis. However, elevated IgE is also found in some chronic inflammatory allergic diseases such as rhinitis, asthma and AD (Immunoglobulin E (IgE) elevation is a useful clinical indicator for atopic dermatitis (AD) and other allergic diseases. Antigen-specific IgE a and AD , 2, and a and AD .AD causes dry, itchy and inflamed skin lesions. Psoriasis is also a chronic inflammatory skin disease with patches of rashes and abundant silvery scales. Both conditions tend to be long-lasting and flare up from time to time. In contrast to the relatively young ages of onset in AD patients, psoriasis is common in adults. The primary immune cell effectors in the two diseases are believed to be different , 5. Manyvia disulfide bonds to form a tetrapeptide chain molecule similar to other monomer immunoglobulin molecules (+ hematopoietic progenitors. Mast cells are long-lived resident cells in tissues that differentiate from other blood-derived progenitor cells (IgE is one of the five immunoglobulins. It consists of two heavy chains (H chains) and two light chains (L chains), which are linked olecules . Structuolecules . The maiolecules . The medolecules , 19. Masor cells . Mast ceor cells . Basophior cells –24.The Th2 immune response plays a central role in activating IgE-mediated activation of mast cells and basophils , 26. In New evidence has suggested that the biological function of IgE is beyond the Type I hypersensitivity reactions. A number of studies have proven that IgE alone can provide survival signals to mast cells even in the absence of allergens , 33 or s+ memory cells have been detected in the blood of allergic patients, but these cells produce only about 0.2% of IgE in serum because of few such cells in the blood. Therefore, it is likely that most IgE-producing and IgE memory cells may exist elsewhere  is a commonly used method to measure the serum IgE in the clinical setting. A previous study showed that RAST was positive in 58 and 22% of patients with chronic plaque psoriasis and active psoriasis, respectively . Several+ cells and FcεRI+ cells are substantially higher in psoriatic lesions relative to non-lesional areas of the skin in the same patient, suggesting that FcεRI+ cells may also be involved in the development of psoriasis , which can significantly reduce IgEease IgE . Howeverease IgE , 96. Risease IgE . Taken tease IgE . Nonbiolease IgE .Despite accumulating evidence showing that an elevated level of total serum IgE is associated with psoriasis, the involvements of IgE in psoriasis pathogenesis are not fully clarified. The elevated IgE level in psoriasis patients suggests a possible shift of Th17 toward Th2 immune responses, which makes it difficult to distinguish between psoriasis and AD. Since the biologics for psoriasis treatment have a profound effect on modulating different subsets of T-helper cells, results from studies on the immunopathogenesis of psoriasis could become more complicated under biologic therapies .Traditionally, AD and psoriasis are considered two distinct diseases by virtue of their immunopathogenic mechanisms, clinical characteristics, and the ages of onset . HoweverCommon genetic predispositions have been noted  for the Clinical symptoms also overlap or coexist between the two diseases, which may present a challenge for clinical diagnosis. Studies have found that patients with overlapping AD and psoriasis have a high incidence of hand involvement . BecauseAD can be categorized into the extrinsic and intrinsic types, in which exogenous AD is frequently associated with activation of Th2 and elevated IgE and intrinsic AD shows activation of Th17 response besides activation of Th2. In adults, extrinsic AD with a decreased Th17 response tends to have nonflexural area involvement . S100A9/AD starts in early childhood but has a tendency to persist throughout life with different clinical manifestations , 124, whS. aureus colonization, perhaps due to a lack of AMPs, damage to the skin barrier and dysbiosis of the microbial ecology. In this case, not only can S. aureus directly disrupt skin barrier function, but it can also cause Th17 polarization by upregulating pro-inflammatory cytokines. This may explain why some AD patients have lesions that are more similar to psoriasis  were expressed at higher levels in AD and psoriasis tissues . The serrophages . However plaques  and in b plaques . However plaques . CD83 is plaques  and mark plaques . The masThe reciprocal regulation of Th1 and Th2 cells is always observed in psoriasis and other infectious diseases, with induction of Th1 cells and release of Th1-associated cytokines IFN-γ and TNF-α resulting in the inhibition of Th2 cells . AccordiAn important mechanism for AD development is the disruption of the skin barrier that increases the transmissibility of external allergens and promotes the Th2 immune response through antigen-presenting cells such as Langerhans cells and dendritic cells, both of which could lead to increased IgE production . In addiIgE is usually associated with type I hypersensitivity reactions and is not present in non-allergic diseases such as psoriasis. Although the roles of IgE in psoriasis pathogenesis remain unclear , the higAlthough AD and psoriasis have traditionally been considered two clinically distinct diseases, it has become clear that overlapping clinical manifestations exist, and even the underlying pathogeneses of these diseases have become somewhat blurred. Clinically, pruritus, as well as other phenotypes of type 2 inflammatory diseases , are typical features of AD and yet are observed in 37-75% of patients with psoriasis. Current evidence supports the notion that AD and psoriasis share some clinical and immunopathogenic characteristics based on the following: (1) some patients are genetically susceptible to both diseases; (2) Th17 cells in psoriasis patients may still have the plasticity to re-differentiate toward Th2 phenotypes under certain conditions; (3) clinical treatments can change the manifestations of either disease. To answer whether AD-like psoriasis is a part of a spectrum that may stretch between psoriasis and AD, we must look further at AD patients with symptoms that overlap with those of psoriasis. Here we have conducted a comprehensive literature review to clarify the impact of Th cell plasticity on different immune inflammatory environments, including high levels of IgE in the progression of AD and psoriasis.LS and CL: elaborated the figures and wrote the manuscript. LS, HX, and DS: reviewed topics and concepts. DS: conceived, reviewed, and discussed concepts in the manuscript. All authors contributed to critical revisions of the article and approved the submitted version.This work was supported in part by grants from the Traditional Chinese Medicine Science and Technology Program of Shandong Province (NM 2021M080) and the Key Research and Development Plan of Jining (NM 2021YXNS121), Shandong, China.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Chaetoceros, while its relative abundance was significantly lower in the sea ice, probably because the cold-surge-induced seawater icing existed only for a short period of time, and Chaetoceros had not yet adapted to the rapid environmental changes in the sea ice. The relative abundance of Bathycoccus in the sea ice was higher, and showed a significant positive correlation with nitrite and silicate, while the relative abundance of Micromonas in the sea ice was also significantly higher than in the seawater, which may be related to the elevated CO2 concentration in the sea ice. This study demonstrates that although the seawater icing due to the cold surge was short, it may have affected the seawater eukaryotic microalgal community, to a certain extent.Climate change has led to frequent cold surges in mid-latitudes, resulting in sudden temperature drops and icing of nearshore seawater, which may be affecting the eukaryotic microalgal community. In this paper, we investigated the differences between a eukaryotic microalgal community in sea ice and in seawater during the seawater freezing, due to the cold surge in Aoshan Bay, Qingdao, China, in January 2021. The results showed that the eukaryotic microalgal community in the sea ice and in the seawater was similar in composition at the phylum and genus levels, but that its relative abundances differed. In the seawater, the eukaryotic microalgal genera were dominated by  This increase in atmospheric CO2 has in turn led to global temperature rise and climate change T were adopted from Dickson (1990)   . The sealted ice .DNA was extracted from 0.2-µm-pore-size polycarbonate filter membranes (Millipore) using DNeasy PowerSoil Kit  . Eukaryot-test, prior to which the data were tested for normality and homogeneity of variance in GraphPad Prism (v 8.4.3). The compositions of the eukaryotic microalgal community in the seawater and in the sea ice at the genus level were calculated, and presented using the vegan and pheatmap packages in R [The raw sequences were demultiplexed and quality-filtered by the bioinformatics analysis software QIIME2 , where ages in R . Differeges in R . The 12 ges in R ,39,40.pCO2 of the seawater at S1 were higher than those in the corresponding sea ice, while the pH and nutrient data were lower than those in the sea ice. The reason that the salinity and pCO2 at S1 were higher than those at the other two stations was probably due to the shallow water depth at this station, and the expulsion of salt during the sea ice formation leading to an increase in the salinity of the seawater under the ice, which in turn led to an increase in pCO2. Salinity, pH, pCO2 and nutrient data at S2 and S3 were all lower than those in the corresponding sea ice.As shown in p < 0.05).The Shannon–Weiner Index of the eukaryotic microalgal community was 2.08, 1.41 and 1.31 in the seawater, and 3.22, 3.09 and 3.42 in the sea ice at S1, S2 and S3, respectively A. The Pip < 0.05), with a relative abundance accounting for 66–82% of the entire eukaryotic microalgal community in the seawater samples, while it only accounted for 33–42% in the sea ice samples, although the relative abundance was still high. The relative abundances of Dinoflagellata and Chlorophyta were higher in the sea ice samples, accounting for 14–24% and 26–43%, respectively, while the relative abundances in the seawater samples were lower, accounting for 4–14% and 11–18%, respectively. In addition, Prymnesiophyceae, Ochrophyta, and Cryptophyceae were also detected in the seawater and sea ice samples, and were more dominant in the sea ice samples, but their relative abundances were lower, accounting for 0.4–1.4%, 4.1–8.2% and 2.4–3.3%, respectively. The relative abundances of Chlorophyta, Ochrophyta and Cryptophyceae in the sea ice samples were significantly higher than those in the seawater (p < 0.05), while no significant difference was seen for Dinoflagellata and Prymnesiophyceae (p > 0.05).A total of six eukaryotic microalgal clades were identified, and the eukaryotic microalgal community in the sea ice and in the seawater was similar in composition at the phylum level A; only DChaetoceros, and the relative abundance of Chaetoceros in the seawater samples was significantly higher than that in the sea ice samples (p < 0.05). The relative abundance of Chaetoceros was highest at S2, at 78%, and decreased sequentially at S1 and S3, at 72% and 67%, respectively. The relative abundance of Chaetoceros in the sea ice decreased compared to that in the seawater, but it was still the most abundant algal genus at S1, at 37%, and lower at S2 