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In this issue of Cell Host & Microbe, Huang et al. determine that an oncogenic bacterium contributes to colorectal cancer progression and resistance to receptor tyrosine kinase inhibitors. These findings highlight the need for an integrative approach for cancer treatment that considers the influence of the microbiome., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Botrytis cinerea is an important fungal pathogen that causes gray mold disease in plants. Previously, Bacillus velezensis TCS001 live culture presented broad-spectrum antifungal activity against various plant pathogenic fungi and oomycetes, particularly B. cinerea . Here, the bioactivity of lipopeptides produced by TCS001 against B. cinerea was investigated. The IC 50 values of the crude lipopeptide extract (CLE) from TCS001 to suppress mycelial growth and conidial germination were 14.20 and 49.39 mg/L, respectively. SEM and TEM imaging revealed that CLE caused morphological deformities and ultrastructural changes in the mycelium. Transcriptomic analyses combined with Δ Bcpsd mutant construction demonstrated that the CLE could confer antifungal activity via suppressing Bcpsd expression in the pathogen. In addition, the CLE activated the plant immune system by increasing the content of defense-related enzymes and the expression of marker genes in immunity signaling pathways in cucumber plants. Therefore, TCS001 CLE could be potentially developed into biopesticides for the biocontrol of gray mold disease.

Preclinical studies are broad and can encompass cellular research, animal trials, and small human trials. Preclinical studies tend to be exploratory and have smaller datasets that often consist of biomarker data. Logistic regression is typically the model of choice for modeling a binary outcome with explanatory variables such as genetic, imaging, and clinical data. Small preclinical studies can have challenging data that may include a complete separation or quasi-complete separation issue that will result in logistic regression inflated coefficient estimates and standard errors. Penalized regression approaches such as Firth's logistic regression are a solution to reduce the bias in the estimates. In this tutorial, a number of examples with separation (complete or quasi-complete) are illustrated and the results from both logistic regression and Firth's logistic regression are compared to demonstrate the inflated estimates from the standard logistic regression model and bias-reduction of the estimates from the penalized Firth's approach. R code and datasets are provided in the supplement., (© 2024 John Wiley & Sons Ltd.)

Cadmium (Cd) is a common environmental pollutant associated with an increased incidence of renal metabolic diseases. Luteolin (Lut), a natural flavonoid, is widely used for its multifaceted therapeutic properties in inflammatory diseases. However, whether Lut protects against Cd-induced nephrotoxicity is still equivocal. The present study investigated the effects of Lut supplementation on renal oxidative stress, inflammation and metabolism and their related mechanisms. Therefore, 40 chickens were treated with Cd and/or Lut with automatic water and free food intake for 1 mo and then the kidney tissues were collected to explore this issue. In this study, Cd exposure induced renal glycolipid metabolism disorders and resultant kidney damage by periodic acid Schiff (PAS) staining, Oil Red O staining, total cholesterol (TC), triglyceride (TG), and glucose (Glu) levels in kidney, which were significantly ameliorated by Lut. Moreover, Lut also normalized the expression levels of factors related to Cd-disturbed glycolipid metabolism, improving metabolic homeostasis, and contributing to alleviating kidney damage. Furthermore, Lut demonstrated therapeutic potential against Cd-induced renal oxidative stress and inflammation by enhancing antioxidant capacity and inhibiting cytokine production in the kidney tissues. Mechanistically, Lut activated the AMPK/SIRT1/FOXO1 signaling pathway, attenuating oxidative stress and inflammatory responses, ameliorating the metabolic disturbance. In conclusion, these observations demonstrate that Lut treatment activates AMPK/SIRT1/FOXO1 signaling pathway, decreases oxidative stress and inflammation response, which may contribute to prevent Cd-induced metabolism disorder and consequent kidney damage., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Chickens are a major source of meat and eggs in human food and have significant economic value. Cadmium (Cd) is a common environmental pollutant that can contaminate feed and drinking water, leading to kidney injury in livestock and poultry, primarily by inducing the generation of free radicals. It is necessary to develop potential medicines to prevent and treat Cd-induced nephrotoxicity in poultry. Luteolin (Lut) is a natural flavonoid compound mainly extracted from peanut shells and has a variety of biological functions to defend against oxidative damage. In this study, we aimed to demonstrate whether Lut can alleviate kidney injury under Cd exposure and elucidate the underlying molecular mechanisms. Renal histopathology and cell morphology were observed. The indicators of renal function, oxidative stress, DNA damage and repair, NAD + content, SIRT1 activity, and autophagy were analyzed. In vitro data showed that Cd exposure increased ROS levels and induced oxidative DNA damage and repair, as indicated by increased 8-OHdG content, increased γ-H2AX protein expression, and the over-activation of the DNA repair enzyme PARP-1. Cd exposure decreased NAD + content and SIRT1 activity and increased LC3 II, ATG5, and particularly p62 protein expression. In addition, Cd-induced oxidative DNA damage resulted in PARP-1 over-activation, reduced SIRT1 activity, and autophagic flux blockade, as evidenced by reactive oxygen species scavenger NAC application. The inhibition of PARP-1 activation with the pharmacological inhibitor PJ34 restored NAD + content and SIRT1 activity. The activation of SIRT1 with the pharmacological activator RSV reversed Cd-induced autophagic flux blockade and cell injury. In vivo data demonstrated that Cd treatment caused the microstructural disruption of renal tissues, reduced creatinine, and urea nitrogen clearance, raised MDA content, and decreased the activities or contents of antioxidants (GSH, T-SOD, CAT, and T-AOC). Cd treatment caused oxidative DNA damage and PARP-1 activation, decreased NAD + content, decreased SIRT1 activity, and impaired autophagic flux. Notably, the dietary Lut supplement observably alleviated these alterations in chicken kidney tissues induced by Cd. In conclusion, the dietary Lut supplement alleviated Cd-induced chicken kidney injury through its potent antioxidant properties by relieving the oxidative DNA damage-activated PARP-1-mediated reduction in SIRT1 activity and repairing autophagic flux blockade.

