Tissue factor binds to and inhibits interferon-α receptor 1 signaling.

Tissue factor (TF), which is a member of the cytokine receptor family, promotes coagulation and coagulation-dependent inflammation. TF also exerts protective effects through unknown mechanisms. Here, we showed that TF bound to interferon-α receptor 1 (IFNAR1) and antagonized its signaling, preventing spontaneous sterile inflammation and maintaining immune homeostasis. Structural modeling and direct binding studies revealed binding of the TF C-terminal fibronectin III domain to IFNAR1, which restricted the expression of interferon-stimulated genes (ISGs). Podocyte-specific loss of TF in mice (PodΔF3) resulted in sterile renal inflammation, characterized by JAK/STAT signaling, proinflammatory cytokine expression, disrupted immune homeostasis, and glomerulopathy. Inhibiting IFNAR1 signaling or loss of Ifnar1 expression in podocytes attenuated these effects in PodΔF3 mice. As a heteromer, TF and IFNAR1 were both inactive, while dissociation of the TF-IFNAR1 heteromer promoted TF activity and IFNAR1 signaling. These data suggest that the TF-IFNAR1 heteromer is a molecular switch that controls thrombo-inflammation.

Activated protein C modulates T-cell metabolism and epigenetic FOXP3 induction via α-ketoglutarate.

A direct regulation of adaptive immunity by the coagulation protease activated protein C (aPC) has recently been established. Preincubation of T cells with aPC for 1 hour before transplantation increases FOXP3+ regulatory T cells (Tregs) and reduces acute graft-versus-host disease (aGVHD) in mice, but the underlying mechanism remains unknown. Because cellular metabolism modulates epigenetic gene regulation and plasticity in T cells, we hypothesized that aPC promotes FOXP3+ expression by altering T-cell metabolism. To this end, T-cell differentiation was assessed in vitro using mixed lymphocyte reaction or plate-bound α-CD3/CD28 stimulation, and ex vivo using T cells isolated from mice with aGVHD without and with aPC preincubation, or analyses of mice with high plasma aPC levels. In stimulated CD4+CD25- cells, aPC induces FOXP3 expression while reducing expression of T helper type 1 cell markers. Increased FOXP3 expression is associated with altered epigenetic markers (reduced 5-methylcytosine and H3K27me3) and reduced Foxp3 promoter methylation and activity. These changes are linked to metabolic quiescence, decreased glucose and glutamine uptake, decreased mitochondrial metabolism (reduced tricarboxylic acid metabolites and mitochondrial membrane potential), and decreased intracellular glutamine and α-ketoglutarate levels. In mice with high aPC plasma levels, T-cell subpopulations in the thymus are not altered, reflecting normal T-cell development, whereas FOXP3 expression in splenic T cells is reduced. Glutamine and α-ketoglutarate substitution reverse aPC-mediated FOXP3+ induction and abolish aPC-mediated suppression of allogeneic T-cell stimulation. These findings show that aPC modulates cellular metabolism in T cells, reducing glutamine and α-ketoglutarate levels, which results in altered epigenetic markers, Foxp3 promoter demethylation and induction of FOXP3 expression, thus favoring a Treg-like phenotype.

Correction: Gupta et al. Neutrophil Extracellular Traps Promote NLRP3 Inflammasome Activation and Glomerular Endothelial Dysfunction in Diabetic Kidney Disease. Nutrients 2022, 14, 2965.

Error in Figure [...].

Activated Protein C Ameliorates Tubular Mitochondrial Reactive Oxygen Species and Inflammation in Diabetic Kidney Disease.