and S3, at 20% and 16%, respectively. The relative abundance of Bathycoccus was only lower than Chaetoceros in the sea ice at S1, at 20%, and was highest at S2 and S3, at 35% and 30%, respectively; in the seawater, its relative abundance was significantly lower (p < 0.05), at 11%, 9% and 9% at S1, S2 and S3, respectively. The relative abundance of Micromonas was significantly higher in the sea ice than in the seawater (p < 0.05), at 6%, 10% and 11% in the sea ice at S1, S2 and S3, respectively, and 5%, 2% and 2% in the seawater, respectively.At the genus level B, the euBased on the Bray–Curtis distance analysis, PCoA was used to evaluate the overall differences between the sea ice and the seawater eukaryotic microalgal community . PCo1 anChaetoceros was relatively more abundant in the seawater, while Prasinophytae, Teleaulax, Subclade_A, Trebouxiophyceae, Bolidomonas, Haptolina, Thalassiosira, Micromonas, Karlodinium, Leptocylindrus, Bathycoccus were more abundant in the sea ice. The most significant differences in dominant genera between the seawater and the sea ice samples were Chaetoceros, Bathycoccus and Micromonas (p < 0.05)  .Chaetocerps showed a significant positive correlation with temperature (p < 0.05); Bathycoccus showed a significant positive correlation with NO2− and SiO32− (p < 0.05), and a highly significant negative correlation with temperature (p < 0.01); Prasinophytae was significantly positively correlated with NH4+ and NO2− (p < 0.05), and highly significantly negatively correlated with temperature (p < 0.01); Subclade_A and Trebouxiophyceae were both negatively correlated with temperature (p < 0.05)  .p < 0.05). The relative abundance of Chaetoceros was greatest in the seawater at the genus level, but decreased significantly in the sea ice (p < 0.05).Cold surges cause nearshore seawater to freeze, and the phase change process of seawater icing can affect the metabolic activities of the eukaryotic microalgae, forcing them to adapt to a new lifestyle in sea ice. In this study, the similarity of eukaryotic microalgal community in the sea ice and in the seawater was higher than the effects arising from the spatial distance of the samples, and the difference in living environments due to the sea ice formation was an important factor in altering the eukaryotic microalgal community. In addition, this study found that the composition differences between the eukaryotic microalgal community in seawater and in sea ice at the phylum and genus levels were not significant, possibly due to the short existence of the sea ice, and because the microalgal cells in the sea ice may have still maintained their metabolic activities in the open water ,42. The Chaetoceros has been found to be a central algal genus in both seawater and sea ice in the Arctic, and the sea ice environment is also favorable for the growth of the sea ice diatom Chaetoceros [Chaetoceros was also the most abundant algal genus in the surveyed area, but the relative abundance of Chaetoceros in the sea ice was significantly lower than in the seawater, which was different from the findings of the polar sea ice study. This difference may have been because our sampling site was located in mid-latitude, where the seawater does not normally freeze, and the diatoms may not have adapted to the dramatic environmental changes in a short period of time, due to the sudden cold surge that caused the freezing of the seawater. Bathycoccus was the dominant genus in the sea ice, and is also the main eukaryotic microalgae in polar regions [Bathycoccus in sea ice, as evidenced by a positive correlation between Bathycoccus and nutrient salt concentrations, especially a significant correlation with nutrient NO2− and SiO32− [Micromonas was significantly higher in the sea ice than in the seawater (p < 0.05), and it is also widely distributed in polar sea ice [2 concentration has been shown to affect the development of the eukaryotic community [Micromonas will significantly increase at high CO2 concentrations [pCO2 in the sea ice was higher than in the seawater (except at S1), Micromonas exhibited a higher relative abundance in the sea ice at high CO2 concentrations.etoceros ,44. In t regions ,46: it p regions ,48. Nutrd SiO32− . In addi sea ice . CO2 conommunity . In parttrations . As pCO2Chaetoceros; and the results of the reduced relative abundance of Chaetoceros in the sea ice differed from those in the Arctic study, probably because the sampling site of this study was in the mid-latitude coastal waters, where the existing time of seawater icing was short, and Chaetoceros may have not yet adapted to the rapid environmental changes. The eukaryotic microalgal community in the sea ice was dominated by Bathycoccus and Micromonas, which had more opportunities to grow in the sea ice with, respectively, higher nutrient and CO2 concentrations. Due to the limited investigation in this study, it is unknown how a eukaryotic microalgal community evolves during the formation and melting of sea ice induced by cold surges. Therefore, we suggest that future studies may consider conducting time series surveys, to understand the effects of cold surges on eukaryotic microalgal communities in mid-latitude waters more comprehensively.Against the background of global climate change, nearshore seawater icing in mid-latitude waters is likely to become the new normal. In this study, we investigated the differences between a eukaryotic microalgal community in seawater and in sea ice in Aoshan Bay, Qingdao, after a cold surge. The compositions of the eukaryotic microalgal community in seawater at the phylum and genus levels were similar to those in the sea ice, but their relative abundances were significantly different, which may be related to the environmental differences caused by the phase change pressure of seawater icing. The eukaryotic microalgal community in the seawater was dominated by the diatom


The alpine insect fauna of Aotearoa/New Zealand is unusual in that many species survive freezing solid at any time of the year. The physiological mechanisms that operate are not well-understood, but we do know the challenges imposed on cells when liquid water expands to form a solid (ice). Insects that are freeze tolerant start freezing at relatively warm temperatures with the help of ice nucleating agents that foster the slow formation of ice crystals outside cells. Here we consider the potential role of microbes living in the guts of alpine Aotearoa/New Zealand insects to facilitate this widespread ability to tolerate freezing. Many bacteria and fungi produce ice-nucleating agents that mediate formation of ice crystals at temperatures just below 0 °C. Some encourage ice formation on foliage with subsequent tissue damage, allowing microbial access to plant nutrients and causing crop damage. Other roles for ice-nucleating agents may include more positive outcomes for insects by facilitating controlled freezing at relatively warm temperatures.Insects that are freeze-tolerant start freezing at high sub-zero temperatures and produce small ice crystals. They do this using ice-nucleating agents that facilitate intercellular ice growth and prevent formation of large crystals where they can damage tissues. In Aotearoa/New Zealand the majority of cold adapted invertebrates studied survive freezing at any time of year, with ice formation beginning in the rich microbiome of the gut. Some freeze-tolerant insects are known to host symbiotic bacteria and/or fungi that produce ice-nucleating agents and we speculate that gut microbes of many New Zealand insects may provide ice-nucleating active compounds that moderate freezing. We consider too the possibility that evolutionary disparate freeze-tolerant insect species share gut microbes that are a source of ice-nucleating agents and so we describe potential transmission pathways of shared gut fauna. Despite more than 30 years of research into the freeze-tolerant mechanisms of Southern Hemisphere insects, the role of exogenous ice-nucleating agents has been neglected. Key traits of three New Zealand freeze-tolerant lineages are considered in light of the supercooling point  of microbial ice-nucleating particles, the initiation site of freezing, and the implications for invertebrate parasites. We outline approaches that could be used to investigate potential sources of ice-nucleating agents in freeze-tolerant insects and the tools employed to study insect microbiomes. Melanoplus sanguinipes grasshoppers in North America overwinter as eggs that enter diapause depending on population-specific environmental conditions [Within their natural ranges, organisms experience temperatures that fluctuate daily and seasonally with varying degrees of predictability . Life cynditions . The rannditions  but specMany insects couple diapause with freeze-avoidance mechanisms in their lifecycle by producing ‘anti-freeze’ proteins to defer mortality as winter approaches. These proteins, such as the β-helical protein of spruce budworm larvae , suppres2O varies with temperature; it is maximal as a liquid at about 4 °C and declines when either heated or cooled. Critically, water undergoes negative thermal expansion when it freezes because the open and ordered hexagonal crystalline structure of ice is less dense (occupying ~9% more volume) than liquid water, and so floats . This is why salt is used to deice roads in winter. However, even when cooled below the influence of solutes, water is only metastable and can remain liquid down to about –40 °C, at which point homogeneous nucleation occurs .Heterogeneous nucleation caused by ice-nucleating agents is common in biological systems and can occur at temperatures just a few degrees below the freezing point depression A. When cC) or nucleation temperature, and approximates to the supercooling point, although TC is usually slightly lower. TC can be measured because the phase change releases heat due to the exothermic nature of the process, and this can be detected as latent heat  agents is unclear. Ice formation in freeze-tolerant insects is typically initiated by ice+ in the lumen of the gut and therefore outside of gut cells, and this facilitates slow ice growth after minimal supercooling [Elevation of the temperature of crystallisation can be detected in haemolymph extracted from insects , but thercooling . Water ircooling ,25,26.Pyrrharctia isabella), and linden bug (Pyrrhocoris apterus) undergo passive ice inoculation from outside their bodies [+), triggering ice formation at temperatures just below 0 °C. For example, snow makers on ski fields use ice+ particles harvested from bacteria  to initiate crystallization of water [The best agent of ice nucleation is another ice crystal, and some freeze-tolerant insects such as the North American woolly bear caterpillar  agents that arrange water molecules into stable embryo crystals at high sub-zero temperatures  using ice-nucleating (iceto −2 °C ) and limto −2 °C . At thesIce recrystallization-inhibiting proteins are also found in some freeze-tolerant animals including some nematodes . These p+ particles produced directly by insects are usually large, heat-sensitive proteins [c) below −6 °C, as recorded by detection of an exotherm . In soma alpina ) remain venaceum ) creatinarticles .