Cadmium (Cd) is a common environmental pollutant and occupational toxicant that seriously affects various mammalian organs, especially the kidney. Iron ion is an essential trace element in the body, and the disorder of iron metabolism is involved in the development of multiple pathological processes. An iron overload can induce a new type of cell death, defined as ferroptosis. However, whether iron metabolism is abnormal in Cd-induced nephrotoxicity and the role of ferroptosis in Cd-induced nephrotoxicity need to be further elucidated. Sprague Dawley male rats were randomly assigned into three groups: a control group, a 50 mg/L CdCl2-treated group, and a 75 mg/L CdCl2-treated group by drinking water for 1 month and 6 months, respectively. The results showed that Cd could induce renal histopathological abnormalities and dysfunction, disrupt the mitochondria's ultrastructure, and increase the ROS and MDA content. Next, Cd exposure caused GSH/GPX4 axis blockade, increased FTH1 and COX2 expression, decreased ACSL4 expression, and significantly decreased the iron content in proximal tubular cells or kidney tissues. Further study showed that the expression of iron absorption-related genes SLC11A2 , CUBN , LRP2 , SLC39A14 , and SLC39A8 decreased in proximal tubular cells or kidneys after Cd exposure, while TFRC and iron export-related gene SLC40A1 did not change significantly. Moreover, Cd exposure increased SLC11A2 gene expression and decreased SLC40A1 gene expression in the duodenum. Finally, NAC or Fer-1 partially alleviated Cd-induced proximal tubular cell damage, while DFO and Erastin further aggravated Cd-induced cell damage. In conclusion, our results indicated that Cd could cause iron deficiency and chronic kidney injury by interfering with the iron metabolism rather than typical ferroptosis. Our findings suggest that an abnormal iron metabolism may contribute to Cd-induced nephrotoxicity, providing a novel approach to preventing kidney disease in clinical practice.

Environmental Cadmium (Cd) is a toxicant with widespread exposure, documented adverse effects on bone homeostasis, and makes the onset of osteoporosis (OP), one of the age-related chronic diseases an enormous burden to modern societies worldwide. Aging is the largest risk factor for a multitude of age-related diseases and osteoblasts senescence reduces bone formation and is a key factor for osteoporosis. Despite anti-aging molecules the nuclear silent information regulator of transcription 1 (SIRT1) actions in chondrocytes and bone cells are critical for normal skeletal development and homeostasis, much less is known about the role of SIRT1 in osteoporosis. Here, we aim to demonstrate that SIRT1 mediates osteoblasts' senescence response to OP caused by Cd. The senescent osteoblasts accumulation and their viability were analyzed after Cd exposure. To explore the effects and mechanism of SIRT1 in Cd-induced osteoblastic senescence, we generated SIRT1-overexpressed osteoblast and SIRT1 conditional overexpression in the rat femur. Meanwhile, the OP rat model was established by removing bilateral ovaries. We found decreased SIRT1 expression and senescent osteoblasts accumulation after Cd exposure. Meanwhile, Cd exposure increased P53, P16INK4a, and P21CIPI proteins level, triggered DNA damage response (DDR) through the phosphorylation of ATM and H2AX, and caused mitochondrial dysfunction by the increased acetylation of SOD2 and excessive mitophagy. SIRT1 overexpression attenuated DDR and mitochondrial dysfunction and downregulated the increase of hall makers senescence caused by Cd in osteoblasts. We found overexpression of osteoblastic SIRT1 protects against Cd-induced senescence, which is likely driven by ATM-mediated DDR and SOD2 ace -mediated mitochondrial dysfunction. Our study demonstrates the mechanism of SIRT1 in mediating bone homeostasis via senescence. Further mechanistic studies using specific SIRT1 mutations elucidating how SIRT1 modulates bone cell senescence, will provide new therapeutic strategies for human osteoporosis., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Cadmium (Cd) can damage bone cells and cause osteoporosis. Osteocytes are the most numerous bone cells and also important target cells for Cd-induced osteotoxic damage. Autophagy plays important role in the progression of osteoporosis. However, osteocyte autophagy in Cd-induced bone injury is not well characterized. Thus, we established a Cd-induced bone injury model in BALB/c mice and a cellular damage model in MLO-Y4 cells. Aqueous Cd exposure for 16 months showed an increase in plasma alkaline phosphatase (ALP) activity and increase in urine calcium (Ca) and phosphorus (P) concentrations in vivo. Moreover, expression level of autophagy-related microtubule-associated protein 1A/1B-light chain 3 II (LC3II) and autophagy-related 5 (ATG5) proteins were induced, and the expression of sequestosome-1 (p62) was reduced, along with Cd-induced trabecular bone damage. In addition, Cd inhibited the phosphorylation of mammalian target of rapamycin (mTOR), protein kinase B (AKT), and phosphatidylinositol 3-kinase (PI3K). In vitro, 80 μM Cd concentrations exposure upregulated LC3II protein expression, and downregulated of p62 protein expression. Similarly, we found that treatment with 80 μM Cd resulted in a reduction in the phosphorylation levels of mTOR, AKT, and PI3K. Further experiments revealed that addition of rapamycin, an autophagy inducer, enhanced autophagy and alleviated the Cd-induced damage to MLO-Y4 cells. The findings of our study reveal for the first time that Cd causes damage to both bone and osteocytes, as well as induces autophagy in osteocytes and inhibits PI3K/AKT/mTOR signaling, which could be a protective mechanism against Cd-induced bone injury., (© 2023 Wiley Periodicals LLC.)

The imbalance between osteogenesis and osteoclastogenesis is a feature of bone metabolic disease. Cadmium (Cd) exposure causes human bone loss and osteoporosis (OP) through bioaccumulation of the food chain. However, the impact of Cd on bone tissues and the underlying molecular mechanisms are not well-characterized. In the current study, we found that the Cd concentration in bone tissues of OP patients was higher than normal subjects; meanwhile, the nuclear silent information regulator of transcription 1 (SIRT1) protein expression level was significantly decreased, which is a new star molecule to treat OP. It is further revealed that SIRT1 activation markedly reprograms bone metabolic and stress-response pathways that incline with osteoblast (OB) apoptosis. Suppressing reactive oxygen species (ROS) release with N -acetyl-l-cysteine (NAC) abolished Cd-induced reduction of SIRT1 protein, deacetylation of P53, OB apoptosis, and attenuated OP. Conversely, overexpression of SIRT1 suppressed Cd-induced ROS release. SIRT1 overexpression in vivo and in vitro dampened PGC-1α protein, acetylation of P53 at lysine 382, and caspase-dependent apoptosis. These results reveal that ROS/SIRT1 controls P53 acetylation and coordinates OB apoptosis involved in the onset of OP.