Diabetic kidney disease (DKD) is an emerging pandemic, paralleling the worldwide increase in obesity and diabetes mellitus. DKD is now the most frequent cause of end-stage renal disease and is associated with an excessive risk of cardiovascular morbidity and mortality. DKD is a consequence of systemic endothelial dysfunction. The endothelial-dependent cytoprotective coagulation protease activated protein C (aPC) ameliorates glomerular damage in DKD, in part by reducing mitochondrial ROS generation in glomerular cells. Whether aPC reduces mitochondrial ROS generation in the tubular compartment remains unknown. Here, we conducted expression profiling of kidneys in diabetic mice (wild-type and mice with increased plasma levels of aPC, APChigh mice). The top induced pathways were related to metabolism and in particular to oxidoreductase activity. In tubular cells, aPC maintained the expression of genes related to the electron transport chain, PGC1-α expression, and mitochondrial mass. These effects were associated with reduced mitochondrial ROS generation. Likewise, NLRP3 inflammasome activation and sterile inflammation, which are known to be linked to excess ROS generation in DKD, were reduced in diabetic APChigh mice. Thus, aPC reduces mitochondrial ROS generation in tubular cells and dampens the associated renal sterile inflammation. These studies support approaches harnessing the cytoprotective effects of aPC in DKD.

ER-Stress and Senescence Coordinately Promote Endothelial Barrier Dysfunction in Diabetes-Induced Atherosclerosis.

Diabetes mellitus is hallmarked by accelerated atherosclerosis, a major cause of mortality among patients with diabetes. Efficient therapies for diabetes-associated atherosclerosis are absent. Accelerated atherosclerosis in diabetic patients is associated with reduced endothelial thrombomodulin (TM) expression and impaired activated protein C (aPC) generation. Here, we directly compared the effects of high glucose and oxidized LDL, revealing that high glucose induced more pronounced responses in regard to maladaptive unfolded protein response (UPR), senescence, and vascular endothelial cell barrier disruption. Ex vivo, diabetic ApoE-/- mice displayed increased levels of senescence and UPR markers within atherosclerotic lesions compared with nondiabetic ApoE-/- mice. Activated protein C pretreatment maintained barrier permeability and prevented glucose-induced expression of senescence and UPR markers in vitro. These data suggest that high glucose-induced maladaptive UPR and associated senescence promote vascular endothelial cell dysfunction, which-however-can be reversed by aPC. Taken together, current data suggest that reversal of glucose-induced vascular endothelial cell dysfunction is feasible.

Podocyte-specific Nlrp3 inflammasome activation promotes diabetic kidney disease.

Efficient therapies for diabetic kidney disease (DKD), now the leading cause of kidney failure, are lacking. One hallmark of DKD is sterile inflammation (inflammation in absence of microorganisms), but the underlying molecular mechanisms remain poorly understood. The NLRP3 inflammasome (innate immune system receptors and sensors regulating activation of caspase-1) is a mechanism of sterile inflammation known to be activated by metabolic stimuli and reactive metabolites associated with DKD, including inflammasome activation in podocytes. However, whether NLRP3 inflammasome activation in podocytes contributes to sterile inflammation and glomerular damage in DKD remains unknown. Here, we found that kidney damage, as reflected by increased albuminuria, glomerular mesangial expansion and glomerular basement membrane thickness was aggravated in hyperglycemic mice with podocyte-specific expression of an Nlrp3 gain-of-function mutant (Nlrp3A350V). In contrast, hyperglycemic mice with podocyte-specific Nlrp3 or Caspase-1 deficiency showed protection against DKD. Intriguingly, podocyte-specific Nlrp3 deficiency was fully protective, while podocyte-specific caspase-1 deficiency was only partially protective. Podocyte-specific Nlrp3, but not caspase-1 deficiency, maintained glomerular autophagy in hyperglycemic mice, suggesting that podocyte Nlrp3 exerts both canonical and non-canonical effects. Thus, podocyte NLRP3 inflammasome activation is both sufficient and required for DKD and supports the concept that podocytes exert some immune cell-like functions. Hence, as podocyte NLRP3 exerts non-canonical and canonical effects, targeting NLRP3 may be a promising therapeutic approach in DKD.

Neutrophil Extracellular Traps Promote NLRP3 Inflammasome Activation and Glomerular Endothelial Dysfunction in Diabetic Kidney Disease.