+ agents are apparently found all over the world in many different habitats, but are they really the same? For example, the cosmopolitan fungus Fusarium is found in soils, and in water, on plants, and in clouds. When more than 100 strains of this fungus were studied to characterise their ability to catalyse ice formation, only ~16% of strains caused water crystallisation above −12 °C [− strains. The macromolecules (<100 kDa) produced by Fusarium are ice+ active and stable after the fungus has died, but they are heat sensitive and lose ice+ activity after reaching 40 °C. The bacterium Pseudomonas syringae is also ubiquitous [+ and ice− strains [Many of the bacteria and fungi acting as icee −12 °C . Strainsiquitous  but incl strains .Zophobas morio) larvae, live Bacillus bacteria that can decompose polystyrene, and free nutrients to the insect [Schistocerca gregaria [+ activity is greater in the gut contents of many insects than in their haemolymph [The concept of the extended phenotype  considere insect . Howevere insect . Some ine insect  and a gugregaria . The congregaria . For exagregaria . In insegregaria ,30,59, aemolymph ,61  collected from the same sub-Antarctic island were both host to gut Pseudomonas sp. with ice+ activity [Hippodamia ladybird beetles freeze at −16 °C but after experimental feeding with ice+ bacteria they froze at just −3.5 °C [+ bacteria to plant leaves. In contrast, freeze-tolerant beetle and moth species play host to specific ice+ microbes that facilitate controlled high-temperature freezing allowing these insects to survive cold conditions [Insects can shape their gut microbiota by ingesting food containing beneficial microbes or ice species . For exaactivity . Experim −3.5 °C   , and to se flies ,68). In se flies . Fusariurpillars  and is sBlattabacterium bacteria via frass [Riptortus pedestris, which does not transmit its Burkholderia symbiont vertically from mother to offspring [+ agents [Bacteria that colonise only the insect gut can nevertheless be transmitted vertically in a number of ways  includinia frass . It is affspring . Thus, t+ agents . There iIn the New Zealand alpine zone, a profusion of separate endemic insect lineages ,75 can sCelatoblatta comprises a radiation of more than a dozen species endemic to New Zealand [This small alpine cockroach of Otago, New Zealand (maximum mass about 130 mg) A lives a Zealand , and sevCelatoblatta quinquemaculata freeze at the relatively high temperature of −5.4 °C and have a lower lethal temperature of −8.9 °C [C. quinquemaculata freeze  is the same as the temperature at which some bacterial ice+ agents ensure ice formation occurs. Although ice+ agents have been found in insect haemolymph, C. quinquemaculata ice+ activity is strongest in the frass, gut contents, and gut [C. quinquemaculata specimens examined had the nematode Blatticola barryi in their gut [C. quinquemaculata is no exception. In the DNA samples we detected high proportions of the expected obligate mutualistic endosymbionts from the family Blattabacteriaceae [ −8.9 °C   that freezes at −4.2 °C  [+ agents. However, New Zealand alpine grasshoppers appear to eat little grass [+ proteins of Chionochloa tussock grasses initiate freezing at a lower temperature than the grasshopper freezes, this is probably not a source of ice+ for S. australis.The alpine grasshoppers of New Zealand can also survive freezing at any time of the year (Pers. Obs.) . Althoug −4.8 °C , suggestnd −3 °C . The lownd −3 °C . New Zeand −6 °C  and potend EAF2)  providinle grass , and as + agents to control and tolerate freezing . Motif matching can identify candidate ice-binding regions in novel sequences; however, with non-model organisms such as these insects there may be few homologues in existing databases and so matching against transcriptomes derived from each group of animals is likely to be important for protein characterisation.If icegenomics , then thechnique  and char+ agents may constitute a relatively small proportion of expressed genes in each sample, enrichment for messenger RNA is an important step. For microbial studies subtractive, hybridization is generally used for depletion of ribosomal RNA, but use of biotinylated oligonucleotides specific to 5S, 16S, and 28S rRNA is an emerging option [+ proteins. Both functional and compositional convergence of the microbiomes of different hosts is possible. Functional information can be extracted by profiling presence/absence and abundance of microbial pathways in a community, and gene ontology analysis will reduce complexity and highlight biological processes. The functional Kyoto Encyclopaedia of Genes and Genomes orthology (KEGG) database provides opportunities for studying functional convergence [RNA extracted and sequenced from gut contents can be used to assemble the meta-transcriptomes of host microbiomes. This allows the study of gene expression across a whole complex of microbial communities within their natural environment. Genes with specific roles can be identified , and theg option . Insect vergence .Meta-transcriptomics is used primarily for functional analysis of genes expressed in the gut microbiome but can also be used for comparing the taxonomic composition of the microbial community. Despite depletion, rRNA sequences are very abundant in all organisms and likely to be represented among data, and these suit taxonomic analyses of community composition ,112.+ agents. By mapping reads from each tissue sample before and after freezing to assembled transcriptomes [Differential gene expression experiments comparing mRNA extracted from a range of tissue samples before and during freezing is a powerful tool for identifying iceriptomes , (after riptomes  and find


Today, an estimated 7.3% (50 million) of all children <5 y of age suffer from wasting, with more burden in African countries including Guinea. Investigating inequalities in childhood wasting is essential for designing efficient programs and interventions, but no related evidence exists in Guinea. This study aimed to examine the trends in the prevalence of childhood wasting and the extent of sex, socio-economic and geographic-based disparities in Guinea.Data from the 1999, 2005 and 2012 Guinea Demographic and Health Surveys and the 2016 Guinea Multiple Indicator Cluster Survey, with a total of 16 137 children <5 y of age were included for analysis. For inequality analysis, we used the 2019 updated World Health Organization Health Equity Assessment Toolkit (HEAT) software. Inequality was measured using four summary measures  for five equity stratifiers . We computed 95% confidence intervals (CIs) around the points estimates to measure statistical significance.The findings revealed a pro-rich (R=1.68 [95% CI 1.11 to 2.24]), pro-urban (PAR=−1.04 [95% CI −1.90 to −0.18]) and subnational region (D=8.11 [95% CI 4.85 to 11.36]) inequalities in childhood wasting across all surveys. Except in 2005, education-based disparities (PAF=−18.2 [95% CI −36.10 to −0.26]) were observed across all survey years, but not sex-based disparities. An approximately constant inequality pattern was seen across all dimensions.This study showed inequalities in childhood wasting in Guinea with a disproportionately higher risk of wasting among children from disadvantaged subpopulations/mothers, including uneducated, poorest/poor, rural residents and regions. Policies that target disadvantaged populations need to be considered in order to ensure social protection, access to a wholesome diet and universal and quality health services. For instance, in 28 May 2013, the Republic of Guinea joined the Scaling Up Nutrition movement with a letter of commitment from three ministers: Health, Agriculture and Social Welfare.14–17 and maternal education.19–22 Socio-economic disparities in childhood wasting are prevalent in SSA,19–22 but little is known about gender and geographic disparities in childhood wasting. In addition, there is a dearth of evidence about inequalities related to childhood wasting in Guinea. Therefore, measuring inequalities between subgroups may inform policies, programs and practices to promote better health among the disadvantaged.24However, the country has not made progress. Therefore, beyond these initiatives, it needs further investigation of other factors, including inequalities. Prior research showed that childhood wasting varies significantly by socio-economic status (SES), including household wealth statusThus the aim of this study was to examine the magnitude and trends in the prevalence of childhood wasting among subpopulations by economic status, education, place of residence, sex and subnational regions in Guinea from 1999 to 2016. Furthermore, we assessed the dynamics of socio-economic, sex and geographic inequalities in childhood wasting in Guinea from 1999 to 2016.25–27 and samples of men and women in their reproductive age, and they provide an adequate representation of urban and rural settings. The surveys also covered all eight administrative regions .The data for this study were from three waves of the Guinea Demographic and Health Survey  and one wave of the Guinea Multiple Indicator Cluster Survey . The GDHS and GMICS were conducted by the National Institute of Statistics of the Ministry of Planning in collaboration with the United States Agency for International Development (USAID) and the United Nations Children's Fund, respectively, with technical assistance from Inner-City Fund International. GDHS and GMICS are highly comparable nationally representative data sources that permit direct comparison between them28–30 The analysis was carried out on 16 137 children <5 y of age preceding the respective surveys.Both the GDHS and GMICS employed a two-stage stratified cluster sampling technique. First, clusters or enumeration areas (EAs) were selected across the entire nation from a list of EAs established in the most recent census. The second stage involved household sampling, where 25–30 households were selected in each cluster.4 For children <5 y of age, a WHZ <−2 SDs from the WHO reference population was coded 1 and a WHZ between −2 SDs and +5 SDs was coded as 0.4 Children with WHZ <−5 SDs or >+5 SDs were considered as having invalid data and were excluded from the analysis. Children who were not weighed and measured and children whose values for weight and height were not recorded were excluded. Children whose month or year of birth was missing or unknown were flagged and excluded. Children whose day of birth was missing or unknown were assigned day 15. Children who were flagged for out-of-range z-scores or invalid z-scores were excluded.32Wasting was the outcome variable and was measured as the weight-for-height z-score (WHZ) <−2 standard deviations (SDs) from the median of the World Health Organization (WHO) child growth standard.33 The selection of these five dimensions of inequality (equity stratifiers) was because these equity stratifiers represent common sources of discrimination and can be widely applied to populations in low- and middle-income countries.34 In the GDHS and GMICS, the wealth index is usually computed using durable goods, household characteristics and basic services, following the methodology explained elsewhere.35Inequality in wasting was measured using five equity stratifiers: economic status, education, place of residence, sex and subnational region. Economic status was approximated by a wealth index.The constructed wealth index was further categorized into five quintiles: from poorest (quintile 1) to richest (quintile 5). Maternal education status was classified as no education, primary education and secondary education or more. Place of residence was classified as urban or rural and child sex was categorized as male or female. The subnational region included the eight regions in the country .37 A detailed discussion of the software is