A pivotal clinical trial is often necessary to assess drug efficacy in the intended to use (IU) population. Ideally, patients should be enrolled based on a positive test result from a well-characterized companion diagnostic (CDx). However, the central challenge is that patients are instead recruited on the basis of a clinical trial assay (CTA) result. This challenge arises because, CTA is available at all local labs; the time delay to enable enrollment based on CDx could result in a significant proportion of patients being unable to participate, adversely affecting precision and/or bias. The difficulty is therefore that patients are recruited on the basis that their CTA result is positive (CTA+) but the goal is to assess the drug efficacy in patients positive by the companion diagnostic (CDx+). In this commentary, we will examine an apparent weakness of a variance formula that is proposed in the context of a sensitivity analysis. We will develop an alternative formula, and argue that this should be used instead.

Various factors cause animal bone malnutrition disease during intensive culture. Osteoclasts play an important role in regulating bone metabolism disease. Osteoprotegerin (OPG) modulates osteoclast function; however, the mechanism underlying this effect is unknown. Therefore, the present study aimed to explore whether OPG affects duck embryo osteoclast function via purinergic receptor P2X7. OPG significantly inhibited duck embryo osteoclast differentiation and bone resorption, and suppressed F-actin formation. In addition, OPG remarkably impaired duck embryo osteoclasts' adhesive structure. After OPG treatment, the expression of P2X7R significantly reduced, the ATP level and Ca 2+ -ATPase activity decreased rapidly, and concomitantly suppressed calcium and MAPK signaling. A438079 (a selective P2X7R inhibitor) significantly inhibited duck embryo osteoclast differentiation and bone resorption, and the phosphorylation of Ca 2+ regulated proteins (CAM, CAMKII, CAMKIV) and MAPKs (ERK, JNK, and P38) were markedly suppressed. Pretreatment of duck embryo osteoclasts with BzATP, a P2X7R agonist, activated Ca 2+ and MAPK signaling. BzATP alleviated OPG-induced duck embryo osteoclast differentiation and adhesive structure damage, and recovered the distribution of adhesion-related proteins in mature duck embryo osteoclasts. Thus, P2RX7-mediated Ca 2+ and MAPK signaling has a key function in OPG-induced duck embryo osteoclast differentiation and adhesive structure damage. P2X7R might be an ideal target to treat bone diseases through regulating bone cell activation., (Copyright © 2022 Elsevier Inc. All rights reserved.)