Diabetes mellitus is a metabolic disease largely due to lifestyle and nutritional imbalance, resulting in insulin resistance, hyperglycemia and vascular complications. Diabetic kidney disease (DKD) is a major cause of end-stage renal failure contributing to morbidity and mortality worldwide. Therapeutic options to prevent or reverse DKD progression are limited. Endothelial and glomerular filtration barrier (GFB) dysfunction and sterile inflammation are associated with DKD. Neutrophil extracellular traps (NETs), originally identified as an innate immune mechanism to combat infection, have been implicated in sterile inflammatory responses in non-communicable diseases. However, the contribution of NETs in DKD remains unknown. Here, we show that biomarkers of NETs are increased in diabetic mice and diabetic patients and that these changes correlate with DKD severity. Mechanistically, NETs promote NLRP3 inflammasome activation and glomerular endothelial dysfunction under high glucose stress in vitro and in vivo. Inhibition of NETs (PAD4 inhibitor) ameliorate endothelial dysfunction and renal injury in DKD. Taken together, NET-induced sterile inflammation promotes diabetes-associated endothelial dysfunction, identifying a new pathomechanism contributing to DKD. Inhibition of NETs may be a promising therapeutic strategy in DKD.

Hypercoagulability Impairs Plaque Stability in Diabetes-Induced Atherosclerosis.

Diabetes mellitus, which is largely driven by nutritional and behavioral factors, is characterized by accelerated atherosclerosis with impaired plaque stability. Atherosclerosis and associated complications are the major cause of mortality in diabetic patients. Efficient therapeutic concepts for diabetes-associated atherosclerosis are lacking. Atherosclerosis among diabetic patients is associated with reduced endothelial thrombomodulin (TM) expression and impaired activated protein C (aPC) generation. Here, we demonstrate that atherosclerotic plaque stability is reduced in hyperglycemic mice expressing dysfunctional TM (TMPro/Pro mice), which have a pro-coagulant phenotype due to impaired thrombin inhibition and markedly reduced aPC generation. The vessel lumen and plaque size of atherosclerotic lesions in the truncus brachiocephalic were decreased in diabetic TMPro/Pro ApoE-/- mice compared to diabetic ApoE-/- mice. While lipid accumulation in lesions of diabetic TMPro/Pro ApoE-/- mice was lower than that in diabetic ApoE-/- mice, morphometric analyses revealed more prominent signs of instable plaques, such as a larger necrotic core area and decreased fibrous cap thickness in diabetic TMPro/Pro ApoE-/- mice. Congruently, more macrophages and fewer smooth muscle cells were observed within lesions of diabetic TMPro/Pro ApoE-/- mice. Thus, impaired TM function reduces plaque stability, a characteristic of hyperglycemia-associated plaques, thus suggesting the crucial role of impaired TM function in mediating diabetes-associated atherosclerosis.

Novel Nongenetic Murine Model of Hyperglycemia and Hyperlipidemia-Associated Aggravated Atherosclerosis.