available elsewhere.37 We used this equity assessment toolkit to examine socio-economic and geographic inequalities in childhood wasting following two steps. First, the prevalence of wasting was disaggregated by five equity stratifiers  across different subpopulations. Second, inequality was assessed using four measures of inequality: difference (D), population attributable risk (PAR), population attributable fraction (PAF) and ratio (R).The analysis was conducted with the latest version of the WHO Health Equity Assessment Toolkit (HEAT) software. This software is used to investigate health inequalities within and between countries for >30 reproductive, maternal, newborn and child health indicators.38 This is deemed essential because of the likelihood of obtaining different and even contrasting conclusions,38 which can lead to bias-informed decisions.38 Details about summary measures and the methods for calculating the summary measures and subsequent interpretation adopted in this study have been described elsewhere39 and are available in 40R and PAF are relative measures, while D and PAR are absolute summary measures. The selection of these simple and complex summary measures was based on evidence that supports the scientific significance of using both absolute and relative measures in studies involving a single health inequality.Ethical approval was not required because the study used publicly available GDHS and GMICS data. All DHS and MICS surveys are approved by Inner City Fund (ICF) International as well as an institutional review board (IRB) in the respective country to ensure that the protocols are in compliance with the US Department of Health and Human Services regulations for the protection of human subjects.In this study, we examined the magnitude and trends in inequality in childhood wasting in Guinea from 1999 to 2016. Overall, a total of 16 137 children <5 y of age were included in all four surveys. Of these, 11 387 (70.6%) were rural residents. More than three-fourths (76.3%) of the respondents had no formal education and 7865 (48.7%) were female.Appendix 2 shows the magnitude and trends of childhood wasting across subpopulations from 1999 to 2016. We observed a lower prevalence among advantaged groups, such as richest, educated mothers, urban residents and from regions such as Mamou (in 2016). For example, significantly higher proportions of childhood wasting were found among children in quintile 1 (poorest) and quintile 2 (poorer) in 2012 and 2016 compared with quintiles 5 (richest) and quintile 4 (richer), respectively , compared with 7.0 pp (95% CI 5.93 to 8.33) among urban children  compared with 7.5 pp (95% CI 6.50 to 8.73) among female children Figure .Furthermore, we found significant differences in the prevalence of childhood wasting across subnational regions in all surveys. For example, in 2016 the prevalence of wasting among children living in the Nzérékoré region was higher by 8.1 pp (95% CI 4.85 to 11.36) when compared with children living in the Mamou region. The pattern of wasting prevalence varied over time across regions. In Kankan region, for instance, the prevalence of wasting decreased from 17.9 pp (95% CI 14.94 to 21.24) in 2012 to 6.8 pp (95% CI 5.27 to 8.72) in 2016. Meanwhile, in the Boke region, it remained fairly constant, as 8.5 pp (95% CI 6.03 to 11.72) was observed in 2012 and 9.2 pp (95% CI 7.53 to 11.22) was observed in 2016  and in 2016 (−2.48 [95% CI −3.74 to −1.23]) indicated significant absolute economic inequality in childhood wasting, with a fairly constant pattern over time. Similarly, the R measure in 2012 (1.76 [95% CI 1.06 to 2.47]) and 2016 (1.68 [95% CI 1.11 to 2.24]) indicated substantial relative wealth-driven inequality in childhood wasting over time.The result showed substantial absolute and relative education-related disparities by the complex measures (PAF and PAR), except in 2005. The simple measures (D and R) did not show education inequality across all surveys, except D in 1999. Generally the overall pattern of education inequality over the last 17 y was fairly constant, although the inequalities disappeared in 2005.Complex measures (PAF and PAR) showed an urban–rural disparity in childhood wasting in 2012 and 2016. For instance, the PAF measure in 2012 and 2016  indicated relative disparities in childhood wasting. Moreover, the PAR measure in 2012 and 2016  indicated absolute residence disparities in childhood wasting.The findings revealed no sex-related absolute or relative inequality in childhood wasting. For instance, the PAR measure in 1999, 2005, 2012 and 2016  supported the absence of sex-related absolute disparities across all the survey years.Subnational region inequality in wasting was observed from 1999 to 2016 using both simple (D and R) and complex (PAF and PAR) measures, with a fairly constant pattern over time. For example, the PAR measures in 1999, 2005, 2012 and 2016  indicated substantial absolute regional inequality in childhood wasting, with a fairly constant pattern over time. Similarly, the R measures in 1999, 2005, 2012 and 2016  indicated significant relative regional disparities in childhood wasting, with a fairly constant pattern over time.20In this study, we assessed the prevalence of childhood wasting across different subpopulations as well as trends in socio-economic, sex and geographic inequalities in childhood wasting, using nationally representative GDHSs and GMICSs from 1999 to 2016. Our findings showed a childhood wasting prevalence of 10.1%, 11.2%, 9.6% and 8.1% in 1999, 2005, 2012 and 2016, respectively. Socio-economic and geographic inequalities in childhood wasting were found across all survey years, but varied on summary measures.13 Ghana,21 Mozambique22 and SSA,41 though the magnitude was small, we found education-related inequality in childhood wasting favouring children born to educated mothers. Parental education, more specifically, maternal education, has been shown to have significant effects on child health.42 Health-seeking behaviour and utilization for promoting child health, including the nutrition status of children, are mostly practiced among educated mothers.44 Previous studies in Mozambique and Uganda showed that maternal education is significantly associated with childhood nutritional status.42 Maternal education may influence childhood nutrition in many ways, including decisions on the allocation of resources for household food consumption and other health-related issues.45Consistent with previous findings from studies in Nigeria.21 Mozambique22 and SSA41 reported similar findings. Differences in household income level, areas of residence, parental education and occupational status and type could explain variations in wasting based on SES.46 Moreover, food security47 in the household, hygiene and sanitation practices and healthcare service accessibility and utilization46 are largely dependent on SES.47 This is supported by previous studies in Latin America and Nigeria,48 where improvement in the SES of women in areas such as education, conditional cash transfer, income level, food security and urbanization has significantly reduced childhood malnutrition, including wasting.48We further found pro-rich inequality in childhood wasting in Guinea over time. Previous studies in Ghana,13 Inequalities in the place of residence in childhood wasting have also been linked with differences in SES.46 Low SES, including poverty, illiteracy and unemployment, are more common in rural settings,4 and residents may be at risk of ill health.49 A higher prevalence of wasting among rural residents may therefore be seen as a deterioration in health conditions and poor access to healthcare services,13 consequently widening the urban–rural gap in health.13 Nonetheless, there is evidence that urban–rural disparities in child health could be reduced when socio-economic inequalities are addressed.50Pro-urban inequalities in childhood wasting were observed, consistent with a previous study in Nigeria.51 The higher proportion of uneducated mothers45 and the urban–rural nature of the provinces52 in the country may explain the variation in childhood wasting prevalence across regions.52 There is evidence that wasting may be caused by a reduced intake of food,53 therefore the regional difference may be partly due to seasonal food insecurity that is related to natural disasters such as extreme poverty, crop damage by locusts and shifts in the practice of agriculture.55 A study conducted in Ethiopia confirmed a strong link between regional variations of malnutrition and dissimilarity in cultivated areas.56 The issue of food insecurity in Guinea is not surprising in light of the Ebola crisis. In 2013, 1 million of Guinea's 11.75 people were food insecure and 2.85 million were borderline food insecure. Food insecurity in Guinea varies between urban and rural residents, with rural residents being three times more likely to be food insecure. For example, in Faranah, 40.6% of households are facing food insecurity.57Finally, we found significant regional inequality in childhood wasting across all the survey years, consistent with a previous study.59However, food security does not always guarantee nutritional security, but it can be a precursor for nutritional improvements so long as the household properly manages the available food. Other factors such as infectious diseases might be another reason for regional variations in the magnitude of childhood wasting.This study has both strengths and limitations. First, the study assessed not only trends of inequality, but also trends of prevalence across different subpopulations using nationally representative data. Second, the study used the recommended WHO Health Equity Monitor database through HEAT software to analyse inequality. Third, we used simple and complex as well as absolute and relative summary measures to assess the inequality of different dimensions. By this approach, policymakers can view problems from different perspectives and provide appropriate interventions. The main limitation of this study was the inability to explore other factors related to disparities in wasting.Our study provides the first evidence of trends in socio-economic, sex and geographic-based inequalities in childhood wasting. With the exception of education-related inequality in 2005, we found absolute and relative socio-economic and geographic-related disparities in childhood wasting that favoured children from advantaged subpopulations, including rich/richest, educated mothers and children living in urban settings and regions such as Mamou in 2016. Although inequalities varied based on different summary measures, the pattern generally remained constant over time. Policies targeting disadvantaged populations need to be considered. This can help to ensure social protection, access to a healthy diet throughout the life course and universal and quality health services, which may in turn reduce wasting by addressing drivers such as poor hygiene and sanitation and household food insecurity that may be aggravated during emergencies, outbreaks of communicable diseases and disasters. Moreover, programs and interventions against childhood wasting need to be strengthened and prioritized for children of uneducated women, those from poor homes, rural residents and those in disadvantaged regions of Guinea. Nutrition education for disadvantaged mothers may have to highlight the need for inexpensive balanced and nutritious meals within their communities.ihac002_Supplemental_FilesClick here for additional data file.