The present study was performed to investigate the effects of Cd exposure on lipid metabolism and mitochondrial dysfunction and to explore the role of mitophagy in Cd-induced dysregulation of lipid metabolism in chicken embryo liver tissues and hepatocytes. To this end, seven-day-old chicken embryos were exposed to different concentrations of Cd for 7 days, and primary chicken embryo hepatocytes were treated with Cd at four different concentrations for 6 h. Furthermore, the mitophagy inhibitor cyclosporine A (CsA) was used to investigate the role of mitophagy in Cd-induced disruption of lipid metabolism. Lipid accumulation, the expression levels of genes involved in lipid metabolism, mitochondrial dysfunction, and mitophagy were measured. The results demonstrated that Cd exposure increases hepatic triglyceride (TG) accumulation and the expression levels of lipogenic genes while decreasing those of lipolytic genes. Furthermore, Cd exposure was observed to alter mitochondrial morphology in terms of reduced size, excessive mitochondrial damage, and the formation of mitophagosomes. The co-localization of lysosome-associated membrane glycoprotein 2 and LC3 puncta was significantly increased in primary chicken embryo hepatocytes after Cd exposure. Moreover, Cd exposure increased LC3, PINK1, and Parkin protein expression levels. CsA effectively alleviated Cd-induced mitochondrial dysfunction, blocked mitochondrial membrane potential collapse, and suppressed PINK1/Parkin-mediated mitophagy. Furthermore, CsA treatment reversed the Cd-induced TG accumulation in liver tissues but further increased it in hepatocytes. Taken together, our findings demonstrate (for the first time) the importance of mitochondrial dysfunction and mitophagy via the PINK1/Parkin pathway in Cd-induced disruption of lipid metabolism., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Cadmium (Cd) is a widespread environmental contaminant that causes severe bone metabolism disease, such as osteoporosis, osteoarthritis, and osteomalacia. The present review aimed to explore the molecular mechanisms of Cd-induced bone injury starting from bone cell function and teeth development. Cd inhibits the differentiation of bone marrow mesenchymal stem cells (BMSCs) into osteoblasts, and directly causes BMSC apoptosis. In the case of osteoporosis, Cd mainly affects the activation of osteoclasts and promotes bone resorption. Cd-induces osteoblast injury and oxidative stress, which causes DNA damage, mitochondrial dysfunction, and endoplasmic reticulum stress, resulting in apoptosis. In addition, the development of osteoarthritis (OA) might be related to Cd-induced chondrocyte damage. The high expression of metallothionein (MT) might reduce Cd toxicity toward osteocytes. The toxicity of Cd toward teeth mainly focuses on enamel development and dental caries. Understanding the effect of Cd on bone cell function and teeth development could contribute to revealing the mechanisms of Cd-induced bone damage. This review explores Cd-induced bone disease from cellular and molecular levels, and provides new directions for removing this heavy metal from the environment., Competing Interests: Declaration of competing interest None., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Cadmium (Cd), an environmental pollutant, induces osteoporosis by directly destroying bone tissue, but its direct damaging effect on bone cells is not fully illustrated. Here, we treated mouse bone marrow stem cells (BMSC) and bone marrow macrophages (BMM) with Cd, and gave BALB/c mice Cd in water. Long-term Cd exposure significantly inhibited BMSC osteogenesis and osteoclast differentiation in vitro, and induced osteoporosis in vivo. Cd exposure also reduced P2X7 expression dramatically. However, P2X7 deletion significantly inhibited osteoblast and osteoclast differentiation; P2X7 overexpression obviously reduced the suppression effect of Cd on osteoblast and osteoclast differentiation. The suppression of P2X7-PI3K-AKT signaling aggravated the effect of Cd. In mice, short-term Cd exposure did not result in osteoporosis, but bone formation was inhibited, RANKL expression was increased, and osteoclasts were significantly increased in vivo. In vitro, short-term Cd exposure not only increased osteoclast numbers, but also promoted osteoclast adhesion function at late-stage osteoclast differentiation. Cd exposure also reduced P2X7 expression in vivo and in vitro. Our results demonstrate that short-term Cd exposure does not affect osteoblast and osteoclast apoptosis in vivo and in vitro, but long-term Cd exposure significantly increases bone tissue apoptosis. Overall, our results describe a novel mechanism for Cd-induced osteoporosis., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Cadmium is a common environmental pollutant that accumulates in the bone and kidneys and causes severe health and social problems. However, the effects of Cd on the occurrence of osteoporosis and its mechanism of action in this process are unclear. To test whether Cd-induced osteoporosis is mediated via P2X7/PI3K/AKT signaling, duck bone marrow mesenchymal stem cells (BMSCs) and bone marrow macrophage cells (BMMs) were treated with Cd for 5 days, and duck embryos were treated with Cd . Micro-CT analysis indicated that Cd-induced osteoporosis occurs in vivo, and histopathology and immunohistochemical analyses also revealed that Cd induced bone damage and the downregulation of osteogenic and bone resorption-related proteins. Cd exposure significantly inhibited the differentiation of BMSCs and BMMs into osteoblasts and osteoclasts in vitro, and promoted osteoblast and osteoclast apoptosis. Cd exposure significantly downregulated the P2X7/PI3K/AKT signaling pathway in vivo and in vitro, and inhibition of this signaling pathway significantly aggravated osteoblast and osteoclast differentiation. Cd exposure also upregulated the OPG/RANKL ratio in vivo and in vitro, further inhibiting osteoclast differentiation. These results demonstrate that Cd causes osteoporosis in duck by inhibiting P2X7/PI3K/AKT signaling and increasing the OPG/RANKL ratio. These results establish a previously unknown mechanism of Cd-induced osteoporosis., Competing Interests: Declaration of competing interest The authors declare that there are no conflicts of interest., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Cadmium (Cd), a heavy metal produced by various industries, contaminates the environment and seriously damages the skeletal system of humans and animals. Recent studies have reported that Cd can affect the viability of cells, including osteoblasts, both in vivo and in vitro. However, the mechanism of Cd-induced apoptosis remains unclear. In the present study, primary rat osteoblasts were used to investigate the Cd-induced apoptotic mechanism. We found that treatment with 2 and 5 μM Cd for 12 h decreased osteoblast viability and increased apoptosis. Furthermore, Cd increased the generation of reactive oxygen species (ROS), and, thus, DNA damage measured via p-H2AX. The level of the nuclear transcription factor p53 was significantly increased, which upregulated the expression of PUMA, Noxa, Bax, and mitochondrial cytochrome c, downregulated the expression of Bcl-2, and increased the level of cleaved caspase-3. However, pretreatment with the ROS scavenger N-acetyl-l-cysteine (NAC) or the p53 transcription specific inhibitor PFT-α suppressed Cd-induced apoptosis. Our results indicate that Cd can induce apoptosis in osteoblasts by increasing the generation of ROS and activating the mitochondrial p53 signaling pathway, and this mechanism requires the transcriptional activation of p53., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Purpose : To investigate the protective effect of naringin (Nar) on H 2 O 2 -induced apoptosis of nucleus pulposus-derived mesenchymal stem cells (NPMSC) and the potential mechanism in this process. Methods : Rat NPMSC were cultured in MSC culture medium or culture medium with different concentrations of H 2 O 2 . Nar or the combination of Nar and LY294002 was added into the culture medium to investigate the effects of Nar. Cell viability was evaluated by cell counting kit-8 (CCK-8) assay. The apoptosis rate was determined using Annexin V/PI dual staining and terminal deoxynucleotide transferase-mediated dUTP nick end labeling (TUNEL) assays. Additionally, the levels of reactive oxygen species (ROS) and mitochondrial membrane potential (MMP) were analyzed by flow cytometry. ATP level in NPMSC was analyzed via ATP detection kit. Mitochondrial ultrastructure change was observed through transmission electron microscope (TEM). Levels of apoptosis-associated molecules (cleaved caspase-3, Bax and Bcl-2) were evaluated via RT-PCR and western blot, respectively. Results : The cells isolated from NP met the criteria for MSC. H 2 O 2 significantly promoted NPMSC apoptosis in a dose and time-dependent manner. Nar showed no cytotoxicity effect on NPMSC up to a concentration of 100 μM for 24 h. Nar exhibited protective effects against H 2 O 2 -induced NPMSC apoptosis including apoptosis rate, expressions of proapoptosis and antiapoptosis related genes and protein. Nar could also alleviate H 2 O 2 -induced mitochondrial dysfunction of increased mitochondrial ROS production, reduced MMP, decreased intracellular ATP and mitochondrial ultrastructure change. However, these protected effects were inhibited after LY294002 treatment. Conclusions : Our results demonstrated that Nar efficiently attenuated H 2 O 2 -induced NPMSC apoptosis and mitochondrial dysfunction. The activation of ROS-mediated PI3K/Akt pathway may be the potential mechanism in this process.