Objective: Atherosclerosis, the main pathology underlying cardiovascular diseases is accelerated in diabetic patients. Genetic mouse models require breeding efforts which are time-consuming and costly. Our aim was to establish a new nongenetic model of inducible metabolic risk factors that mimics hyperlipidemia, hyperglycemia, or both and allows the detection of phenotypic differences dependent on the metabolic stressor(s). Methods and Results: Wild-type mice were injected with gain-of-function PCSK9D377Y (proprotein convertase subtilisin/kexin type 9) mutant adeno-associated viral particles (AAV) and streptozotocin and fed either a high-fat diet (HFD) for 12 or 20 weeks or a high-cholesterol/high-fat diet (Paigen diet, PD) for 8 weeks. To evaluate atherosclerosis, two different vascular sites (aortic sinus and the truncus of the brachiocephalic artery) were examined in the mice. Combined hyperlipidemic and hyperglycemic (HGHCi) mice fed a HFD or PD displayed characteristic features of aggravated atherosclerosis when compared to hyperlipidemia (HCi HFD or PD) mice alone. Atherosclerotic plaques of HGHCi HFD animals were larger, showed a less stable phenotype (measured by the increased necrotic core area, reduced fibrous cap thickness, and less α-SMA-positive area) and had more inflammation (increased plasma IL-1ß level, aortic pro-inflammatory gene expression, and MOMA-2-positive cells in the BCA) after 20 weeks of HFD. Differences between the HGHCi and HCi HFD models were confirmed using RNA-seq analysis of aortic tissue, revealing that significantly more genes were dysregulated in mice with combined hyperlipidemia and hyperglycemia than in the hyperlipidemia-only group. The HGHCi-associated genes were related to pathways regulating inflammation (increased Cd68, iNos, and Tnfa expression) and extracellular matrix degradation (Adamts4 and Mmp14). When comparing HFD with PD, the PD aggravated atherosclerosis to a greater extent in mice and showed plaque formation after 8 weeks. Hyperlipidemic and hyperglycemic mice fed a PD (HGHCi PD) showed less collagen (Sirius red) and increased inflammation (CD68-positive cells) within aortic plaques than hyperlipidemic mice (HCi PD). HGHCi-PD mice represent a directly inducible hyperglycemic atherosclerosis model compared with HFD-fed mice, in which atherosclerosis is severe by 8 weeks. Conclusion: We established a nongenetically inducible mouse model allowing comparative analyses of atherosclerosis in HCi and HGHCi conditions and its modification by diet, allowing analyses of multiple metabolic hits in mice.

Speaking Fundamental Frequency of Native Urdu Speakers-A Pilot Study.

OBJECTIVES: This study aimed to find the normative fundamental frequency values for adult native Urdu speakers from Pakistan. The age and gender differences were also examined. METHOD: A total of 71 participants aged between 21 and 50 years old (Men = 34, Women = 37) were recruited from University of the Punjab, Lahore, Pakistan. The participants were further divided into subgroups based on age: Group 1 (21-30 years), Group 2 (31-40 years), Group 3 (41-50 years). At least three voice samples were recorded using Praat in a sound-proof lab for each participant in his/her habitual voice. An average speaking fundamental frequency value was computed for each participant. RESULTS: Two-way ANOVA was employed to investigate age and gender differences in the SF0 values. The findings revealed significant gender differences with females having significantly higher SF0 values as compared to males. The findings were further compared with similar data from other populations with different ethnic backgrounds. CONCLUSION: This study provides baseline fundamental frequency data for Native Urdu speakers. The clinical implications of the findings are also discussed.

Phonological Development in Urdu-Speaking Children: A Systematic Review.

Background Speech and language researchers study phonological acquisition and evaluate the errors that children make to understand this complex process. This information provides clinicians with a scientific reference for better assessment and intervention services to children with articulation/phonological disorders. Urdu is a language spoken by more than 200 million speakers worldwide. However, research on Urdu phonological development is in its infancy. Purpose This systematic review aimed to identify relevant studies and provide a comprehensive review of which aspects of Urdu phonological development have been targeted, along with the reported findings. Method Five phases of the Preferred Reporting Items for Systematic Review and Meta-Analyses guidelines were followed. All retrieved studies published between January 1980 and March 2020 and focusing on Urdu speech sound acquisition and/or phonological processes in typically developing native Urdu speakers aged up to 8 years were included in the review. The final search was conducted on May 4, 2020. Results A total of 873 records were identified from five databases and a manual search. Nine studies fulfilling the inclusion criteria were included in the final review. All studies were published in the English language, five of which were unpublished master's theses and four were peer-reviewed journal articles. Four of these studies focused on consonant acquisition, whereas five explored phonological processes. No study focused on the acquisition of Urdu vowels, diphthongs, or consonant clusters. These shortlisted studies were reviewed in detail to determine participants' demographic characteristics, focused areas of phonological acquisition, data elicitation methods and contexts, transcription systems, and major findings. Conclusion This study provides a comprehensive review of the available literature on Urdu phonological development and highlights areas for future research.

CHOP-ASO Ameliorates Glomerular and Tubular Damage on Top of ACE Inhibition in Diabetic Kidney Disease.