Left ventricular non-compaction cardiomyopathy (LVNC) is one of the most common inherited cardiovascular diseases. The genetic backgrounds of most LVNC patients are not fully understood. We collected clinical data, family histories, and blood samples and performed genetic analysis using next-generation sequencing (NGS) from a Chinese family of 15 subjects. Clinically LVNC affected subjects showed marked cardiac phenotype heterogeneity. We found that these subjects with LVNC carried a missense heterozygous genetic mutation c.905G>A (p.R302Q) in γ2 subunit of AMP-activated protein kinase (PRKAG2) gene through NGS. Individuals without this mutation showed no symptoms or cardiac structural abnormalities related to LVNC. One subject was the victim of sudden cardiac death. To sum up, PRKAG2 mutation c.905G>A (p.R302Q) caused familial LVNC. Our results described a potentially pathogenic mutation associated with LVNC, which may further extend the spectrum of LVNC phenotypes related to PRKAG2 gene mutations.The online version contains supplementary material available at 10.1186/s12920-022-01361-2. Left ventricular non-compaction cardiomyopathy (LVNC) is a rare disease caused by abnormalities in the normal myocardial compaction process. Both sporadic and familial LVNC have been described , 2. In f2 subunit of the AMP-activated protein kinase (AMPK)  which might to be the potentially pathogenic mutation. To our knowledge, this is the first study to describe the association of this mutation with LVNC.LVNC, first described by Grant in 1926 , is a heThe American Heart Association (AHA) classifies LVNC as a separate genetic cardiomyopathy, but the European Society of Cardiology (ESC) defines it as an unclassified entity . It is sAmong familial diseases, LVNC is a genetically heterogeneous disease that usually shares mutations in genes encoding for sarcomere proteins . CurrentThe proband’s echocardiography showed uneven thickening of the left ventricular wall, most notably in the basal segment of the ventricular septum. The lateral wall of the left ventricle middle segment and the lateral wall of the apical part of the heart were rich in myocardial trabeculae and were increased and thickened. Previous trials have collected a total of 242 children diagnosed with isolated LVNC, and 66 patients (27%) presented with hypertrophic . HypertrThe proband and her young sister presented with short PR and sinus bradycardia. PRKAG2 cardiac syndrome may include conduction system disease, and an increased risk of SCD. It was reported that the short PR interval was largely attributed to excessive glycogen accumulation in myocytes and presented in 68% of PRKAG2 syndrome patients . Sinus bThe proband’s sister and son (II-4 and III-1) manifesting with LVNC carried the mutation. The proband’s brother (II-5) died of SCD, thus we concluded that the proband’s brother may have suffered from LVNC and carried the same mutation. Our predictive genetic testing identified an asymptomatic individual (III-3) who was 6 years old and carried the disease-causing mutation, but unfortunately, he was so young that he could not cooperate to complete the CMR. His associated clinical symptoms have not yet expressed. Therefore, on the basis of our genetic diagnosis of LVNC in this family, we will facilitate better disease management and follow-up by clinicians before the appearance of symptoms. Bioinformatic prediction can provide us with some useful information about the pathogenicity of the PRKAG2 mutation c.905G>A (p.R302Q). However, our experiments do not reflect the true pathology of this mutant in cardiac myocytes. Due to the inaccessibility of human heart tissues, we were unable to obtain sufficient numbers of patient heart tissues. The creation of mutant mice using CRISPR/Cas9 gene editing methods or the use of patient-specific induced pluripotent stem cell (iPSC) derived cardiomyocytes are state-of-the-art methods to directly and reproducibly study the pathogenicity of human mutants. In the future, we will further investigate the pathogenic role of the mutation using cardiomyocytes derived from patient-specific iPSCs.We reported a missense heterozygous mutation of PRKAG2 [c.905G>A (p.R302Q)] in a Chinese family that presented with LVNC. The present study confirmed the genotype-phenotype correlation of the PRKAG2 mutation and provided further insight into the genetic factors underlying LVNC pathology. In addition, our findings demonstrate that CMR and NGS assays are effective methods for identifying pathogenic mutations associated with hereditary cardiomyopathies.Below is the link to the electronic supplementary material.Supplementary Material 1


Z)-5-(substituted benzylidene)-3-phenyl-2-thioxothiazolidin-4-one ((Z)-BPTT) analogs were designed based on the structural features of two potent tyrosinase inhibitors, viz. (Z)-5-(3-hydroxy-4-methoxybenzylidene)-2-thioxothiazolidin-4-one (5-HMT) and (Z)-2-benzoimidazothiazol-3(2H)-one (compound I). The trisubstituted double bond geometry of the (Z)-BPTT analogs that were generated by Knoevenagel condensation was determined using vicinal 1H and 13C coupling constants in 13C NMR spectra. Four analogs, numbers 1–3 and 6, inhibited mushroom tyrosinase 9 to 29 times more potently than kojic acid did. Kinetic study results indicated that these four analogs inhibited mushroom tyrosinase competitively and this was supported by docking simulation. Also, docking results using human tyrosinase suggested that analogs 2 and 3 might be potent human tyrosinase inhibitors. In vitro studies using B16F10 cells (a melanoma cell line) showed that analogs 1, 2, 3, and 6 inhibited cellular tyrosinase and melanin production more than kojic acid did, without perceptible cytotoxicity. In particular, analog 2, which possesses a catechol group, exerted an extremely potent anti-melanogenic effect. In addition, analog 2 showed strong scavenging activity against DPPH and ABTS radicals. Furthermore, analog 2 not only reduced ROS levels, which induce melanogenesis, but it also suppressed tyrosinase and MITF  protein levels and the expressions of melanogenesis-related genes. These results suggest that analog 2 is an efficient tyrosinase inhibitor that alleviates melanogenesis by dual mechanisms of (i) the inhibition of melanogenesis-related proteins and genes and (ii) the direct inhibition of tyrosinase activity.The rate-determining role of tyrosinase makes it a critical component in the mechanism that is responsible for melanogenesis. Thirteen ( The mode of action of each analog was determined using the convergence point of the four plot lines.The Michael constants   was downloaded from the Protein Data Bank (PDB) database into Maestro 12.4 Protein Preparation Wizard. The crystal structure of mTYR was processed and the unwanted protein chains were removed. In order to optimize the structure, hydrogen atoms were added and water molecules more than 3 Å away from the enzyme were removed. The Glide grid and the active site of the enzyme was determined using the binding site of the ligand (tropolone) as described by the PDB and literature , incubated on ice for 20 min, and then centrifuged at 12,000 rpm for 10 min at 4 °C. The supernatants that were obtained contained cytosolic fractions. The pellets were resuspended in buffer B , incubated on ice for 30 min, and then centrifuged at 12,000 rpm for 20 min at 4 °C. The resultant supernatants contained nuclear fractions.B16F10 cells were treated with five concentrations  of analog ® West Pico Chemiluminescent Substrate kit  and the Davinch-Chemi™ . The western blot data were quantified using CS Analyser 3.2 (Densitograph) image analysis software .Protein concentrations were determined using a BCA™ protein assay kit . Briefly, lysed samples were boiled for 10 min in gel-loading buffer  at a volume ratio of 1:1. The total protein equivalents were separated by SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) using 9% acrylamide gels and then transferred to polyvinylidene fluoride (PVDF) membranes  at 25 V for 10 min using a semi-dry transfer system . The membranes were immediately placed in blocking buffer (5% non-fat milk) in TBST (10 mM Tris (pH 7.5), 100 mM NaCl, and 0.1% Tween-20 at room temperature for 1 h. The membranes were then incubated with the appropriate specific primary antibodies at 4 °C overnight and treated with horseradish peroxidase-conjugated anti-mouse or anti-goat antibodies (1:5000) for 1 h at 25 °C. The specific antibodies for tyrosinase, MITF, β-actin, and TFIIB were purchased from Santa Cruz Biotechnology . The protein bands were visualized using the SuperSignal® No-ROX dye  and a CFX Connect System . The qRT-PCR primers that are specifically for tyrosinase, TRP-1, TRP-2, and β-actin were obtained from Bioneer Inc. . The relative gene expressions were calculated using the standard curve method and β-actin as the internal control. The primer