Cadmium (Cd) is one of worldwide environmental pollutants that causes bone homeostasis, which depends on the resorption of bones by osteoclasts and formation of bones by the osteoblasts (OB). However, the Cd toxicity on OB and its mechanism are unclear. Autophagy is an evolutionarily conserved degradation process in which domestic intracellular components are selectively digested for the recycling of nutrients and energy. This process is indispensable for cell homeostasis maintenance and stress responses. Dysregulation at the level of autophagic activity consequently disturbs the balance between bone formation and bone resorption and mediates the onset and progression of multiple bone diseases, including osteoporosis. TAK1 has been recently emerged as an activator of AMPK and hence an autophagy inducer. AMPK is a key molecule that induces autophagy and regulates cellular metabolism to maintain energy homeostasis. Conversely, autophagy is inhibited by mTORC1. In this study, we found that Cd treatment caused the formation of autophagosomes, LC3-II lipidation and p62 downregulation, and the increased autophagic flux, indicating that Cd treatment induced autophagy in OBs. Cd treatment induced TAK1 activation mediated AMPK phosphorylation, which promoted autophagy via phosphorylation of ULK1 at S317. Meanwhile, Cd treatment dramatically decreased mTORC1, S6K1, 4E-BP1, S6, ULK1 S555 and ULK1 S757 phosphorylation, suggesting that mTORC1 activity was inhibited and inactive mTORC1 prevents ULK1 activation by phosphorylating ULK1 at SerS555 and Ser757. Our data strongly suggest that TAK1 mediates AMPK activation, which activates ULK1 by phosphorylating ULK1 S317 and suppressing mTORC1-mediated ULK1 S555 and ULK1 S757 phosphorylation. Our study has revealed a signaling mechanism for TAK1 in Cd-induced autophagy in OBs., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Calcium is essential to various physiological and pathophysiological cellular processes. Calcium-sensing receptor (CasR), a seven-transmembrane-spanning protein that responds to changes in extracellular Ca 2+ , partly modulates calcium homeostasis, thereby influencing bone metabolism. In this study, we aimed to elucidate the role of CasR in Cd-induced calcium homeostasis disruption and OB apoptosis, and the underlying mechanisms. Cd treatment dramatically increased the protein expression of CasR and elevated the intracellular calcium concentration. Meanwhile, OBs apoptosis rate and caspase-dependent apoptosis protein levels, including cleaved caspase 3, cleaved caspase 9 and the ratio of Bax/Bcl-2 were increased. However, downregulation of CasR by CasR siRNA effectively suppressed the effects of Cd on theses phenomena. At the same time, we illustrated that CasR siRNA pretreatment blocked Cd-inhibited the phosphorylation of PKC and decreased Cd-induced the phosphorylation of PI3K/AKT. Our results suggested that CasR-mediated PKC and PI3K/AKT signaling pathways involve in calcium oscillation and apoptosis in OB caused by Cd maybe responsible for the bone homeostasis., Competing Interests: Declaration of Competing Interest The authors declare that they have no conflict of interest. I would like to declare on behalf of my co-authors that the work described was original research that has not been published previously, and not under consideration for publication elsewhere, in whole or in part., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Environmental cadmium (Cd) pollution can ultimately lead to chronic toxicity via food consumption. Previous studies have demonstrated that long-term low-dose Cd exposure decreases bone mineral density and bone mineralization. Cd may increase receptor activator of nuclear factor-κ B ligand (RANKL) expression by osteoclasts, and inhibit the expression of osteoprotegerin. However, the molecular mechanism underlying Cd toxicity toward osteoclasts is unclear. In this study, bone marrow monocytes were isolated from C57BL/6 mice and treated with macrophage colony-stimulating factor and RANKL to induce the formation of osteoclasts. The results show that low-dose Cd exposure induced osteoclast differentiation. Cd also increased the intracellular calcium concentration of osteoclasts by triggering release of calcium ions from the endoplasmic reticulum into the cytoplasm. Furthermore, the elevation of intracellular calcium levels was shown to activate the calmodulin (CaM)/calmodulin-dependent protein kinase (CaMK) pathway. NFATc1 is a downstream protein of CaM/CaMK signaling, as well as a key player in osteoclast differentiation. Overall, we conclude that Cd activates the CaM/CaMK/NFATc1 pathway and regulates osteoclast differentiation by increasing intracellular calcium concentration. Our data provide new insights into the mechanisms underlying osteoclast differentiation following Cd exposure. This study provides a theoretical basis for future investigations into the therapeutic application of CaMK inhibitors in osteoporosis induced by Cd exposure., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Salmonella enterica serovar Typhimurium (S. Typhimurium) is a facultative intracellular pathogen that damages gastrointestinal tissue and causes severe diarrhoea. The mechanisms by which Salmonella disrupts epithelial barrier and increases the paracellular permeability are incompletely understood. Our present study aims to determine the role of Gli1, a transcription factor activated in the sonic hedgehog (Shh) pathway, in decreasing the levels of apical junction proteins in a Salmonella-infected human colonic epithelial cancer cell line, Caco-2, and in the intestinal tissue of Salmonella-infected mice. Here, we report that S. Typhimurium increased the mRNA and protein levels of Gli1 and Snail, a downstream transcription factor that plays an important role in the epithelial-to-mesenchymal transition (EMT). S. Typhimurium also decreased the levels of E-cadherin and three tight junction proteins (ZO-1, claudin-1, and occludin). Gli1 siRNA and GANT61, a Gli1-specific inhibitor, blocked S. Typhimurium-induced Snail expression, restored the levels of E-cadherin and tight junction proteins, and prevented S. Typhimurium-increased paracellular permeability. Further study showed that Gli1 was cross-activated by the MAP and PI-3 kinase pathways. S. Typhimurium devoid of sopB, an effector of the Type 3 secretion system (T3SS) responsible for AKT activation, was unable to induce Snail expression and to decrease the expression of apical junction proteins. Our study uncovered a novel role of Gli1 in mediating the Salmonella-induced disruption of the intestinal epithelial barrier., (© 2020 John Wiley & Sons Ltd.)