BACKGROUND: Maladaptive endoplasmic reticulum stress signaling in diabetic kidney disease (DKD) is linked to increased glomerular and tubular expression of the cell-death-promoting transcription factor C/EBP homologous protein (CHOP). Here, we determined whether locked nucleic acid (LNA)-modified antisense oligonucleotides (ASOs) targeting CHOP ameliorate experimental DKD. METHODS: We determined the efficacy of CHOP-ASO in the early and late stages of experimental DKD (in 8- or 16-week-old db/db mice, respectively) alone or with an angiotensin-converting enzyme inhibitor (ACEi), after an in vivo dose-escalation study. We used renal functional parameters and morphologic analyses to assess the effect of CHOP-ASO and renal gene-expression profiling to identify differentially regulated genes and pathways. Several human CHOP-ASOs were tested in hyperglycemia-exposed human kidney cells. RESULTS: CHOP-ASOs efficiently reduced renal CHOP expression in diabetic mice and reduced markers of DKD at the early and late stages. Early combined intervention (CHOP-ASO and ACEi) efficiently prevented interstitial damage. At the later timepoint, the combined treatment reduced indices of both glomerular and tubular damage more efficiently than either intervention alone. CHOP-ASO affected a significantly larger number of genes and disease pathways, including reduced sodium-glucose transport protein 2 (Slc5a2) and PROM1 (CD133). Human CHOP-ASOs efficiently reduced glucose-induced CHOP and prevented death of human kidney cells in vitro . CONCLUSIONS: The ASO-based approach efficiently reduced renal CHOP expression in a diabetic mouse model, providing an additional benefit to an ACEi, particularly at later timepoints. These studies demonstrate that ASO-based therapies efficiently reduce maladaptive CHOP expression and ameliorate experimental DKD.

Corrigendum to "Data for the narrative skills of Urdu-speaking Preschoolers" [Data in Brief, 34 (2021) 106,737].

[This corrects the article DOI: 10.1016/j.dib.2021.106737.].

Data for the narrative skills of Urdu-speaking preschoolers.

A total of 80 participants were recruited from three private middle SES schools of Rahim Yar Khan, Pakistan to explore the narrative skills development in Urdu-speaking preschoolers aged between 4-and-5 years. Data collection was completed using a two-stage sampling technique (convenience and purposive). After obtaining parental consent, the participants were screened for their intellectual functioning. Three personal narrative samples, using conversational maps, were collected from the participants who passed the screening. The narrative data were analysed on both macro and microstructural levels by using high point analysis and use of cohesions (conjunctive and referential), mean length of utterances in words (MLUw), the total number of words (TNW), and the number of different words (NDW). The data presented here include age and gender of participants, their scores on the verbal IQ test, the scores obtained on macro-and microstructural levels. The results based on differences and relationships of the data obtained are published somewhere else [1]. The data can be reused by comparing the obtained figures with the adult population, children from different age bands, children with different developmental disorders, and at cross-linguistic levels as narratives are said to be significantly influenced by cultures.

Methanolic extract of Citrullus colocynthis suppresses growth and proliferation of breast cancer cells through regulation of cell cycle.

Breast cancer is a major cause of cancer related deaths in women worldwide. Available treatments pose serious limitations such as systemic toxicity, metastasis, tumor recurrence, off-target effects, and drug resistance. In recent years, phytochemicals such as secondary metabolites due to their effective anticancer potential at very low concentration have gained attention. Aim of the study was to evaluate anticancer potential of Citrullus colocynthis and its possible molecular targets on MCF-7, a human breast cancer cell line. Methanolic extract of leaves was prepared and fractionated by solvents (n-hexane, chloroform, ethyl acetate and n-butanol) with increasing polarity. Bioassays and gene expression regulation was conducted to evaluate the anticancer activity, proliferation rate and cell cycle regulation of breast cancer cells treated with extract and its fractions, separately. Results showed a significant anticancer activity of methanolic extract of C. colocynthis and two of its fractions prepared with chloroform and ethyl acetate. Bioassays depicted significant decrease in proliferation and growth potential along with cell cycle arrest of treated cells compared to control untreated cells. Expression regulation of genes further confirmed the cell cycle arrest through significant upregulation of cyclin-CDK inhibitors (p21 and p27) and cell cycle checkpoint regulators (HUS1, RAD1, ATM) followed by downregulation of downstream cell cycle progression genes (Cyclin A, Cyclin E, CDK2). It is concluded that C. colocynthis arrests cell cycle in human breast cancer cells through expression regulation of cyclin-CDK inhibitors and with further research can be proposed for therapeutic interventions.