sequences are displayed in The total RNAs from the cells were purified using the RiboEx Total RNA solution  and the cDNA was synthesized from the total RNA (2 µg) using SuPrimeScript RT Premix with random hexamer , according to the manufacturers’ instructions. Real-time qPCR was performed using SensiFASTTM SYBRp-values of <0.05 were considered to be statistically significant.A one-way analysis of variance (ANOVA) followed by the Newman–Keuls test was used in order to assess significant differences between the treatments. The analysis was conducted using GraphPad Prism 5 software . The results are the means ± the standard errors of means (SEMs) and two-sided Z)-BPTT analogs that were examined have been previously synthesized from 3-phenyl-2-thioxothiazolidin-4-one  with the appropriate benzaldehydes [C-(dithiocarboxy)formic acid [Z)-BPTT analogs using the former method  generated analog 3 in a 30% yield. The double bond geometries of the (Z)-BPTT analogs were assigned using 13C NMR 1H,13C coupling constants that were obtained in proton-coupled mode. Nair et al. reported that vicinal 1H,13C-coupling constants could be used in order to determine the configuration of trisubstituted exocyclic C, C-double bonds [1H,13C-coupling constants of N-methyl-N,2,3-triphenylacrylamide depended on double bond geometry ((E)-isomer: 3cisJC(1), H-C(3))-isomer: 3transJC(1), H-C(3))( = 11.5 Hz) -BPTT analog 3 was performed in proton-coupled 13C mode and the vicinal 1H,13C-coupling constant (3JC(1), H-C(3)) of C1 was 6.6 Hz (3 had a (Z)-configuration.The  . Furthers 6.6 Hz , which i50 values of the (Z)-BPTT analogs were determined using the tyrosinase-inhibitory activities of the test compounds at ≥three different concentrations. All 13 of the (Z)-BPTT analogs inhibited tyrosinase activity in a concentration-dependent manner. The IC50 values are shown in The tyrosinase-inhibitory activities were screened against mushroom tyrosinase using L-tyrosine as a substrate, as previously described . Kojic a50 = 17.05 ± 3.82 μM). Interestingly, analog 1 (IC50 = 1.45 ± 0.04 μM), with a 4-hydroxyl group on the β-phenyl ring of the β-phenyl-α,β-unsaturated carbonyl (PUSC) scaffold, inhibited mushroom tyrosinase 12-fold more than kojic acid. Analogs 12 (IC50 = 65.54 ± 9.40 μM) and 13 (IC50 = 78.75 ± 8.63 μM)  had significantly lower tyrosinase-inhibitory activities than analog 1. Similarly, the introduction of an additional 3-methoxyl to the β-phenyl of analog 1 also reduced the tyrosinase-inhibitory activity . The insertion of an ethoxy group at position 3 of the β-phenyl of analog 1 further reduced the tyrosinase-inhibitory activity . Analog 4 moderately inhibited mushroom tyrosinase, but exchanging the 4-hydroxyl and 3-methoxyl groups on its β-phenyl ring greatly enhanced its inhibitory activity  by 43-fold. Notably, the insertion of additional alkoxyl or bromo substituents on the β-phenyl ring of analog 1 diminished its tyrosinase-inhibitory potency, whereas the introduction of an additional hydroxyl substituent at positions 2 or 3 of the β-phenyl ring of analog 1 either retained or enhanced its inhibitory activity . Analogs 7 and 10  with no hydroxyl group did not exhibit tyrosinase-inhibitory activity, whereas analogs 8 (IC50 = 94.29 ± 7.70 μM) and 9 (IC50 = 22.15 ± 4.23 μM), in which all of the hydroxyl groups of analogs 2 and 3 were replaced by methoxyl groups, exhibited weak to moderate activity. Of the 13 synthesized (Z)-BPTT analogs, analog 3 most potently inhibited mushroom tyrosinase and was 29-fold more potent than kojic acid. Four analogs, numbers 1, 2, 3, and 6, inhibited mushroom tyrosinase more than kojic acid.Kojic acid had potent tyrosinase-inhibitory activity (ICZ)-BPTT analogs are absorbed by skin, their log p (partition coefficient) values were examined using ChemDraw Ultra Ver. 12.0. The log p values of the 13 analogs are provided in p value of –2.45, whereas the (Z)-BPTT analogs had log p values ranging from 3.16 to 5.20. Analogs 2 and 3 with two hydroxyl groups on the β-phenyl ring of the PUSC scaffold had partition coefficients that were 105-fold greater than that of kojic acid (log p: 3.16 vs. –2.45). These results suggest that all 13 of the analogs are better absorbed by skin than kojic acid.In order to determine how well the  of each analog was independent of the analog’s concentration. On the other hand, the Michaelis–Menten constants (KM) increased upon the increase of the analogs’ concentrations  for the mushroom tyrosinase–analog complexes, the Lineweaver–Burk plots were converted into their corresponding Dixon plots -BPTT analogs 1–3 and 6 using the crystal structure of mushroom tyrosinase and a human tyrosinase (hTYR) homology model. Docking studies were conducted using Schrodinger Suite (release 2021-1). As the software did not provide docking results for analog 6, only the docking results for analogs 1–3 are provided in In silico studies were performed on  was imported from the Protein Data Bank database and docked with analogs 1–3 or kojic acid. The binding interactions between these compounds and mushroom tyrosinase are shown in 1–3 occupied the same active binding pocket as kojic acid. Analogs 2  and 3  had higher binding affinities than kojic acid , whereas analog 1  had a higher docking score than analog 2, analog 3, and kojic acid. Kojic acid and analog 2 interacted with two copper ions. The hydroxyl of kojic acid coordinated with Cu400 and Cu401 and the phenolic 4-OH group on the β-phenyl ring of analog 2 formed salt bridges with Cu400 and Cu401 at distances of 2.19 Å and 2.24 Å, respectively. Also, in the same manner as kojic acid, the phenyl ring of analog 2 interacted with His263 by pi–pi stacking. The phenolic 4-OH group of analog 3 also formed salt bridges with Cu400 and Cu401 at distances of 2.30 Å and 2.34 Å, respectively, and the β-phenyl ring of analog 3 formed two pi–pi interactions with histidine residues His259 and His263. In addition, the N-phenyl moiety of analog 3 formed a pi–cation interaction with Arg268. In analog 1, the β-phenyl ring formed a pi–pi interaction with His259, which led to a lower binding affinity than those of the kojic acid or analogs 2 or 3.In order to determine the binding affinities of analogs 1, 2, or 3 indicated that the interaction between the 4-OH of the β-phenyl ring of 1, 2, or 3 and the two copper ions contributed much to the binding affinity with mushroom tyrosinase. Also, the additional OH on the β-phenyl ring of analogs 2 and 3 at positions 2 or 3 appeared to enhance their binding affinity. Overall, (Z)-BPTT analogs 1, 2, and 3 appeared to inhibit mushroom tyrosinase directly by acting in the same manner as kojic acid at the active site of the mushroom tyrosinase.Comparisons of the binding interactions between mushroom tyrosinase and kojic acid or analogs 1–3 and kojic acid. The binding interactions are shown in 2D and 3D formats in 1–3 occupied the same active binding pocket as kojic acid. As was observed for mushroom tyrosinase, analogs 2  and 3  had lower docking scores than kojic acid  with the active site of the human tyrosinase homology model. Analog 1  had a much higher docking score than analog 2, analog 3, and kojic acid for the binding pocket of the human tyrosinase homology model. In kojic acid, the branched hydroxymethyl group coordinated with zinc (Zn7), the pyranone ring formed a pi–pi stacking interaction with His367, and 3-OH formed a hydrogen bond with Ser375. On the other hand, the 4-hydroxyl group on the β-phenyl ring of analog 2 formed two salt bridges with Zn6 and Zn7 at distances of 2.24 and 2.65 Å, respectively. Like kojic acid, the β-phenyl ring of analog 2 also interacted with His367 by pi–pi stacking and the 3-hydroxyl group on the β-phenyl ring formed a hydrogen bond with Ser380. Analog 3, like analog 2, formed two salt bridges with the two zinc ions of the human tyrosinase model at 2.21 Å and 2.32 Å, which were shorter than the salt bridges that were generated by analog 2. These shorter salt bridge distances may explain why analog 3 had slightly greater binding affinity than analog 2. In addition, analog 3 formed a pi–pi stacking interaction with His367, which was also used in the same manner by kojic acid and analog 2. As compared with analogs 2 and 3, analog 1 showed a reversed conformation in the active site of the human tyrosinase model. The β-phenyl rings of analogs 2 and 3 were located close to the zinc ions; whereas, in analog 1, the N-phenyl ring was located near these ions. Analog 1 only formed a pi–pi stacking interaction with His367 using its N-phenyl ring, which indicated that analog 1 has a lower binding affinity for human tyrosinase than analogs 2 or 3 or kojic acid. These results suggest that (Z)-BPTT analogs 2 and 3 are potential human tyrosinase inhibitors.Since no crystal structure was available for human tyrosinase in the PDB database, we first created a homology model of human