Motivation: Single-cell RNA-sequencing (scRNA-seq) has become an important tool to unravel cellular heterogeneity, discover new cell (sub)types, and understand cell development at single-cell resolution. However, one major challenge to scRNA-seq research is the presence of 'drop-out' events, which usually is due to extremely low mRNA input or the stochastic nature of gene expression. In this article, we present a novel single-cell RNA-seq drop-out correction (scDoc) method, imputing drop-out events by borrowing information for the same gene from highly similar cells., Results: scDoc is the first method that directly involves drop-out information to accounting for cell-to-cell similarity estimation, which is crucial in scRNA-seq drop-out imputation but has not been appropriately examined. We evaluated the performance of scDoc using both simulated data and real scRNA-seq studies. Results show that scDoc outperforms the existing imputation methods in reference to data visualization, cell subpopulation identification and differential expression detection in scRNA-seq data., Availability and Implementation: R code is available at https://github.com/anlingUA/scDoc., Supplementary Information: Supplementary data are available at Bioinformatics online., (© The Author(s) 2020. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Background: When conservative treatments do not work, TKA may be the best option for patients with knee osteoarthritis, although a relatively large proportion of individuals do not have clinically important improvement after TKA. Evidence also suggests that women are less likely to benefit from TKA than men, but the reasons are unclear. Widespread pain disproportionately affects women and has been associated with worse outcomes after joint arthroplasty, yet it is unknown if the effect of widespread pain on TKA outcomes differs by patient gender., Questions/purposes: (1) Does the association between widespread pain and no clinically important improvement in osteoarthritis-related pain and disability 2 years after TKA differ between men and women? (2) Does the use of pain medications 2 years after TKA differ between those with widespread pain and those without widespread pain before surgery?, Methods: Osteoarthritis Initiative (https://nda.nih.gov/oai/) study participants were followed annually from March 2005 until October 2015. Participants who underwent TKA up to the 7-year follow-up visit with pain/disability assessment at the protocol-planned visit before TKA and at the second planned annual visit after surgery were included in the analysis. Among 4796 study participants, 391 had a confirmed TKA, including 315 with pain/disability assessment at the protocol-planned visit before TKA. Overall, 95% of participants (298) had the required follow-up assessment; 5% (17) did not have follow-up data. Widespread pain was defined based on the modified American College of Rheumatology criteria. Symptoms were assessed using the WOMAC pain (range 0 to 20; higher score, more pain) and disability (range 0 to 68; higher score, more disability) scores, and the Knee Injury and Osteoarthritis Outcome Score for pain (range 0 to 100; higher score, less pain). Improvements in pain and disability were classified based on improvement from established clinically important differences (decrease in WOMAC pain ≥ 1.5; decrease in WOMAC disability ≥ 6.0; increase in Knee Injury and Osteoarthritis Outcome Score for pain ≥ 9). At baseline, more women presented with widespread pain than men (45% [84 of 184] versus 32% [36 of 114]). Probability and the relative risk (RR) of no clinically important improvement were estimated using a logistic regression analysis in which participants with widespread pain and those without were compared. The analyses were done for men and women separately, then adjusted for depression and baseline outcome scores., Results: Among women, preoperative widespread pain was associated with an increased risk of no clinically important improvement 2 years after TKA, based on WOMAC pain scores (13.5% versus 4.6%; RR 2.93 [95% CI 1.18 to 7.30]; p = 0.02) and the Knee Injury and Osteoarthritis Outcome Score for pain (16.5% versus 4.9%; RR 3.39 [95% CI 1.34 to 8.59]; p = 0.02). Given the lower and upper limits of the confidence intervals, our data are compatible with a broad range of disparate associations between widespread pain and lack of clinically important improvement in WOMAC pain scores (RR 0.77 [95% CI 0.22 to 2.70]; p = 0.68) and the Knee Injury and Osteoarthritis Outcome Score for pain (RR 1.37 [95% CI 0.47 to 4.00]; p = 0.57) among men, as well as clinically important improvement in WOMAC disability scores among men (RR 0.72 [95% CI 0.20 to 2.55]; p = 0.61) and women (RR 1.98 [95% CI 0.92 to 4.26]; p = 0.08). Participants presenting with widespread pain before TKA were more likely than those without widespread pain to use medication for symptoms of knee osteoarthritis most days for at least 1 month 2 years after TKA (51% [61 of 120] versus 32% [57 of 178]; mean difference, 18.8 [95% CI 7.3 to 30.1]; p < 0.01)., Conclusions: Widespread pain before TKA was associated with an increased risk of no clinically important improvement in knee pain 2 years postoperatively among women. Because of the small number of men with widespread pain in the sample, the results for men were inconclusive. In clinical practice, screening TKA candidates for widespread pain may be useful, and expectations of surgical outcomes may need to be tempered if patients have a concurrent diagnosis of widespread pain. Future studies should include more men with widespread pain and investigate if treatment of widespread pain before or concurrent with TKA surgery may improve surgical outcomes., Level of Evidence: Level III, therapeutic study.

Data normalization is vital to single-cell sequencing, addressing limitations presented by low input material and various forms of bias or noise present in the sequencing process. Several such normalization methods exist, some of which rely on spike-in genes, molecules added in known quantities to serve as a basis for a normalization model. Depending on available information and the type of data, some methods may express certain advantages over others. We compare the effectiveness of seven available normalization methods designed specifically for single-cell sequencing using two real data sets containing spike-in genes and one simulation study. Additionally, we test those methods not dependent on spike-in genes using a real data set with three distinct cell-cycle states and a real data set under the 10X Genomics GemCode platform with multiple cell types represented. We demonstrate the differences in effectiveness for the featured methods using visualization and classification assessment and conclude which methods are preferable for normalizing a certain type of data for further downstream analysis, such as classification or differential analysis. The comparison in computational time for all methods is addressed as well., (Copyright © 2020 Lytal, Ran and An.)

Human SCGB1A1 protein has been shown to be significantly reduced in BAL, sputum, and serum from humans with asthma as compared with healthy individuals. However, the mechanism of this reduction and its functional impact have not been entirely elucidated. By mining online datasets, we found that the mRNA of SCGB1A1 was significantly repressed in brushed human airway epithelial cells from individuals with asthma, and this repression appeared to be associated with reduced expression of FOXA2. Consistently, both Scgb1A1 and FoxA2 were downregulated in an ovalbumin-induced mouse model of asthma. Furthermore, compared with wild-type mice, Scgb1a1 knockout mice had increased airway hyperreactivity and inflammation when they were exposed to ovalbumin, confirming the antiinflammatory role of Scgb1a1 in protection against asthma phenotypes. To search for potential asthma-related stimuli of SCGB1A1 repression, we tested T-helper cell type 2 cytokines. Both IL-4 and IL-13 repressed epithelial expression of SCGB1A1 and FOXA2. Importantly, infection of epithelial cells with human rhinovirus similarly reduced expression of these two genes, which suggests that FOXA2 may be the common regulator of SCGB1A1. To establish the causal role of reduced FOXA2 in SCGB1A1 repression, we demonstrated that FOXA2 was required for SCGB1A1 expression at baseline. FOXA2 overexpression was sufficient to drive promoter activity and expression of SCGB1A1 and was also able to restore the repressed SCGB1A1 expression in IL-13-treated or rhinovirus-infected cells. Taken together, these findings suggest that low levels of epithelial SCGB1A1 in asthma are caused by reduced FOXA2 expression.