In Vivo Evaluation of Histopathological Alterations and Trace Metals Estimation of the Small Intestine in Bisphenol A-Intoxicated Rats.

BPA, a ubiquitously used plasticizer, has become one of the contaminants of emerging concern and causes many serious health implications in humans due to multiple exposure pathways. The current study was aimed at investigating the deformities of structures that arise by exposure of the small intestine to BPA through trace elements estimation of tissues as well as the study of serum profile. Two major groups of Wistar rats were established: one control group and the other experimental group, which was further divided into four groups based on dose (10 mg/kg/bodyweight and 25 mg/kg/bodyweight, respectively) and duration of exposure (6 and 12 weeks, respectively). Histological study of the small intestine showed the distorted structures in the experimental groups. The special staining performed illustrated the accumulation of calcium deposits in the small intestinal tissue in treated groups. Trace metals estimation showed a significant increase in the metallic content of sodium and iron and a decrease in the calcium content in the experimental groups (p=0.05). Serum profiling illustrated an increase in total iron-binding capacity and glucose levels and a decrease in the serum total iron level (p=0.05). An increased expression of a proinflammatory cytokine (IFN-α) was observed in the liver. From all these findings, it was inferred that BPA caused many structural alterations in the small intestinal tissue, which further affected its functioning. The calcium deposits seen through special staining affected the motility of the small intestine and caused its dysfunction. It was also induced from serum profiling that BPA affected the homeostasis of iron and glucose and caused its imbalance. Also, as BPA got absorbed from the small intestine and reached the liver via the blood stream, it caused hepatoxicity in the liver and led to increased inflammatory response by IFN-α against the toxicant.

Acoustic Analysis of Normal Voice Patterns in Pakistani Adults.

This study was designed to find out normative acoustic measures of adults in Pakistan. Age and gender differences in these parameters were also studied. The study variables include fundamental frequency, absolute jitter, relative jitter, relative average perturbation, 5-point period perturbation quotient perturbation quotient, jitter average absolute difference between consecutive periods, divided by the average period, shimmer local, shimmer decibel, three-point amplitude perturbation quotient, five-point amplitude perturbation quotient, average absolute difference of consecutive differences between the consecutive periods' amplitudes, and harmonics-to-noise ratio. Participants (N = 71; men = 34; women = 37) were recruited through two-stage sampling; convenience and purposive sampling. Participants were selected from students, staff, and faculty of the University of the Punjab, caretakers of the clients coming for clinical services, and researchers' friends, family, and acquaintances. Gender groups were further divided into three subgroups based on the age bands of 21-30, 31-40, and 41-50 years. After screening for health issues, the voice samples were recorded in a sound-proof acoustic laboratory by using a stand-held microphone and Praat software. Praat was also used for voice analyses. Means and standard deviations were conducted for the participants. Age and gender differences were investigated by using the two-way analysis of variance. Normative values were also measured for men and women between the age bracket of 21 and 50 years. Age differences were not significant for any variable. Gender differences were significant in fundamental frequency, absolute jitter, relative jitter, jitter period perturbation quotient, shimmer five-point amplitude perturbation quotient, and harmonics-to-noise ratio. The interaction between age and gender was significant in all the jitter measures. The interaction showed an increase in all these measures between the ages of 31 and 40 years and a decrease between the ages of 41 and 50 years in women, whereas men showed a reverse effect both in the age ranges of 31-40 and 41-50 years.