tyrosinase based on tyrosinase-related protein-1 (TRP-1) so as to estimate the binding affinities between human tyrosinase and analogs Z)-BPTT analogs 1, 2, 3, and 6 exhibited greater mushroom tyrosinase-inhibitory activity than kojic acid, we selected these analogs for further study in cellular experiments. The cell viabilities of analogs 1–3 and 6 were evaluated in B16F10 murine melanoma cells. An EZ-Cytox analysis was performed in order to assess the cells’ viabilities. B16F10 cells were treated with six different concentrations  of each analog and incubated for 24 and 48 h in a humidified 5% CO2 atmosphere before adding the EZ-Cytox solution. The optical densities of the wells were measured at 450 nm using a microplate reader.Since the four  and 3-isobutyl-1-methylxanthine  were added sequentially in order to enhance melanin production. After incubation for 48 h at 37 °C, the melanin contents were assessed by measuring the well optical densities at 405 nm using a microplate reader. Kojic acid (10 or 20 µM) was used as a positive control.B16F10 cells were seeded in 6-well culture plates and pretreated with analogs 1–3 and 6 on melanin production are shown in 1, 3, and 6 at 5 µM were as effective as kojic acid at 20 µM and at 20 µM they reduced the α-MSH plus IBMX-induced increases in the melanin contents by 56.3, 61.3, and 50.9%, respectively, while kojic acid at 20 µM reduced the induced increase in the melanin content by only 22.9%. Analog 2 at 2.5 µM suppressed α-MSH plus IBMX-induced melanogenesis as effectively as kojic acid at 10 µM, and at 10 µM decreased α-MSH plus IBMX-induced increase in melanin content by 89.6%, which was 4.1-fold higher than the suppression that was yielded from kojic acid at 10 µM, which reduced the α-MSH plus IBMX-induced increase in melanin content by 21.8%. Notably, although analog 3 inhibited mushroom tyrosinase activity slightly more than analog 2, analog 2 inhibited melanogenesis much more than analog 3. These results suggest that analog 2 is a promising inhibitor of melanogenesis in mammalian cells.The inhibitory effects of analogs Z)-BPTT analogs on melanin production were due to the inhibition of cellular tyrosinase activity, we also examined the inhibitory effects of analogs 1, 2, 3, and 6 on cellular tyrosinase activity in B16F10 melanoma cells. Briefly, B16F10 cells were seeded in 6-well culture plates and pretreated with four different concentrations  of each of the four analogs for 1 h, and then the cells were co-stimulated with α-MSH and IBMX for 48 h at 37 °C so as to increase their cellular tyrosinase activity. Their optical densities (ODs) were then measured using a microplate reader at 492 nm in order to quantify the dopachrome production. The inhibitory effect of each analog on cellular tyrosinase activity was evaluated using these ODs. Kojic acid (20 µM) was used as a positive control.In order to determine whether the inhibitory effects of the . Notably, analog 3 inhibited mushroom tyrosinase-inhibitory activity slightly more than analog 2, while analog 2 inhibited the activity of B16F10 tyrosinase much more than analog 3. The tyrosinase inhibition pattern was similar to the melanin content reduction pattern that is shown in Z)-BPTT analogs 1–3 and 6 are mainly due to their tyrosinase-inhibitory activities.The measured tyrosinase-inhibitory activities are shown in Z)-BPTT analogs were investigated using l-ascorbic acid (vitamin C) as a positive control. The final concentrations of the analogs and of the positive control in the test samples and in the DPPH methanol solution were 1 and 0.18 mM, respectively. The scavenging activities of the analogs were assessed by measuring their optical densities at 517 nm after standing the samples in the dark for 30 min. Four (Z)-BPTT analogs, analogs 2–5, exhibited moderate to strong DPPH radical scavenging activities as compared with l-ascorbic acid . However, the insertion of an additional 3-alkoxyl group on the β-phenyl ring greatly increased the radical scavenging activity to 78.7% for analog 4 and 77.5% for analog 5. The radical scavenging activity was slightly improved when additional methoxyl groups were introduced at positions 3 and 5 of the β-phenyl ring of analog 1. On the other hand, bromo groups at position 3 or positions 3 and 5 on the β-phenyl ring of analog 1 did not influence DPPH radical scavenging activity. Of the 13 (Z)-BPTT analogs, analog 2 with a catechol  moiety exhibited the strongest DPPH radical scavenging activity at 92.2%, comparable to L-ascorbic acid’s 96.9% scavenging activity. In addition, analog 3 with a resorcinol  moiety exerted potent radical scavenging activity (57.0% inhibition). Analogs 7–10 with no hydroxyl group were found to have no or weak DPPH radical scavenging effect. Notably, analog 4 with a 4-hydroxy-3-methoxyl group on the β-phenyl ring exhibited strong radical scavenging activity, whereas analog 6, in which the positions of the 4-hydroxyl group and the 3-methoxyl group of analog 4 were exchanged, had poor radical scavenging activity (27.2%). These results suggest that phenolic hydroxyl groups and their positions on the β-phenyl ring importantly influence DPPH radical scavenging activity.The DPPH  radical scavenging abilities of the ) free radical scavenging activity were investigated using trolox as a positive reference control. The concentrations of the analogs and trolox in the test samples were 100 µM. The free radical scavenging activities were assessed by measuring the optical densities at 734 nm after standing in the dark for 2 min. The ABTS free radical scavenging activities of the analogs are provided in Z)-BPTT analogs were similar to the DPPH radical scavenging results. Five analogs, 2–5 and 11, exhibited potent radical scavenging activities as compared with the other analogs, as was observed for the DPPH radical scavenging activities. Analogs 4, 5, and 11 exhibited moderate ABTS scavenging activities at 61.5%, 51.2%, and 57.2%, respectively. Analog 3, with a resorcinol moiety, had moderate DPPH radical scavenging ability but exhibited strong ABTS radical scavenging activity (82.0% inhibition). As was observed for the DPPH radical scavenging activity, analog 2 (96.9% inhibition), with a catechol moiety, exhibited the strongest ABTS free radical scavenging activity and was comparable to trolox (99.8% inhibition) in this respect. Analogs 7–10, with no hydroxyl groups on the β-phenyl ring, had weak ABTS and DPPH radical scavenging activities. Analog 1, with a 4-hydroxyl group on the β-phenyl ring, also had weak ABTS free radical scavenging activity, but the insertion of an additional 3,5-dimethoxyl group on the β-phenyl ring greatly increased its radical scavenging activity . However, the introduction of a bromine into positions 3 and 5 of the β-phenyl ring of analog 1 slightly decreased its ABTS free radical scavenging activity  as compared with analog 1. However, the introduction of one bromo substituent at position 3 of analog 1 did not influence the ABTS free radical scavenging activity .The effects of analogs Z)-BPTT analogs 1–3 and 6 were investigated in vitro using DCFH-DA. SIN-1 (3-morpholinosydnonimine) and trolox were used as a RNS/ROS generator [1 and 6 did not show ROS scavenging activity at concentrations ≤ 20 µM, but analog 3, which possessed a resorcinol moiety, at 20 µM had a ROS scavenging effect that was similar to that of trolox. On the other hand, analog 2, with a catechol moiety, significantly and concentration-dependently reduced SIN-1-induced increases in the intracellular ROS activity and it did so to a greater extent than trolox. These results suggested that (Z)-BPTT analogs, especially analog 2, effectively reduced SIN-1-induced increases in intracellular ROS levels. A comparison of the structures and ROS scavenging activities of the (Z)-BPTT analogs revealed that the catechol moiety  markedly influenced intracellular ROS scavenging activity, a finding which concurs with the findings of previous reports [2 is probably, at least in part, due to the melanogenesis inhibition that is caused by intracellular ROS scavenging.We investigated the effects of the analogs on intracellular ROS (reactive oxygen species) because, reportedly, ROS might regulate melanogenesis in melanoma cells . The prienerator  and a po reports ,56,57. T2 inhibited melanogenesis in B16F10 cells more than analogs 1, 3, and 6, we investigated the mechanism that is responsible for its anti-melanogenic effect. Western blotting was used to determine the cytosolic tyrosinase protein levels and nuclear MITF levels in total cell lysates , MITF expression instead increased at the same concentrations. Due to this discrepancy, we examined the effect of analog 2 on MITF target gene expressions such as tyrosinase, TRP-1, and TRP-2, which are required for melanin biosynthesis [2-treated B16F10 cells with those of non-treated controls. The expression levels