Ca 2+ is an important ion in various intracellular metabolic pathways. Endoplasmic reticulum (ER) is a major intracellular calcium store and ER calcium homeostasis plays a key part in the regulation of apoptosis. We have previously shown that Cadmium (Cd) induces apoptosis in osteoblasts (OBs), accompany by increased cytoplasmic calcium. As the role of calcium in OBs apoptosis induced by Cd has not been clarified we investigated the effects of Cd exposure in rat OBs on intracellular Ca 2+ , CaMKII phosphorylation, and the pathways implicated in inducing apoptosis. The results showed that cadmium(Cd) induced elevation of intracellular Ca 2+ ([Ca 2+ ] i ) in OBs by the release of Ca 2+ from ER and the inflow of Ca 2+ from the extracellular matrix. Cd induced [Ca 2+ ] i elevation and phosphorylation of CaMKII which might be involved in activation of MAPKs and participated in Cd-induced mitochondrial apoptosis through the alteration of the ratio of Bax/Bcl-2 expression. Meanwhile, CaMKII phosphorylation activated unfolded protein response (UPR) during cadmium treatment and could enable the ER apoptosis pathway through the activation of caspase-12. These results indicated that CaMKII plays an important role in Cd induced ER apoptosis and MAPK activation. Our data provide new insights into the mechanisms underlying apoptosis in OBs following Cd exposure. This provides a theoretical basis for future investigations into the clinical therapeutic application of CaMKⅡ inhibitors in osteoporosis induced by Cd exposure., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Early endosperm development presents a unique system in which to uncover epigenetic regulatory mechanisms because the contributing maternal and paternal genomes possess differential epigenetic modifications. In Arabidopsis ( Arabidopsis thaliana ), the initiation of endosperm coenocytic growth upon fertilization and the transition to endosperm cellularization are regulated by the FERTILIZATION-INDEPENDENT SEED (FIS)-Polycomb Repressive Complex 2 (PRC2), a putative H3K27 methyltransferase. Here, we address the possible role of the FIS-PRC2 complex in regulating the type I MADS-box gene family, which has been shown previously to regulate early endosperm development. We show that a subclass of type I MADS-box genes (C2 genes) was expressed in distinct domains of the coenocytic endosperm in wild-type seeds. Furthermore, the C2 genes were mostly up-regulated biallelically during the extended coenocytic phase of endosperm development in the FIS-PRC2 mutant background. Using allele-specific expression analysis, we also identified a small subset of C2 genes subjected to FIS-PRC2-dependent maternal or FIS-PRC2-independent paternal imprinting. Our data support a dual role for the FIS-PRC2 complex in the regulation of C2 type I MADS-box genes, as evidenced by a generalized role in the repression of gene expression at both alleles associated with endosperm cellularization and a specialized role in silencing the maternal allele of imprinted genes., (© 2018 American Society of Plant Biologists. All Rights Reserved.)

Objectives: Characterize radiographic worsening in knee osteoarthritis (KOA) by race and sex over 4 years and evaluate the role of established risk factors in observed race/sex differences., Methods: Whites (WHs) (694 males and 929 females) and African-Americans (AAs) (92 males and 167 females) at risk for radiographic KOA were eligible. Cox shared frailty models were used to estimate race and sex group differences in radiographic worsening, defined by Kellgren-Lawrence (K-L) and OARSI joint space narrowing (JSN). Mixed effect models for repeated measures were used to estimate race- and sex-specific mean medial and lateral fixed joint space width (fJSW) over 4 years of follow-up, as well as annual loss of fJSW., Results: Risk of OARSI medial JSN grade worsening was higher among AA males than WH females [HR = 2.28, (95% CI: 1.14-4.57)], though adjustment for KOA risk factors attenuated the association. Compared to WH females, WH males had lower risk of K-L grade worsening [adjusted HR = 0.75 (95% CI: 0.58-0.96)]. Mean baseline medial fJSW (mm) was 6.49 in WH and AA males, 5.42 in WH females, and 5.41 in AA females. Annual change in mean medial fJSW was greater in AA males (-0.19mm/year) than in other subgroups (-0.09 WH males, -0.07 WH females, -0.10 AA females, p < 0.0001). Compared to WHs, AAs had less lateral fJSW at baseline and throughout follow-up., Conclusions: Compared to WHs and AA females, AA males experienced higher risk of medial joint space loss. Controlling for established risk factors attenuated associations between race/sex and disease worsening, suggesting that risk factors such as obesity, history of knee injury, and bony finger joint enlargements largely explain race/sex variations in rates of KOA development and progression., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Rapidly decreasing cost of next-generation sequencing has led to the recent availability of large-scale RNA-seq data, that empowers the analysis of gene expression variability, in addition to gene expression means. In this paper, we present the MDSeq, based on the coefficient of dispersion, to provide robust and computationally efficient analysis of both gene expression means and variability on RNA-seq counts. The MDSeq utilizes a novel reparametrization of the negative binomial to provide flexible generalized linear models (GLMs) on both the mean and dispersion. We address challenges of analyzing large-scale RNA-seq data via several new developments to provide a comprehensive toolset that models technical excess zeros, identifies outliers efficiently, and evaluates differential expressions at biologically interesting levels. We evaluated performances of the MDSeq using simulated data when the ground truths are known. Results suggest that the MDSeq often outperforms current methods for the analysis of gene expression mean and variability. Moreover, the MDSeq is applied in two real RNA-seq studies, in which we identified functionally relevant genes and gene pathways. Specifically, the analysis of gene expression variability with the MDSeq on the GTEx human brain tissue data has identified pathways associated with common neurodegenerative disorders when gene expression means were conserved., (© The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Objective: To determine if severity of osteoarthritis-related knee pain is associated with a willingness to undergo total knee replacement (TKR) and whether this association is confounded or modified by components of socioeconomic status and health care coverage., Methods: Cross-sectional analysis was conducted among 3,530 Osteoarthritis Initiative study participants. Logistic regression models were used to assess the effect of knee pain severity (where 0 = none, 1-3 = mild, 4-7 = moderate, and 8-10 = severe) on willingness to undergo TKR. Stratified analyses were conducted to evaluate whether socioeconomic status and health care coverage modify the effect of knee pain severity on willingness., Results: Participants with severe knee pain, compared to participants without pain, were less willing to undergo TKR (odds ratio [OR] 0.73, 95% confidence interval [95% CI] 0.57-0.93). This association was attenuated when adjusted for age, sex, comorbidity, depression, health insurance coverage, prescription medicine coverage, health care source, education, income, employment, race, and marital status (adjusted OR 0.92, 95% CI 0.68-1.24). The odds of willingness to undergo TKR were significantly lower in those with the highest level of pain, compared to those without pain, among participants without health insurance (adjusted OR 0.08, 95% CI 0.01-0.56), but not among those with health insurance (adjusted OR 1.03, 95% CI 0.73-1.38), when adjusted for demographic, clinical, health care access, and socioeconomic factors (P = 0.015). However, <5% of participants were without health insurance., Conclusion: Among participants without health insurance, severe knee pain was paradoxically associated with less willingness to undergo TKR. Policies that improve access to quality health care may affect patient preferences and increase utilization of TKR surgery among vulnerable populations., (© 2016, American College of Rheumatology.)

The aim of the present study was to investigate the efficacy of a novel surgical intervention, excisional keratectomy combined with focal cryotherapy and amniotic membrane inlay (EKCAI), for the treatment of recalcitrant filamentary fungal keratitis. A retrospective analysis was performed of patients who underwent excisional keratectomy combined with conjunctival flap inlay (EKCFI), EKCAI or therapeutic penetrating keratoplasty (TPK) from January 2006 to January 2011. Recalcitrance was determined as being unresponsive to standard medical antifungal therapy for at ≥1 week. Outcome measures among the three intervention modalities were compared. A total of 128 patients had a follow-up of ≥1 year after the primary intervention. The success rates of interventions at 1-year follow-up were 58.33% in the EKCFI group, 88.37% in the EKCAI group and 93.44% in the TPK group (P<0.0002). The preoperative visual acuity of the three groups were similar (P=0.6458), while the postoperative best-corrected visual acuity (BCVA) of patients without recurrence was significantly different among the three groups 3 months after surgery. The best postoperative BCVA was found in the TPK group, while the worst was in the EKCFI group. In conclusion, EKCAI does not require donor cornea, is straightforward surgically, and has a favorable success rate compared with EKCFI.

Objective: Evaluate the relationship between patient preferences for total knee replacement (TKR) with receipt of TKR, and assess participant characteristics that may influence change in willingness to undergo TKR., Methods: Structured interviews of knee osteoarthritis (OA) patients were conducted. Logistic regression models were conducted to assess the association between baseline willingness and eventual receipt of TKR, adjusted for sociodemographic and clinical variables. Mixed models for repeated measures were used to estimate the effects of sex, race, social support, Δ WOMAC, and orthopedic consult on change in willingness., Results: A total of 589 participants were willing, and 215 participants were unwilling to undergo TKR. Willing participants, compared to others, were more often White (69.4% vs. 48.4%), with more than a high school education (60.8% vs. 47.0%) and employed (39.1% vs. 26.5%). At follow-up, the odds of having TKR were twice as high among those who were willing to have the procedure at baseline, but this was no longer significant when adjusted for demographic variables (adjusted OR = 1.82, 95% CI: 0.89-3.69). Willingness to undergo TKR declined over 2 years. Among those who were willing to undergo TKR at baseline but did not obtain one, only 66.5% were still willing at the 2-year follow-up. This decline was less among those who had a greater increase (>median) in WOMAC disability (adjusted Δ = -0.34, 95% CI: -0.47 to -0.20) than those who had minimal change in their WOMAC disability (p = 0.08). The decline in willingness was also less among those who had seen an orthopedic surgeon (adjusted Δ = -0.32, 95% CI: -0.46 to -0.17) than those who did not (p = 0.05)., Conclusions: Preference for TKR was consistent with TKR surgery utilization, but not after controlling for patient demographic characteristics. Willingness to undergo TKR declined over time, but this decrease was mitigated by worsening OA-related disability and by consultation with an orthopedic surgeon., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Objective: To establish the transgenic mouse of cTnT(R141W) gene to make an animal model of dilated cardiomyopathy., Methods: A transgenic plasmid was constructed by inserting the cTnT(R141W) gene driven by the alpha-MHC promoter. The expression level of the gene was determined with Northern blotting. Pathologic changes were observed by light microscopy and transmission electronic microscopy and analyzed with echocardiography. The localization of the mutant human cTnT protein was detected by immunohistochemistry. The hypertrophy markers were analyzed by RT-PCR., Results: Transgenic mice carrying the cTnT(R141W) mutation were established. The cTnT(R141W) was expressed by 1.5- to 2.0-fold that of the endogenous cTnT gene and was showed to assemble in the sarcomere. The transgenic heart exhibited a thinner ventricular wall and an enlarged ventricular chamber. Interstitial fibrosis and the elongated and lysed myofrils were also observed in the transgenic heart tissue. The function on EF%, FS% and movement of the ventricular wall was significantly decreased. The immature death occurred after 4 months of age and the immature death rate was 11.1% before 8 months of age in the cTnT(R141W) mice. The increased NPPB, ACTA1 and decreased ATP2A2 were detected in the transgenic heart., Conclusions: The expression of mutant cTnT(R141W) in the mouse heart caused ventricular chamber enlargement, systolic dysfunction, myocardial hypertrophy, and interstitial fibrosis, suggesting that the cTnT(R141W) gene is a causal factor for DCM and that the cTnT(R141W) transgenic mouse is a useful animal model for the study of human DCM.