of all three of the enzymes were significantly and concentration-dependently reduced by analog 2, which suggests that analog 2 downregulated MITF expression and, thus, suppressed the expressions of tyrosinase, TRP-1, and TRP-2 -BPTT analogs were synthesized based on the structural characteristics of two potent tyrosinase inhibitors. The analogs’ structures were determined by 1H and 13C NMR and low- and high-resolution mass spectroscopy. In particular, the double bond geometries were elucidated using 1H,13C-coupling constants. The 13 analogs were evaluated for mushroom tyrosinase-inhibitory activity and four analogs  were found to be more potent than kojic acid. Kinetic studies performed using Lineweaver–Burk plots indicated that all four of these analogs were competitive mushroom tyrosinase inhibitors and this was supported by docking results. A human tyrosinase homology model was prepared and docked with analogs 1–3 and analogs 2 and 3 were found to have greater binding affinity than kojic acid. Four analogs, 1–3 and 6, dose-dependently inhibited cellular tyrosinase activity and melanin production in B16F10 cells more potently than kojic acid did. In particular, analog 2, which had a catechol moiety, was much more potent than the other three analogs and kojic acid. In addition, five analogs showed moderate to strong radical scavenging activities against DPPH and ABTS and analog 2 showed the greatest radical scavenging activity, which was comparable to those of the positive controls, l-ascorbic acid and trolox. Furthermore, analog 2 more potently scavenged ROS than trolox and it significantly and concentration-dependently decreased tyrosinase and MITF protein levels and the expressions of melanogenesis-related genes viz. tyrosinase and TRP-1 and -2. These results have demonstrated that analog 2 has anti-melanogenic properties that are derived from dual modes of action, namely, (i) by directly inhibiting tyrosinase activity and (ii) by suppressing the expressions of melanogenesis-related proteins and genes.Thirteen (


Sik1 mRNA. In contrast, SIK inhibition had no effects on FSH receptor expression. Next, we determined whether SIK inhibition enhances the effect of several sequential direct activators of the FSH signaling pathway. The findings revealed that SIK inhibition stimulates the induction of steroidogenic genes by forskolin, cAMP, protein kinase A (PKA), and cAMP-response element-binding protein (CREB). Strikingly, FSH stimulation of CREB and AKT phosphorylation was not affected by SIK inhibition. Therefore, we analyzed the expression and activation of putative CREB cofactors and demonstrated that GCs express CREB-regulated transcriptional coactivators (CRTC2) and that FSH treatment and SIK inhibition increase the nuclear expression of this factor. We concluded that SIKs target the FSH pathway by affecting factors located between cAMP/PKA and CREB and propose that SIKs control the activity of CRTC2 in ovarian GCs. The findings demonstrate for the first time that SIKs blunt the response of GCs to FSH, cAMP, PKA, and CREB, providing further evidence for a crucial role for SIKs in regulating ovarian function and female fertility.The optimal development of preovulatory follicles needs follicle-stimulating hormone (FSH). Recent findings revealed that salt-inducible kinases (SIKs) inhibit FSH actions in humans and rodents. This report seeks to increase our understanding of the molecular mechanisms controlled by SIKs that participate in the inhibition of FSH actions in primary rat granulosa cells (GCs). The results showed that FSH causes a transient induction of  Therefore, SIKs are key regulators of the response of GCs to gonadotropins and consequently control ovulation efficiency and fertility. These results motivate studies aimed at determining the specific signaling mechanisms targeted by SIKs in GCs.Ovulation is the pinnacle of folliculogenesis, a process requiring granulosa cell (GC) proliferation and differentiation, both needed for preovulatory follicle formation. The optimal development of preovulatory follicles needs follicle-stimulating hormone (FSH). We recently revealed novel roles for a family of kinases named salt-inducible kinase (SIKs) in regulating folliculogenesis and ovulation in rodents and controlling GC differentiation in humans and rodents . In partThe three SIK isoforms  are serine/threonine protein kinases of the AMP-activated kinases family known to regulate metabolism, cancer, melanocytes, and bone formation , 5. NeveCyp19a1   (Cyp11a1 (most commonly referred to as P450scc)  activity and the production of cyclic adenosine 3′,5′-monophosphate (cAMP). cAMP leads to the activation of protein kinase A (PKA) and cAMP response element-binding protein (CREB), leading to the induction of markers of GC differentiation, including omatase) –12, steras StAR) , and CypP450scc) . Here, win vitro model for examining GC differentiation and the mechanisms involved in the regulation of GCs by FSH (Cells – GCs were isolated from 23-25 days old estradiol-treated immature rats and cultured as described previously –17. The s by FSH . Cells wRpl19). Then, the expression of each GOI is reported as the ratio between the number of copies of the GOI and Rpl19.RNA isolation and quantification – Total RNA was isolated using TRIzol  and reverse-transcribed using anchored oligo-dT primers  and Moloney Murine Leukemia Virus reverse transcriptase (Invitrogen). Intron-spanning primers were used to amplify the gene of interest (GOI) along with a standard curve containing serial dilutions of the cDNA of the GOI. Real-time PCR amplifications were performed with Brilliant II qPCR SYBR master mix  using an AriaMx instrument (Agilent). For each sample, the number of cDNA copies corresponding to 10 ng of total RNA was computed for each GOI and ribosomal protein L19 (Promoter Reporter Assays - The CRE-Luc reporter was generated by cloning three copies of the cAMP response element (TGACGTCA) followed by the firefly luciferase cDNA (CRE-Luc). Lentiviruses containing this construct were generated using 293FT cells (Invitrogen) as previously described . Cells w-DN were added directly to the cells 2 h after plating at a multiplicity of infection of 20 and cultured for 24 hours before the initiation of the treatments described in each figure.Overexpression experiments – Expression plasmids encoding constitutively active PKA  or C2/CR- Cytosolic and nuclear extracts were prepared as described previously . Differences between two groups were determined by Student’s in vitro and in vivo that SIK inhibition enhances FSH-induction of steroidogenic genes and estradiol production in human and rodent GCs  of HG-9-91-01 (HG). HG-9-91-01 is an effective SIK inhibitor, which has been shown to selectively target SIK proteins (Fshr) expression : After observing that SIK inhibition has no effect on FSH receptor expression, we hypothesized that it might affect signaling downstream of the FSH receptor. Since the FSH receptor activates the AC, we treated GCs with forskolin, a specific AC activator, in the presence or absence of 0.3, 1, or 3 micromolar  of HG-9-91-01 (HG). These concentrations are based on our previous publications on GCs , the lowest concentration tested. In good agreement with our previous report , a cell-permeable analog of cAMP, which alone induced Cyp19a1, Stard1, and Cyp11a1 expression. As with FSH and forskolin, SIK inhibition augmented the stimulatory effect of dbcAMP on gene expression in a concentration-dependent manner  and PKA . TherefoCyp19a1, Stard1, and Cyp11a1 expression an effect that was significantly enhanced by SIK inhibition  protein, which carries His87Gln and Trp196Arg mutations rendering it insensitive to the regulatory units . We firshibition Figure 4PKA activates AKT and CREB in GCs , 30, 31;Since inhibition of SIK activity did not augment CREB phosphorylation, we next investigated the effect of SIK inhibition on CREB activity. For this purpose, we created a cAMP response element reporter . As expected, CRE-Luc activity increased significantly after treatment with FSH. HG treatment enhanced FSH-induced CRE-Luc activity and alone increased CRE-Luc activity significantly Figure 6Cyp19a1, Stard1, and Cyp11a1 expression. Inhibition of SIK activity in cells overexpressing C2/CREB augmented Cyp19a1, Stard1, and Cyp11a1 expression to levels that were significantly higher than those observed with C2/CREB alone  . As thisCRTCs activity is mainly regulated by their translocation to the nucleus, where they contribute to activating gene expression . TherefoCyp19a1, Stard1, and Cyp11a1 expression by FSH or by the combination of FSH plus HG , which has been shown to block the effects of all CRTCs , 24. Expin vitro and in vivo  for the financial support grant R01HD097202 to CS.The authors thank Dr. Esfandyari for her technical support.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.